Patent Grant
9617268
The technology described herein has not been made with U.S. Government support.
PCT Therapeutics, Inc. and F. Hoffmann-La Roche AG.
The subject matter disclosed was developed and the claimed invention was made by, or on behalf of, one or more parties to a joint research agreement that was in effect on or before the effective filing date of the claimed invention;
the claimed invention was made as a result of activities undertaken within the scope of the joint research agreement; and
the application for patent for the claimed invention discloses or is amended to disclose the names of the parties to the joint research agreement.
Provided herein are compounds, compositions thereof and uses therewith for treating Spinal Muscular Atrophy.
Spinal muscular atrophy (SMA), in its broadest sense, describes a collection of inherited and acquired central nervous system (CNS) diseases characterized by progressive motor neuron loss in the spinal cord and brainstem causing muscle weakness and muscle atrophy. The most common form of SMA is caused by mutations in the Survival Motor Neuron (SMN) gene and manifests over a wide range of severity affecting infants through adults (Crawford and Pardo, Neurobiol. Dis., 1996, 3:97).
Infantile SMA is the most severe form of this neurodegenerative disorder. Symptoms include muscle weakness, poor muscle tone, weak cry, limpness or a tendency to flop, difficulty sucking or swallowing, accumulation of secretions in the lungs or throat, feeding difficulties, and increased susceptibility to respiratory tract infections. The legs tend to be weaker than the arms and developmental milestones, such as lifting the head or sitting up, cannot be reached. In general, the earlier the symptoms appear, the shorter the lifespan. As the motor neuron cells deteriorate, symptoms appear shortly afterward. The severe forms of the disease are fatal and all forms have no known cure. The course of SMA is directly related to the rate of motor neuron cell deterioration and the resulting severity of weakness. Infants with a severe form of SMA frequently succumb to respiratory disease due to weakness in the muscles that support breathing. Children with milder forms of SMA live much longer, although they may need extensive medical support, especially those at the more severe end of the spectrum. The clinical spectrum of SMA disorders has been divided into the following five groups.
(a) Type 0 SMA (In Utero SMA) is the most severe form of the disease and begins before birth. Usually, the first symptom of Type 0 SMA is reduced movement of the fetus that can first be observed between 30 and 36 weeks of pregnancy. After birth, newborns have little movement and difficulties with swallowing and breathing.
(b) Type 1 SMA (Infantile SMA or Werdnig-Hoffmann disease) presents the first symptoms between 0 and 6 months: This type of SMA is also very severe. Patients never achieve the ability to sit, and death usually occurs within the first 2 years without respiratory support.
(c) Type 2 SMA (Intermediate SMA) has an age of onset at 7-18 months. Patients achieve the ability to sit unsupported, but never stand or walk unaided. Prognosis in this group is largely dependent on the degree of respiratory involvement.
(d) Type 3 SMA (Juvenile SMA or Kugelberg-Welander disease) is generally diagnosed after 18 months. Type 3 SMA individuals are able to walk independently at some point during the course of the disease but often become wheelchair-bound during youth or adulthood.
(e) Type 4 SMA (Adult onset SMA). Weakness usually begins in late adolescence in the tongue, hands or feet, then progresses to other areas of the body. The course of adult onset SMA is much slower and has little or no impact on life expectancy.
The SMN gene has been mapped by linkage analysis to a complex region in chromosome 5q. In humans, this region contains an approximately 500 thousand base pairs (kb) inverted duplication resulting in two nearly identical copies of the SMN gene. SMA is caused by an inactivating mutation or deletion of the telomeric copy of the gene (SMN1) in both chromosomes, resulting in the loss of SMN1 gene function. However, all patients retain the centromeric copy of the gene (SMN2), and the copy number of the SMN2 gene in SMA patients generally correlates inversely with the disease severity; i.e., patients with less severe SMA have more copies of SMN2. Nevertheless, SMN2 is unable to compensate completely for the loss of SMN1 function due to alternative splicing of exon 7 caused by a translationally silent C to T mutation in exon 7. As a result, the majority of transcripts produced from SMN2 lack exon 7 (SMN2 Δ7), and encode a truncated Smn protein that has an impaired function and is rapidly degraded.
Smn is thought to play a role in RNA processing and metabolism, having a well characterized function of mediating the assembly of a specific class of RNA-protein complexes termed snRNPs. Smn may have other functions in motor neurons, however its role in preventing the selective degeneration of motor neurons is not well established.
In most cases, SMA is diagnosed based on clinical symptoms and by the presence of at least on copy of the SMN1 gene test. However, in approximately 5% of cases SMA is caused by mutation in genes other than the inactivation of SMN1, some known and others not yet defined. In some cases, when the SMN1 gene test is not feasible or does not show any abnormality, other tests such as an electromyography (EMG) or muscle biopsy may be indicated.
Medical care for SMA patients at present is limited to supportive therapy including respiratory, nutritional and rehabilitation care; there is no drug known to address the cause of the disease. Current treatment for SMA consists of prevention and management of the secondary effects of chronic motor unit loss. The major management issue in Type 1 SMA is the prevention and early treatment of pulmonary problems, which are the cause of death in the majority of the cases. While some infants afflicted with SMA grow to be adults, those with Type 1 SMA have a life expectancy of less than two years.
Several mouse models of SMA have been developed. In particular, the SMNΔ7 model (Le et al., Hum. Mol. Genet., 2005, 14:845) carries both the SMN2 gene and several copies of the SMN2Δ7 cDNA and recapitulates many of the phenotypic features of Type 1 SMA. The SMNΔ7 model can be used for both SMN2 expression studies as well as the evaluation of motor function and survival. The C/C-allele mouse model (Jackson Laboratory strain #008714) provides a less severe SMA disease model, with mice having reduced levels of both SMN2 FL mRNA and Smn protein. The C/C-allele mouse phenotype has the SMN2 gene and a hybrid mSmn1-SMN2 gene that undergoes alternative splicing, but does not have overt muscle weakness. The C/C-allele mouse model is used for SMN2 expression studies.
As a result of improved understanding of the genetic basis for SMA, several strategies for treatment have been explored, but none have yet demonstrated success in the clinic.
Gene replacement of SMN1, using viral delivery vectors, and cell replacement, using differentiated SMN1+/+ stem cells, have demonstrated efficacy in animal models of SMA. More research is needed to determine the safety and immune response and to address the requirement for the initiation of treatment at the neonatal stage before these approaches can be applied to humans.
Correction of alternative splicing of SMN2 in cultured cells has also been achieved using synthetic nucleic acids as therapeutic agents: (i) antisense oligonucleotides that target sequence elements in SMN2 pre-mRNA and shift the outcome of the splicing reaction toward the generation of full length SMN2 mRNA (Passini et al., Sci. Transl. Med., 2011, 3:72ra18; and, Hua et al., Nature, 2011, 478:123) and (ii) trans-splicing RNA molecules that provide a fully functional RNA sequence that replace the mutant fragment during splicing and generate a full length SMN1 mRNA (Coady and Lorson, J Neurosci., 2010, 30:126).
Other approaches under exploration include searching for drugs that increase Smn levels, enhance residual Smn function, or compensate for loss of Smn. Aminoglycosides have been shown to enhance expression of stabilized Smn produced from SMN2 Δ7 mRNA by promoting the translational read-through of the aberrant stop codon, but have poor central nervous system penetration and are toxic after repeated dosing. Chemotherapeutic agents, such as aclarubicin, have been shown to increase Smn in cell culture; however, the toxicity profile of these drugs prohibits long-term use in SMA patients. Some drugs under clinical investigation for the treatment of SMA include transcription activators such as histone deacetylase (“HDAC”) inhibitors (e.g., butyrates, valproic acid, and hydroxyurea), and mRNA stabilizers (mRNA decapping inhibitor RG3039 from Repligen), intended to increase the amount of total RNA transcribed from the SMN2 gene. However, the use of HDAC inhibitors or mRNA stabilizers does not address the underlying cause of SMA and may result in a global increase in transcription and gene expression with potential safety problems in humans.
In an alternative approach, neuroprotective agents such as olesoxime have been chosen for investigation. Such strategies are not aimed at producing functional Smn for the treatment of SMA, but instead are being explored to protect the Smn-deficient motor neurons from neurodegeneration.
A system designed to identify compounds that increase the inclusion of exon 7 of SMN into RNA transcribed from the SMN2 gene and certain benzooxazole and benzoisoxazole compounds identified thereby have been described in International Application PCT/US2009/003238 filed May 27, 2009 (published as International Publication Number WO2009/151546 and United States Publication Number US2011/0086833). A system designed to identify compounds that produce a stabilized Smn protein from SMN2 Δ7 mRNA and certain isoindolinone compounds identified thereby have been described in International Application PCT/US2009/004625 filed Aug. 13, 2009 (published as International Publication Number WO2010/019236 and United States Publication Number US2011/0172284). Each of the foregoing documents is herein incorporated in their entirety and for all purposes.
All other documents referred to herein are incorporated by reference into the present application as though fully set forth herein.
Despite the progress made in understanding the genetic basis and pathophysiology of SMA, there remains a need to identify compounds that alter the course of spinal muscular atrophy, one of the most devastating childhood neurological diseases.
In one aspect, provided herein are compounds of Formula (I):
or a form thereof, wherein: w1, w2, Ra and Rb are as defined herein. In one embodiment, provided herein is a pharmaceutical composition comprising a compound of Formula (I) or a form thereof, and a pharmaceutically acceptable carrier, excipient or diluent. In a specific embodiment, provided herein is a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof for treating spinal muscular atrophy (SMA).
SMA is caused by deletion or mutation of the SMN1 gene, resulting in selective degeneration of Smn-deficient motor neurons. Although human subjects retain several copies of the SMN2 gene, the small amount of functional Smn protein expressed from SMN2 does not fully compensate for the loss of Smn that would have been expressed from the SMN1 gene. The compounds, compositions thereof and uses therewith described herein are based, in part, on the Applicants discovery that a compound of Formula (I) increases the inclusion of exon 7 of SMN2 into mRNA that is transcribed from an SMN2 minigene. The minigene reproduces the alternative splicing reaction of exon 7 of SMN2 which results in the loss of exon 7 in the majority of SMN2 transcripts. Thus, compounds of Formula (I) or a form thereof may be used to modulate inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene. Applicants have also discovered that a compound of Formula (I) increases the inclusion of exon 7 of SMN1 into mRNA that is transcribed from an SMN1 minigene. Thus, compounds of Formula (I) or a form thereof may be used to modulate the inclusion of exon 7 of SMN1 into mRNA that is transcribed from the SMN1 gene.
In a specific embodiment, provided herein are compounds of Formula (I) or a form thereof that may be used to modulate the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene. In another specific embodiment, provided herein are compounds of Formula (I) or a form thereof that may be used to modulate the inclusion of exon 7 of SMN1 into mRNA that is transcribed from the SMN1 gene. In yet another embodiment, provided herein are compounds of Formula (I) or a form thereof that may be used to modulate the inclusion of exon 7 of SMN1 and SMN2 into mRNA that is transcribed from the SMN1 and SMN2 genes, respectively.
In another aspect, provided herein is the use of a compound of Formula (I) or a form thereof for treating SMA. In a specific embodiment, provided herein is a method for treating SMA in a human subject in need thereof, comprising administering to the subject an effective amount of a compound of Formula (I) or a form thereof. The compound of Formula (I) or a form thereof is preferably administered to a human subject in a pharmaceutical composition. In another specific embodiment, provided herein is the use of a compound of Formula (I) for treating SMA, wherein the compound enhances the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene. Without being limited by theory, compounds of Formula (I) enhance inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene and increase levels of Smn protein produced from the SMN2 gene, and thus can be used to treat SMA in a human subject in need thereof.
In another aspect, provided herein are primers and/or probes described below in the Biological Examples (e.g., SMN primers such as SEQ ID NO. 1, 7, 8, 11 or 13, and/or SEQ ID NO. 2, 9 or 12, and/or SMN probes such as a SEQ ID NO. 3 or 10) and the use of those primers and/or probes. In a specific embodiment, provided herein is an isolated nucleotide sequence comprising SEQ ID NOs: 1, 2, 3, 7, 8, 9, 10, 11, 12 or 13. In another specific embodiment, provided herein is an isolated nucleotide sequence consisting essentially of SEQ ID NOs: 1, 2, 3, 7, 8, 9, 10, 11, 12 or 13. In another specific embodiment, provided herein is an isolated nucleotide sequence consisting of SEQ ID NOs: 1, 2, 3, 7, 8, 9, 10, 11, 12 or 13.
In certain embodiments, the amount of mRNA that is transcribed from the SMN1 gene and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 may be used as a biomarker for SMA, such as disclosed herein. In other embodiments, the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 may be used as a biomarker for treating a patient with a compound, such as disclosed herein. In a specific embodiment, the patient is an SMA patient.
In certain embodiments, the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 as well as the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 may be used as biomarkers for treating a patient with a compound, such as disclosed herein. In a specific embodiment, the patient is an SMA patient.
In accordance with these embodiments, an SMN primer(s) and/or an SMN probe described below may be used in assays, such as PCR (e.g., qPCR), rolling circle amplification, and RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR) to assess and/or quantify the amount of mRNA that is transcribed from the SMN1 gene and/or SMN2 gene and does or does not include exon 7 of SMN1 and/or SMN2.
In a specific embodiment, a primer and/or probe described below in the Biological Examples (e.g., SMN primers such as SEQ ID NO. 1, 7, 8, 11 or 13 and/or SEQ ID NO. 2, 9 or 12, and/or SMN probes such as a SEQ ID NO. 3 or 10) is used in an assay, such as RT-PCR, RT-qPCR, endpoint RT-PCR, PCR, qPCR, rolling circle amplification, Northern blot or Southern blot (e.g., an assay such as described below in the Biological Examples), to determine whether a compound (e.g., a compound of Formula (I) or a form thereof) enhances the inclusion of exon 7 of SMN2 into mRNA that is transcribed from an SMN2 gene.
In a specific embodiment, a primer and/or probe described below in the Biological Examples (e.g., SMN primers such as SEQ ID NO. 1, 7, 8, 11 or 13 and/or SEQ ID NO. 2, 9 or 12, and/or SMN probes such as a SEQ ID NO. 3 or 10) is used in an assay, such as RT-PCR, RT-qPCR, endpoint RT-PCR, PCR, qPCR, rolling circle amplification, Northern blot or Southern blot (e.g., an assay such as described below in the Biological Examples), to determine whether a compound (e.g., a compound of Formula (I) or a form thereof) enhances the inclusion of exon 7 of SMN1 into mRNA that is transcribed from an SMN1 gene.
In a specific embodiment, a primer and/or probe described below in the Biological Examples (e.g., SMN primers such as SEQ ID NO. 1, 7, 8, 11 or 13 and/or SEQ ID NO. 2, 9 or 12, and/or SMN probes such as a SEQ ID NO. 3 or 10) is used in an assay, such as RT-PCR, RT-qPCR, endpoint RT-PCR, PCR, qPCR, rolling circle amplification, Northern blot or Southern blot (e.g., an assay such as described below in the Biological Examples), to determine whether a compound (e.g., a compound of Formula (I) or a form thereof) enhances the inclusion of exon 7 of SMN1 and/or SMN2 into mRNA that is transcribed from an SMN1 and/or SMN2 gene.
In another embodiment, a primer and/or probe described below in the Biological Examples (e.g., SMN primers such as SEQ ID NO. 7, 11 or 13 and/or SEQ ID NO. 9 or 12, and/or SMN probes such as a SEQ ID NO. 3 or 10) is used in an assay, such as RT-PCR, RT-qPCR, endpoint RT-PCR, PCR, qPCR, rolling circle amplification, Northern blot or Southern blot (e.g., an assay such as described below in the Biological Examples), to monitor the amount of mRNA that is transcribed from the SMN2 gene and includes exon 7 of SMN2 in a patient sample. In a specific embodiment, the patient is an SMA patient.
In another embodiment, a primer and/or probe described below in the Biological Examples (e.g., SMN primers such as SEQ ID NO. 7, 11 or 13 and/or SEQ ID NO. 9 or 12, and/or SMN probes such as a SEQ ID NO. 3 or 10) is used in an assay, such as RT-PCR, RT-qPCR, endpoint RT-PCR, PCR, qPCR, rolling circle amplification, Northern blot or Southern blot (e.g., an assay such as described below in the Biological Examples), to monitor the amount of mRNA that is transcribed from the SMN1 gene and includes exon 7 of SMN1 in a patient sample. In a specific embodiment, the patient is an SMA patient.
In another embodiment, a primer and/or probe described below in the Biological Examples (e.g., SMN primers such as SEQ ID NO. 7, 11 or 13 and/or SEQ ID NO. 9 or 12, and/or SMN probes such as a SEQ ID NO. 3 or 10) is used in an assay, such as RT-PCR, RT-qPCR, endpoint RT-PCR, PCR, qPCR, rolling circle amplification, Northern blot or Southern blot (e.g., an assay such as described below in the Biological Examples), to monitor the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in a patient sample. In a specific embodiment, the patient is an SMA patient.
In another embodiment, a primer and/or probe described below in the Biological Examples (e.g., SMN primers such as SEQ ID NO. 7, 8, 11 or 13 and/or SEQ ID NO. 9 or 12, and/or SMN probes such as a SEQ ID NO. 3 or 10) is used in an assay, such as RT-PCR, RT-qPCR, endpoint RT-PCR, PCR, qPCR, rolling circle amplification, Northern blot or Southern blot (e.g., an assay such as described below in the Biological Examples), to monitor a patient's response to a compound (e.g., a compound of Formula (I) or a form thereof). In a specific embodiment, the patient is an SMA patient.
In another embodiment, provided herein is a method for determining whether a compound (e.g., a compound of Formula (I) disclosed herein) enhances the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene, comprising (a) contacting mRNA that is transcribed from an SMN2 minigene described herein or in International Application PCT/US2009/004625, filed Aug. 13, 2009 (published as International Publication Number WO2010/019236) or United States Publication Number US2011/0172284 in the presence of a compound (e.g., a compound of Formula (I) disclosed herein) with a primer(s) described herein (e.g., SEQ ID NO. 1 and/or 2) along with applicable components for, e.g., RT-PCR, RT-qPCR, PCR, endpoint RT-PCR, qPCR or rolling circle amplification; and (b) detecting the amount of mRNA that is transcribed from the minigene and includes exon 7 of the SMN2, wherein (1) an increase in the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN2 in the presence of the compound relative to the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN2 in the absence of the compound indicates that the compound enhances inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN2 in the presence of the compound relative to the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN2 in the absence of the compound indicates that the compound does not enhance the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene.
In another embodiment, provided herein is a method for determining whether a compound (e.g., a compound of Formula (I) disclosed herein) enhances the inclusion of exon 7 of SMN1 into mRNA that is transcribed from the SMN1 gene, comprising (a) contacting mRNA that is transcribed from an SMN1 minigene described in International Application PCT/US2009/004625, filed Aug. 13, 2009 (published as International Publication Number WO2010/019236) or United States Publication Number US2011/0172284 in the presence of a compound (e.g., a compound of Formula (I) disclosed herein) with a primer(s) described herein (e.g., SEQ ID NO. 1 and/or 2) along with applicable components for, e.g., RT-PCR, RT-qPCR, PCR, endpoint RT-PCR, qPCR or rolling circle amplification; and (b) detecting the amount of mRNA that is transcribed from the minigene and includes exon 7 of the SMN1, wherein (1) an increase in the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN1 in the presence of the compound relative to the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN1 in the absence of the compound indicates that the compound enhances inclusion of exon 7 of SMN1 into mRNA that is transcribed from the SMN1 gene; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN1 in the presence of the compound relative to the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN1 in the absence of the compound indicates that the compound does not enhance the inclusion of exon 7 of SMN1 into mRNA that is transcribed from the SMN1 gene.
In another embodiment, provided herein is a method for determining whether a compound (e.g., a compound of Formula (I) disclosed herein) enhances the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene, comprising (a) contacting mRNA that is transcribed from an SMN2 minigene described herein or in International Application PCT/US2009/004625, filed Aug. 13, 2009 (published as International Publication Number WO2010/019236) or United States Publication Number US2011/0172284 in the presence of a compound (e.g., a compound of Formula (I) disclosed herein) with a probe described herein (e.g., SEQ ID NO. 3 or 10) along with applicable components for, e.g., RT-PCR, RT-qPCR, endpoint RT-PCR, PCR, qPCR, rolling circle amplification and, as applicable, Northern blot or Southern blot; and (b) detecting the amount of mRNA that is transcribed from the minigene and includes exon 7 of the SMN2, wherein (1) an increase in the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN2 in the presence of the compound relative to the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN2 in the absence of the compound indicates that the compound enhances inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN2 in the presence of the compound relative to the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN2 in the absence of the compound indicates that the compound does not enhance the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene.
In another embodiment, provided herein is a method for determining whether a compound (e.g., a compound of Formula (I) disclosed herein) enhances the inclusion of exon 7 of SMN1 into mRNA that is transcribed from the SMN1 gene, comprising (a) contacting mRNA that is transcribed from an SMN1 minigene described in International Application PCT/US2009/004625, filed Aug. 13, 2009 (published as International Publication Number WO2010/019236) or United States Publication Number US2011/0172284 in the presence of a compound (e.g., a compound of Formula (I) disclosed herein) with a probe described herein (e.g., SEQ ID NO. 3 or 10) along with applicable components for, e.g., RT-PCR, RT-qPCR, endpoint RT-PCR, PCR, qPCR, rolling circle amplification and, as applicable, Northern blot or Southern blot; and (b) detecting the amount of mRNA that is transcribed from the minigene and includes exon 7 of the SMN1, wherein (1) an increase in the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN1 in the presence of the compound relative to the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN1 in the absence of the compound indicates that the compound enhances inclusion of exon 7 of SMN1 into mRNA that is transcribed from the SMN1 gene; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN1 in the presence of the compound relative to the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN1 in the absence of the compound indicates that the compound does not enhance the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene.
In another embodiment, provided herein is a method for determining whether a compound (e.g., a compound of Formula (I) disclosed herein) enhances the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene, comprising (a) contacting mRNA that is transcribed from an SMN2 minigene described herein or in International Application PCT/US2009/004625, filed Aug. 13, 2009 (published as International Publication Number WO2010/019236) or United States Publication Number US2011/0172284 in the presence of a compound (e.g., a compound of Formula (I) disclosed herein) with a primer(s) (e.g., SEQ ID NO. 1 or 2) and/or a probe described herein (e.g., SEQ ID NO. 3 or 10) along with applicable components for, e.g, RT-PCR, RT-qPCR, endpoint RT-PCR, PCR, qPCR, rolling circle amplification and, as applicable, Northern blot or Southern blot; and (b) detecting the amount of mRNA that is transcribed from the minigene and includes exon 7 of the SMN2, wherein (1) an increase in the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN2 in the presence of the compound relative to the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN2 in the absence of the compound indicates that the compound enhances inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN2 in the presence of the compound relative to the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN2 in the absence of the compound indicates that the compound does not enhance the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene.
In another embodiment, provided herein is a method for determining whether a compound (e.g., a compound of Formula (I) disclosed herein) enhances the inclusion of exon 7 of SMN1 into mRNA that is transcribed from the SMN1 gene, comprising (a) contacting mRNA that is transcribed from an SMN1 minigene described in International Application PCT/US2009/004625, filed Aug. 13, 2009 (published as International Publication Number WO2010/019236) or United States Publication Number US2011/0172284 in the presence of a compound (e.g., a compound of Formula (I) disclosed herein) with a primer(s) (e.g., SEQ ID NO. 1 or 2) and/or a probe described herein (e.g., SEQ ID NO. 3 or 10) along with applicable components for, e.g, RT-PCR, RT-qPCR, endpoint RT-PCR, PCR, qPCR, rolling circle amplification and, as applicable, Northern blot or Southern blot; and (b) detecting the amount of mRNA that is transcribed from the minigene and includes exon 7 of the SMN1, wherein (1) an increase in the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN1 in the presence of the compound relative to the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN1 in the absence of the compound indicates that the compound enhances inclusion of exon 7 of SMN1 into mRNA that is transcribed from the SMN1 gene; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN1 in the presence of the compound relative to the amount of mRNA that is transcribed from the minigene and includes exon 7 of SMN1 in the absence of the compound indicates that the compound does not enhance the inclusion of exon 7 of SMN1 into mRNA that is transcribed from the SMN1 gene.
In another aspect, provided herein are kits comprising a primer and/or probe described below in the Biological Examples (e.g., SMN primers such as SEQ ID NO. 1, 7, 8, 11 or 13 and/or SEQ ID NO. 2, 9 or 12, and/or SMN probes such as a SEQ ID NO. 3 or 10) and the use thereof.
8147-8266: 3′UTR (deg).
Provided herein are compounds of Formula (I):
or a form thereof, wherein:
In one embodiment of a compound of Formula (I), R1 is C1-8alkyl, amino, C1-8alkyl-amino, (C1-8alkyl)2-amino, C1-8alkoxy-C1-8alkyl-amino, (C1-8alkoxy-C1-8alkyl)2-amino, (C1-8alkoxy-C1-8alkyl)(C1-8alkyl)amino, amino-C1-8alkyl, C1-8alkyl-amino-C1-8alkyl, (C1-8alkyl)2-amino-C1-8alkyl, C1-8alkoxy-C1-8alkyl-amino-C1-8alkyl, (C1-8alkoxy-C1-8alkyl)2-amino-C1-8alkyl, (C1-8alkoxy-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl, amino-C1-8alkyl-amino, (amino-C1-8alkyl)2-amino, (amino-C1-8alkyl)(C1-8alkyl)amino, C1-8alkyl-amino-C1-8alkyl-amino, (C1-8alkyl-amino-C1-8alkyl)2-amino, (C1-8alkyl-amino-C1-8alkyl)(C1-8alkyl)amino, (C1-8alkyl)2-amino-C1-8alkyl-amino, [(C1-8alkyl)2-amino-C1-8alkyl](C1-8alkyl)amino, amino-C1-8alkoxy, C1-8alkyl-amino-C1-8alkoxy, (C1-8alkyl)2-amino-C1-8alkoxy, C1-8alkoxy-C1-8alkyl-amino-C1-8alkoxy, (C1-8alkoxy-C1-8alkyl)2-amino-C1-8alkoxy, (C1-8alkoxy-C1-8alkyl)(C1-8alkyl)amino-C1-8alkoxy, amino-C2-8alkenyl, C1-8alkyl-amino-C2-8alkenyl, (C1-8alkyl)2-amino-C2-8alkenyl, amino-C2-8alkynyl, C1-8alkyl-amino-C2-8alkynyl, (C1-8alkyl)2-amino-C2-8alkynyl, halo-C1-8alkyl-amino, (halo-C1-8alkyl)2-amino, (halo-C1-8alkyl)(C1-8alkyl)amino, hydroxy-C1-8alkyl, hydroxy-C1-8alkoxy-C1-8alkyl, hydroxy-C1-8alkyl-amino, (hydroxy-C1-8alkyl)2-amino, (hydroxy-C1-8alkyl)(C1-8alkyl)amino, hydroxy-C1-8alkyl-amino-C1-8alkyl, (hydroxy-C1-8alkyl)2-amino-C1-8alkyl, (hydroxy-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl, hydroxy-C1-8alkyl-amino-C1-8alkoxy, (hydroxy-C1-8alkyl)2-amino-C1-8alkoxy, (hydroxy-C1-8alkyl)(C1-8alkyl)amino-C1-8alkoxy, hydroxy-C1-8alkyl-amino-C1-8alkyl-amino, (hydroxy-C1-8alkyl-amino-C1-8alkyl)2-amino, (hydroxy-C1-8alkyl-amino-C1-8alkyl)(C1-8alkyl)amino, (hydroxy-C1-8alkyl)2-amino-C1-8alkyl-amino, (hydroxy-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl-amino, [(hydroxy-C1-8alkyl)2-amino-C1-8alkyl](C1-8alkyl)amino, [(hydroxy-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl](C1-8alkyl)amino, heterocyclyl, heterocyclyl-C1-8alkyl, heterocyclyl-C1-8alkoxy, heterocyclyl-amino, (heterocyclyl)(C1-8alkyl)amino, heterocyclyl-amino-C1-8alkyl, heterocyclyl-C1-8alkyl-amino, (heterocyclyl-C1-8alkyl)2-amino, (heterocyclyl-C1-8alkyl)(C1-8alkyl)amino, heterocyclyl-C1-8alkyl-amino-C1-8alkyl, (heterocyclyl-C1-8alkyl)2-amino-C1-8alkyl, (heterocyclyl-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl, heterocyclyl-oxy, heterocyclyl-carbonyl, heterocyclyl-carbonyl-oxy, aryl-C1-8alkyl-amino, (aryl-C1-8alkyl)2-amino, (aryl-C1-8alkyl)(C1-8alkyl)amino, aryl-C1-8alkyl-amino-C1-8alkyl, (aryl-C1-8alkyl)2-amino-C1-8alkyl, (aryl-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl, heteroaryl, heteroaryl-C1-8alkyl, heteroaryl-C1-8alkoxy, heteroaryl-amino, heteroaryl-C1-8alkyl-amino, (heteroaryl-C1-8alkyl)2-amino, (heteroaryl-C1-8alkyl)(C1-8alkyl)amino, heteroaryl-C1-8alkyl-amino-C1-8alkyl, (heteroaryl-C1-8alkyl)2-amino-C1-8alkyl or (heteroaryl-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl; wherein, each instance of heterocyclyl and heteroaryl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is C1-8alkoxy-C1-8alkyl-amino, (C1-8alkoxy-C1-8alkyl)2-amino, (C1-8alkoxy-C1-8alkyl)(C1-8alkyl)amino, amino-C1-8alkyl, C1-8alkyl-amino-C1-8alkyl, (C1-8alkyl)2-amino-C1-8alkyl, C1-8alkoxy-C1-8alkyl-amino-C1-8alkyl, (C1-8alkoxy-C1-8alkyl)2-amino-C1-8alkyl, (C1-8alkoxy-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl, amino-C1-8alkyl-amino, (amino-C1-8alkyl)2-amino, (amino-C1-8alkyl)(C1-8alkyl)amino, C1-8alkyl-amino-C1-8alkyl-amino, (C1-8alkyl-amino-C1-8alkyl)2-amino, (C1-8alkyl-amino-C1-8alkyl)(C1-8alkyl)amino, (C1-8alkyl)2-amino-C1-8alkyl-amino, [(C1-8alkyl)2-amino-C1-8alkyl](C1-8alkyl)amino, amino-C1-8alkoxy, C1-8alkyl-amino-C1-8alkoxy, (C1-8alkyl)2-amino-C1-8alkoxy, C1-8alkoxy-C1-8alkyl-amino-C1-8alkoxy, (C1-8alkoxy-C1-8alkyl)2-amino-C1-8alkoxy, (C1-8alkoxy-C1-8alkyl)(C1-8alkyl)amino-C1-8alkoxy, amino-C2-8alkenyl, C1-8alkyl-amino-C2-8alkenyl, (C1-8alkyl)2-amino-C2-8alkenyl, amino-C2-8alkynyl, C1-8alkyl-amino-C2-8alkynyl, (C1-8alkyl)2-amino-C2-8alkynyl, halo-C1-8alkyl-amino, (halo-C1-8alkyl)2-amino, hydroxy-C1-8alkyl, hydroxy-C1-8alkoxy-C1-8alkyl, hydroxy-C1-8alkyl-amino, (hydroxy-C1-8alkyl)2-amino, (hydroxy-C1-8alkyl)(C1-8alkyl)amino, hydroxy-C1-8alkyl-amino-C1-8alkyl, (hydroxy-C1-8alkyl)2-amino-C1-8alkyl, (hydroxy-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl, hydroxy-C1-8alkyl-amino-C1-8alkoxy, (hydroxy-C1-8alkyl)2-amino-C1-8alkoxy, (hydroxy-C1-8alkyl)(C1-8alkyl)amino-C1-8alkoxy, hydroxy-C1-8alkyl-amino-C1-8alkyl-amino, (hydroxy-C1-8alkyl-amino-C1-8alkyl)2-amino, (hydroxy-C1-8alkyl-amino-C1-8alkyl)(C1-8alkyl)amino, (hydroxy-C1-8alkyl)2-amino-C1-8alkyl-amino, (hydroxy-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl-amino, [(hydroxy-C1-8alkyl)2-amino-C1-8alkyl](C1-8alkyl)amino, [(hydroxy-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl](C1-8alkyl)amino, heterocyclyl, heterocyclyl-C1-8alkyl, heterocyclyl-C1-8alkoxy, heterocyclyl-amino, (heterocyclyl)(C1-8alkyl)amino, heterocyclyl-amino-C1-8alkyl, heterocyclyl-C1-8alkyl-amino, (heterocyclyl-C1-8alkyl)2-amino, (heterocyclyl-C1-8alkyl)(C1-8alkyl)amino, heterocyclyl-C1-8alkyl-amino-C1-8alkyl, (heterocyclyl-C1-8alkyl)2-amino-C1-8alkyl, (heterocyclyl-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl, heterocyclyl-oxy, heterocyclyl-carbonyl, heterocyclyl-carbonyl-oxy, aryl-C1-8alkyl-amino, (aryl-C1-8alkyl)2-amino, (aryl-C1-8alkyl)(C1-8alkyl)amino, aryl-C1-8alkyl-amino-C1-8alkyl, (aryl-C1-8alkyl)2-amino-C1-8alkyl, (aryl-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl, heteroaryl, heteroaryl-C1-8alkyl, heteroaryl-C1-8alkoxy, heteroaryl-amino, heteroaryl-C1-8alkyl-amino, (heteroaryl-C1-8alkyl)2-amino, (heteroaryl-C1-8alkyl)(C1-8alkyl)amino, heteroaryl-C1-8alkyl-amino-C1-8alkyl, (heteroaryl-C1-8alkyl)2-amino-C1-8alkyl or (heteroaryl-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl; wherein, each instance of heterocyclyl and heteroaryl is optionally substituted.
In another embodiment of a compound of Formula (I), R1 is heterocyclyl selected from azetidinyl, tetrahydrofuranyl, pyrrolidinyl, piperidinyl, piperazinyl, 1,4-diazepanyl, 1,2,5,6-tetrahydropyridinyl, 1,2,3,6-tetrahydropyridinyl, hexahydropyrrolo[3,4-b]pyrrol-(1H)-yl, (3aS,6aS)-hexahydropyrrolo[3,4-b]pyrrol-(1H)-yl, (3aR,6aR)-hexahydropyrrolo[3,4-b]pyrrol-(1H)-yl, hexahydropyrrolo[3,4-b]pyrrol-(2H)-yl, (3aS,6aS)-hexahydropyrrolo[3,4-b]pyrrol-(2H)-yl, hexahydropyrrolo[3,4-c]pyrrol-(1H)-yl, (3aR,6aS)-hexahydropyrrolo[3,4-c]pyrrol-(1H)-yl, octahydro-5H-pyrrolo[3,2-c]pyridinyl, octahydro-6H-pyrrolo[3,4-b]pyridinyl, (4aR,7aR)-octahydro-6H-pyrrolo[3,4-b]pyridinyl, (4aS,7aS)-octahydro-6H-pyrrolo[3,4-b]pyridinyl, hexahydropyrrolo[1,2-a]pyrazin-(1H)-yl, (7R,8aS)-hexahydropyrrolo[1,2-a]pyrazin-(1H)-yl, (8aS)-hexahydropyrrolo[1,2-a]pyrazin-(1H)-yl, (8aR)-hexahydropyrrolo[1,2-a]pyrazin-(1H)-yl, (8aS)-octahydropyrrolo[1,2-a]pyrazin-(1H)-yl, (8aR)-octahydropyrrolo[1,2-a]pyrazin-(1H)-yl, hexahydropyrrolo[1,2-a]pyrazin-(2H)-one, octahydro-2H-pyrido[1,2-a]pyrazinyl, 3-azabicyclo[3.1.0]hexyl, (1R,5S)-3-azabicyclo[3.1.0]hexyl, 8-azabicyclo[3.2.1]octyl, (1R,5S)-8-azabicyclo[3.2.1]octyl, 8-azabicyclo[3.2.1]oct-2-enyl, (1R,5S)-8-azabicyclo[3.2.1]oct-2-enyl, 9-azabicyclo[3.3.1]nonyl, (1R,5S)-9-azabicyclo[3.3.1]nonyl, 2,5-diazabicyclo[2.2.1]heptyl, (1S,4S)-2,5-diazabicyclo[2.2.1]heptyl, 2,5-diazabicyclo[2.2.2]octyl, 3,8-diazabicyclo[3.2.1]octyl, (1R,5S)-3,8-diazabicyclo[3.2.1]octyl, 1,4-diazabicyclo[3.2.2]nonyl, azaspiro[3.3]heptyl, 2,6-diazaspiro[3.3]heptyl, 2,7-diazaspiro[3.5]nonyl, 5,8-diazaspiro[3.5]nonyl, 2,7-diazaspiro[4.4]nonyl or 6,9-diazaspiro[4.5]decyl; wherein, each instance of heterocyclyl is optionally substituted.
In another embodiment of a compound of Formula (I), R1 is heterocyclyl selected from azetidin-1-yl, tetrahydrofuran-3-yl, pyrrolidin-1-yl, piperidin-1-yl, piperidin-4-yl, piperazin-1-yl, 1,4-diazepan-1-yl, 1,2,5,6-tetrahydropyridin-3-yl, 1,2,3,6-tetrahydropyridin-4-yl, hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl, (3aS,6aS)-hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl, (3aS,6aS)-hexahydropyrrolo[3,4-b]pyrrol-5(1H)-yl, (3aR,6aR)-hexahydropyrrolo[3,4-b]pyrrol-5(1H)-yl, hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl, (3aR,6aS)-hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl, octahydro-5H-pyrrolo[3,2-c]pyridin-5-yl, octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl, (4aR,7aR)-octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl, (4aS,7aS)-octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl, hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, hexahydropyrrolo[1,2-a]pyrazin-6(2H)-one, (7R,8aS)-hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, (8aS)-hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, (8aR)-hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, (8aS)-octahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, (8aR)-octahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, octahydro-2H-pyrido[1,2-a]pyrazin-2-yl, 3-azabicyclo[3.1.0]hex-3-yl, 8-azabicyclo[3.2.1]oct-3-yl, (1R,5S)-8-azabicyclo[3.2.1]oct-3-yl, 8-azabicyclo[3.2.1]oct-2-en-3-yl, (1R,5S)-8-azabicyclo[3.2.1]oct-2-en-3-yl, 9-azabicyclo[3.3.1]non-3-yl, (1R,5S)-9-azabicyclo[3.3.1]non-3-yl, 2,5-diazabicyclo[2.2.1]hept-2-yl, (1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl, 2,5-diazabicyclo[2.2.2]oct-2-yl, 3,8-diazabicyclo[3.2.1]oct-3-yl, (1R,5S)-3,8-diazabicyclo[3.2.1]oct-3-yl, 1,4-diazabicyclo[3.2.2]non-4-yl, azaspiro[3.3]hept-2-yl, 2,6-diazaspiro[3.3]hept-2-yl, 2,7-diazaspiro[3.5]non-7-yl, 5,8-diazaspiro[3.5]non-8-yl, 2,7-diazaspiro[4.4]non-2-yl or 6,9-diazaspiro[4.5]dec-9-yl; wherein, each instance of heterocyclyl is optionally substituted.
In another embodiment of a compound of Formula (I), R1 is substituted heterocyclyl selected from (3aS,6aS)-1-methylhexahydropyrrolo[3,4-b]pyrrol-5(1H)-yl, (3aS,6aS)-5-methylhexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl, (3aR,6aR)-1-methylhexahydropyrrolo[3,4-b]pyrrol-5(1H)-yl, (3aR,6aS)-5-methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl, (3aR,6aS)-5-(2-hydroxyethyl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl, (3aR,6aS)-5-(propan-2-yl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl, (3aR,6aS)-5-ethylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl, (4aR,7aR)-1-methyloctahydro-6H-pyrrolo[3,4-b]pyridin-6-yl, (4aR,7aR)-1-ethyloctahydro-6H-pyrrolo[3,4-b]pyridin-6-yl, (4aR,7aR)-1-(2-hydroxyethyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl, (4aS,7aS)-1-methyloctahydro-6H-pyrrolo[3,4-b]pyridin-6-yl, (4aS,7aS)-1-(2-hydroxyethyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl, (7R,8aS)-7-hydroxyhexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, (8aS)-8a-methyloctahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, (8aR)-8a-methyloctahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, (1R,5S,6s)-6-(dimethylamino)-3-azabicyclo[3.1.0]hex-3-yl, (1R,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl, 9-methyl-9-azabicyclo[3.3.1]non-3-yl, (3-exo)-9-methyl-9-azabicyclo[3.3.1]non-3-yl, (1R,5S)-9-methyl-9-azabicyclo[3.3.1]non-3-yl, (1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]hept-2-yl or (1S,4S)-5-ethyl-2,5-diazabicyclo[2.2.1]hept-2-yl.
In one embodiment of a compound of Formula (I), R1 is heterocyclyl-C1-8alkyl, wherein heterocyclyl is selected from morpholinyl, piperidinyl, piperazinyl, imidazolyl or pyrrolidinyl; and, wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heterocyclyl-C1-8alkyl selected from morpholin-4-yl-methyl, morpholin-4-yl-ethyl, morpholin-4-yl-propyl, piperidin-1-yl-methyl, piperazin-1-yl-methyl, piperazin-1-yl-ethyl, piperazin-1-yl-propyl, piperazin-1-yl-butyl, imidazol-1-yl-methyl, imidazol-1-yl-ethyl, imidazol-1-yl-propyl, imidazol-1-yl-butyl, pyrrolidin-1-yl-methyl, pyrrolidin-1-yl-ethyl, pyrrolidin-1-yl-propyl or pyrrolidin-1-yl-butyl; wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heterocyclyl-C1-8alkoxy, wherein heterocyclyl is selected from pyrrolidinyl, piperidinyl or morpholinyl; and, wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heterocyclyl-C1-8alkoxy selected from pyrrolidin-2-yl-methoxy, pyrrolidin-2-yl-ethoxy, pyrrolidin-1-yl-methoxy, pyrrolidin-1-yl-ethoxy, piperidin-1-yl-methoxy, piperidin-1-yl-ethoxy, morpholin-4-yl-methoxy or morpholin-4-yl-ethoxy; wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heterocyclyl-amino, wherein heterocyclyl is selected from azetidinyl, pyrrolidinyl, piperidinyl, 9-azabicyclo[3.3.1]nonyl or (1R,5S)-9-azabicyclo[3.3.1]nonyl; and, wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heterocyclyl-amino selected from azetidin-3-yl-amino, pyrrolidin-3-yl-amino, piperidin-4-yl-amino, 9-azabicyclo[3.3.1]non-3-yl-amino, (1R,5S)-9-azabicyclo[3.3.1]non-3-yl-amino, 9-methyl-9-azabicyclo[3.3.1]non-3-yl-amino, (3-exo)-9-methyl-9-azabicyclo[3.3.1]non-3-yl-amino or (1R,5S)-9-methyl-9-azabicyclo[3.3.1]non-3-yl-amino; wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is (heterocyclyl)(C1-8alkyl)amino, wherein heterocyclyl is selected from pyrrolidinyl or piperidinyl; and, wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is (heterocyclyl)(C1-8alkyl)amino selected from (pyrrolidin-3-yl)(methyl)amino or (piperidin-4-yl)(methyl)amino; wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heterocyclyl-amino-C1-8alkyl, wherein heterocyclyl is selected from tetrahydrofuranyl; and, wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heterocyclyl-amino-C1-8alkyl, selected from 3-(tetrahydrofuran-3-yl-amino)propyl; wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heterocyclyl-C1-8alkyl-amino-C1-8alkyl, wherein heterocyclyl is selected from tetrahydrofuranyl, thienyl or pyridinyl; and, wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heterocyclyl-C1-8alkyl-amino-C1-8alkyl, selected from 3-[(tetrahydrofuran-2-ylmethyl)amino]propyl, 3-[(thiophenyl-3-ylmethyl)amino]propyl, 3-[(pyridin-2-ylmethyl)amino]propyl or 3-[(pyridin-4-ylmethyl)amino]propyl; wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heterocyclyl-oxy, wherein heterocyclyl is selected from pyrrolidinyl or piperidinyl; and, wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heterocyclyl-oxy selected from pyrrolidin-3-yl-oxy or piperidin-4-yl-oxy; wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heterocyclyl-carbonyl, wherein heterocyclyl is selected from piperazinyl; and, wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heterocyclyl-carbonyl selected from piperazin-1-yl-carbonyl; wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heterocyclyl-carbonyl-oxy, wherein heterocyclyl is selected from piperazinyl; and, wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heterocyclyl-carbonyl-oxy selected from piperazin-1-yl-carbonyl-oxy; wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is aryl-C1-8alkyl-amino-C1-8alkyl, wherein aryl is selected from phenyl; wherein, each instance of aryl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is aryl-C1-8alkyl-amino-C1-8alkyl selected from 3-(benzylamino)propyl; wherein, each instance of aryl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heteroaryl, wherein heteroaryl is selected from pyridinyl; and, wherein, each instance of heteroaryl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heteroaryl selected from pyridin-4-yl; wherein, each instance of heteroaryl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heteroaryl-C1-8alkyl, wherein heteroaryl is selected from 1H-imidazolyl; and, wherein, each instance of heteroaryl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heteroaryl-C1-8alkyl selected from 1H-imidazol-1-yl-methyl; wherein, each instance of heteroaryl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is (heteroaryl-C1-8alkyl)(C1-8alkyl)amino, wherein heteroaryl is selected from pyridinyl; and, wherein, each instance of heteroaryl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is (heteroaryl-C1-8alkyl)(C1-8alkyl)amino selected from (pyridin-3-yl-methyl)(methyl)amino; wherein, each instance of heteroaryl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heteroaryl-C1-8alkyl-amino-C1-8alkyl, wherein heteroaryl is selected from thienyl or pyridinyl; and, wherein, each instance of heteroaryl is optionally substituted.
In one embodiment of a compound of Formula (I), R1 is heteroaryl-C1-8alkyl-amino-C1-8alkyl selected from thien-3-yl-methyl-amino-propyl, pyridin-2-yl-methyl-amino-propyl, pyridin-3-yl-methyl-amino-propyl or pyridin-4-yl-methyl-amino-propyl; wherein, each instance of heteroaryl is optionally substituted.
In one embodiment of a compound of Formula (I), R3 is selected from cyano, halogen, hydroxy, oxo, C1-8alkyl, halo-C1-8alkyl, C1-8alkyl-carbonyl, C1-8alkoxy, halo-C1-8alkoxy, C1-8alkoxy-C1-8alkyl, C1-8alkoxy-carbonyl, amino, C1-8alkyl-amino, (C1-8alkyl)2-amino, amino-C1-8alkyl, C1-8alkyl-amino-C1-8alkyl, (C1-8alkyl)2-amino-C1-8alkyl, amino-C1-8alkyl-amino, C1-8alkyl-amino-C1-8alkyl-amino, (C1-8alkyl)2-amino-C1-8alkyl-amino, C1-8alkoxy-C1-8alkyl-amino, C1-8alkyl-carbonyl-amino, C1-8alkoxy-carbonyl-amino, hydroxy-C1-8alkyl, hydroxy-C1-8alkoxy-C1-8alkyl, hydroxy-C1-8alkyl-amino, (hydroxy-C1-8alkyl)(C1-8alkyl)amino or (hydroxy-C1-8alkyl)2-amino.
In one embodiment of a compound of Formula (I), R3 is selected from cyano, halogen, hydroxy, oxo, C1-8alkyl, halo-C1-8alkyl, C1-8alkoxy, C1-8alkoxy-C1-8alkyl, C1-8alkoxy-carbonyl, amino, C1-8alkyl-amino, (C1-8alkyl)2-amino, amino-C1-8alkyl, C1-8alkyl-amino-C1-8alkyl, (C1-8alkyl)2-amino-C1-8alkyl, C1-8alkyl-amino-C1-8alkyl-amino, C1-8alkoxy-C1-8alkyl-amino, C1-8alkoxy-carbonyl-amino, hydroxy-C1-8alkyl, hydroxy-C1-8alkoxy-C1-8alkyl, hydroxy-C1-8alkyl-amino, (hydroxy-C1-8alkyl)(C1-8alkyl)amino or (hydroxy-C1-8alkyl)2-amino.
In one embodiment of a compound of Formula (I), R3 is C1-8alkyl selected from methyl, ethyl, propyl, isopropyl or tert-butyl.
In one embodiment of a compound of Formula (I), R3 is C1-8alkyl selected from ethyl, propyl, isopropyl or tert-butyl.
In one embodiment of a compound of Formula (I), R3 is halo-C1-8alkyl selected from trihalo-methyl, dihalo-methyl, halo-methyl, trihalo-ethyl, dihalo-ethyl, halo-ethyl, trihalo-propyl, dihalo-propyl or halo-propyl; wherein, halo is selected from fluoro, chloro, bromo or iodo.
In one embodiment of a compound of Formula (I), R3 is halo-C1-8alkyl selected from trihalo-methyl, dihalo-methyl, halo-methyl, trihalo-ethyl, dihalo-ethyl, trihalo-propyl or dihalo-propyl; wherein, halo is selected from fluoro, chloro, bromo or iodo.
In one embodiment of a compound of Formula (I), R3 is hydroxy-C1-8alkyl selected from hydroxy-methyl, hydroxy-ethyl, hydroxy-propyl, dihydroxy-propyl, hydroxy-butyl or dihydroxy-butyl.
In one embodiment of a compound of Formula (I), R3 is hydroxy-C1-8alkyl selected from hydroxy-methyl, dihydroxy-propyl, hydroxy-butyl or dihydroxy-butyl.
In one embodiment of a compound of Formula (I), R3 is C1-8alkoxy selected from methoxy, ethoxy, propoxy or isopropoxy.
In one embodiment of a compound of Formula (I), R3 is halo-C1-8alkoxy selected from trihalo-methoxy, dihalo-methoxy, halo-methoxy, trihalo-ethoxy, dihalo-ethoxy, halo-ethoxy, trihalo-propoxy, dihalo-propoxy or halo-propoxy; wherein, halo is selected from fluoro, chloro, bromo or iodo.
In one embodiment of a compound of Formula (I), R3 is C1-8alkoxy-carbonyl-amino selected from methoxy-carbonyl-amino, ethoxy-carbonyl-amino, propoxy-carbonyl-amino, isopropoxy-carbonyl-amino, tert-butoxy-carbonyl-amino.
In one embodiment of a compound of Formula (I), R4 is C3-14cycloalkyl selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl; wherein, each instance of C3-14cycloalkyl is optionally substituted.
In one embodiment of a compound of Formula (I), R4 is C3-8cycloalkyl selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl; wherein, each instance of C3-8cycloalkyl is optionally substituted.
In one embodiment of a compound of Formula (I), R4 is C3-14cycloalkyl-C1-8alkyl, wherein C3-14cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl; and, wherein, each instance of C3-14cycloalkyl is optionally substituted.
In one embodiment of a compound of Formula (I), R4 is C3-8cycloalkyl-C1-8alkyl, wherein C3-8cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl; and, wherein, each instance of C3-8cycloalkyl is optionally substituted.
In one embodiment of a compound of Formula (I), R4 is C3-14cycloalkyl-amino, wherein C3-14cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl; and, wherein, each instance of C3-14cycloalkyl is optionally substituted.
In one embodiment of a compound of Formula (I), R4 is C3-8cycloalkyl-amino, wherein C3-8cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl; and, wherein, each instance of C3-8cycloalkyl is optionally substituted.
In one embodiment of a compound of Formula (I), R4 is aryl-C1-8alkyl, aryl-C1-8alkoxy-carbonyl or aryl-sulfonyloxy-C1-8alkyl, wherein each instance of aryl is selected from phenyl; and, wherein, each instance of aryl is optionally substituted.
In one embodiment of a compound of Formula (I), R4 is aryl-C1-8alkyl or aryl-C1-8alkoxy-carbonyl, wherein each instance of aryl is selected from phenyl; and, wherein, each instance of aryl is optionally substituted.
In one embodiment of a compound of Formula (I), R4 is heterocyclyl selected from oxetanyl, pyrrolidinyl, piperidinyl, piperazinyl, 1,3-dioxanyl or morpholinyl; wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R4 is heterocyclyl selected from oxetan-3-yl, pyrrolidin-1-yl, piperidin-1-yl, piperazin-1-yl, 1,3-dioxan-5-yl or morpholin-4-yl; wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R4 is heterocyclyl-C1-8alkyl, wherein each instance of heterocyclyl is selected from pyrrolidinyl or piperidinyl; and, wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R4 is heterocyclyl-C1-8alkyl selected from pyrrolidin-1-yl-C1-8alkyl or piperidin-1-yl-C1-8alkyl; wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R5 is selected from halogen, hydroxy, cyano, nitro, halo-C1-8alkyl, C1-8alkoxy, halo-C1-8alkoxy, amino, C1-8alkyl-amino, (C1-8alkyl)2-amino or C1-8alkyl-thio.
In one embodiment of a compound of Formula (I), R5 is hydroxy.
In one embodiment of a compound of Formula (I), R5 is C1-8alkyl selected from methyl, ethyl, propyl, isopropyl, n-butyl or tert-butyl.
In one embodiment of a compound of Formula (I), R5 is C1-8alkyl selected from ethyl, propyl, isopropyl or tert-butyl.
In one embodiment of a compound of Formula (I), R5 is halo-C1-8alkyl selected from trihalo-methyl, dihalo-methyl, halo-methyl, trihalo-ethyl, dihalo-ethyl, halo-ethyl, trihalo-propyl, dihalo-propyl or halo-propyl; wherein, halo is selected from fluoro, chloro, bromo or iodo.
In one embodiment of a compound of Formula (I), R5 is C1-8alkoxy selected from methoxy, ethoxy, propoxy or isopropoxy.
In one embodiment of a compound of Formula (I), R5 is halo-C1-8alkoxy selected from trihalo-methoxy, dihalo-methoxy, halo-methoxy, trihalo-ethoxy, dihalo-ethoxy, halo-ethoxy, trihalo-propoxy, dihalo-propoxy or halo-propoxy; wherein, halo is selected from fluoro, chloro, bromo or iodo.
In one embodiment of a compound of Formula (I), R2 is aryl selected from phenyl; wherein, each instance of aryl is optionally substituted.
In one embodiment of a compound of Formula (I), R2 is aryl-amino, wherein aryl is selected from phenyl; and, wherein, each instance of aryl is optionally substituted.
In one embodiment of a compound of Formula (I), R2 is aryl-amino selected from phenyl-amino; wherein, each instance of aryl is optionally substituted.
In one embodiment of a compound of Formula (I), R2 is aryl-amino-carbonyl, wherein aryl is selected from phenyl; wherein, each instance of aryl is optionally substituted.
In one embodiment of a compound of Formula (I), R2 is aryl-amino-carbonyl selected from phenyl-amino-carbonyl; wherein, each instance of aryl is optionally substituted.
In one embodiment of a compound of Formula (I), R2 is heterocyclyl selected from 1,2,3,6-tetrahydropyridinyl, 1,3-benzodioxolyl, 3a,7a-dihydrooxazolo[4,5-b]pyridinyl or 2,3-dihydro-1,4-benzodioxinyl; wherein, each instance of heterocyclyl is optionally substituted.
In another embodiment of a compound of Formula (I), R2 is heterocyclyl selected from 1,2,3,6-tetrahydropyridin-4-yl, 1,3-benzodioxol-5-yl or 2,3-dihydro-1,4-benzodioxin-6-yl; wherein, each instance of heterocyclyl is optionally substituted.
In one embodiment of a compound of Formula (I), R2 is heteroaryl selected from thienyl, 1H-pyrazolyl, 1H-imidazolyl, 1,3-thiazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, pyridinyl, pyrimidinyl, indolyl, 1H-indazolyl, 2H-indazolyl, indolizinyl, benzofuranyl, benzothienyl, 1H-benzimidazolyl, 1,3-benzothiazolyl, 1,3-benzooxazolyl, 9H-purinyl, furo[3,2-b]pyridinyl, furo[3,2-c]pyridinyl, furo[2,3-c]pyridinyl, thieno[3,2-c]pyridinyl, thieno[2,3-d]pyrimidinyl, 1H-pyrrolo[2,3-b]pyridinyl, 1H-pyrrolo[2,3-c]pyridinyl, pyrrolo[1,2-a]pyrimidinyl, pyrrolo[1,2-a]pyrazinyl, pyrrolo[1,2-b]pyridazinyl, pyrazolo[1,5-a]pyridinyl, pyrazolo[1,5-a]pyrazinyl, imidazo[1,2-a]pyridinyl, [1,3]oxazolo[4,5-b]pyridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-b]pyridazinyl, imidazo[1,2-a]pyrazinyl, imidazo[2,1-b][1,3]thiazolyl, imidazo[2,1-b][1,3,4]thiadiazolyl or quinoxalinyl; wherein, each instance of heteroaryl is optionally substituted.
In another embodiment of a compound of Formula (I), R2 is heteroaryl selected from thien-2-yl, thien-3-yl, 1H-pyrazol-3-yl, 1H-pyrazol-4-yl, 1H-pyrazol-5-yl, 1H-imidazol-1-yl, 1H-imidazol-4-yl, 1,3-thiazol-2-yl, 1,2,4-oxadiazol-3-yl, 1,3,4-oxadiazol-2-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrimidin-4-yl, 1H-indol-3-yl, 1H-indol-4-yl, indol-5-yl, indol-6-yl, 1H-indazol-5-yl, 2H-indazol-5-yl, indolizin-2-yl, benzofuran-2-yl, benzothien-2-yl, benzothien-3-yl, 1H-benzimidazol-2-yl, 1H-benzimidazol-6-yl, 1,3-benzoxazol-2-yl, 1,3-benzoxazol-5-yl, 1,3-benzoxazol-6-yl, 1,3-benzothiazol-2-yl, 1,3-benzothiazol-5-yl, 1,3-benzothiazol-6-yl, 9H-purin-8-yl, furo[3,2-b]pyridin-2-yl, furo[3,2-c]pyridin-2-yl, furo[2,3-c]pyridin-2-yl, thieno[3,2-c]pyridin-2-yl, thieno[2,3-d]pyrimidin-6-yl, 1H-pyrrolo[2,3-b]pyridin-5-yl, 1H-pyrrolo[2,3-c]pyridin-4-yl, pyrrolo[1,2-a]pyrimidin-7-yl, pyrrolo[1,2-a]pyrazin-7-yl, pyrrolo[1,2-b]pyridazin-2-yl, pyrrolo[1,2-b]pyridazin-6-yl, pyrazolo[1,5-a]pyridin-2-yl, pyrazolo[1,5-a]pyrazin-2-yl, imidazo[2,1-b][1,3]thiazol-6-yl, imidazo[2,1-b][1,3,4]thiadiazol-6-yl, [1,3]oxazolo[4,5-b]pyridin-2-yl imidazo[1,2-a]pyridin-2-yl, imidazo[1,2-a]pyridin-6-yl, imidazo[1,2-a]pyrimidin-2-yl, imidazo[1,2-a]pyrimidin-6-yl, imidazo[1,2-c]pyrimidin-2-yl, imidazo[1,2-b]pyridazin-2-yl, imidazo[1,2-a]pyrazin-2-yl or quinoxalin-2-yl; wherein, each instance of heteroaryl is optionally substituted.
In another embodiment of a compound of Formula (I), R2 is substituted heteroaryl selected from 4-methylthiophen-2-yl, 1-methyl-1H-pyrazol-3-yl, 4-methyl-1H-pyrazol-3-yl, 1-phenyl-1H-pyrazol-3-yl, 1-phenyl-1H-imidazol-4-yl, 2-methyl-1-(pyridin-2-yl)-1H-imidazol-4-yl, 4-methyl-1,3-thiazol-2-yl, 4-(trifluoromethyl)-1,3-thiazol-2-yl, 4-phenyl-1,3-thiazol-2-yl, 5-phenyl-1,2,4-oxadiazol-3-yl, 3-fluoropyridin-4-yl, 6-fluoropyridin-2-yl, 2-chloropyridin-4-yl, 4-chloropyridin-3-yl, 5-chloropyridin-2-yl, 6-methylpyridin-3-yl, 2-(trifluoromethyl)pyridin-3-yl, 4-(trifluoromethyl)pyridin-2-yl, 6-(trifluoromethyl)pyridin-2-yl, 2-methoxypyridin-4-yl, 4-methoxypyridin-3-yl, 6-methoxypyridin-2-yl, 2-ethoxypyridin-3-yl, 6-ethoxypyridin-2-yl, 6-(propan-2-yloxy)pyridin-2-yl, 6-(dimethylamino)pyridin-3-yl, 6-(methylsulfanyl)pyridin-2-yl, 6-(cyclobutyloxy)pyridin-2-yl, 6-(pyrrolidin-1-yl)pyridin-2-yl, 2-methylpyrimidin-4-yl, 2-(propan-2-yl)pyrimidin-4-yl, 2-cyclopropylpyrimidin-4-yl, 1-methyl-1H-indol-3-yl, 2-methyl-2H-indazol-5-yl, 1-methyl-1H-benzimidazol-2-yl, 4-methyl-1H-benzimidazol-2-yl 5-fluoro-1H-benzimidazol-2-yl, 4-fluoro-1,3-benzoxazol-2-yl, 5-fluoro-1,3-benzoxazol-2-yl, 4-chloro-1,3-benzoxazol-2-yl, 4-iodo-1,3-benzoxazol-2-yl, 2-methyl-1,3-benzoxazol-6-yl, 4-methyl-1,3-benzoxazol-2-yl, 4-(trifluoromethyl)-1,3-benzoxazol-2-yl, 7-(trifluoromethyl)-1,3-benzoxazol-2-yl, 4-chloro-1,3-benzothiazol-2-yl, 7-chloro-1,3-benzothiazol-2-yl, 2-methyl-1,3-benzothiazol-2-yl, 4-(trifluoromethyl)-1,3-benzothiazol-2-yl, 5-methylfuro[3,2-b]pyridin-2-yl, 4,6-dimethylfuro[3,2-c]pyridin-2-yl, 5,7-dimethylfuro[2,3-c]pyridin-2-yl, 4,6-dimethylthieno[3,2-c]pyridin-2-yl, 2,4-dimethylthieno[2,3-d]pyrimidin-6-yl, 1-methylpyrrolo[1,2-a]pyrazin-7-yl, 3-methylpyrrolo[1,2-a]pyrazin-7-yl, 1,3-dimethylpyrrolo[1,2-a]pyrazin-7-yl, 2-methylpyrrolo[1,2-b]pyridazin-6-yl, 5-methylpyrazolo[1,5-a]pyridin-2-yl, 4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl, 2-chloroimidazo[2,1-b][1,3]thiazol-6-yl, 2-methylimidazo[2,1-b][1,3]thiazol-6-yl, 3-methylimidazo[2,1-b][1,3]thiazol-6-yl, 2-ethylimidazo[2,1-b][1,3]thiazol-6-yl, 2-methylimidazo[2,1-b][1,3,4]thiadiazol-6-yl, 6-cyanoimidazo[1,2-c]pyridin-2-yl (also referred to as 2-imidazo[1,2-a]pyridine-6-carbonitrile), 6-fluoroimidazo[1,2-a]pyridin-2-yl, 8-fluoroimidazo[1,2-a]pyridin-2-yl, 6,8-difluoroimidazo[1,2-a]pyridin-2-yl, 7-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl, 8-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl, 6-chloroimidazo[1,2-a]pyridin-2-yl, 7-chloroimidazo[1,2-a]pyridin-2-yl, 8-chloroimidazo[1,2-a]pyridin-2-yl, 8-bromoimidazo[1,2-a]pyridin-2-yl, 2-methylimidazo[1,2-a]pyridin-2-yl, 5-methylimidazo[1,2-a]pyridin-2-yl, 6-methylimidazo[1,2-a]pyridin-2-yl, 7-methylimidazo[1,2-a]pyridin-2-yl, 8-methylimidazo[1,2-a]pyridin-2-yl, 7-ethylimidazo[1,2-a]pyridin-2-yl, 8-ethylimidazo[1,2-a]pyridin-2-yl, 6,8-dimethylimidazo[1,2-a]pyridin-2-yl, 8-ethyl-6-methylimidazo[1,2-a]pyridin-2-yl, 7-methoxyimidazo[1,2-a]pyridin-2-yl, 8-methoxyimidazo[1,2-a]pyridin-2-yl, 6-fluoro-8-methylimidazo[1,2-a]pyridin-2-yl, 8-fluoro-6-methylimidazo[1,2-a]pyridin-2-yl, 8-chloro-6-methylimidazo[1,2-a]pyridin-2-yl, 6-methyl-8-nitroimidazo[1,2-a]pyridin-2-yl, 8-cyclopropylimidazo[1,2-a]pyridin-2-yl, 2-methylimidazo[1,2-a]pyridin-6-yl, 2-ethylimidazo[1,2-a]pyridin-6-yl, 2,3-dimethylimidazo[1,2-a]pyridin-6-yl, 2,8-dimethylimidazo[1,2-a]pyridin-6-yl, 2-(trifluoromethyl)imidazo[1,2-a]pyridin-6-yl, 8-chloro-2-methylimidazo[1,2-a]pyridin-6-yl, 8-fluoro-2-methylimidazo[1,2-a]pyridin-6-yl, 6-fluoroimidazo[1,2-a]pyrimidin-2-yl, 6-chloroimidazo[1,2-a]pyrimidin-2-yl, 6-methylimidazo[1,2-a]pyrimidin-2-yl, 7-methylimidazo[1,2-a]pyrimidin-2-yl, 2-methylimidazo[1,2-a]pyrimidin-6-yl, 6-methylimidazo[1,2-b]pyridazin-2-yl, 2-methyl-3-(1,2,3,6-tetrahydropyridin-4-yl)imidazo[1,2-b]pyridazin-6-yl, 6-methylimidazo[1,2-a]pyrazin-2-yl, 8-methylimidazo[1,2-a]pyrazin-2-yl, 6,8-dimethylimidazo[1,2-c]pyrazin-2-yl, 6-chloro-8-methylimidazo[1,2-a]pyrazin-2-yl, 6-methyl-8-(trifluoromethyl)imidazo[1,2-a]pyrazin-2-yl or 8-(methylsulfanyl)imidazo[1,2-a]pyrazin-2-yl.
In one embodiment of a compound of Formula (I), R2 is heteroaryl-amino, wherein heteroaryl is selected from pyridinyl or pyrimidinyl; and, wherein, each instance of heteroaryl is optionally substituted.
In another embodiment of a compound of Formula (I), R2 is heteroaryl-amino selected from pyridin-2-yl-amino, pyridin-3-yl-amino or pyrimidin-2-yl-amino; wherein, each instance of heteroaryl is optionally substituted.
In one embodiment of a compound of Formula (I), R6 is selected from halogen, hydroxy, cyano, nitro, C1-8alkyl, halo-C1-8alkyl, hydroxy-C1-8alkyl, C1-8alkoxy, halo-C1-8alkoxy, (C1-8alkyl)2-amino or C1-8alkyl-thio
In one embodiment of a compound of Formula (I), R6 is C1-8alkyl selected from methyl, ethyl, propyl, isopropyl or tert-butyl.
In one embodiment of a compound of Formula (I), R6 is C1-8alkyl selected from ethyl, propyl, isopropyl or tert-butyl.
In one embodiment of a compound of Formula (I), R6 is C2-8alkenyl selected from ethenyl, allyl or buta-1,3-dienyl.
In one embodiment of a compound of Formula (I), R6 is C2-8alkenyl selected from ethenyl or allyl.
In one embodiment of a compound of Formula (I), R6 is halo-C1-8alkyl selected from trihalo-methyl, dihalo-methyl, halo-methyl, trihalo-ethyl, dihalo-ethyl, halo-ethyl, trihalo-propyl, dihalo-propyl or halo-propyl; wherein, halo is selected from fluoro, chloro, bromo or iodo.
In one embodiment of a compound of Formula (I), R6 is hydroxy-C1-8alkyl selected from hydroxy-methyl, hydroxy-ethyl, hydroxy-propyl, dihydroxy-propyl, hydroxy-butyl or dihydroxy-butyl.
In one embodiment of a compound of Formula (I), R6 is hydroxy-C1-8alkyl selected from hydroxy-methyl, dihydroxy-propyl, hydroxy-butyl or dihydroxy-butyl.
In one embodiment of a compound of Formula (I), R6 is C1-8alkoxy selected from methoxy, ethoxy, propoxy or isopropoxy.
In one embodiment of a compound of Formula (I), R6 is halo-C1-8alkoxy selected from trihalo-methoxy, dihalo-methoxy, halo-methoxy, trihalo-ethoxy, dihalo-ethoxy, halo-ethoxy, trihalo-propoxy, dihalo-propoxy or halo-propoxy; wherein, halo is selected from fluoro, chloro, bromo or iodo.
In one embodiment of a compound of Formula (I), R7 is C3-14cycloalkyl, C3-14cycloalkyl-oxy, aryl, heterocyclyl or heteroaryl; wherein C3-14cycloalkyl is selected from cyclopropyl or cyclobutoxy; wherein aryl is selected from phenyl; wherein heterocyclyl is selected from pyrrolidinyl or 1,2,3,6-tetrahydropyridinyl; and, wherein heteroaryl is selected from thienyl or pyridinyl.
In one embodiment of a compound of Formula (I), R7 is C3-14cycloalkyl or C3-14cycloalkyl-oxy, wherein each instance of C3-14cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.
In one embodiment of a compound of Formula (I), R7 is C3-8cycloalkyl or C3-8cycloalkyl-oxy, wherein each instance of C3-8cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.
In one embodiment of a compound of Formula (I), R7 is aryl selected from phenyl.
In one embodiment of a compound of Formula (I), R7 is heterocyclyl selected from pyrrolidinyl or 1,2,3,6-tetrahydropyridinyl.
In one embodiment of a compound of Formula (I), R7 is heterocyclyl selected from pyrrolidin-1-yl or 1,2,3,6-tetrahydropyridin-4-yl.
In one embodiment of a compound of Formula (I), R7 is heteroaryl selected from thienyl or pyridinyl.
In one embodiment of a compound of Formula (I), R7 is heteroaryl selected from pyridinyl.
In one embodiment of a compound of Formula (I), R7 is heteroaryl selected from thien-2-yl or pyridin-2-yl.
In one embodiment of a compound of Formula (I), R7 is heteroaryl selected from pyridin-2-yl.
In one embodiment of a compound of Formula (I), the compound is selected from Formula (Ia) or Formula (Ib):
or a form thereof, wherein all variables are as previously defined.
In one embodiment of a compound of Formula (I), when w1 is C—R1, w2 is C—R2 and R1 is selected from (methyl)2-amino, then R2 is 1H-benzoimidazol-2-yl substituted with one, two or three R6 substituents and one additional, optional R7 substituent.
In one embodiment of a compound of Formula (I), when w1 is C—R1, w2 is C—R2 and R1 is selected from (methyl)2-amino and R2 is 1H-benzoimidazol-2-yl substituted with one R6 substituent, then R6 is other than methyl.
In certain embodiments, the compound of Formula (I) is other than:
Further provided herein are compounds of Formula (I):
or a form thereof, wherein:
In one embodiment of a compound of Formula (I), the compound is selected from the group consisting of:
or a form thereof.
Terminology
The chemical terms used above and throughout the description herein, unless specifically defined otherwise, shall be understood by one of ordinary skill in the art to have the following indicated meanings.
As used herein, the term “C1-8alkyl” generally refers to saturated hydrocarbon radicals having from one to eight carbon atoms in a straight or branched chain configuration, including, but not limited to, methyl, ethyl, n-propyl (also referred to as propyl or propanyl), isopropyl, n-butyl (also referred to as butyl or butanyl), isobutyl, sec-butyl, tert-butyl, n-pentyl (also referred to as pentyl or pentanyl), n-hexyl (also referred to as hexyl or hexanyl), n-heptyl (also referred to as heptyl or heptanyl), n-octyl and the like. In some embodiments, C1-8alkyl includes, but is not limited to, C1-6alkyl, C1-4alkyl and the like. A C1-8alkyl radical is optionally substituted with substituent species as described herein where allowed by available valences.
As used herein, the term “C2-8alkenyl” generally refers to partially unsaturated hydrocarbon radicals having from two to eight carbon atoms in a straight or branched chain configuration and one or more carbon-carbon double bonds therein, including, but not limited to, ethenyl (also referred to as vinyl), allyl, propenyl and the like. In some embodiments, C2-8alkenyl includes, but is not limited to, C2-6alkenyl, C2-4alkenyl and the like. A C2-8alkenyl radical is optionally substituted with substituent species as described herein where allowed by available valences.
As used herein, the term “C2-8alkynyl” generally refers to partially unsaturated hydrocarbon radicals having from two to eight carbon atoms in a straight or branched chain configuration and one or more carbon-carbon triple bonds therein, including, but not limited to, ethynyl, propynyl and the like. In some embodiments, C2-8alkynyl includes, but is not limited to, C2-6alkynyl, C2-4alkynyl and the like. A C2-8alkynyl radical is optionally substituted with substituent species as described herein where allowed by available valences.
As used herein, the term “C1-8alkoxy” generally refers to saturated hydrocarbon radicals having from one to eight carbon atoms in a straight or branched chain configuration of the formula: —O—C1-8alkyl, including, but not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, n-hexoxy and the like. In some embodiments, C1-8alkoxy includes, but is not limited to, C1-6alkoxy, C1-4alkoxy and the like. A C1-8alkoxy radical is optionally substituted with substituent species as described herein where allowed by available valences.
As used herein, the term “C3-14cycloalkyl” generally refers to a saturated monocyclic, bicyclic or polycyclic hydrocarbon radical, including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 1H-indanyl, indenyl, tetrahydro-naphthalenyl and the like. In some embodiments, C3-14cycloalkyl includes, but is not limited to, C3-8cycloalkyl, C5-8cycloalkyl, C3-10cycloalkyl and the like. A C3-14cycloalkyl radical is optionally substituted with substituent species as described herein where allowed by available valences.
As used herein, the term “aryl” generally refers to a monocyclic, bicyclic or polycyclic aromatic carbon atom ring structure radical, including, but not limited to, phenyl, naphthyl, anthracenyl, fluorenyl, azulenyl, phenanthrenyl and the like. An aryl radical is optionally substituted with substituent species as described herein where allowed by available valences.
As used herein, the term “heteroaryl” generally refers to a monocyclic, bicyclic or polycyclic aromatic carbon atom ring structure radical in which one or more carbon atom ring members have been replaced, where allowed by structural stability, with one or more heteroatoms, such as an O, S or N atom, including, but not limited to, furanyl (also referred to as furyl), thienyl (also referred to as thiophenyl), pyrrolyl, 2H-pyrrolyl, 3H-pyrrolyl, pyrazolyl, 1H-pyrazolyl, imidazolyl, 1H-imidazolyl, isoxazolyl, isothiazolyl, oxazolyl, 1,3-thiazolyl, triazolyl (such as 1H-1,2,3-triazolyl and the like), oxadiazolyl (such as 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl and the like), thiadiazolyl, tetrazolyl (such as 1H-tetrazolyl, 2H-tetrazolyl and the like), pyridinyl (also referred to as pyridyl), pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, indolyl, indazolyl, 1H-indazolyl, 2H-indazolyl, indolizinyl, isoindolyl, benzofuranyl, benzothienyl (also referred to as benzothiophenyl), benzoimidazolyl, 1H-benzoimidazolyl, 1,3-benzothiazolyl, 1,3-benzoxazolyl (also referred to as 1,3-benzooxazolyl), purinyl, 9H-purinyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, 1,3-diazinyl, 1,2-diazinyl, 1,2-diazolyl, 1,4-diazanaphthalenyl, acridinyl, furo[3,2-b]pyridinyl, furo[3,2-c]pyridinyl, furo[2,3-c]pyridinyl, 6H-thieno[2,3-b]pyrrolyl, thieno[3,2-c]pyridinyl, thieno[2,3-d]pyrimidinyl, 1H-pyrrolo[2,3-b]pyridinyl, 1H-pyrrolo[2,3-c]pyridinyl, 1H-pyrrolo[3,2-b]pyridinyl, pyrrolo[1,2-a]pyrimidinyl, pyrrolo[1,2-a]pyrazinyl, pyrrolo[1,2-b]pyridazinyl, pyrazolo[1,5-a]pyridinyl, pyrazolo[1,5-a]pyrazinyl, imidazo[1,2-a]pyridinyl, 3H-imidazo[4,5-b]pyridinyl, [1,3]oxazolo[4,5-b]pyridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-c]pyrimidinyl, imidazo[1,2-b]pyridazinyl, imidazo[1,2-a]pyrazinyl, imidazo[2,1-b][1,3]thiazolyl, imidazo[2,1-b][1,3,4]thiadiazolyl, [1,2,4]triazolo[1,5-a]pyridinyl, [1,2,4]triazolo[4,3-a]pyridinyl and the like. A heteroaryl radical is optionally substituted on a carbon or nitrogen atom ring member with substituent species as described herein where allowed by available valences.
As used herein, the term “heterocyclyl” generally refers to a saturated or partially unsaturated monocyclic, bicyclic or polycyclic carbon atom ring structure radical in which one or more carbon atom ring members have been replaced, where allowed by structural stability, with a heteroatom, such as an O, S or N atom, including, but not limited to, oxiranyl, oxetanyl, azetidinyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, isoxazolinyl, isoxazolidinyl, isothiazolinyl, isothiazolidinyl, oxazolinyl, oxazolidinyl, thiazolinyl, thiazolidinyl, triazolinyl, triazolidinyl, oxadiazolinyl, oxadiazolidinyl, thiadiazolinyl, thiadiazolidinyl, tetrazolinyl, tetrazolidinyl, pyranyl, dihydro-2H-pyranyl, thiopyranyl, 1,3-dioxanyl, 1,2,5,6-tetrahydropyridinyl, 1,2,3,6-tetrahydropyridinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, 1,4-diazepanyl, 1,3-benzodioxolyl (also referred to as benzo[d][1,3]dioxolyl), 1,4-benzodioxanyl, 2,3-dihydro-1,4-benzodioxinyl (also referred to as 2,3-dihydrobenzo[b][1,4]dioxinyl), hexahydropyrrolo[3,4-b]pyrrol-(1H)-yl, (3aS,6aS)-hexahydropyrrolo[3,4-b]pyrrol-(1H)-yl, (3aR,6aR)-hexahydropyrrolo[3,4-b]pyrrol-(1H)-yl, hexahydropyrrolo[3,4-b]pyrrol-(2H)-yl, (3aS,6aS)-hexahydropyrrolo[3,4-b]pyrrol-(2H)-yl, (3aR,6aR)-hexahydropyrrolo[3,4-b]pyrrol-(2H)-yl, hexahydropyrrolo[3,4-c]pyrrol-(1H)-yl, (3aR,6aS)-hexahydropyrrolo[3,4-c]pyrrol-(1H)-yl, (3aR,6aR)-hexahydropyrrolo[3,4-c]pyrrol-(1H)-yl, octahydro-5H-pyrrolo[3,2-c]pyridinyl, octahydro-6H-pyrrolo[3,4-b]pyridinyl, (4aR,7aR)-octahydro-6H-pyrrolo[3,4-b]pyridinyl, (4aS,7aS)-octahydro-6H-pyrrolo[3,4-b]pyridinyl, hexahydropyrrolo[1,2-a]pyrazin-(1H)-yl, (7R,8aS)-hexahydropyrrolo[1,2-a]pyrazin-(1H)-yl, (8aS)-hexahydropyrrolo[1,2-a]pyrazin-(1H)-yl, (8aR)-hexahydropyrrolo[1,2-a]pyrazin-(1H)-yl, (8aS)-octahydropyrrolo[1,2-a]pyrazin-(1H)-yl, (8aR)-octahydropyrrolo[1,2-a]pyrazin-(1H)-yl, hexahydropyrrolo[1,2-a]pyrazin-(2H)-one, octahydro-2H-pyrido[1,2-a]pyrazinyl, 3-azabicyclo[3.1.0]hexyl, (1R,5S)-3-azabicyclo[3.1.0]hexyl, 8-azabicyclo[3.2.1]octyl, (1R,5S)-8-azabicyclo[3.2.1]octyl, 8-azabicyclo[3.2.1]oct-2-enyl, (1R,5S)-8-azabicyclo[3.2.1]oct-2-enyl, 9-azabicyclo[3.3.1]nonyl, (1R,5S)-9-azabicyclo[3.3.1]nonyl, 2,5-diazabicyclo[2.2.1]heptyl, (1S,4S)-2,5-diazabicyclo[2.2.1]heptyl, 2,5-diazabicyclo[2.2.2]octyl, 3,8-diazabicyclo[3.2.1]octyl, (1R,5S)-3,8-diazabicyclo[3.2.1]octyl, 1,4-diazabicyclo[3.2.2]nonyl, azaspiro[3.3]heptyl, 8-azabicyclo[3.2.1]oct-2-enyl, 2,6-diazaspiro[3.3]heptyl, 2,7-diazaspiro[3.5]nonyl, 5,8-diazaspiro[3.5]nonyl, 2,7-diazaspiro[4.4]nonyl or 6,9-diazaspiro[4.5]decyl and the like. A heterocyclyl radical is optionally substituted on a carbon or nitrogen atom ring member with substituent species as described herein where allowed by available valences.
As used herein, the term “C1-8alkoxy-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-O—C1-8alkyl.
As used herein, the term “C1-8alkoxy-C1-8alkyl-amino” refers to a radical of the formula: —NH—C1-8alkyl-O—C1-8alkyl.
As used herein, the term “(C1-8alkoxy-C1-8alkyl)2-amino” refers to a radical of the formula: —N(C1-8alkyl-O—C1-8alkyl)2.
As used herein, the term “(C1-8alkoxy-C1-8alkyl)(C1-8alkyl)amino” refers to a radical of the formula: —N(C1-8alkyl)(C1-8alkyl-O—C1-8alkyl).
As used herein, the term “C1-8alkoxy-C1-8alkyl-amino-C1-8alkoxy” refers to a radical of the formula: —O—C1-8alkyl-NH—C1-8alkyl-O—C1-8alkyl.
As used herein, the term “(C1-8alkoxy-C1-8alkyl)2-amino-C1-8alkoxy” refers to a radical of the formula: —O—C1-8alkyl-N(C1-8alkyl-O—C1-8alkyl)2.
As used herein, the term “(C1-8alkoxy-C1-8alkyl)(C1-8alkyl)amino-C1-8alkoxy” refers to a radical of the formula: —O—C1-8alkyl-N(C1-8alkyl)(C1-8alkyl-O—C1-8alkyl).
As used herein, the term “C1-8alkoxy-C1-8alkyl-amino-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-NH—C1-8alkyl-O—C1-8alkyl.
As used herein, the term “(C1-8alkoxy-C1-8alkyl)2-amino-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-N(C1-8alkyl-O—C1-8alkyl)2.
As used herein, the term “(C1-8alkoxy-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-N(C1-8alkyl)(C1-8alkyl-O—C1-8alkyl).
As used herein, the term “C1-8alkoxy-carbonyl” refers to a radical of the formula: —C(O)—O—C1-8alkyl.
As used herein, the term “C1-8alkoxy-carbonyl-amino” refers to a radical of the formula: —NH—C(O)—O—C1-8alkyl.
As used herein, the term “C1-8alkyl-amino” refers to a radical of the formula: —NH—C1-8alkyl.
As used herein, the term “(C1-8alkyl)2-amino” refers to a radical of the formula: —N(C1-8alkyl)2.
As used herein, the term “C1-8alkyl-amino-C2-8alkenyl” refers to a radical of the formula: —C2-8alkenyl-NH—C1-8alkyl.
As used herein, the term “(C1-8alkyl)2-amino-C2-8alkenyl” refers to a radical of the formula: —C2-8alkenyl-N(C1-8alkyl)2.
As used herein, the term “C1-8alkyl-amino-C1-8alkoxy” refers to a radical of the formula: —O—C1-8alkyl-NH—C1-8alkyl.
As used herein, the term “(C1-8alkyl)2-amino-C1-8alkoxy” refers to a radical of the formula: —O—C1-8alkyl-N(C1-8alkyl)2.
As used herein, the term “C1-8alkyl-amino-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-NH—C1-8alkyl.
As used herein, the term “(C1-8alkyl)2-amino-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-N(C1-8alkyl)2.
As used herein, the term “C1-8alkyl-amino-C1-8alkyl-amino” refers to a radical of the formula: —NH—C1-8alkyl-NH—C1-8alkyl.
As used herein, the term “(C1-8alkyl)2-amino-C1-8alkyl-amino” refers to a radical of the formula: —NH—C1-8alkyl-N(C1-8alkyl)2.
As used herein, the term “(C1-8alkyl-amino-C1-8alkyl)2-amino” refers to a radical of the formula: —N(C1-8alkyl-NH—C1-8alkyl)2.
As used herein, the term “(C1-8alkyl-amino-C1-8alkyl)(C1-8alkyl)amino” refers to a radical of the formula: —N(C1-8alkyl)(C1-8alkyl-NH—C1-8alkyl).
As used herein, the term “[(C1-8alkyl)2-amino-C1-8alkyl](C1-8alkyl)amino” refers to a radical of the formula: —N(C1-8alkyl) [C1-8alkyl-N(C1-8alkyl)2].
As used herein, the term “C1-8alkyl-amino-C2-8alkynyl” refers to a radical of the formula: —C2-8alkynyl-NH—C1-8alkyl.
As used herein, the term “(C1-8alkyl)2-amino-C2-8alkynyl” refers to a radical of the formula: —C2-8alkynyl-N(C1-8alkyl)2.
As used herein, the term “C1-8alkyl-carbonyl” refers to a radical of the formula: —C(O)—C1-8alkyl.
As used herein, the term “C1-8alkyl-carbonyl-amino” refers to a radical of the formula: —NH—C(O)—C1-8alkyl.
As used herein, the term “C1-8alkyl-thio” refers to a radical of the formula: —S—C1-8alkyl.
As used herein, the term “amino-C2-8alkenyl” refers to a radical of the formula: —C2-8alkenyl-NH2.
As used herein, the term “amino-C1-8alkoxy” refers to a radical of the formula: —O—C1-8alkyl-NH2.
As used herein, the term “amino-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-NH2.
As used herein, the term “amino-C1-8alkyl-amino” refers to a radical of the formula: —NH—C1-8alkyl-NH2.
As used herein, the term “(amino-C1-8alkyl)2-amino” refers to a radical of the formula: —N(C1-8alkyl-NH2)2.
As used herein, the term “(amino-C1-8alkyl)(C1-8alkyl)amino” refers to a radical of the formula: —N(C1-8alkyl)(C1-8alkyl-NH2).
As used herein, the term “amino-C2-8alkynyl” refers to a radical of the formula: —C2-8alkynyl-NH2.
As used herein, the term “aryl-C1-8alkoxy-carbonyl” refers to a radical of the formula: —C(O)—O—C1-8alkyl-aryl.
As used herein, the term “aryl-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-aryl.
As used herein, the term “aryl-C1-8alkyl-amino” refers to a radical of the formula: —NH—C1-8alkyl-aryl.
As used herein, the term “(aryl-C1-8alkyl)2-amino” refers to a radical of the formula: —N(C1-8alkyl-aryl)2.
As used herein, the term “(aryl-C1-8alkyl)(C1-8alkyl)amino” refers to a radical of the formula: —N(C1-8alkyl)(C1-8alkyl-aryl).
As used herein, the term “aryl-C1-8alkyl-amino-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-NH—C1-8alkyl-aryl.
As used herein, the term “(aryl-C1-8alkyl)2-amino-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-N(C1-8alkyl-aryl)2.
As used herein, the term “(aryl-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-N(C1-8alkyl)(C1-8alkyl-aryl).
As used herein, the term “aryl-amino” refers to a radical of the formula: —NH-aryl.
As used herein, the term “aryl-amino-carbonyl” refers to a radical of the formula: —C(O)—NH-aryl.
As used herein, the term “aryl-sulfonyloxy-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-O—SO2-aryl.
As used herein, the term “benzoxy-carbonyl” refers to a radical of the formula: —C(O)O—CH2-phenyl.
As used herein, the term “C3-14cycloalkyl-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-C3-14cycloalkyl.
As used herein, the term “C3-14cycloalkyl-amino” refers to a radical of the formula: —NH—C3-14cycloalkyl.
As used herein, the term “C3-14cycloalkyl-oxy” refers to a radical of the formula: —O—C3-14cycloalkyl.
As used herein, the term “halo” or “halogen” generally refers to a halogen atom radical, including fluoro, chloro, bromo and iodo.
As used herein, the term “halo-C1-8alkoxy” refers to a radical of the formula: —O—C1-8alkyl-halo, wherein C1-8alkyl is partially or completely substituted with one or more halogen atoms where allowed by available valences.
As used herein, the term “halo-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-halo, wherein C1-8alkyl is partially or completely substituted with one or more halogen atoms where allowed by available valences.
As used herein, the term “halo-C1-8alkyl-amino” refers to a radical of the formula: —NH—C1-8alkyl-halo.
As used herein, the term “(halo-C1-8alkyl)(C1-8alkyl)amino” refers to a radical of the formula: —N(C1-8alkyl)(C1-8alkyl-halo).
As used herein, the term “(halo-C1-8alkyl)2-amino” refers to a radical of the formula: —N(C1-8alkyl-halo)2.
As used herein, the term “heteroaryl-C1-8alkoxy” refers to a radical of the formula: —O—C1-8alkyl-heteroaryl.
As used herein, the term “heteroaryl-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-heteroaryl.
As used herein, the term “heteroaryl-C1-8alkyl-amino” refers to a radical of the formula: —NH—C1-8alkyl-heteroaryl.
As used herein, the term “(heteroaryl-C1-8alkyl)2-amino” refers to a radical of the formula: —N(C1-8alkyl-heteroaryl)2.
As used herein, the term “(heteroaryl-C1-8alkyl)(C1-8alkyl)amino” refers to a radical of the formula: —N(C1-8alkyl)(C1-8alkyl-heteroaryl).
As used herein, the term “heteroaryl-C1-8alkyl-amino-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-NH—C1-8alkyl-heteroaryl.
As used herein, the term “(heteroaryl-C1-8alkyl)2-amino-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-N(C1-8alkyl-heteroaryl)2.
As used herein, the term “(heteroaryl-C1-8alkyl(C1-8alkyl)amino-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-N(C1-8alkyl)(C1-8alkyl-heteroaryl).
As used herein, the term “heteroaryl-amino” refers to a radical of the formula: —NH-heteroaryl.
As used herein, the term “heterocyclyl-C1-8alkoxy” refers to a radical of the formula: —O—C1-8alkyl-heterocyclyl.
As used herein, the term “heterocyclyl-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-heterocyclyl.
As used herein, the term “(heterocyclyl)(C1-8alkyl)amino” refers to a radical of the formula: —N(C1-8alkyl)(heterocyclyl).
As used herein, the term “heterocyclyl-C1-8alkyl-amino” refers to a radical of the formula: —NH—C1-8alkyl-heterocyclyl.
As used herein, the term “(heterocyclyl-C1-8alkyl)2-amino” refers to a radical of the formula: —N(C1-8alkyl-heterocyclyl)2.
As used herein, the term “(heterocyclyl-C1-8alkyl)(C1-8alkyl)amino” refers to a radical of the formula: —N(C1-8alkyl)(C1-8alkyl-heterocyclyl).
As used herein, the term “heterocyclyl-C1-8alkyl-amino-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-NH—C1-8alkyl-heterocyclyl.
As used herein, the term “(heterocyclyl-C1-8alkyl)2-amino-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-N(C1-8alkyl-heterocyclyl)2.
As used herein, the term “(heterocyclyl-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-N(C1-8alkyl)(C1-8alkyl-heterocyclyl).
As used herein, the term “heterocyclyl-amino” refers to a radical of the formula: —NH-heterocyclyl.
As used herein, the term “heterocyclyl-amino-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-NH-heterocyclyl.
As used herein, the term “heterocyclyl-carbonyl” refers to a radical of the formula: —C(O)-heterocyclyl.
As used herein, the term “heterocyclyl-carbonyl-oxy” refers to a radical of the formula: —O—C(O)-heterocyclyl.
As used herein, the term “heterocyclyl-oxy” refers to a radical of the formula: —O-heterocyclyl.
As used herein, the term “hydroxy” refers to a radical of the formula: —OH.
As used herein, the term “hydroxy-C1-8alkoxy-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-O—C1-8alkyl-OH.
As used herein, the term “hydroxy-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-OH, wherein C1-8alkyl is partially or completely substituted with one or more hydroxy radicals where allowed by available valences.
As used herein, the term “(hydroxy-C1-8alkyl)(C1-8alkyl)amino” refers to a radical of the formula: —N(C1-8alkyl)(C1-8alkyl-OH).
As used herein, the term “hydroxy-C1-8alkyl-amino” refers to a radical of the formula: —NH—C1-8alkyl-OH.
As used herein, the term “(hydroxy-C1-8alkyl)2-amino” refers to a radical of the formula: —N(C1-8alkyl-OH)2.
As used herein, the term “hydroxy-C1-8alkyl-amino-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-NH—C1-8alkyl-OH.
As used herein, the term “(hydroxy-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-N(C1-8alkyl)(C1-8alkyl-OH).
As used herein, the term “(hydroxy-C1-8alkyl)2-amino-C1-8alkyl” refers to a radical of the formula: —C1-8alkyl-N(C1-8alkyl-OH)2.
As used herein, the term “hydroxy-C1-8alkyl-amino-C1-8alkoxy” refers to a radical of the formula: —O—C1-8alkyl-NH—C1-8alkyl-OH.
As used herein, the term “(hydroxy-C1-8alkyl)(C1-8alkyl)amino-C1-8alkoxy” refers to a radical of the formula: —O—C1-8alkyl-N(C1-8alkyl)(C1-8alkyl-OH).
As used herein, the term “(hydroxy-C1-8alkyl)2-amino-C1-8alkoxy” refers to a radical of the formula: —O—C1-8alkyl-N(C1-8alkyl-OH)2.
As used herein, the term “hydroxy-C1-8alkyl-amino-C1-8alkyl-amino” refers to a radical of the formula: —NH—C1-8alkyl-NH—C1-8alkyl-OH.
As used herein, the term “(hydroxy-C1-8alkyl-amino-C1-8alkyl)2-amino” refers to a radical of the formula: —N(C1-8alkyl-NH—C1-8alkyl-OH)2.
As used herein, the term “(hydroxy-C1-8alkyl)2-amino-C1-8alkyl-amino” refers to a radical of the formula: —NH—C1-8alkyl-N(C1-8alkyl-OH)2.
As used herein, the term “(hydroxy-C1-8alkyl-amino-C1-8alkyl)(C1-8alkyl)amino” refers to a radical of the formula: —N(C1-8alkyl)(C1-8alkyl-NH—C1-8alkyl-OH).
As used herein, the term “[(hydroxy-C1-8alkyl)2-amino-C1-8alkyl](C1-8alkyl)amino” refers to a radical of the formula: —N(C1-8alkyl)[C1-8alkyl-N(C1-8alkyl-OH)2].
As used herein, the term “(hydroxy-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl-amino” refers to a radical of the formula: —NH—C1-8alkyl-N(C1-8alkyl,C1-8alkyl-OH).
As used herein, the term “[(hydroxy-C1-8alkyl)(C1-8alkyl)amino-C1-8alkyl](C1-8alkyl)amino” refers to a radical of the formula: —N(C1-8alkyl)[C1-8alkyl-N(C1-8alkyl)(C1-8alkyl-OH)].
As used herein, the term “substituent” means positional variables on the atoms of a core molecule that are attached at a designated atom position, replacing one or more hydrogen atoms on the designated atom, provided that the atom of attachment does not exceed the available valence or shared valences, such that the substitution results in a stable compound. Accordingly, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds. It should also be noted that any carbon as well as heteroatom with a valence level that appears to be unsatisfied as described or shown herein is assumed to have a sufficient number of hydrogen atom(s) to satisfy the valences described or shown.
For the purposes of this description, where one or more substituent variables for a compound of Formula (I) encompass functionalities incorporated into a compound of Formula (I), each functionality appearing at any location within the disclosed compound may be independently selected, and as appropriate, independently and/or optionally substituted.
As used herein, the terms “independently selected,” or “each selected” refer to functional variables in a substituent list that may be attached more than once on the structure of a core molecule, where the pattern of substitution at each occurrence is independent of the pattern at any other occurrence. Further, the use of a generic substituent on a core structure for a compound provided herein is understood to include the replacement of the generic substituent with specie substituents that are included within the particular genus, e.g., aryl may be independently replaced with phenyl or naphthalenyl (also referred to as naphthyl) and the like, such that the resulting compound is to be included within the scope of the compounds described herein.
As used herein, the term “each instance of” when used in a phrase such as “ . . . aryl, aryl-C1-8alkyl, heterocyclyl and heterocyclyl-C1-8alkyl, wherein each instance of aryl and heterocyclyl is optionally substituted with one or two substituents . . . ” is intended to include optional, independent substitution on each of the aryl and heterocyclyl rings and on the aryl and heterocyclyl portions of aryl-C1-8alkyl and heterocyclyl-C1-8alkyl.
As used herein, the term “optionally substituted” means that the specified substituent variables, groups, radicals or moieties represent the scope of the genus and may be independently chosen as needed to replace one or more hydrogen atoms on the designated atom of attachment of a core molecule.
As used herein, the terms “stable compound” or “stable structure” mean a compound that is sufficiently robust to be isolated to a useful degree of purity from a reaction mixture and formulations thereof into an efficacious therapeutic agent.
Compound names provided herein were obtained using ACD Labs Index Name software provided by ACD Labs and/or ChemDraw Ultra software provided by CambridgeSoft®. When the compound name disclosed herein conflicts with the structure depicted, the structure shown will supercede the use of the name to define the compound intended. Nomenclature for substituent radicals defined herein may differ slightly from the chemical name from which they are derived; one skilled in the art will recognize that the definition of the substituent radical is intended to include the radical as found in the chemical name.
The term “SMN,” unless otherwise specified herein, refers to the human SMN1 gene, DNA or RNA, and/or human SMN2 gene, DNA or RNA. In a specific embodiment, the term “SMN1” refers to the human SMN1 gene, DNA or RNA. In another specific embodiment, the term “SMN2” refers to the human SMN2 gene, DNA or RNA.
Nucleic acid sequences for the human SMN1 and SMN2 genes are known in the art. For nucleic acid sequences of human SMN1, see, e.g., GenBank Accession Nos. DQ894095, NM_000344, NM_022874, and BC062723. For nucleic acid sequences of human SMN2, see, e.g., NM_022875, NM_022876, NM_022877, NM_017411, DQ894734 (Life Technologies, Inc. (formerly Invitrogen), Carlsbad, Calif.), BC000908, BC070242, CR595484, CR598529, CR609539, U21914, and BC015308.
The SMN1 gene can be found on the forward strand of human chromosome 5 from approximately nucleotide 70,220,768 to approximately nucleotide 70,249,769. The approximate locations of exons 6, 7 and 8 and introns 6 and 7 of SMN1 on human chromosome 5 are as follows:
70,241,893 to 70,242,003 exon 6;
70,242,004 to 70,247,767 intron 6;
70,247,768 to 70,247,821 exon 7;
70,247,822 to 70,248,265 intron 7; and,
70,248,266 to 70,248,839 exon 8.
The SMN2 gene can be found on the forward strand of human chromosome 5 from approximately nucleotide 69,345,350 to approximately nucleotide 69,374,349.
The approximate locations of exons 6, 7 and 8 and introns 6 and 7 of SMN2 on human chromosome 5 are as follows:
69,366,468 to 69,366,578 exon 6;
69,366,579 to 69,372,347 intron 6;
69,372,348 to 69,372,401 exon 7;
69,372,402 to 69,372,845 intron 7; and,
69,372,846 to 69,373,419 exon 8.
In specific embodiments, the nucleotide sequences delineated above for exons 6, 7 and 8 and introns 6 and 7 of SMN1 are used in the SMN1 minigene nucleic acid constructs described herein. In other specific embodiments, the nucleotide sequences of exons 6, 7 and 8 and introns 6 and 7 of SMN2 in the examples provided herein are used in the SMN2 minigene nucleic acid constructs described herein.
The term “Smn” or “Smn protein,” unless otherwise specified herein, refers to a human Smn protein that contains the amino acid residues encoded by exons 1 through 7 of the SMN1 gene and/or SMN2 gene. In a specific embodiment, the Smn protein is stable and functional in vitro and/or in vivo as assessed by methods known to one of skill in the art. In another specific embodiment, the Smn protein is the full-length protein encoded by the human SMN1 gene and/or SMN2 gene. In another specific embodiment, the Smn protein has the amino acid sequence found at GenBank Accession No. NP_000335, AAC50473.1, AAA66242.1, or NP_059107.
As used herein, the term “enhances the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene,” and analogous terms, unless otherwise specified herein, refers to the inclusion of the complete, intact, non-truncated sequence of exon 7 of SMN2 into the mature mRNA that is transcribed from the SMN2 gene (i.e., resulting in the production of full-length SMN2 mRNA) in vitro and/or in vivo, as assessed by methods known to one of skill in the art, such that increased levels of Smn protein are produced from the SMN2 gene in vitro and/or in vivo, as assessed by methods known to one of skill in the art; or, that increased expression of stable and functional Smn protein is produced from the SMN2 gene in vitro and/or in vivo, as assessed by methods known to one of skill in the art; or, that expression of the fusion protein encoded by the minigene is increased in vitro, as assessed by methods known to one of skill in the art; or, that expression of Smn protein produced from the SMN2 gene in a subject (e.g., an animal model for SMA or a human subject) in need thereof is increased.
As used herein, the term “enhances the inclusion of exon 7 of SMN1 into mRNA that is transcribed from the SMN1 gene,” and analogous terms, unless otherwise specified herein, refers to the inclusion of the complete, intact, non-truncated sequence of exon 7 of SMN1 into the mature mRNA that is transcribed from the SMN1 gene (i.e., resulting in the production of full-length SMN1 mRNA) in vitro and/or in vivo, as assessed by methods known to one of skill in the art, such that increased levels of Smn protein are produced from the SMN1 gene in vitro and/or in vivo, as assessed by methods known to one of skill in the art; or, that increased expression of stable and functional Smn protein is produced from the SMN1 gene in vitro and/or in vivo, as assessed by methods known to one of skill in the art; or, that expression of the fusion protein encoded by the minigene is increased in vitro, as assessed by methods known to one of skill in the art; or, that expression of Smn protein produced from the SMN1 gene in a subject (e.g., an animal model for SMA or a human subject) in need thereof is increased.
As used herein, the term “substantial change” in the context of the amount of mRNA means that the amount of mRNA changes by a statistically significant amount, e.g., a p value less than a value selected from 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001.
As used herein, the terms “subject” and “patient” are used interchangeably to refer to an animal or any living organism having sensation and the power of voluntary movement, and which requires for its existence oxygen and organic food. Nonlimiting examples include members of the human, equine, porcine, bovine, rattus, murine, canine and feline species. In some embodiments, the subject is a mammal or a warm-blooded vertebrate animal. In certain embodiments, the subject is a non-human animal. In specific embodiments, the subject is a human.
As used herein, the term “elderly human” refers to a human 65 years old or older.
As used herein, the term “human adult” refers to a human that is 18 years or older.
As used herein, the term “human child” refers to a human that is 1 year to 18 years old.
As used herein, the term “human infant” refers to a newborn to 1 year old year human.
As used herein, the term “human toddler” refers to a human that is 1 year to 3 years old.
Compound Forms
As used herein, the terms “a compound of Formula (Ia)” and “a compound of Formula (Ib)” refer to sub-genuses of the compound of Formula (I) or a form thereof and are defined herein. Rather than repeat embodiments for a compound of Formula (Ia) or a compound of Formula (Ib), in certain embodiments, the term “a compound(s) of Formula (I) or a form thereof” is used to refer to either a compound of Formula (Ia) or a form thereof, a compound of Formula (Ib) or a form thereof, or both. Thus, embodiments and references to “a compound of Formula (I)” are intended to include compounds of Formula (Ia) and Formula (Ib).
As used herein, the term “form” means a compound of Formula (I) selected from a free acid, free base, salt, isotopologue, stereoisomer, racemate, enantiomer, diastereomer, or tautomer thereof.
In certain embodiments described herein, the form of the compound of Formula (I) is a selected from a salt, isotopologue, stereoisomer, racemate, enantiomer, diastereomer or tautomer thereof.
In certain embodiments described herein, the form of the compound of Formula (I) is a selected from a free acid, isotopologue, stereoisomer, racemate, enantiomer, diastereomer or tautomer thereof.
In certain embodiments described herein, the form of the compound of Formula (I) is a selected from a free base, isotopologue, stereoisomer, racemate, enantiomer, diastereomer or tautomer thereof.
In certain embodiments described herein, the form of the compound of Formula (I) is a free acid, free base or salt thereof.
In certain embodiments described herein, the form of the compound of Formula (I) is an isotopologue thereof.
In certain embodiments described herein, the form of the compound of Formula (I) is a stereoisomer, racemate, enantiomer or diastereomer thereof.
In certain embodiments described herein, the form of the compound of Formula (I) is a tautomer thereof.
In certain embodiments described herein, the form of the compound of Formula (I) is a pharmaceutically acceptable form.
In certain embodiments described herein, the compound of Formula (I) or a form thereof is isolated for use.
As used herein, the term “isolated” means the physical state of a compound of Formula (I) or a form thereof after being isolated and/or purified from a synthetic process (e.g., from a reaction mixture) or natural source or combination thereof according to an isolation or purification process or processes described herein or which are well known to the skilled artisan (e.g., chromatography, recrystallization and the like) in sufficient purity to be characterizable by standard analytical techniques described herein or well known to the skilled artisan.
As used herein, the term “protected” means that a functional group on a compound of Formula (I) is in a form modified to preclude undesired side reactions at the protected site when the compound is subjected to a reaction. Suitable protecting groups will be recognized by those with ordinary skill in the art as well as by reference to standard textbooks such as, for example, T. W. Greene et al, Protective Groups in Organic Synthesis (1991), Wiley, New York.
Prodrugs of a compound of Formula (I) or a form thereof are also contemplated herein.
As used herein, the term “prodrug” means that a functional group on a compound of Formula (I) is in a form (e.g., acting as an active or inactive drug precursor) that is transformed in vivo to yield an active or more active compound of Formula (I) or a form thereof. The transformation may occur by various mechanisms (e.g., by metabolic and/or non-metabolic chemical processes), such as, for example, by hydrolysis and/or metabolism in blood, liver and/or other organs and tissues. A discussion of the use of prodrugs is provided by V. J. Stella, et. al., “Biotechnology: Pharmaceutical Aspects, Prodrugs: Challenges and Rewards,” American Association of Pharmaceutical Scientists and Springer Press, 2007.
In one example, when a compound of Formula (I) or a form thereof contains a carboxylic acid functional group, a prodrug can comprise an ester formed by the replacement of the hydrogen atom of the acid group with a functional group such as alkyl and the like. In another example, when a compound of Formula (I) or a form thereof contains an alcohol functional group, a prodrug can be formed by the replacement of the hydrogen atom of the alcohol group with a functional group such as alkyl or substituted carbonyl and the like. In another example, when a compound of Formula (I) or a form thereof contains an amine functional group, a prodrug can be formed by the replacement of one or more amine hydrogen atoms with a functional group such as alkyl or substituted carbonyl. In another example, when a compound of Formula (I) or a form thereof contains a hydrogen substituent, a prodrug can be formed by the replacement of one or more hydrogen atoms with an alkyl substituent.
Pharmaceutically acceptable prodrugs of compounds of Formula (I) or a form thereof include those compounds substituted with one or more of the following groups: carboxylic acid esters, sulfonate esters, amino acid esters phosphonate esters, mono-, di- or triphosphate esters or alkyl substituents where appropriate. As described herein, it is understood by a person of ordinary skill in the art that one or more of such substituents may be used to provide a compound of Formula (I) or a form thereof for use as a prodrug.
One or more compounds described herein may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and the description herein is intended to embrace both solvated and unsolvated forms.
As used herein, the term “solvate” means a physical association of a compound described herein with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. As used herein, “solvate” encompasses both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like.
One or more compounds described herein may optionally be converted to a solvate. Preparation of solvates is generally known. A typical, non-limiting process involves dissolving a compound in a desired amount of the desired solvent (organic or water or mixtures thereof) at a higher than ambient temperature, and cooling the solution at a rate sufficient to form crystals which are then isolated by standard methods. Analytical techniques such as, for example infrared spectroscopy, show the presence of the solvent (or water) in the crystals as a solvate (or hydrate).
As used herein, the term “hydrate” means a solvate wherein the solvent molecule is water.
The compounds of Formula (I) can form salts which are intended to be included within the scope of this description. Reference to a compound of Formula (I) herein is understood to include reference to salts thereof, unless otherwise indicated. The term “salt(s)”, as employed herein, denotes acidic salts formed with inorganic and/or organic acids, as well as basic salts formed with inorganic and/or organic bases. In addition, when a compound of Formula (I) contains both a basic moiety, such as, but not limited to a pyridine or imidazole, and an acidic moiety, such as, but not limited to a carboxylic acid, zwitterions (“inner salts”) may be formed and are included within the term “salt(s)” as used herein.
The term “pharmaceutically acceptable salt(s)”, as used herein, means those salts of compounds described herein that are safe and effective (i.e., non-toxic, physiologically acceptable) for use in mammals and that possess biological activity, although other salts are also useful. Salts of the compounds of Formula (I) may be formed, for example, by reacting a compound of Formula (I) with an amount of acid or base, such as an equivalent or stoichiometric amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.
Pharmaceutically acceptable salts include one or more salts of acidic or basic groups present in compounds described herein. Embodiments of acid addition salts include, and are not limited to, acetate, acid phosphate, ascorbate, benzoate, benzenesulfonate, bisulfate, bitartrate, borate, butyrate, chloride, citrate, camphorate, camphorsulfonate, ethanesulfonate, formate, fumarate, gentisinate, gluconate, glucaronate, glutamate, hydrobromide, hydrochloride, dihydrochloride, hydroiodide, isonicotinate, lactate, maleate, methanesulfonate, naphthalenesulfonate, nitrate, oxalate, pamoate, pantothenate, phosphate, propionate, saccharate, salicylate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate (also known as tosylate), trifluoroacetate salts and the like. Certain embodiments of mono-acid, di-acid or tri-acid addition salts include a chloride, hydrochloride, dihydrochloride, trihydrochloride, hydrobromide, acetate, diacetate or trifluoroacetate salt. More particular embodiments include a chloride, hydrochloride, dihydrochloride, hydrobromide or trifluoroacetate salt.
Additionally, acids which are generally considered suitable for the formation of pharmaceutically useful salts from basic pharmaceutical compounds are discussed, for example, by P. Stahl et al, Camille G. (eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use. (2002) Zurich: Wiley-VCH; S. Berge et al, Journal of Pharmaceutical Sciences (1977) 66(1) 1-19; P. Gould, International J. of Pharmaceutics (1986) 33, 201-217; Anderson et al, The Practice of Medicinal Chemistry (1996), Academic Press, New York; and in The Orange Book (Food & Drug Administration, Washington, D.C. on their website). These disclosures are incorporated herein by reference thereto.
Suitable basic salts include, but are not limited to, aluminum, ammonium, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts. Certain compounds described herein can also form pharmaceutically acceptable salts with organic bases (for example, organic amines) such as, but not limited to, dicyclohexylamines, tert-butyl amines and the like, and with various amino acids such as, but not limited to, arginine, lysine and the like. Basic nitrogen-containing groups may be quarternized with agents such as lower alkyl halides (e.g., methyl, ethyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, and dibutyl sulfates), long chain halides (e.g., decyl, lauryl, and stearyl chlorides, bromides and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides), and others.
All such acid salts and base salts are intended to be pharmaceutically acceptable salts within the scope of the description herein and all acid and base salts are considered equivalent to the free forms of the corresponding compounds for the purposes described herein.
Compounds of Formula I and forms thereof may further exist in a tautomeric form (for example, as a keto or enol form such as an embedded enone system). All such tautomeric forms are contemplated herein as part of the present description.
The compounds of Formula (I) may contain asymmetric or chiral centers, and, therefore, may exist in different stereoisomeric forms. The present description is intended to include all stereoisomeric forms of the compounds of Formula (I) as well as mixtures thereof, including racemic mixtures.
The compounds of Formula (I) described herein may include one or more chiral centers, and as such may exist as racemic mixtures (R/S) or as substantially pure enantiomers and diastereomers. The compounds may also exist as substantially pure (R) or (S) enantiomers (when one chiral center is present). In one embodiment, the compounds of Formula (I) described herein are (S) isomers and may exist as enantiomerically pure compositions substantially comprising only the (S) isomer. In another embodiment, the compounds of Formula (I) described herein are (R) isomers and may exist as enantiomerically pure compositions substantially comprising only the (R) isomer. As one of skill in the art will recognize, when more than one chiral center is present, the compounds of Formula (I) described herein may also include portions described as an (R,R), (R,S), (S,R) or (S,S) isomer, as defined by IUPAC Nomenclature Recommendations.
As used herein, the term “substantially pure” refers to compounds consisting substantially of a single isomer in an amount greater than or equal to 90%, in an amount greater than or equal to 92%, in an amount greater than or equal to 95%, in an amount greater than or equal to 98%, in an amount greater than or equal to 99%, or in an amount equal to 100% of the single isomer.
In one aspect, a compound of Formula (I) is a substantially pure (S) enantiomer present in an amount greater than or equal to 90%, in an amount greater than or equal to 92%, in an amount greater than or equal to 95%, in an amount greater than or equal to 98%, in an amount greater than or equal to 99%, or in an amount equal to 100%.
In one aspect, a compound of Formula (I) is a substantially pure (R) enantiomer present in an amount greater than or equal to 90%, in an amount greater than or equal to 92%, in an amount greater than or equal to 95%, in an amount greater than or equal to 98%, in an amount greater than or equal to 99%, or in an amount equal to 100%.
As used herein, a “racemate” is any mixture of isometric forms that are not “enantiomerically pure”, including mixtures such as, without limitation, in a ratio of about 50/50, about 60/40, about 70/30, about 80/20, about 85/15 or about 90/10.
In addition, the present description embraces all geometric and positional isomers. For example, if a compound of Formula (I) incorporates a double bond or a fused ring, both the cis- and trans-forms, as well as mixtures, are embraced within the scope of the description herein.
Diastereomeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods well known to those skilled in the art, such as, for example, by chromatography and/or fractional crystallization. Enantiomers can be separated by use of chiral HPLC column or other chromatographic methods known to those skilled in the art.
Enantiomers can also be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., chiral auxiliary such as a chiral alcohol or Mosher's acid chloride), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. Also, some of the compounds of Formula (I) may be atropisomers (e.g., substituted biaryls) and are considered part of this description.
It is also possible that the compounds of Formula (I) may exist in different tautomeric forms, and all such forms are embraced within the scope of this description. Accordingly, all keto-enol and imine-enamine forms of a compound of Formula (I) are included in the description herein.
All stereoisomer forms (for example, geometric isomers, optical isomers, positional isomers and the like) of the present compounds (including salts, solvates, esters and prodrugs and transformed prodrugs thereof) which may exist due to asymmetric carbons on various substituents, including enantiomeric forms (which may exist even in the absence of asymmetric carbons), rotameric forms, atropisomers, diastereomeric forms and regioisomeric forms are contemplated within the scope of the description herein. For example, if a compound of Formula (I) incorporates a double bond or a fused ring, both the cis- and trans-forms, as well as mixtures thereof, are embraced within the scope of the description herein. Also, for example, all keto-enol and imine-enamine tautomeric forms of the compounds are included in the description herein. Individual stereoisomers of the compounds of Formula (I) described herein may, for example, be substantially free of other isomers, or may be present in a racemic mixture, as described supra.
The use of the terms “salt,” “prodrug” and “transformed prodrug” are intended to equally apply to the salts, prodrugs and transformed prodrugs of all contemplated isotopologues, stereoisomers, racemates or tautomers of the instant compounds.
The term “isotopologue” refers to isotopically-enriched compounds which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds described herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as H2, H3, C13, C14, N15, O18, O17, P31, P32, S35, F18, Cl35 and Cl36, respectively, each of which is also within the scope of this description.
Certain isotopically-enriched compounds described herein (e.g., those labeled with H3 and C14) are useful in compound and/or substrate tissue distribution assays. Tritiated (i.e., H3) and carbon-14 (i.e., C14) isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (i.e., H2) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances. Isotopically-enriched compounds of Formula (I) can generally be prepared using procedures known to persons of ordinary skill in the art by substituting an appropriate isotopically-enriched reagent for a non-isotopically-enriched reagent.
When the compounds are enriched with deuterium, the deuterium-to-hydrogen ratio in the deuterated areas of the molecules substantially exceeds the naturally occurring deuterium-to-hydrogen ratio.
An embodiment described herein may include a compound of Formula (I) and forms thereof, wherein the isotopologue is deuterium.
An embodiment described herein may include a compound of Formula (I) and forms thereof, wherein a carbon atom may have from 1 to 3 hydrogen atoms optionally replaced with deuterium.
Polymorphic crystalline and amorphous forms of the compounds of Formula (I), and of the salts, solvates, esters and prodrugs of the compounds of Formula (I), are further intended to be included in the scope of the compounds described herein.
Compound Uses
Compounds of Formula (I) or a form thereof that enhance inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene are described herein. Such compounds of Formula (I) or a form thereof have been shown to enhance the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene using the assays described herein (see Biological example section, infra). Accordingly, compounds of Formula (I) or a form thereof have utility as enhancers for the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene.
Compounds of Formula (I) or a form thereof for enhancing inclusion of exon 7 of SMN1 into mRNA that is transcribed from the SMN1 gene are described herein. Such compounds of Formula (I) or a form thereof may enhance inclusion of exon 7 of SMN1 into mRNA that is transcribed from the SMN1 gene using, e.g., an SMN1 minigene assay. Accordingly, compounds of Formula (I) or a form thereof may have utility as enhancers for the inclusion of exon 7 of SMN1 into mRNA that is transcribed from the SMN1 gene.
In one aspect, provided herein are methods for modulating the inclusion of exon 7 of SMN2 into RNA transcribed from the SMN2 gene, comprising contacting a A method for enhancing the inclusion of exon 7 of SMN2 into mRNA transcribed from the SMN2 gene, comprising contacting a human cell with a compound of Formula (I) or a form thereof. In a specific embodiment, provided herein are methods for modulating the inclusion of exon 7 of SMN2 into RNA transcribed from the SMN2 gene, comprising contacting a human cell with a compound of Formula (I) or a form thereof that modulates the expression of an SMN2 minigene described herein or in International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833, each of which is incorporated herein by reference in its entirety. In one embodiment, the minigene is a minigene described in the Examples of International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833. In another embodiment, the minigene is the minigene described in Biological Example 1, infra. The human cell can be contacted with a compound of Formula (I) or a form thereof in vitro, in a non-human animal or in a human. In a specific embodiment, the human cell is in a human. In another specific embodiment, the human cell is in a human SMA patient. In another specific embodiment, the human cell is in a human SMA patient, wherein SMA is caused by an inactivating mutation or deletion in the SMN1 gene on both chromosomes, resulting in a loss of SMN1 gene function. In another embodiment, the human cell is a human cell from a human SMA patient. In certain embodiments, the human cell is from a cell line, such as GM03813, GM00232, GM09677, and/or GM23240 (available from Coriell Institute).
In a specific embodiment, provided herein is a method for enhancing the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene, comprising contacting a human cell with a compound of Formula (I) or a form thereof. In another embodiment, provided herein is a method for enhancing the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene, comprising contacting a human cell with a compound of Formula (I) or a form thereof that enhances the expression of an SMN2 minigene described herein or in International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833, each of which is incorporated herein by reference in its entirety. In one embodiment, the minigene is a minigene described in the Examples of International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833. In another embodiment, the minigene is the minigene described in Biological Example 1, infra. The human cell can be contacted with a compound of Formula (I) or a form thereof in vitro, in a non-human animal or in a human. In a specific embodiment, the human cell is in a human. In another specific embodiment, the human cell is in a human SMA patient. In another specific embodiment, the human cell is in a human SMA patient, wherein SMA is caused by an inactivating mutation or deletion in the SMN1 gene on both chromosomes, resulting in a loss of SMN1 gene function. In another embodiment, the human cell is a human cell from a human SMA patient. In certain embodiments, the human cell is from a cell line, such as GM03813, GM00232, GM09677, and/or GM23240 (available from Coriell Institute).
In another aspect, provided herein are methods for enhancing the inclusion of exon 7 of SMN1 into RNA transcribed from the SMN1 gene, comprising contacting a human cell with a compound of Formula (I) or a form thereof. In a specific embodiment, provided herein are methods for enhancing the inclusion of exon 7 of SMN1 into RNA transcribed from the SMN1 gene, comprising contacting a human cell with a compound of Formula (I) or a form thereof. In another specific embodiment, provided herein are methods for enhancing the inclusion of exon 7 of SMN1 into RNA transcribed from the SMN1 gene, comprising contacting a human cell with a compound of Formula (I) or a form thereof that modulates the expression of an SMN1 minigene described in International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833, each of which is incorporated herein by reference in its entirety. In one embodiment, the minigene is a minigene described in the Examples of International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833. The human cell can be contacted with a compound of Formula (I) or a form thereof in vitro, in a non-human animal or in a human. In a specific embodiment, the human cell is in a human. In another specific embodiment, the human cell is in a human SMA patient.
In specific embodiments, provided herein are methods for enhancing the inclusion of exon 7 of SMN1 and SMN2 into RNA transcribed from the SMN1 and SMN2 genes, comprising contacting a human cell with a compound of Formula (I) or a form thereof. The human cell can be contacted with a compound of Formula (I) or a form thereof in vitro, in a non-human animal or in a human. In a specific embodiment, the human cell is in a human. In another specific embodiment, the human cell is in a human SMA patient.
In another aspect, provided herein is a method for modulating the inclusion of exon 7 of SMN2 into RNA transcribed from the SMN2 gene, comprising administering to a non-human animal model for SMA a compound of Formula (I) or a form thereof. In a specific embodiment, provided herein is a method for modulating the inclusion of exon 7 of SMN2 into RNA transcribed from the SMN2 gene, comprising administering to a non-human animal model for SMA a compound of Formula (I) or a form thereof that modulates the expression of an SMN2 minigene described herein or in International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833, each of which is incorporated herein by reference in its entirety. In one embodiment, the minigene is a minigene described in the Examples of International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833. In another embodiment, the minigene is the minigene described in Biological Example 1, infra.
In a specific embodiment, provided herein is a method for enhancing the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene, comprising administering to a non-human animal model for SMA a compound of Formula (I) or a form thereof. In another specific embodiment, provided herein is a method for enhancing the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene, comprising administering to a non-human animal model for SMA a compound of Formula (I) or a form thereof that enhances the expression of an SMN2 minigene described herein or in International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833, each of which is incorporated herein by reference in its entirety. In one embodiment, the minigene is a minigene described in the Examples of International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833. In another embodiment, the minigene is the minigene described in Biological Example 1, infra.
In another aspect, provided herein is a method for enhancing the inclusion of exon 7 of SMN1 into RNA transcribed from the SMN1 gene, comprising administering to a non-human animal model for SMA a compound of Formula (I) or a form thereof. In a specific embodiment, provided herein is a method for enhancing the inclusion of exon 7 of SMN1 into RNA transcribed from the SMN1 gene, comprising administering to a non-human animal model for SMA a compound of Formula (I) or a form thereof that modulates the expression of an SMN1 minigene described herein or in International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833, each of which is incorporated herein by reference in its entirety. In one embodiment, the minigene is a minigene described in the Examples of International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833. In specific embodiments, provided herein is a method for enhancing the inclusion of exon 7 of SMN1 and SMN2 into RNA transcribed from the SMN1 and SMN2 genes, comprising administering to a non-human animal model for SMA a compound of Formula (I) or a form thereof.
In another aspect, provided herein is a method for increasing the amount of Smn protein, comprising contacting a human cell with a compound of Formula (I) or a form thereof. In a specific embodiment, provided herein is a method for increasing the amount of Smn protein, comprising contacting a human cell with a compound of Formula (I) that enhances the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene. In another specific embodiment, provided herein is a method for increasing the amount of Smn protein, comprising contacting a human cell with a compound of Formula (I) that enhances the inclusion of exon 7 of SMN1 and/or SMN2 into mRNA that is transcribed from the SMN1 and/or SMN2 gene. The human cell can be contacted with a compound of Formula (I) or a form thereof in vitro, in a non-human animal or in a human. In a specific embodiment, the human cell is in a human. In another specific embodiment, the human cell is in a human SMA patient. In another specific embodiment, the human cell is in a human SMA patient, wherein SMA is caused by an inactivating mutation or deletion in the SMN1 gene on both chromosomes, resulting in a loss of SMN1 gene function. In another embodiment, the human cell is a human cell from a human SMA patient. In certain embodiments, the human cell is from a cell line, such as GM03813, GM00232, GM09677, and/or GM23240 (available from Coriell Institute).
In another aspect, provided herein is a method for increasing the amount of Smn protein, comprising administering to a non-human animal model for SMA a compound of Formula (I) or a form thereof. In a specific embodiment, provided herein is a method for increasing the amount of Smn protein, comprising administering to a non-human animal model for SMA a compound of Formula (I) that enhances the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene in, e.g., a cell-based or cell-free assay, such as described in the Biological Examples, infra. In another specific embodiment, provided herein is a method for increasing the amount of Smn protein, comprising administering to a non-human animal model for SMA a compound of Formula (I) that enhances the inclusion of exon 7 of SMN1 and/or SMN2 into mRNA that is transcribed from the SMN1 and/or SMN2 gene in, e.g., a cell-based or cell-free assay.
In one embodiment, the compound of Formula (I) enhances the expression of a minigene described herein or in International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833, each of which is incorporated herein by reference in its entirety. In a specific embodiment, the compound of Formula (I) enhances the expression of a minigene described in the Examples of International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833. In another specific embodiment, the compound of Formula (I) enhances the expression of a minigene described in Biological Example 1, infra.
In one embodiment, provided herein is the use of a compound of Formula (I) or a form thereof for the preparation of a medicament that enhances the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene. In another embodiment, provided herein is the use of a compound of Formula (I) or a form thereof for the preparation of a medicament that enhances the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene, thereby increasing expression of Smn protein in a human subject in need thereof. In a particular embodiment, the compound of Formula (I) or a form thereof enhances the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene in an assay described herein (see, e.g., the Biological Examples, infra).
In one embodiment, provided herein is the use of a compound of Formula (I) or a form thereof for the preparation of a medicament that enhances the inclusion of exon 7 of SMN1 and/or SMN2 into mRNA that is transcribed from the SMN1 and/or SMN2 gene. In another embodiment, provided herein is the use of a compound of Formula (I) or a form thereof for the preparation of a medicament that enhances the inclusion of exon 7 of SMN1 and/or SMN2 into mRNA that is transcribed from the SMN1 and/or SMN2 gene, thereby increasing expression of Smn protein in a human subject in need thereof.
In another aspect, provided herein are methods for enhancing the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene in a human subject in need thereof, comprising administering to the human subject an effective amount of a compound of Formula (I) or a form thereof. In a specific embodiment, provided herein is a method for enhancing the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene in a human subject in need thereof, comprising administering to the human subject an effective amount a compound of Formula (I) or a form thereof that enhances the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene as determined in an assay described herein (see, e.g., the Biological Examples, infra). In specific embodiments, the effective amount of the compound of Formula (I) or a form thereof is administered to the human subject in a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent. In a particular embodiment, the compound of Formula (I) or a form thereof enhances the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene in an assay described herein (see, e.g., the Biological Examples, infra). In a specific embodiment, the human subject is a human SMA patient. In another specific embodiment, the human subject is a human SMA patient, wherein SMA is caused by an inactivating mutation or deletion in the SMN1 gene on both chromosomes, resulting in a loss of SMN1 gene function.
In another aspect, provided herein are methods for enhancing the inclusion of exon 7 of SMN1 into mRNA that is transcribed from the SMN1 gene in a human subject in need thereof, comprising administering to the human subject an effective amount of a compound of Formula (I) or a form thereof. In a particular embodiment, the compound of Formula (I) or a form thereof enhances the inclusion of exon 7 of SMN1 into mRNA that is transcribed from the SMN1 gene in an assay described in International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833. In specific embodiments, the effective amount of the compound of Formula (I) or a form thereof is administered to the human subject in a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent. In a specific embodiment, the human subject is a human SMA patient.
In another aspect, provided herein is a method for enhancing the inclusion of exon 7 of SMN1 and SMN2 into mRNA that is transcribed from the SMN1 and SMN2 genes in a human subject in need thereof, comprising administering to the human subject an effective amount a compound of Formula (I) or a form thereof. In a particular embodiment, the compound of Formula (I) or a form thereof enhances the inclusion of exon 7 of SMN1 into mRNA that is transcribed from the SMN1 gene in an assay(s) described in International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833 (see, e.g., the Examples in those publications), each of which is incorporated herein by reference in its entirety. In specific embodiments, the effective amount of the compound of Formula (I) or a form thereof is administered to the human subject in a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent. In a specific embodiment, the human subject is a human SMA patient. In another specific embodiment, the human subject is a human SMA patient, wherein SMA is caused by an inactivating mutation or deletion in the SMN1 gene on both chromosomes, resulting in a loss of SMN1 gene function.
In another aspect, provided herein are methods for enhancing the expression of Smn protein in a human subject in need thereof, comprising administering to the human subject an effective amount of a compound of Formula (I) or a form thereof. In a specific embodiment, provided herein is a method for enhancing the expression of Smn protein in a human subject in need thereof, comprising administering to the human subject an effective amount a compound of Formula (I) or a form thereof that enhances the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene. In another specific embodiment, provided herein is a method for enhancing the expression of Smn protein in a human subject in need thereof, comprising administering to the human subject an effective amount a compound of Formula (I) or a form thereof that enhances the inclusion of exon 7 of SMN1 and/or SMN2 into mRNA that is transcribed from the SMN1 and/or SMN2 gene. In specific embodiments, the effective amount of the compound of Formula (I) or a form thereof is administered to the human subject in a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent. In a particular embodiment, the compound of Formula (I) or a form thereof enhances the inclusion of exon 7 of SMN1 and/or SMN2 into mRNA that is transcribed from the SMN1 and/or SMN2 gene in an assay described herein (see, e.g., the Biological Examples, infra) or in International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833 (see, e.g., the Examples in those publications), each of which is incorporated herein by reference in its entirety.
In a specific embodiment, the human subject is a human SMA patient. In another specific embodiment, the human subject is a human SMA patient, wherein SMA is caused by an inactivating mutation or deletion in the teleomeric copy of the SMN1 gene in both chromosomes, resulting in a loss of SMN1 gene function.
In another embodiment, provided herein is the use of a compound of Formula (I) or a form thereof for the preparation of a medicament that enhances expression of Smn protein in a human subject in need thereof. In a particular embodiment, the compound of Formula (I) or a form thereof enhances the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene as determined in an assay described herein (see, e.g., the Biological Examples, infra). In another embodiment, the compound of Formula (I) or a form thereof enhances the inclusion of exon 7 of SMN1 and/or SMN2 into mRNA that is transcribed from the SMN1 and/or SMN2 gene as determined in an assay described herein (see, e.g., the Biological Examples, infra) or in International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833 (see, e.g., the Examples in those publications), each of which is incorporated herein by reference in its entirety.
In another aspect, provided herein are methods for treating spinal muscular atrophy (SMA), comprising administering to a subject an effective amount of a compound of Formula (I) or a form thereof. In a specific embodiment, provided herein is a method for treating SMA in a human subject in need thereof, comprising administering to the subject an effective amount of a compound of Formula (I) or a form thereof. In another specific embodiment, provided herein is a method for treating SMA in a human subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising an effective amount of a compound of Formula (I) or a form thereof, and a pharmaceutically acceptable carrier, excipient or diluent.
In another embodiment, provided herein is a method for treating SMA in a human subject in need thereof, comprising administering to the subject an effective amount of a compound of Formula (I) or a form thereof that enhances the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene. In a specific embodiment, provided herein is a method for treating SMA in a human subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising an effective amount of a compound of Formula (I) or a form thereof that enhances the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene, and a pharmaceutically acceptable carrier, excipient or diluent. In another specific embodiment, provided herein is a method for treating SMA in a human subject in need thereof, comprising administering to the human subject a pharmaceutical composition comprising an effective amount of a compound of Formula (I) or a form thereof that enhances the inclusion of exon 7 of SMN1 and/or SMN2 into mRNA that is transcribed from the SMN1 and/or SMN2 gene, and a pharmaceutically acceptable carrier, excipient or diluent. In a particular embodiment, the compound of Formula (I) or a form thereof enhances the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene in an assay described herein (see, e.g., the Biological Examples, infra). In another embodiment, the compound of Formula (I) or a form thereof enhances the inclusion of exon 7 of SMN1 and/or SMN2 into mRNA that is transcribed from the SMN1 and/or SMN2 gene as determined in an assay described herein (see, e.g., the Biological Examples, infra) or in International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833 (see, e.g., the Examples in those publications), each of which is incorporated herein by reference in its entirety.
In another embodiment, provided herein is the use of a compound of Formula (I) or a form thereof in the manufacture of a medicament for treating SMA in a human subject in need thereof. In a particular embodiment, the compound of Formula (I) or a form thereof enhances the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene as determined in an assay described herein (see, e.g., the Biological Examples, infra). In another embodiment, the compound of Formula (I) or a form thereof enhances the inclusion of exon 7 of SMN1 and/or SMN2 into mRNA that is transcribed from the SMN1 and/or SMN2 gene as determined in an assay described herein (see, e.g., the Biological Examples, infra) or in International Publication No. WO2009/151546 or U.S. Patent Application Publication No. 2011/0086833 (see, e.g., the Examples in those publications), each of which is incorporated herein by reference in its entirety.
In an embodiment of a use or method provided herein, compounds of Formula (I) or a form thereof are used in combination with one or more additional agents. A compound(s) of Formula (I) or a form thereof can be administered to a subject or contacted with a cell prior to, concurrently with, or subsequent to administering to the subject or contacting the cell with an additional agent(s). A compound(s) of Formula (I) or a form thereof and an additional agent(s) can be administered to a subject or contacted with a cell in single composition or different compositions. In a specific embodiments, a compound(s) of Formula (I) or a form thereof is used in combination with gene replacement of SMN1 (using, e.g., viral delivery vectors). In another specific embodiments, a compound(s) of Formula (I) or a form thereof are used in combination with cell replacement using differentiated SMN1+/+ and/or SMN2+/+ stem cells. In another specific embodiments, a compound(s) of Formula (I) or a form thereof are used in combination with cell replacement using differentiated SMN1+/+ stem cells. In another specific embodiments, a compound(s) of Formula (I) or a form thereof are used in combination with cell replacement using differentiated SMN2+/+ stem cells. In another specific embodiment, a compound(s) of Formula (I) or a form thereof are used in combination with aclarubicin. In another specific embodiment, a compound(s) of Formula (I) or a form thereof are used in combination with a transcription activator such as a histone deacetylase (“HDAC”) inhibitor (e.g., butyrates, valproic acid, and hydroxyurea), and mRNA stabilizers (e.g., mRNA decapping inhibitor RG3039 from Repligen).
In one embodiment, provided herein is the use of compounds of Formula (I) or a form thereof in combination with supportive therapy, including respiratory, nutritional or rehabilitation care.
In certain embodiments, treating SMA with a compound of Formula (I) or a form thereof (alone or in combination with an additional agent) has a therapeutic effect and/or beneficial effect. In a specific embodiment, treating SMA with a compound of Formula (I) or a form thereof (alone or in combination with an additional agent) results in one, two or more of the following effects: (i) reduces or ameliorates the severity of SMA; (ii) delays onset of SMA; (iii) inhibits the progression of SMA; (iv) reduces hospitalization of a subject; (v) reduces hospitalization length for a subject; (vi) increases the survival of a subject; (vii) improves the quality of life of a subject; (viii) reduces the number of symptoms associated with SMA; (ix) reduces or ameliorates the severity of a symptom(s) associated with SMA; (x) reduces the duration of a symptom associated with SMA; (xi) prevents the recurrence of a symptom associated with SMA; (xii) inhibits the development or onset of a symptom of SMA; and/or (xiii) inhibits of the progression of a symptom associated with SMA.
Symptoms of SMA include muscle weakness, poor muscle tone, weak cry, weak cough, limpness or a tendency to flop, difficulty sucking or swallowing, difficulty breathing, accumulation of secretions in the lungs or throat, clenched fists with sweaty hand, flickering/vibrating of the tongue, head often tilted to one side, even when lying down, legs that tend to be weaker than the arms, legs frequently assuming a “frog legs” position, feeding difficulties, increased susceptibility to respiratory tract infections, bowel/bladder weakness, lower-than-normal weight, inability to sit without support, failure to walk, failure to crawl, and hypotonia, areflexia, and multiple congenital contractures (arthrogryposis) associated with loss of anterior horn cells.
In a specific embodiment, treating SMA with a compound of Formula (I) or a form thereof (alone or in combination with an additional agent) results in one, two or more of the following effects: (i) a reduction in the loss of muscle strength; (ii) an increase in muscle strength; (iii) a reduction in muscle atrophy; (iv) a reduction in the loss of motor function; (v) an increase in motor neurons; (vii) a reduction in the loss of motor neurons; (viii) protection of SMN deficient motor neurons from degeneration; (ix) an increase in motor function; (x) an increase in pulmonary function; and/or (xi) a reduction in the loss of pulmonary function.
In another embodiment, treating SMA with a compound of Formula (I) or a form thereof (alone or in combination with an additional agent) results in the functional ability or helps retain the functional ability for a human infant or a human toddler to sit up. In another embodiment, treating SMA with a compound of Formula (I) or a form thereof (alone or in combination with an additional agent) results in the functional ability or helps retain the functional ability for a human infant, a human toddler, a human child or a human adult to stand up unaided. In another embodiment, treating SMA with a compound of Formula (I) or a form thereof (alone or in combination with an additional agent) results in the functional ability or helps retain the functional ability for a human infant, a human toddler, a human child or a human adult to walk unaided. In another embodiment, treating SMA with a compound of Formula (I) or a form thereof (alone or in combination with an additional agent) results in the functional ability or helps retain the functional ability for a human infant, a human toddler, a human child or a human adult to run unaided. In another embodiment, treating SMA with a compound of Formula (I) or a form thereof (alone or in combination with an additional agent) results in the functional ability or helps retain the functional ability for a human infant, a human toddler, a human child or a human adult to breathe unaided. In another embodiment, treating SMA with a compound of Formula (I) or a form thereof (alone or in combination with an additional agent) results in the functional ability or helps retain the functional ability for a human infant, a human toddler, a human child or a human adult to turn during sleep unaided. In another embodiment, treating SMA with a compound of Formula (I) or a form thereof (alone or in combination with an additional agent) results in the functional ability or helps retain the functional ability for a human infant, a human toddler, a human child or a human adult to swallow unaided.
In certain embodiments, a primer and/or probe described below in the Biological Examples (e.g., SMN primers such as SEQ ID NO. 1, 7, 8, 11 or 13 and/or SEQ ID NO. 2, 9 or 12, and SMN probes such as a SEQ ID NO. 3 or 10) is used in an assay, such as RT-PCR, RT-qPCR, endpoint RT-PCR, PCR, qPCR, rolling circle amplification, Northern blot or Southern blot, to determine whether a compound of Formula (I) or a form thereof enhances the inclusion of exon 7 of SMN1 and/or SMN2 into mRNA that is transcribed from an SMN1 and/or SMN2 gene. In some embodiments, a primer and/or probe described below in the Biological Examples (e.g., SMN primers such as SEQ ID NO. 1, 7, 8, 11 or 13 and/or SEQ ID NO. 2, 9 or 12, and SMN probes such as a SEQ ID NO. 3 or 10) is used in an assay, such as RT-PCR, RT-qPCR, endpoint RT-PCR, PCR, qPCR, rolling circle amplification, Northern blot or Southern blot, or a pharmaceutical or assay kit as described infra, to monitor patient responses to a compound of Formula (I) or a form thereof.
In a specific embodiment, a compound of Formula (I):
In another specific embodiment, the compound of Formula (I) used in accordance with a method described herein is a compound selected from Formula (Ia) or Formula (Ib):
or a form thereof, wherein all variables are as previously defined.
Patient Population
In some embodiments, a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof is administered to a subject suffering from SMA. In other embodiments, a compound of Formula (I) or a form thereof, is administered to a subject predisposed or susceptible to SMA. In a specific embodiment, a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof is administered to a human subject, wherein the subject has SMA caused by an inactivating mutation or deletion in the SMN1 gene on both chromosomes, resulting in a loss of SMN1 gene function. In certain embodiments, the human subject is genotyped prior to administration of a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof to determine whether the subject has an inactivating mutation or deletion in the teleomeric copy of the SMN1 gene in both chromosomes, which results in a loss of SMN1 gene function. In some embodiments, a compound of Formula (I) or a form thereof, or pharmaceutical composition thereof is administered to a subject with Type 0 SMA. In some embodiments, a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof is administered to a subject with Type 1 SMA. In other embodiments, a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof is administered to a subject with Type 2 SMA. In other embodiments, a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof is administered to a subject with Type 3 SMA. In some embodiments, a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof is administered to a subject with Type 4 SMA.
In certain embodiments, a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof is administered to a subject that will or might benefit from enhanced inclusion of exon 7 of SMN1 and/or SMN2 into mRNA that is transcribed from the SMN1 and/or SMN2 gene. In specific embodiments, a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof is administered to a subject that will or may benefit from enhanced Smn protein expression.
In certain embodiments, a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof is administered to a human that has an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.
In some embodiments, a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof is administered to a human infant. In other embodiments, a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof is administered to a human toddler. In other embodiments, a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof is administered to a human child. In other embodiments, a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof is administered to a human adult. In yet other embodiments, a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof is administered to an elderly human.
In some embodiments, a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof, is administered to a patient to prevent the onset of SMA in a patient at risk of developing SMA. In other embodiments, an effective amount of a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof, is administered to a patient to prevent the onset of SMA in a patient at risk of developing SMA. In other embodiments, a prophylactically effective amount of a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof, is administered to a patient to prevent the onset of SMA in a patient at risk of developing SMA. In other embodiments, a therapeutically effective amount of a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof, is administered to a patient to prevent the onset of SMA in a patient at risk of developing SMA. In a specific embodiment, the patient is an SMA patient.
In some embodiments, a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof, is administered to a patient to treat or ameliorate SMA in an SMA patient. In other embodiments, an effective amount of a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof, is administered to a patient to treat or ameliorate SMA in an SMA patient. In other embodiments, a prophylactically effective amount of a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof, is administered to a patient to prevent advancement of SMA in an SMA patient. In other embodiments, a therapeutically effective amount of a compound of Formula (I) or a form thereof, or a pharmaceutical composition thereof, is administered to a patient to treat or ameliorate SMA in an SMA patient. In a specific embodiment, the patient is an SMA patient.
In some embodiments, a compound of Formula (I) or a form thereof, or a medicament thereof is administered to a subject suffering from SMA. In other embodiments, a compound of Formula (I) or a form thereof, is administered to a subject predisposed or susceptible to SMA. In a specific embodiment, a compound of Formula (I) or a form thereof, or a medicament thereof is administered to a human subject, wherein the subject has SMA caused by an inactivating mutation or deletion in the SMN1 gene on both chromosomes, resulting in a loss of SMN1 gene function. In certain embodiments, the human subject is genotyped prior to administration of a compound of Formula (I) or a form thereof, or a medicament thereof to determine whether the subject has an inactivating mutation or deletion in the teleomeric copy of the SMN1 gene in both chromosomes, which results in a loss of SMN1 gene function. In some embodiments, a compound of Formula (I) or a form thereof, or medicament thereof is administered to a subject with Type 0 SMA. In some embodiments, a compound of Formula (I) or a form thereof, or a medicament thereof is administered to a subject with Type 1 SMA. In other embodiments, a compound of Formula (I) or a form thereof, or a medicament thereof is administered to a subject with Type 2 SMA. In other embodiments, a compound of Formula (I) or a form thereof, or a medicament thereof is administered to a subject with Type 3 SMA. In some embodiments, a compound of Formula (I) or a form thereof, or a medicament thereof is administered to a subject with Type 4 SMA.
In certain embodiments, a compound of Formula (I) or a form thereof, or a medicament thereof is administered to a subject that will or might benefit from enhanced inclusion of exon 7 of SMN1 and/or SMN2 into mRNA that is transcribed from the SMN1 and/or SMN2 gene. In specific embodiments, a compound of Formula (I) or a form thereof, or a medicament thereof is administered to a subject that will or may benefit from enhanced Smn protein expression.
In certain embodiments, a compound of Formula (I) or a form thereof, or a medicament thereof is administered to a human that has an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.
In some embodiments, a compound of Formula (I) or a form thereof, or a medicament thereof is administered to a human infant. In other embodiments, a compound of Formula (I) or a form thereof, or a medicament thereof is administered to a human toddler. In other embodiments, a compound of Formula (I) or a form thereof, or a medicament thereof is administered to a human child. In other embodiments, a compound of Formula (I) or a form thereof, or a medicament thereof is administered to a human adult. In yet other embodiments, a compound of Formula (I) or a form thereof, or a medicament thereof is administered to an elderly human.
In some embodiments, a compound of Formula (I) or a form thereof, or a medicament thereof is administered to a patient to prevent the onset of SMA in a patient at risk of developing SMA. In other embodiments, an effective amount of a compound of Formula (I) or a form thereof, or a medicament thereof, is administered to a patient to prevent the onset of SMA in a patient at risk of developing SMA. In other embodiments, a prophylactically effective amount of a compound of Formula (I) or a form thereof, or a medicament thereof, is administered to a patient to prevent the onset of SMA in a patient at risk of developing SMA. In other embodiments, a therapeutically effective amount of a compound of Formula (I) or a form thereof, or a medicament thereof, is administered to a patient to prevent the onset of SMA in a patient at risk of developing SMA. In a specific embodiment, the patient is an SMA patient.
In some embodiments, a compound of Formula (I) or a form thereof, or a medicament thereof, is administered to a patient to treat or ameliorate SMA in an SMA patient. In other embodiments, an effective amount of a compound of Formula (I) or a form thereof, or a medicament thereof, is administered to a patient to treat or ameliorate SMA in an SMA patient. In other embodiments, a prophylactically effective amount of a compound of Formula (I) or a form thereof, or a medicament thereof, is administered to a patient to prevent advancement of SMA in an SMA patient. In other embodiments, a therapeutically effective amount of a compound of Formula (I) or a form thereof, or a medicament thereof, is administered to a patient to treat or ameliorate SMA in an SMA patient. In a specific embodiment, the patient is an SMA patient.
Mode of Administration
When administered to a patient, a compound of Formula (I) or a form thereof is preferably administered as a component of a composition that optionally comprises a pharmaceutically acceptable carrier, excipient or diluent. The composition can be administered orally, or by any other convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa) and may be administered together with another biologically active agent. Administration can be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, and can be used to administer the compound. In a specific embodiment, the patient is an SMA patient.
Methods of administration include but are not limited to parenteral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin. The mode of administration is left to the discretion of the practitioner. In most instances, administration will result in the release of a compound into the bloodstream. In a specific embodiment, a compound is administered orally.
Dosage and Dosage Forms
The amount of a compound of Formula (I) or a form thereof that will be effective in the treatment of SMA depend, e.g., on the route of administration, the type of SMA, the general health of the subject, ethnicity, age, weight, and gender of the subject, diet, time, and the severity of SMA, and should be decided according to the judgment of the practitioner and each patient's or subject's circumstances.
In specific embodiments, an “effective amount,” “prophylactically effective amount” or “therapeutically effective amount” in the context of the administration of a compound of Formula (I) or a form thereof, or composition or medicament thereof refers to an amount of a compound of Formula (I) which has a therapeutic effect and/or beneficial effect. In certain specific embodiments, an “effective amount,” “prophylactically effective amount” or “therapeutically effective amount” in the context of the administration of a compound of Formula (I) or a form thereof, or composition or medicament thereof results in one, two or more of the following effects: (i) reduces or ameliorates the severity of SMA; (ii) delays onset of SMA; (iii) inhibits the progression of SMA; (iv) reduces hospitalization of a subject; (v) reduces hospitalization length for a subject; (vi) increases the survival of a subject; (vii) improves the quality of life of a subject; (viii) reduces the number of symptoms associated with SMA; (ix) reduces or ameliorates the severity of a symptom(s) associated with SMA; (x) reduces the duration of a symptom associated with SMA; (xi) prevents the recurrence of a symptom associated with SMA; (xii) inhibits the development or onset of a symptom of SMA; and/or (xiii) inhibits of the progression of a symptom associated with SMA. In certain embodiments, an effective amount of a compound of Formula (I) or a form thereof is an amount effective to enhance inclusion of exon 7 of SMN2 into SMN2 mRNA that is transcribed from the SMN2 gene and increases the levels of Smn protein produced from the SMN2 gene and thus producing a desired beneficial effect in a subject in need thereof. In some instances, the desired effect can be determined by analyzing or quantifying: (1) the inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene; or (2) the levels of Smn protein produced from the SMN2 gene. Non-limiting examples of effective amounts of a compound of Formula (I) or a form thereof are described herein.
For example, the effective amount may be the amount required to treat SMA in a human subject in need thereof, or the amount required to enhance inclusion of exon 7 of SMN2 into mRNA that is transcribed from the SMN2 gene in a human subject in need thereof, or the amount required to increase levels of Smn protein produced from the SMN2 gene in a human subject in need thereof.
In general, the effective amount will be in a range of from about 0.001 mg/kg/day to about 500 mg/kg/day for a patient or subject having a weight in a range of between about 1 kg to about 200 kg. The typical adult subject is expected to have a median weight in a range of between about 70 and about 100 kg.
Within the scope of the present description, the “effective amount” of a compound of Formula (I) or a form thereof for use in the manufacture of a medicament, the preparation of a pharmaceutical kit or in a method for treating SMA in a human subject in need thereof, is intended to include an amount in a range of from about 0.001 mg to about 35,000 mg. In a specific embodiment, the human subject is an SMA patient.
The compositions described herein are formulated for administration to the subject via any drug delivery route known in the art. Nonlimiting examples include oral, ocular, rectal, buccal, topical, nasal, ophthalmic, subcutaneous, intramuscular, intravenous (bolus and infusion), intracerebral, transdermal, and pulmonary routes of administration.
Pharmaceutical Compositions
Embodiments described herein include the use of a compound of Formula (I) or a form thereof in a pharmaceutical composition. In a specific embodiment, described herein is the use of a compound of Formula (I) or a form thereof in a pharmaceutical composition for treating SMA in a human subject in need thereof comprising administering an effective amount of a compound of Formula (I) or a form thereof in admixture with a pharmaceutically acceptable carrier, excipient or diluent. In a specific embodiment, the human subject is an SMA patient.
A compound of Formula (I) or a form thereof may optionally be in the form of a composition comprising the compound or a form thereof and an optional carrier, excipient or diluent. Other embodiments provided herein include pharmaceutical compositions comprising an effective amount of a compound of Formula (I) or a form thereof and a pharmaceutically acceptable carrier, excipient, or diluent. In a specific embodiment, the pharmaceutical compositions are suitable for veterinary and/or human administration. The pharmaceutical compositions provided herein can be in any form that allows for the composition to be administered to a subject.
In a specific embodiment and in this context, the term “pharmaceutically acceptable carrier, excipient or diluent” means a carrier, excipient or diluent approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a specific carrier for intravenously administered pharmaceutical compositions. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
Typical compositions and dosage forms comprise one or more excipients. Suitable excipients are well-known to those skilled in the art of pharmacy, and non limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a patient and the specific active ingredients in the dosage form. Further provided herein are anhydrous pharmaceutical compositions and dosage forms comprising one or more compounds of Formula (I) or a form thereof as described herein. The compositions and single unit dosage forms can take the form of solutions or syrups (optionally with a flavoring agent), suspensions (optionally with a flavoring agent), emulsions, tablets (e.g., chewable tablets), pills, capsules, granules, powder (optionally for reconstitution), taste-masked or sustained-release formulations and the like.
Pharmaceutical compositions provided herein that are suitable for oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets, caplets, capsules, granules, powder, and liquids. Such dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art.
Examples of excipients that can be used in oral dosage forms provided herein include, but are not limited to, binders, fillers, disintegrants, and lubricants.
Biomarkers
In certain embodiments, the amount of mRNA that is transcribed from the SMN1 gene and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 is used as a biomarker for SMA. In certain embodiments, the amount of mRNA that is transcribed from the SMN1 gene and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 is used as a biomarker for SMA. In a specific embodiment, the patient is an SMA patient.
In other embodiments, the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 is used as a biomarker for an SMA patient being treated with a compound, such as disclosed herein. In other embodiments, the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 is used as a biomarker for an SMA patient being treated with a compound, such as disclosed herein. In a specific embodiment, the patient is an SMA patient.
In some embodiments, a change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 and a corresponding change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 is a biomarker for a patient being treated with a compound, such as disclosed herein. In a specific embodiment, the patient is an SMA patient.
In a specific embodiment, an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 and a corresponding decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, after the administration of a compound (e.g., a compound of Formula (I) disclosed herein), indicates that the compound may be effective to treat SMA. In another specific embodiment, a decrease in the amount of mRNA that is transcribed from the SMN2 gene and includes exon 7 of SMN2 and a corresponding increase in the amount of mRNA that is transcribed from the SMN2 gene and does not include exon 7 of SMN2, after the administration of a compound (e.g., a compound of Formula (I) disclosed herein), indicates that the compound will not be effective to treat SMA. In accordance with these embodiments, an SMN primer(s) and/or an SMN probe described below can be used in assays, such as PCR (e.g., qPCR) and RT-PCR (e.g., RT-qPCR or endpoint RT-PCR) to assess and/or quantify the amount of mRNA that is transcribed from the SMN1 gene and/or SMN2 gene that does or does not include exon 7 of SMN1 and/or SMN2.
In one embodiment, provided herein are SMN primers and/or SMN probes (e.g., a forward primer having the nucleotide sequence of SEQ ID NO. 1, 7, 8, 11 or 13; and/or a reverse primer having the nucleotide sequence of SEQ ID NO. 9 or 12; and/or an SMN probe such as a SEQ ID NO. 3 or 10) for amplifying nucleic acids encoding or encoded by human SMN1 and/or SMN2. These primers can be used as primers in, e.g., RT-PCR (such as RT-PCR, endpoint RT-PCR and/or RT-qPCR as described herein or as known to one skilled in the art), PCR (such as qPCR) or rolling circle amplification, and as probes in hybridization assays, such as a Northern blot and/or a Southern blot assay. As utilized in the Biological Examples herein, endpoint RT-PCR is a reverse transcription-polymerase chain reaction that is carried out for a certain number of amplification cycles (or until starting materials are exhausted) following by a quantification of each of the DNA products using, e.g., gel electrophoretic separation, staining with a fluorescent dye, quantification of fluorescence and the like.
SEQ ID NO. 1 hybridizes to DNA or RNA comprising nucleotides corresponding to nucleotides 22 to 40 of exon 7 of SMN1 and/or SMN2, SEQ ID NO. 2 hybridizes to DNA or RNA comprising nucleotides corresponding to nucleotides 4 to 26 of the firefly luciferase coding sequence; SEQ ID NO. 7 hybridizes to nucleic acid sequences (e.g., the sense strand of DNA) comprising nucleotides corresponding to nucleotides 32 to 54 of exon 7 of SMN1 and/or SMN2 and nucleotides 1 to 4 of exon 8 of SMN1 and/or SMN2, SEQ ID NO. 8 hybridizes to nucleic acid sequences (e.g., the sense strand of DNA) comprising nucleotides corresponding, in order, to nucleotides 87 to 111 of exon 7 of SMN1 and/or SMN2 and nucleotides 1 to 3 of exon 8 of SMN1 and/or SMN2, SEQ ID NO. 9 hybridizes to nucleic acid sequences (e.g., the antisense strand of DNA or RNA) comprising nucleotides corresponding to nucleotides 39 to 62 of exon 8 of SMN1 and/or SMN2, SEQ ID NO. 11 hybridizes to nucleic acid sequences (e.g., the sense strand of DNA) comprising nucleotides corresponding to nucleotides 43 to 63 of exon 6 of SMN1 and/or SMN2, SEQ ID NO. 12 hybridizes to nucleic acid sequences (e.g., the antisense strand of DNA or RNA) comprising nucleotides corresponding to nucleotides 51 to 73 of exon 8 of SMN1 and/or SMN2, and SEQ ID NO. 13 hybridizes to nucleic acid sequence (e.g., the sense strand of DNA) comprising nucleotides corresponding to nucleotides 22 to 46 of exon 6 of SMN1 and/or SMN2.
Accordingly, an oligonucleotide corresponding to SEQ ID NO. 9, 11, 12 and/or 13 can be used in an amplification reaction to amplify nucleic acids encoding or encoded by human SMN1 and/or SMN2 lacking exon 7 of human SMN1 and/or SMN2 and nucleic acid encoding or encoded by human SMN1 and/or SMN2 and includes exon 7 of human SMN1 and/or SMN2. In contrast, an oligonucleotide corresponding to SEQ ID NO. 8 in conjunction with a downstream reverse primer (e.g., SEQ ID NO. 9 or 12) can be used to amplify nucleic acids encoding or encoded by human SMN1 and/or SMN2 lacking exon 7 of human SMN1 and/or SMN2 and an oligonucleotide corresponding to SEQ ID NO. 1 and 7 in conjunction with a downstream reverse primer (e.g., SEQ ID NO. 9 or 12) can be used to amplify nucleic acids encoding or encoded by human SMN1 and/or human SMN2 and includes exon 7 of SMN1 and/or SMN2.
SEQ ID NO. 3 hybridizes to nucleic acid sequences (e.g., the sense strand of DNA) comprising nucleotides corresponding, in order, to nucleotides 50 to 54 of exon 7 of human SMN1 and/or SMN2 and nucleotides 1 to 21 of exon 8 of human SMN1 and/or SMN2, and SEQ ID NO. 10 hybridizes to nucleic acid sequences (e.g., the sense strand of DNA) comprising nucleotides corresponding to nucleotides 7 to 36 of exon 8 of human SMN1 and/or SMN2. SEQ ID NO. 3 is useful as a probe to detect mRNA that is transcribed from the minigene and includes exon 7 of SMN1 and/or SMN2, described herein or described in International Publication No. WO 2009/151546 or U.S. Patent Application Publication No. 2011/0086833 (each of which is incorporated herein by reference in its entirety) and to detect mRNA that is transcribed from human SMN1 and/or SMN2 and includes exon 7 of SMN1 and/or SMN2. In addition, SEQ ID NO. 10 is useful as a probe to detect mRNA that is transcribed from the minigene that does or does not include exon 7 of SMN1 and/or SMN2 and to detect mRNA that is transcribed from human SMN1 and/or SMN2, described herein or as described in International Publication No. WO 2009/151546 or U.S. Patent Application Publication No. 2011/0086833, each of which is incorporated herein by reference in its entirety.
In a specific embodiment, a primer and/or probe described below in the Biological Examples (e.g., SMN primers such as SEQ ID NO. 1, 7, 11 or 13 and/or SEQ ID NO. 2, 9 or 12, and/or SMN probes such as a SEQ ID NO. 3 or 10) is used in an assay, such as RT-PCR, RT-qPCR, endpoint RT-PCR, PCR, qPCR, rolling circle amplification and, as applicable, Northern blot or Southern blot (e.g., an assay such as described below in the Biological Examples), to determine whether a compound (e.g., a compound of Formula (I) or a form thereof) enhances the inclusion of exon 7 of SMN1 and/or SMN2 into mRNA that is transcribed from an SMN1 and/or SMN2 gene.
In another embodiment, a primer and/or probe described below in the Biological Examples (e.g., SMN primers such as SEQ ID NO. 1, 7, 11 or 13 and/or SEQ ID NO. 9 or 12, and/or SMN probes such as a SEQ ID NO. 3 or 10) is used in an assay, such as RT-PCR, RT-qPCR, endpoint RT-PCR, PCR, qPCR, rolling circle amplification and, as applicable, Northern blot or Southern blot (e.g., an assay such as described below in the Biological Examples), to monitor the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in a patient sample. In a specific embodiment, the patient is an SMA patient.
In another embodiment, a primer and/or probe described below in the Biological Examples (e.g., SMN primers such as SEQ ID NO. 1, 7, 11 or 13 and/or SEQ ID NO. 9 or 12, and/or SMN probes such as a SEQ ID NO. 3 or 10) is used in an assay, such as RT-PCR, RT-qPCR, endpoint RT-PCR, PCR, qPCR, rolling circle amplification and, as applicable, Northern blot or Southern blot (e.g., an assay such as described below in the Biological Examples), to monitor a patient's response to a compound (e.g., a compound of Formula (I) or a form thereof). In a specific embodiment, the patient is an SMA patient.
A sample (e.g., a blood sample, PBMC sample, or tissue sample, such as a skin or muscle tissue sample) from a patient can be obtained using techniques known to one skilled in the art and the primers and/or probes described in the Biological Examples below can be used in assays (e.g., PCR, RT-PCR, RT-qPCR, qPCR, endpoint RT-PCR, rolling circle amplification, Northern blot and Southern blot) to determine the amount of mRNA that is transcribed from the SMN1 and/or SMN2 genes (e.g., the amount of mRNA that includes exon 7 of SMN2 transcribed from the SMN2 gene). A sample derived from a patient refers to a sample that is processed and/or manipulated after being obtained from the patient using techniques known to one skilled in the art. For example, a sample from a patient can be processed to, e.g., extract RNA, using techniques known to one of skill in the art. A sample from a patient can be processed to, e.g., extract RNA and the RNA is reversed transcribed to produce cDNA. In a specific embodiment, the patient is an SMA patient.
In a specific embodiment, provided herein is a method for detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2, comprising: (a) contacting a patient sample (e.g., blood sample or tissue sample) or a sample derived from a patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with a forward SMN primer described below (e.g., SEQ ID NO. 1, 7, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) along with applicable components for, e.g., an RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification; and (b) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2. In certain embodiments, the sample is from or derived from a patient administered a compound, such as a compound of Formula (I) or a form thereof as described herein. In a specific embodiment, the patient is an SMA patient.
In another specific embodiment, provided herein is a method for detecting the amount of mRNA that is transcribed from the SMN1 and SMN2 genes, comprising: (a) contacting a patient sample (e.g., blood sample or tissue sample) or a sample derived from a patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with a forward SMN primer described below (e.g., SEQ ID NO. 1, 7, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) along with applicable components for, e.g., an RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification; and (b) detecting the amount of mRNA that is transcribed from the SMN1 and SMN2 genes. In certain embodiments, the sample is from or derived from a patient administered a compound, such as a compound of Formula (I) or a form thereof as described herein. In a specific embodiment, the patient is an SMA patient.
The amount of mRNA that is transcribed from the human SMN1 and SMN2 genes and includes exon 7 of SMN1 and SMN2 and the amount of mRNA that is transcribed from the human SMN1 and SMN2 genes and does not include exon 7 of SMN1 and SMN2 can be differentiated from each other by, e.g., size of the RNA or DNA fragment generated from SMN1 and SMN2 mRNA that includes exon 7 of SMN1 and SMN2 and from SMN1 and SMN2 mRNA that does not include exon 7 of SMN1 and SMN2.
In another specific embodiment, provided herein is a method for detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, comprising: (a) contacting a patient sample (e.g., blood sample or tissue sample) or a sample derived from a patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with a forward SMN primer described below (e.g., SEQ ID NO. 8, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) along with applicable components for, e.g., an RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification; and (b) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2. In certain embodiments, the sample is from or derived from a patient administered a compound, such as a compound of Formula (I) or a form thereof as described herein. In a specific embodiment, the patient is an SMA patient.
In another specific embodiment, provided herein is a method for detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2, comprising: (a) contacting a patient sample (e.g., blood sample or tissue sample) or a sample derived from a patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with an SMN probe described below (e.g., SEQ ID NO. 3 or 10) along with applicable components, e.g., of an RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR), rolling circle amplification and, as applicable, Northern blot or Southern blot; and (b) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2. In certain embodiments, the sample is from or derived from a patient administered a compound, such as a compound of Formula (I) or a form thereof as described herein. In a specific embodiment, the patient is an SMA patient.
In another specific embodiment, provided herein is a method for detecting the amount of mRNA that is transcribed from the SMN1 and SMN2 genes, comprising: (a) contacting a patient sample (e.g., blood sample or tissue sample) or a sample derived from a patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with an SMN probe described below (e.g., SEQ ID NO. 3 or 10) along with applicable components for, e.g., an RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR), rolling circle amplification and, as applicable, Northern blot or Southern blot; and (b) detecting the amount of mRNA that is transcribed from the SMN1 and SMN2 genes. In a specific embodiment, the patient is an SMA patient.
The amount of mRNA that is transcribed from the human SMN1 and SMN2 genes and includes exon 7 of SMN1 and SMN2 and the amount of mRNA that is transcribed from the human SMN1 and SMN2 genes and does not include exon 7 of SMN1 and SMN2 can be differentiated from each other by, e.g., size of the RNA or DNA fragment generated from SMN1 and SMN2 mRNA that includes exon 7 of SMN1 and SMN2 and from SMN1 and SMN2 mRNA that does not include exon 7 of SMN1 and SMN2. In certain embodiments, the sample is from or derived from a patient administered a compound, such as a compound of Formula (I) or a form thereof as described herein. In a specific embodiment, the patient is an SMA patient.
In another specific embodiment, provided herein is a method for detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, comprising: (a) contacting a patient sample (e.g., blood sample or tissue sample) or a sample derived from a patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with an SMN probe described below (e.g., SEQ ID NO. 10) along with applicable components for, e.g., an RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR), rolling circle amplification, or Northern blot or Southern blot; and (b) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2. In certain embodiments, the sample is from or derived from a patient administered a compound, such as a compound of Formula (I) or a form thereof as described herein. In a specific embodiment, the patient is an SMA patient.
In a specific embodiment, provided herein is a method for detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2, comprising: (a) contacting a patient sample (e.g., blood sample or tissue sample) or a sample derived from a patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with a forward SMN primer described below (e.g., SEQ ID NO. 1, 7, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) and/or an SMN probe described herein (e.g., SEQ ID NO. 3 or 10) along with applicable components for e.g., an RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification; and (b) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2. In certain embodiments, the sample is from or derived from a patient administered a compound, such as a compound of Formula (I) or a form thereof as described herein. In a specific embodiment, the patient is an SMA patient.
In a specific embodiment, provided herein is a method for detecting the amount of mRNA that is transcribed from the SMN1 and SMN2 genes, comprising: (a) contacting a patient sample (e.g., blood sample or tissue sample) or a sample derived from a patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with a forward SMN primer described below (e.g., SEQ ID NO. 1, 7, 8, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) and/or an SMN probe described herein (e.g., SEQ ID NO. 3 or 10) along with applicable components for e.g., an RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification, as applicable; and (b) detecting the amount of mRNA that is transcribed from the SMN1 and SMN2 genes. In a specific embodiment, the patient is an SMA patient.
The amount of mRNA that is transcribed from the human SMN1 and SMN2 genes and includes exon 7 of SMN1 and SMN2 and the amount of mRNA that is transcribed from the human SMN1 and SMN2 genes and does not include exon 7 of SMN1 and SMN2 can be differentiated from each other by, e.g., size of the RNA or DNA fragment generated from SMN1 and SMN2 mRNA that includes exon 7 of SMN1 and SMN2 and from SMN1 and SMN2 mRNA that does not include exon 7 of SMN1 and SMN2. In certain embodiments, the sample is from or derived from a patient administered a compound, such as a compound of Formula (I) or a form thereof as described herein. In a specific embodiment, the patient is an SMA patient.
In a specific embodiment, provided herein is a method for detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, comprising: (a) contacting a patient sample (e.g., blood sample or tissue sample) or a sample derived from a patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with a forward SMN primer described below (e.g., SEQ ID NO. 8) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) and/or an SMN probe described herein (e.g., SEQ ID NO. 10) along with applicable components for e.g., an RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification; and (b) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2. In certain embodiments, the sample is from or derived from a patient administered a compound, such as a compound of Formula (I) or a form thereof as described herein. In a specific embodiment, the patient is an SMA patient.
In a specific embodiment, provided herein is a method for assessing an SMA patient's response to a compound, comprising: (a) contacting an SMA patient sample (e.g., blood sample or tissue sample) or a sample derived from an SMA patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with a forward SMN primer described below (e.g., SEQ ID NO. 1, 7, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) along with applicable components for e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification, wherein the sample is from or derived from an SMA patient administered a compound (e.g., a compound described herein); and (b) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2, wherein (1) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound indicates that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound indicates that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is assessed 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In another specific embodiment, provided herein is a method for assessing an SMA patient's response to a compound, comprising: (a) administering a compound to an SMA patient; (b) contacting a sample (e.g., blood sample or tissue sample) obtained or derived from the patient with a forward SMN primer described below (e.g., SEQ ID NO. 1, 7, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) along with applicable components for e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification; and (c) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN2, wherein (1) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound indicates that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound indicates that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is assessed 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In a specific embodiment, provided herein is a method for assessing an SMA patient's response to a compound, comprising: (a) contacting an SMA patient sample (e.g., blood sample or tissue sample) or a sample derived from an SMA patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with a forward SMN primer described below (e.g., SEQ ID NO. 1, 7, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) and/or an SMN probe (e.g., SEQ ID NO. 3 or 10) along with applicable components for e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification, wherein the sample is from or derived from an SMA patient administered a compound (e.g., a compound of Formula (I) or a form thereof as described herein); and (b) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2, wherein (1) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound indicates that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound indicates that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is assessed 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In another specific embodiment, provided herein is a method for assessing an SMA patient's response to a compound, comprising: (a) administering a compound to an SMA patient; (b) contacting a sample (e.g., blood sample or tissue sample) obtained or derived from the patient with a forward SMN primer described below (e.g., SEQ ID NO. 1, 7, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) and/or an SMN probe (e.g., SEQ ID NO. 3 or 10) along with applicable components for e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification; and (c) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2, wherein (1) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound indicates that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound indicates that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is assessed 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In a specific embodiment, provided herein is a method for assessing an SMA patient's response to a compound, comprising: (a) contacting an SMA patient sample (e.g., blood sample or tissue sample) or a sample derived from an SMA patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with a forward SMN primer described below (e.g., SEQ ID NO. 8, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) along with applicable components for e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification, wherein the sample is from or derived from an SMA patient administered a compound (e.g., a compound of Formula (I) or a form thereof as described herein); and (b) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound indicates that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound indicates that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is assessed 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In another specific embodiment, provided herein is a method for assessing an SMA patient's response to a compound, comprising: (a) administering a compound to an SMA patient; (b) contacting a sample (e.g., blood sample or tissue sample) obtained or derived from the patient with a forward SMN primer described below (e.g., SEQ ID NO. 8, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) along with applicable components for e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification; and (c) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound indicates that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound indicates that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is assessed 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In a specific embodiment, provided herein is a method for assessing an SMA patient's response to a compound, comprising: (a) contacting an SMA patient sample (e.g., blood sample or tissue sample) or a sample derived from an SMA patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with a forward SMN primer described below (e.g., SEQ ID NO. 8, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) and/or an SMN probe (e.g., SEQ ID NO. 10) along with applicable components for e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification, wherein the sample is from or derived from an SMA patient administered a compound (e.g., a compound of Formula (I) or a form thereof as described herein); and (b) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound indicates that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound indicates that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is assessed 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In another specific embodiment, provided herein is a method for assessing an SMA patient's response to a compound, comprising: (a) administering a compound to an SMA patient; (b) contacting a sample (e.g., blood sample or tissue sample) obtained or derived from the patient with a forward SMN primer described below (e.g., SEQ ID NO. 8, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) and/or an SMN probe (e.g., SEQ ID NO. 10) along with applicable components for e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification; and (c) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound indicates that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound indicates that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is assessed 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In a specific embodiment, provided herein is a method for assessing an SMA patient's response to a compound, comprising: (a) contacting an SMA patient sample (e.g., blood sample or tissue sample) or a sample derived from an SMA patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with a forward SMN primer described below (e.g., SEQ ID NO. 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) along with applicable components for e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification, wherein the sample is from or derived from an SMA patient administered a compound (e.g., a compound of Formula (I) or a form thereof as described herein); and (b) detecting the amount of mRNA transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 and the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) (i) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound, and (ii) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound, indicate that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) (i) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound, and (ii) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound, indicates that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is assessed 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In another specific embodiment, provided herein is a method for assessing an SMA patient's response to a compound, comprising: (a) administering a compound to an SMA patient; (b) contacting a sample (e.g., blood sample or tissue sample) obtained or derived from the patient with a forward SMN primer described below (e.g., SEQ ID NO. 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) along with applicable components for e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification; and (c) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 and the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) (i) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound, and (ii) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound, indicate that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) (i) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound, and (ii) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound, indicate that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is assessed 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In a specific embodiment, provided herein is a method for assessing an SMA patient's response to a compound, comprising: (a) contacting an SMA patient sample (e.g., blood sample or tissue sample) or a sample derived from an SMA patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with an SMN probe (e.g., SEQ ID NO. 10) along with applicable components for e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification, wherein the sample is from or derived from a patient administered a compound (e.g., a compound of Formula (I) or a form thereof as described herein); and (b) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 and the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) (i) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound, and (ii) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound, indicate that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) (i) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound, and (ii) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound, indicate that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is assessed 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In another specific embodiment, provided herein is a method for assessing an SMA patient's response to a compound, comprising: (a) administering a compound to an SMA patient; (b) contacting a sample (e.g., blood sample or tissue sample) obtained or derived from the patient with an SMN probe (e.g., SEQ ID NO. 10) along with applicable components for e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification; and (c) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 and the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) (i) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound, and (ii) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound, indicate that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) (i) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound, and (ii) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound, indicate that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is assessed 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In a specific embodiment, provided herein is a method for assessing an SMA patient's response to a compound, comprising: (a) contacting an SMA patient sample (e.g., blood sample or tissue sample) or a sample derived from an SMA patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with a forward SMN primer described below (e.g., SEQ ID NO. 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) and/or an SMN probe (e.g., SEQ ID NO. 10) along with applicable components for e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR) or PCR (e.g., qPCR), wherein the sample is from or derived from a patient administered a compound (e.g., a compound of Formula (I) or a form thereof as described herein); and (b) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 and the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) (i) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound, and (ii) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound, indicate that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) (i) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound, and (ii) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound, indicate that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is assessed 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In another specific embodiment, provided herein is a method for assessing an SMA patient's response to a compound, comprising: (a) administering a compound to an SMA patient; (b) contacting a sample (e.g., blood sample or tissue sample) obtained or derived from the patient with a forward SMN primer described below (e.g., SEQ ID NO. 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) and/or an SMN probe (e.g., SEQ ID NO. 10) along with applicable components for, e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification; and (c) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 and the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) (i) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound, and (ii) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound, indicate that the SMN1 and/or patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) (i) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound, and (ii) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound, indicate that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is assessed 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In a specific embodiment, provided herein is a method for monitoring an SMA patient's responsiveness to a compound, comprising: (a) contacting an SMA patient sample (e.g., blood sample or tissue sample) or a sample derived from an SMA patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with a forward SMN primer described below (e.g., SEQ ID NO. 1, 7, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) along with applicable components for e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification, wherein the sample is from or derived from a patient administered a compound (e.g., a compound of Formula (I) or a form thereof as described herein); and (b) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2, wherein (1) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to the administration of the compound or a certain number of doses of the compound, or a certain earlier date indicates that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to the administration of the compound or a certain number of doses of the compound, or a certain earlier date indicates that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is monitored 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the patient has received 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more doses of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the administration of 1-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40-50, or 50-100 doses of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In another specific embodiment, provided herein is a method for monitoring an SMA patient's responsiveness to a compound, comprising: (a) administering a compound to an SMA patient; (b) contacting a sample (e.g., blood sample or tissue sample) obtained or derived from the patient with a forward SMN primer described below (e.g., SEQ ID NO. 1, 7, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) along with applicable components for, e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification; and (c) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2, wherein (1) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to the administration of the compound or a certain number of doses of the compound, or a certain earlier date indicates that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to the administration of the compound or a certain number of doses of the compound, or a certain earlier date indicates that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is monitored 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the patient has received 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more doses of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the administration of 1-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40-50, or 50-100 doses of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In a specific embodiment, provided herein is a method for monitoring an SMA patient's responsiveness to a compound, comprising: (a) contacting an SMA patient sample (e.g., blood sample or tissue sample) or a sample derived from an SMA patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with a forward SMN primer described below (e.g., SEQ ID NO. 1, 7, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) and/or an SMN probe (e.g., SEQ ID NO. 3 or 10) along with applicable components for e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification, wherein the sample is from or derived from a patient administered a compound (e.g., a compound of Formula (I) or a form thereof as described herein); and (b) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2, wherein (1) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to the administration of the compound or a certain number of doses of the compound, or a certain earlier date indicates that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to the administration of the compound or a certain number of doses of the compound, or a certain earlier date indicates that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is monitored 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the patient has received 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more doses of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the administration of 1-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40-50, or 50-100 doses of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In another specific embodiment, provided herein is a method for monitoring an SMA patient's responsiveness to a compound, comprising: (a) administering a compound to an SMA patient; (b) contacting a sample (e.g., blood sample or tissue sample) obtained or derived from the patient with a forward SMN primer described below (e.g., SEQ ID NO. 1, 7, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) and/or an SMN probe (e.g., SEQ ID NO. 3 or 10) along with applicable components for, e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification; and (c) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2, wherein (1) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to the administration of the compound or a certain number of doses of the compound, or a certain earlier date indicates that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to the administration of the compound or a certain number of doses of the compound, or a certain earlier date indicates that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is monitored 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the patient has received 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more doses of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the administration of 1-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40-50, or 50-100 doses of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In a specific embodiment, provided herein is a method for monitoring an SMA patient's responsiveness to a compound, comprising: (a) contacting an SMA patient sample (e.g., blood sample or tissue sample) or a sample derived from an SMA patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with a forward SMN primer described below (e.g., SEQ ID NO. 8, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) along with applicable components for, e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification, wherein the sample is from or derived from a patient administered a compound (e.g., a compound of Formula (I) or a form thereof as described herein); and (b) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to the administration of the compound or a certain number of doses of the compound, or a certain earlier date indicates that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to the administration of the compound or a certain number of doses of the compound, or a certain earlier date indicates that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is monitored 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the patient has received 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more doses of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the administration of 1-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40-50, or 50-100 doses of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In another specific embodiment, provided herein is a method for monitoring an SMA patient's responsiveness to a compound, comprising: (a) administering a compound to an SMA patient; (b) contacting a sample (e.g., blood sample or tissue sample) obtained or derived from the patient with a forward SMN primer described below (e.g., SEQ ID NO. 8, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) along with applicable components for, e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification; and (c) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to the administration of the compound or a certain number of doses of the compound, or a certain earlier date indicates that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to the administration of the compound or a certain number of doses of the compound, or a certain earlier date indicates that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is monitored 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the patient has received 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more doses of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the administration of 1-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40-50, or 50-100 doses of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In a specific embodiment, provided herein is a method for monitoring an SMA patient's responsiveness to a compound, comprising: (a) contacting an SMA patient sample (e.g., blood sample or tissue sample) or a sample derived from an SMA patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with a forward SMN primer described below (e.g., SEQ ID NO. 8, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) and/or an SMN probe (e.g., SEQ ID NO. 10) along with applicable components for, e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification, wherein the sample is from or derived from a patient administered a compound (e.g., a compound of Formula (I) or a form thereof as described herein); and (b) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to the administration of the compound or a certain number of doses of the compound, or a certain earlier date indicates that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to the administration of the compound or a certain number of doses of the compound, or a certain earlier date indicates that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is monitored 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the patient has received 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more doses of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the administration of 1-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40-50, or 50-100 doses of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In another specific embodiment, provided herein is a method for monitoring a SMA patient's responsiveness to a compound, comprising: (a) administering a compound to a SMA patient; (b) contacting a sample (e.g., blood sample or tissue sample) obtained or derived from the patient with a forward SMN primer described below (e.g., SEQ ID NO. 8, 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) and/or an SMN probe (e.g., SEQ ID NO. 10) along with applicable components for, e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification; and (c) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to the administration of the compound or a certain number of doses of the compound, or a certain earlier date indicates that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to the administration of the compound or a certain number of doses of the compound, or a certain earlier date indicates that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is monitored 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the patient has received 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more doses of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the administration of 1-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40-50, or 50-100 doses of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In a specific embodiment, provided herein is a method for monitoring an SMA patient's response to a compound, comprising: (a) contacting an SMA patient sample (e.g., blood sample or tissue sample) or a sample derived from an SMA patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with a forward SMN primer described below (e.g., SEQ ID NO. 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) along with applicable components for, e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification, wherein the sample is from or derived from a patient administered a compound (e.g., a compound of Formula (I) or a form thereof as described herein); and (b) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 and the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) (i) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, and (ii) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, indicate that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) (i) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, and (ii) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, indicate that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is monitored 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the patient has received 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more doses of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the administration of 1-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40-50, or 50-100 doses of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In another specific embodiment, provided herein is a method for monitoring an SMA patient's response to a compound, comprising: (a) administering a compound to an SMA patient; (b) contacting a sample (e.g., blood sample or tissue sample) obtained or derived from the patient with a forward SMN primer described below (e.g., SEQ ID NO. 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) along with applicable components for, e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR), or rolling circle amplification; and (c) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 and the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) (i) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, and (ii) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, indicate that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) (i) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, and (ii) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, indicate that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is monitored 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the patient has received 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more doses of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the administration of 1-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40-50, or 50-100 doses of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In a specific embodiment, provided herein is a method for monitoring an SMA patient's response to a compound, comprising: (a) contacting an SMA patient sample (e.g., blood sample or tissue sample) or a sample derived from an SMA patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with an SMN probe (e.g., SEQ ID NO. 10) along with applicable components for, e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification, wherein the sample is from or derived from a patient administered a compound (e.g., a compound of Formula (I) or a form thereof as described herein); and (b) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 and the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) (i) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, and (ii) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, indicate that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) (i) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, and (ii) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, indicate that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is monitored 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the patient has received 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more doses of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the administration of 1-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40-50, or 50-100 doses of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In another specific embodiment, provided herein is a method for monitoring an SMA patient's response to a compound, comprising: (a) administering a compound to an SMA patient; (b) contacting a sample (e.g., blood sample or tissue sample) obtained or derived from the patient with an SMN probe (e.g., SEQ ID NO. 10) along with applicable components for, e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification; and (c) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 and the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) (i) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, and (ii) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, indicate that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) (i) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, and (ii) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, indicate that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is monitored 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the patient has received 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more doses of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the administration of 1-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40-50, or 50-100 doses of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In a specific embodiment, provided herein is a method for monitoring an SMA patient's response to a compound, comprising: (a) contacting an SMA patient sample (e.g., blood sample or tissue sample) or a sample derived from an SMA patient (e.g., a blood sample or tissue sample that has been processed to extract RNA) with a forward SMN primer described below (e.g., SEQ ID NO. 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) and/or an SMN probe (SEQ ID NO. 10) along with applicable components for, e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification, wherein the sample is from or derived from a patient administered a compound (e.g., a compound of Formula (I) or a form thereof as described herein); and (b) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 and the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) (i) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, and (ii) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, indicate that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) (i) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, and (ii) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, indicate that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is monitored 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the patient has received 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more doses of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the administration of 1-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40-50, or 50-100 doses of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In another specific embodiment, provided herein is a method for monitoring an SMA patient's response to a compound, comprising: (a) administering a compound to an SMA patient; (b) contacting a sample (e.g., blood sample or tissue sample) obtained or derived from the patient with a forward SMN primer described below (e.g., SEQ ID NO. 11 or 13) and/or a reverse SMN primer described herein (e.g., SEQ ID NO. 9 or 12) and/or an SMN probe (SEQ ID NO. 10) along with applicable components for, e.g., RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification; and (c) detecting the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 and the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, wherein (1) (i) an increase in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, and (ii) a decrease in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., from the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, indicate that the patient is responsive to the compound and that the compound may be or is beneficial and/or of therapeutic value to the patient; and (2) (i) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, and (ii) no change or no substantial change in the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in the patient sample relative to the amount of mRNA that is transcribed from the SMN2 gene and does not include exon 7 of SMN1 and/or SMN2 in an analogous sample (e.g., the same type of tissue sample) from the patient prior to administration of the compound or a certain number of doses of the compound, or a certain earlier date, indicate that the patient is not responsive to the compound and that the compound is not beneficial and/or of therapeutic value to the patient. In certain embodiments, the patient's response is monitored 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months or more after administration of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the patient has received 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more doses of a compound, such as a compound of Formula (I) or a form thereof as described herein. In some embodiments, the patient's response is monitored after the administration of 1-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40-50, or 50-100 doses of a compound, such as a compound of Formula (I) or a form thereof as described herein.
In specific embodiments, the SMA in the patient is caused by an inactivating mutation or deletion in the SMN1 gene on both chromosomes, resulting in a loss of SMN1 gene function.
Kits
In one aspect, provided herein are pharmaceutical or assay kits comprising an SMN primer or probe described herein, in one or more containers, and instructions for use. In one embodiment, a pharmaceutical or assay kit comprises, in a container, one or more SMN reverse primers (e.g., SEQ ID NO. 2, 9 and/or 12) and/or one or more SMN forward primers (SEQ ID NO. 1, 7, 8, 11 and/or 13)) and instructions for use. In another embodiment, a pharmaceutical or assay kit comprises, in one container, an SMN reverse primer (e.g., SEQ ID NO. 2, 9 or 12), an SMN forward primer (SEQ ID NO. 1, 7, 8, 11 or 13)) and instructions for use.
In one embodiment, a pharmaceutical or assay kit comprises, in separate containers, one SMN reverse primer (e.g., SEQ ID NO. 2, 9 or 12) in one container, another SMN forward primer (e.g., SEQ ID NO. 1, 7, 8, 11 or 13)) in another container, and instructions for use.
In certain embodiments, applicable components needed for a PCR (e.g., qPCR), RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR) or rolling circle amplification, such as polymerase, deoxynucleoside triphosphates, etc., are included in such kits. In some embodiments, components needed for hybridization are included in such kits. A pharmaceutical or assay kit containing such primers can be used in PCR and RT-PCR to, e.g.,: (i) assess whether a therapeutic agent (e.g., a compound of Formula (I) or a form thereof) enhances inclusion of exon 7 of SMN1 and/or SMN2 into mRNA that is transcribed from the SMN1 and/or SMN2 gene, (ii) monitor the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 and the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, and/or (iii) monitor a subject's response to a therapeutic agent (e.g., a compound of Formula (I) or a form thereof).
In a specific embodiment, a pharmaceutical or assay kit comprises the forward primer with the sequence found in SEQ ID NO. 1, in a container, and the reverse primer with the sequence found in SEQ ID NO. 2, in another container. In certain embodiments, these primers are used in RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification for amplifying nucleotide sequences encoded by a human SMN1 minigene or human SMN2 minigene, such as described those described herein or in International Publication No. WO 2009/151546 or U.S. Patent Application Publication No. 2011/0086833, each of which is incorporated herein by reference in its entirety. In other embodiments, these primers are used as probes in, e.g., hybridization assays, such as Southern blot or Northern blot.
In a specific embodiment, a pharmaceutical or assay kit comprises the forward primer with the nucleotide sequence found in SEQ ID NO. 7, in a container, and the reverse primer with the nucleotide sequence found in SEQ ID NO. 9, in another container. In certain embodiments, these primers are used in RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification for amplifying nucleotide sequences encoded by endogenous human SMN1 and SMN2 genes. In other embodiments, these primers are used as probes in, e.g., hybridization assays, such as Southern blot or Northern blot.
In another specific embodiment, a pharmaceutical or assay kit comprises the forward primer with the nucleotide sequence found in SEQ ID NO. 8, in a container, and the reverse primer with the nucleotide sequence found in SEQ ID NO. 9, in another container. In certain embodiments, these primers are used in RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification for amplifying nucleotide sequences encoded by the endogenous human SMN2 gene. In other embodiments, these primers are used as probes in, e.g., hybridization assays, such as Southern blot or Northern blot.
In a specific embodiment, a pharmaceutical or assay kit comprises the forward primer with the nucleotide sequence found in SEQ ID NO. 7, in a container, the forward primer with the nucleotide sequence found in SEQ ID NO. 8, in another container, and the reverse primer with the nucleotide sequence found in SEQ ID NO. 9, in another container. In certain embodiments, these primers are used in RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification for amplifying nucleotide sequences encoded by endogenous human SMN1 and SMN2 genes. In other embodiments, these primers are used as probes in, e.g., hybridization assays, such as Southern blot or Northern blot.
In a specific embodiment, a pharmaceutical or assay kit comprises the forward primer with the nucleotide sequence found in SEQ ID NO. 11, in a container, and the reverse primer with the nucleotide sequence found in SEQ ID NO. 12, in another container. In certain embodiments, these primers are used in RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification for amplifying nucleotide sequences encoded by endogenous human SMN1 and SMN2 genes. In other embodiments, these primers are used as probes in, e.g., hybridization assays, such as Southern blot or Northern blot.
In a specific embodiment, a pharmaceutical or assay kit comprises the forward primer with the nucleotide sequence found in SEQ ID NO. 11, in a container, and the reverse primer with the nucleotide sequence found in SEQ ID NO. 9, in another container. In certain embodiments, these primers are used in RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification for amplifying nucleotide sequences encoded by endogenous human SMN1 and SMN2 genes. In other embodiments, these primers are used as probes in, e.g., hybridization assays, such as Southern blot or Northern blot.
In a specific embodiment, a pharmaceutical or assay kit comprises the forward primer with the nucleotide sequence found in SEQ ID NO. 13, in a container, and the reverse primer with the nucleotide sequence found in SEQ ID NO. 12, in another container. In certain embodiments, these primers are used in RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification for amplifying nucleotide sequences encoded by endogenous human SMN1 and SMN2 genes. In other embodiments, these primers are used as probes in, e.g., hybridization assays, such as Southern blot or Northern blot.
In a specific embodiment, a pharmaceutical or assay kit comprises the forward primer with the nucleotide sequence found in SEQ ID NO. 13, in a container, and the reverse primer with the nucleotide sequence found in SEQ ID NO. 9, in another container. In certain embodiments, these primers are used in RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification for amplifying nucleotide sequences encoded by endogenous human SMN1 and SMN2 genes. In other embodiments, these primers are used as probes in, e.g., hybridization assays, such as Southern blot or Northern blot.
In a specific embodiment, a pharmaceutical or assay kit comprises the forward primer with the nucleotide sequence found in SEQ ID NO. 1, in a container, and the reverse primer with the nucleotide sequence found in SEQ ID NO. 9, in another container. In certain embodiments, these primers are used in RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification for amplifying nucleotide sequences encoded by endogenous human SMN1 and SMN2 genes. In other embodiments, these primers are used as probes in, e.g., hybridization assays, such as Southern blot or Northern blot.
In a specific embodiment, a pharmaceutical or assay kit comprises the forward primer with the nucleotide sequence found in SEQ ID NO. 1, in a container, and the reverse primer with the nucleotide sequence found in SEQ ID NO. 12, in another container. In certain embodiments, these primers are used in RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR), PCR (e.g., qPCR) or rolling circle amplification for amplifying nucleotide sequences encoded by endogenous human SMN1 and SMN2 genes. In other embodiments, these primers are used as probes in, e.g., hybridization assays, such as Southern blot or Northern blot.
In another embodiment, a pharmaceutical or assay kit comprises an SMN probe described herein (e.g., SEQ ID NO. 3 or 10), in one container. In other embodiments, the probe is used in, e.g., a hybridization assay, such as a Southern blot or Northern blot. In a specific embodiment, the probe is used in RT-qPCR or qPCR. In certain embodiments, components needed for a PCR (e.g., qPCR), RT-PCR (e.g., endpoint RT-PCR and/or RT-qPCR) or rolling circle amplification, such as polymerase, deoxynucleoside triphosphates, primers, etc., are included in such kits. In some embodiments, components needed for hybridization are included in such kits.
In one embodiment, a pharmaceutical or assay kit comprises an SMN reverse primer (e.g., SEQ ID NO. 2, 9 or 12) in one container, an SMN forward primer (e.g., SEQ ID NO. 1, 7, 8, 11 or 13) in another container, and an SMN probe (e.g., SEQ ID NO. 3 or 10) in another container, and instructions for use. In another embodiment, a pharmaceutical or assay kit comprises one or more SMN reverse primers (e.g., SEQ ID NO. 2, 9 and/or 12) in one container, one or more SMN forward primers (e.g., SEQ ID NO. 1, 7, 8, 11 and/or 13) in another container, and one or more SMN probe (e.g., SEQ ID NO. 3 and/or 10) in another container, and instructions for use.
In certain embodiments, components needed to run a PCR, RT-PCR or rolling circle amplification, such as polymerase, deoxynucleoside triphosphates, etc., are included in such kits. A pharmaceutical or assay kit containing such probes and/or primers can be used in PCR and RT-PCR to, e.g.,: (i) assess whether a therapeutic agent (e.g., a compound of Formula (I) or a form thereof) enhances inclusion of exon 7 of SMN1 and/or SMN2 into mRNA that is transcribed from the SMN1 and/or SMN2 gene, (ii) monitor the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and includes exon 7 of SMN1 and/or SMN2 and the amount of mRNA that is transcribed from the SMN1 and/or SMN2 gene and does not include exon 7 of SMN1 and/or SMN2, and/or (iii) monitor a subject's response to a therapeutic agent (e.g., a compound of Formula (I) or a form thereof).
In another aspect, provided herein is a pharmaceutical kit comprising a compound of Formula (I) or a form thereof, in a container, and instructions for use of the compound or form thereof. In a specific embodiment, provided herein is a pharmaceutical kit comprising a pharmaceutical composition comprising a compound of Formula (I) or a form thereof and a pharmaceutically acceptable carrier, excipient or diluent, and instructions for use. In another specific embodiment, provided herein is a pharmaceutical kit comprising a pharmaceutical composition comprising an effective amount of a compound of Formula (I) or a form thereof and a pharmaceutically acceptable carrier, excipient or diluent, and instructions for use. In one embodiment, the instructions for use explain one, two or more of the following: the dose, route of administration, frequency of administration and side effects of administration of a compound of Formula (I) or a form thereof to a subject.
General Synthetic Methods
As disclosed herein, general methods for preparing the compounds of Formula (I) or a form thereof as described herein are available via standard, well-known synthetic methodology. Many of the starting materials are commercially available or, when not available, can be prepared using the routes described below using techniques known to those skilled in the art. The synthetic schemes provided herein comprise multiple reaction steps, each of which is intended to stand on its own and can be carried out with or without any preceding or succeeding step(s). In other words, each of the individual reactions steps of the synthetic schemes provided herein in isolation is contemplated.
Compounds of Formula (I), wherein R2 is an aryl or heteroaryl monocyclic or bicyclic ring system, can be prepared as described in Scheme A below.
Compound A1 (where X represents various reactive groups which may be used to prepare R1 substituents via functional group substitution reactions using techniques known to a person of ordinary skill in the art) can be regioselectively formylated by treatment with a Lewis acid (such as MgCl2 and the like) and paraformaldehyde in a suitable solvent (such as acetonitrile or THF and the like) to afford Compound A2. Compound A2 is reacted with Compound A3, where R is a C1-4alkyl group (such as methyl, ethyl, t-butyl and the like), and in the presence of condensation reagents (such as piperidine/acetic acid and the like) will undergo Knoevenagel condensation followed by lactone formation to afford Compound A4.
Compound A3 can be prepared by combining a mixture of acetic acid ester (such as t-butyl acetate and the like) and a base (such as LiHMDS and the like) in a suitable solvent (such as THF and the like) with Compound A5, wherein R2 represents an aryl, heterocycle or heteroaryl and L represents a leaving group.
Compounds of Formula (I), wherein R2 is a bicyclic heteroaryl ring system, can be prepared as described in Scheme B below.
Compound B2, an optionally substituted monocyclic heteroaryl ring system containing an amidine-like moiety (such as but not limited to 2-aminopyridine, 2-aminopyrimidine, 2-aminopyrazine, 3-aminopyridazine, 2-aminothiazole, 4-aminothiazole, 4-aminopyrimidine and the like) is reacted with Compound B1 (where R represents a C1-4alkyl group such as methyl, ethyl and the like) in a suitable solvent (such as EtOH and the like) to give Compound B3. Compound B3, is reacted with Compound A2, and in the presence of condensation reagents (such as piperidine/acetic acid and the like), will undergo Knoevenagel condensation followed by lactone formation to afford Compound B4.
Compounds of Formula (I), wherein R2 is a bicyclic heteroaryl ring system, can be prepared as described in Scheme C below.
Compound C1 (where R represents a C1-4alkyl group such as methyl, ethyl and the like) is reacted with Compound C2, an optionally substituted aniline (where Y can be OH, NH2, or SH; and, where the aniline ring may have one or more carbon atom ring members replaced with one or more nitrogen atoms, thus making Compound C2 an optionally substituted ring system such as a pyridine, pyrimidine, pyrazine and the like), and in a suitable solvent (such as EtOH or acetonitrile and the like) affords Compound C3. Compound C3 is reacted with Compound A2, and in the presence of condensation reagents (such as piperidine/acetic acid and the like), will undergo Knoevenagel condensation followed by lactone formation to afford Compound C4.
Compounds of Formula (I), wherein R2 is a monocyclic heteroaryl ring system, can be prepared as described in Scheme D below.
Compound D1 (where R represents a C1-4alkyl group such as methyl, ethyl and the like) is reacted with hydrogen sulfide in the presence of an organic base (such as triethylamine and the like) and a suitable solvent (such as pyridine and the like) to give Compound D2. Compound D2 is reacted with Compound D3, an α-bromoketone (where W represents a C1-4alkyl or halo-C1-4alkyl group such as methyl, ethyl, trifluoromethyl and the like), and in an appropriate solvent (such as DMF and the like), undergoes a tandem alkylation dehydrative condensation to give Compound D4. Compound D4 is reacted with Compound A2, and in the presence of condensation reagents (such as piperidine/acetic acid and the like), will undergo Knoevenagel condensation followed by lactone formation to afford Compound D5.
Compounds of Formula (I), wherein R2 is a monocyclic or bicyclic aryl or heteroaryl ring system, can be prepared as described in Scheme E below.
Compound A2 is reacted with acetic anhydride, and in the presence of an organic base (such as triethylamine and the like), undergoes Aldol condensation/lactone formation to afford Compound E1. Compound E1 is brominated with an appropriate brominating reagent (such as Br2 or NBS) to afford Compound E2. Compound E2 is reacted with a boronic acid (where Z represents B(OH)2 and the like) or a trialkyl stannane (where Z represents SnBu3 and the like), and in the presence of a palladium catalyst (such as tetrakis(triphenylphosphine)palladium(0), bis(triphenylphosphine)palladium(II) dichloride, palladium acetate and the like) and an appropriate phospine ligand will undergo Suzuki or Stille cross coupling to give Compound A4.
Compounds of Formula (I), wherein R2 is a monocyclic or bicyclic aryl-amino or heteroaryl-amino, can be prepared as described in Scheme F below.
Compound E2 is reacted with Compound F1, where Ar represents an optionally substituted monocyclic or bicyclic aryl or heteroaryl ring system such as an optionally substituted aniline or amino-heteroaryl, in the presence of a palladium catalyst (such as tris(dibenzylideneacetone)dipalladium(0) and the like), phosphine ligand (such as xantphos and the like), and an inorganic base (such as cesium carbonate and the like) in an appropriate solvent (such as 1,4-dioxane or toluene and the like) to afford Compound F2.
Compounds of Formula (I), wherein R2 is a bicyclic heteroaryl ring system, can be prepared as described in Scheme G below.
Compound A2 is reacted with ethyl acetoacetate, and in the presence of condensation reagents (such as piperidine/acetic acid and the like), will undergo Knoevenagel condensation followed by lactone formation to afford Compound G1. The α-methyl group of Compound G1 can be selectively brominated with an appropriate brominating reagent (such as Br2 or NBS and the like) to afford Compound G2. Compound G2 is reacted with Compound B2, an optionally substituted monocyclic heteroaryl ring system containing an amidine-like moiety (such as but not limited to 2-aminopyridine, 2-aminopyrimidine, 2-aminopyrazine, 3-aminopyridazine, 2-aminothiazole, 4-aminothiazole, 4-aminopyrimidine and the like) in a suitable solvent (such as acetonitrile and the like) to give Compound B4.
Compounds of Formula (I), wherein R2 is a bicyclic heteroaryl ring system, can be prepared as described in Scheme H below.
Compound G2 is reacted with Compound H1, an optionally substituted monocyclic heteroaryl ring system containing a ketimine-like moiety (such as but not limited to 2-methylpyridine, 2-methylpyrimidine, 2-methylpyrazine, 3-methylpyridazine and the like), and in a suitable solvent (such as acetonitrile and the like), undergoes a tandem alkylation dehydrative cyclization reaction to give Compound H2.
Compounds of Formula (I), wherein R2 is a bicyclic heteroaryl ring system, can be prepared as described in Scheme I below.
Compound E2 is reacted with trimethylsilylacetylene and an organic base (such as triethylamine and the like) in the presence of copper(I) iodide and a palladium catalyst (such as tetrakis(triphenylphosphine)palladium(0), bis(triphenylphosphine)palladium(II) dichloride, palladium acetate and the like) and, in the presence of an appropriate phospine ligand undergoes a Sonogashira coupling. The resulting trimethylsilylacetylene product when treated with an inorganic base (such as potassium carbonate and the like) in an appropriate solvent (such as methanol and the like) yields Compound I1. Compound I1 can undergo an additional Sonogashira coupling with Compound I2, an iodo-hydroxy-substituted monocyclic heteroaryl ring system (where the heteroaryl ring may have one or more additional nitrogen atom ring members, thus making Compound I2 an iodo-hydroxy-substituted ring system such as a pyridine, pyrimidine, pyrazine and the like, and where the iodo and hydroxy substituents are in an ortho orientation with respect to one another, such as 2-iodopyridin-3-ol, 4-iodopyridin-3-ol and the like) to give Compound I3.
Compounds of Formula (I), wherein R2 is a monocyclic or bicyclic heteroaryl ring system, can be prepared as described in Scheme J below.
Compound I1 is reacted with Compound J1, a chloro-iodo-substituted monocyclic heteroaryl ring system (where the heteroaryl ring may have one or more additional nitrogen atom ring members, thus making Compound J1 a chloro-iodo-substituted ring system such as a pyridine, pyrimidine, pyrazine and the like, and where the chloro- and iodo-substituents are in an ortho orientation with respect to one another, such as 2-chloro-3-iodopyridine or 4-chloro-3-iodopyridine and the like), and an organic base (such as triethylamine and the like) in the presence of copper(I) iodide and a palladium catalyst (such as tetrakis(triphenylphosphine) palladium(0), bis(triphenylphosphine)palladium(II) dichloride, palladium acetate and the like) and, in the presence of an appropriate phospine ligand undergoes a Sonogashira coupling to afford Compound J2. Compound J2 treated with sodium hydrosulfide in a suitable solvent (such as EtOH and the like) affords Compound J3.
Compounds of Formula (I), wherein R2 is an optionally substituted 1,2,4-oxadiazole ring system, can be prepared as described in Scheme K below.
Compound A2 is reacted with ethyl cyanoacetate, and in the presence of condensation reagents (such as piperidine/acetic acid and the like) will undergo Knoevenagel condensation followed by lactone formation to afford Compound K1. Compound K1 is reacted with hydroxylamine in a suitable solvent (such as CH2Cl2) to give Compound K2. Compound K2 is reacted with Compound K3 (where W represents a C1-4alkyl, aryl or heteroaryl group), and in the presence of an organic base (such as triethylamine and the like), affords an O-acyl-hydroxyamidine intermediate, that undergoes dehydrative cyclization at elevated temperatures (>100° C.) to yield Compound K4.
Compounds of Formula (I), wherein R2 is a monocyclic heteroaryl ring system, can be prepared as described in Scheme L below.
Compound G1 is reacted with dimethylformamide dimethyl acetal and an organic base (such as pyrrolidine and the like) to give an enaminone intermediate, which is then reacted with hydrazine in the presence of an organic acid (such as acetic acid and the like) to afford Compound L1. Compound L1 is reacted with Compound L2 (where W represents a C1-4alkyl, aryl, or heteroaryl group and L represents a leaving group (such as I or Br and the like), in a suitable solvent (such as DMF and the like), in the presence of an inorganic base (such as Cs2CO3 and the like), and an optional catalyst (such as CuI and the like) to afford Compound L3.
Compounds of Formula (I), wherein R2 is a monocyclic heteroaryl ring system, can be prepared as described in Scheme M below.
Compound E1 can be regioselectively iodinated with an appropriate iodinating agent (such as iodine or bis(trifluoroacetoxy)iodo]benzene and the like) in an appropriate solvent (such as CHCl3 and the like). Compound M1, when treated with hexabutylditin in the presence of a palladium catalyst (such as tetrakis(triphenylphosphine)palladium(0), bis(triphenylphosphine) palladium(II) dichloride, palladium acetate and the like) in an appropriate solvent (such as 1,4-dioxane or toluene), affords Compound M2. Compound M2 is reacted with 5-iodoimidazole, in the presence of a catalyst (such as tetrakis(triphenylphosphine)palladium(0), bis(triphenylphosphine)palladium(II) dichloride, palladium acetate and the like) and a cocatalyst (such as CuI and the like), in an appropriate solvent (such as 1,4-dioxane or toluene and the like) to afford Compound M3. Compound M3 is reacted with Compound L2 (where W represents a C1-4alkyl, aryl, or heteroaryl group and L represents a leaving group (such as I or Br and the like), in a suitable solvent (such as DMF and the like), in the presence of an inorganic base (such as Cs2CO3 and the like), and an optional catalyst (such as CuI and the like) to afford Compound M4.
Compounds of Formula (I), wherein R2 is a monocyclic or bicyclic aryl or heteroaryl ring system and R3 is hydrogen or alkyl, can be prepared as described in Scheme N below.
Compound N1 is treated under the conditions for ester hydrolysis (such as aqueous NaOH), to afford Compound N2. Compound N2 is reacted with Compound N3 (where R represents a hydrogen or C1-4alkyl group), and in the presence of a coupling reagent (such as N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and the like) and an organic base (such as triethylamine and the like), undergoes ester formation followed by Knoevenagel condensation to give Compound N4.
To describe in more detail and assist in understanding, the following non-limiting examples are offered to more fully illustrate the scope of compounds described herein and are not to be construed as specifically limiting the scope thereof. Such variations of the compounds described herein that may be now known or later developed, which would be within the purview of one skilled in the art to ascertain, are considered to fall within the scope of the compounds as described herein and hereinafter claimed. These examples illustrate the preparation of certain compounds. Those of skill in the art will understand that the techniques described in these examples represent techniques, as described by those of ordinary skill in the art, that function well in synthetic practice, and as such constitute preferred modes for the practice thereof. However, it should be appreciated that those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific methods that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present description.
Other than in the following examples of the embodied compounds, unless indicated to the contrary, all numbers expressing quantities of ingredients, reaction conditions, experimental data, and so forth used in the specification and claims are to be understood as being modified by the term “about”. Accordingly, all such numbers represent approximations that may vary depending upon the desired properties sought to be obtained by a reaction or as a result of variable experimental conditions. Therefore, within an expected range of experimental reproducibility, the term “about” in the context of the resulting data, refers to a range for data provided that may vary according to a standard deviation from the mean. As well, for experimental results provided, the resulting data may be rounded up or down to present data consistently, without loss of significant figures. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and rounding techniques used by those of skill in the art.
While the numerical ranges and parameters setting forth the broad scope of the present description are approximations, the numerical values set forth in the examples set forth below are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
As used above, and throughout the present description, the following abbreviations, unless otherwise indicated, shall be understood to have the following meanings:
A mixture of 2-aminobenzenethiol (5.34 mL, 50 mmol) and 3-ethoxy-3-iminopropanoate hydrochloride (9.75 g, 50 mmol) in EtOH (50 mL) was heated at 70° C. for 16 h. The mixture was partitioned in EtOAc (200 mL) and water (200 mL). The organic layer was washed with brine, dried over MgSO4, filtered and concentrated. The residue was purified by silica gel column chromatography (10% EtOAc in hexanes) to give the title compound (6.0 g, 54%) as a yellow oil. MS m/z 222.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.05 (1H, d, J=8.1 Hz), 7.91 (1H, d, J=8.0 Hz), 7.51 (1H, t, J=8 Hz), 7.43 (1H, t, J=8 Hz), 4.28 (2H, q, J=7.2 Hz), 4.22 (2H, s), 1.33 (3H, t, J=7.1 Hz).
A mixture of 4-fluoro-2-hydroxybenzaldehyde (10 g, 71.4 mmol), 1-boc-piperazine (15.3 g, 82.2 mmol), and DMSO (100 mL) was heated at 100° C. for 27 h. The reaction mixture was diluted in an aqueous K2CO3 solution and extracted with EtOAc. The organic layer was washed with H2O and brine, dried over MgSO4, filtered, and concentrated under vacuum. The residue was triturated with hexane/ether (1:1), yielding the title compound (18.8 g, 86%) as a yellow solid. MS m/z 307.2 [M+H]+; 1H NMR (500 MHz, CDCl3): δ 11.50 (1H, s), 9.60 (1H, s), 7.36 (1H, d, J=9 Hz), 6.27 (1H, d, J=2 Hz), 6.45 (1H, dd, J=9 Hz, 2 Hz), 3.58 (4H, m), 3.42 (4H, m), 1.49 (9H, s).
Step A: tert-Butyl 4-(4-formyl-3-hydroxyphenyl)piperazine-1-carboxylate (49 mg, 0.16 mmol) and ethyl 2-(benzo[d]thiazol-2-yl)acetate (35 mg, 0.16 mmol) were combined with piperidine (10 μL, 0.1 mmol) and acetic acid (6 μL, 0.1 mmol) in EtOH (1 mL). The mixture was heated at reflux for 1 h. After cooling the mixture to room temperature, a precipitate formed. The solid was collected by vacuum filtration, washed with 1:1 EtOH:H2O (1 mL) and dried under vacuum to afford tert-butyl 4-(3-(benzo[d]thiazol-2-yl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate.
Step B: tert-butyl 4-(3-(benzo[d]thiazol-2-yl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate was suspended in 4N HCl in 1,4-dioxane (1 mL). After stirring the mixture for 30 min at room temperature, the solvent was removed with a stream of nitrogen, to give the title compound (40 mg, 69%) as a yellow powder: m.p. 250° C. (decomp.); MS m/z 364.4 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 9.26 (2H, br s), 9.14 (1H, s), 8.16 (1H, d, J=7.9 Hz), 8.04 (1H, d, J=8.1 Hz), 7.93 (1H, d, J=9.0 Hz), 7.56 (1H, m), 7.47 (1H, m), 7.16 (1H, dd, J=8.9 Hz, 2.3 Hz), 7.09 (1H, d, J=2.3 Hz), 3.76-3.74 (4H, m), 3.25-3.23 (4H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 1 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: To a solution of 1-(3-chlorophenyl)thiourea (5.09 g, 27.2 mmol) in acetic acid (100 mL) was added bromine (1.82 mL, 35.4 mmol) dropwise at 60° C. The mixture was heated at 80° C. for 2 h and the solvent was removed under reduced pressure. Diethyl ether was added to the mixture to produce a precipitate. The solid was collected and dried to give 4-chlorobenzo[d]thiazol-2-amine (5.7 g, 79%). MS m/z 185.9 [M+H]+.
Step B: To a mixture of 4-chlorobenzo[d]thiazol-2-amine (4.78 g, 25.8 mmol) and copper(II) chloride (4.16 g, 31 mmol) in CH3CN (25 mL) was added t-butyl nitrite (4.61 mL, 38.8 mmol) at room temperature. The reaction mixture was heated at 60° C. for 30 min, then the solvent was removed from the mixture. The residue was suspended in water, collected by filtration and dried to give 2,4-dichlorobenzo[d]thiazole. (5.3 g, 81%). MS m/z 205.9 [M+H]+.
Step C: To a mixture of t-butyl acetate (4.93 mL, 36.6 mmol) and 2,4-dichlorobenzo[d]thiazole (5 g, 24.4 mmol) in toluene (20 mL) was added lithium bis(trimethylsilyl)amide (1M in THF, 66 mL, 66 mmol) at 0° C. The mixture was stirred at room temperature overnight. Excess reagent was quenched with the addition of aqueous saturated NH4Cl. The aqueous mixture was extracted with EtOAc. The organic layer was concentrated and purified by silica gel column chromatography (0-5% EtOAC in hexanes) to give the title compound (5.9 g, 85%) as a yellow oil. MS m/z 282.1 [M−H]−. 1H NMR (500 MHz, CDCl3): δ 7.75 (1H, d, J=8.2 Hz), 7.47 (1H, d, J=7.7 Hz), 7.29 (1H, t, J=7.9 Hz), 4.15 (2H, s), 1.48 (9H, s).
Step A: Following the procedure found in Example 1, Part 3, tert-Butyl 4-(4-formyl-3-hydroxyphenyl)piperazine-1-carboxylate (49 mg, 0.16 mmol), tert-butyl 2-(4-chlorobenzo[d]thiazol-2-yl)acetate (35 mg, 0.16 mmol), piperidine (10 μL, 0.1 mmol) and acetic acid (6 μL, 0.1 mmol) in EtOH (1 mL) gave tert-butyl 4-(3-(4-chlorobenzo[d]thiazol-2-yl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate.
Step B: Following the procedure found in Example 1, Part 3, tert-butyl 4-(3-(4-chlorobenzo [d]thiazol-2-yl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate and 4N HCl in 1,4-dioxane (1 mL) gave the title compound (62 mg, 97%) as a yellow powder: m.p. 290° C. (decomp.); MS m/z 398.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 9.18 (2H, br s), 9.09 (1H, s), 8.14 (1H, dd, J=8.0 Hz, 1.0 Hz), 7.99 (1H, d, J=9.2 Hz), 7.65 (1H, dd, J=7.7 Hz, 1.0 Hz), 7.44 (1H, t, J=7.8 Hz), 7.17 (1H, dd, J=9.0 Hz, 2.4 Hz), 7.09 (1H, d, J=2.2 Hz), 3.77-3.74 (4H, m), 3.25-3.23 (4H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 2 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 3-chloro-2-nitrophenol (18.95 g, 100 mmol) and Pd/C (10%, 0.50 g) in MeOH (300 mL) was stirred under H2 (1 atm). After 15 h, the mixture was filtered through Celite. The filtrate was concentrated to give a brown solid, which was washed with CH2Cl2 to give 2-amino-3-chlorophenol (7.39 g, 52%) as a light brown solid. MS m/z 144.1 [M+H]+.
Step B: To a solution of 2-amino-3-chlorophenol (2.0 g, 14 mmol) in EtOH (30 mL) was added ethyl 3-ethoxy-3-iminopropanoate hydrochloride (3.01 g, 15.4 mmol). After heating at 80° C. for 2 d, the mixture was concentrated. The residue was partitioned between EtOAc and water. The organic layer was concentrated and purified by silica gel column chromatography (CH2Cl2) to give ethyl 2-(4-chlorobenzo[d]oxazol-2-yl)acetate (3.17 g, 94%) as an off-white solid. MS m/z 240.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 7.75 (1H, dd, J=8.0 Hz, 0.9 Hz), 7.49 (1H, dd, J=8.0 Hz, 0.9 Hz), 7.43 (1H, t, J=8.0 Hz), 4.28 (2H, s), 4.16 (2H, q, J=7.2 Hz), 1.21 (3H, t, J=7.2 Hz).
To a solution of ethyl 2-(4-chlorobenzo[d]oxazol-2-yl)acetate (72 mg, 0.3 mmol, prepared according to Example 1) and 2-hydroxy-4-(4-methylpiperazin-1-yl)benzaldehyde (66 mg, 0.3 mmol, prepared following the procedure in Example 1, Part 2) in CH3CN (0.5 mL) were added piperidine (3 uL, 0.03 mmol) and AcOH (3.4 uL, 0.06 mmol). After heating at 90° C. for 2 h, the mixture was cooled to room temperature. The product was collected by vacuum filtration, washed with CH3CN and dried to give the title compound (92 mg, 78%) as a yellow solid: m.p. 229-231° C.; MS m/z 396.2, 398.2 [M+H]+; 1H NMR (500 MHz, CDCl3): δ 8.91 (1H, s), 8.79 (1H, d, J=9.1 Hz), 7.76 (1H, dd, J=8.2 Hz, 0.9 Hz), 7.50 (1H, dd, J=8.0 Hz, 0.8 Hz), 7.42 (1H, t, J=8.0 Hz), 7.08 (1H, dd, J=9.0 Hz, 2.4 Hz), 6.90 (1H, d, J=2.3 Hz), 3.49 (4H, m), 2.43 (4H, m), 2.26 (3H, s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 3 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of ethyl 2-(benzo[d]thiazol-2-yl)acetate (0.53 g, 2.4 mmol, prepared in Example 1, Part 1), 4-fluoro-2-hydroxybenzaldehyde (0.336 g, 2.4 mmol), piperidine (80 μL, 0.8 mmol) and acetic acid (92 μL, 0.16 mmol) in CH3CN (2 mL) was heated at 60° C. for 1 h. The mixture was filtered. The solid material was washed with CH3CN and dried to give 3-(benzo[d]thiazol-2-yl)-7-fluoro-2H-chromen-2-one (0.57 g, 80%) as a yellow solid. MS m/z 298.1 [M+H]+.
Step B: A mixture of 3-(benzo[d]thiazol-2-yl)-7-fluoro-2H-chromen-2-one (89 mg, 0.3 mmol), 1-methyl-1,4-diazepane (75 μL, 0.6 mmol), N,N-diisopropylethylamine (78 μL, 0.45 mmol) in CH3CN (1 mL) was heated at 90° C. After 15 h, the mixture was cooled to room temperature and filtered. The solid material was washed with CH3CN to give the title compound (110 mg, 94%) as a yellow solid: m.p. 217-220° C.; MS m/z 392.2 [M+H]+; 1H NMR (500 MHz, CDCl3): δ 9.04 (1H, s), 8.12 (1H, d, J=7.7 Hz), 7.99 (1H, d, J=8.1 Hz), 7.79 (1H, d, J=9.1), 7.54-7.51 (1H, m), 7.43-7.40 (1H, m), 6.93 (1H, dd, J=9.0 Hz, 2.4 Hz), 6.76 (1H, d, J=2.2 Hz), 3.69 (2H, m), 3.61 (2H, t, J=6.2 Hz), 2.64 (2H, m), 2.46 (2H, m), 2.26 (3H, s), 1.91 (2H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 4 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: tert-Butyl 4-(4-formyl-3-hydroxyphenyl)piperazine-1-carboxylate (918 mg, 3 mmol, prepared in Example 1, Part 2), 2,2-dimethyl-1,3-dioxane-4,6-dione (648 mg, 4.5 mmol) and triethylamine (0.14 mL, 1 mmol) were combined in EtOH (6 mL). The mixture was heated at 60° C. for 4 h. The mixture was cooled to room temperature and filtered. The collected material was washed with EtOH and dried under vacuum to afford 7-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-oxo-2H-chromene-3-carboxylic acid (1.05 g, 94%) as a yellow powder. MS m/z 373.2 [M−H]−.
Step B: 7-(4-(tert-Butoxycarbonyl)piperazin-1-yl)-2-oxo-2H-chromene-3-carboxylic acid (60 mg, 0.16 mmol) was combined with aniline (22 μL, 0.24 mmol), (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (100 mg, 0.19 mmol) and triethylamine (45 μL, 0.32 mmol) in DMF (1 mL). The mixture was stirred at room temperature for 2 h. A solution of 4:1 MeOH:H2O (1 mL) was added to the mixture. A precipitate formed and was collected by vacuum filtration. The solid was washed with MeOH:H2O (4:1) and dried under vacuum to afford tert-butyl 4-(2-oxo-3-(phenylcarbamoyl)-2H-chromen-7-yl)piperazine-1-carboxylate.
Step C: A mixture of tert-butyl 4-(2-oxo-3-(phenylcarbamoyl)-2H-chromen-7-yl)piperazine-1-carboxylate in trifluoroacetic acid (1 mL) was stirred at room temperature for 20 min, then the solvent was removed with a stream of nitrogen to afford the title compound (75 mg, 99%) as a yellow powder: MS m/z 350.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 10.73 (1H, s), 8.81 (1H, s), 7.78 (1H, d, J=9.1 Hz), 7.72 (2H, d, J=8.6 Hz), 7.38 (2H, m), 7.13 (1H, t, J=7.4 Hz), 7.09 (1H, dd, J=9.1 Hz, 2.5 Hz), 6.93 (1H, d, J=2.3 Hz), 3.43 (4H, m), 2.81 (4H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 5 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: 3,5-Difluorophenol (2.6 g, 20 mmol) was dissolved in CH3CN (50 mL) with triethylamine (14 mL, 100 mmol). Magnesium chloride (3.8 g, 40 mmol) and paraformaldehyde (6.4 g, 200 mmol) were added sequentially. The heterogeneous mixture was stirred vigorously at 60° C. for 16 h. The mixture was diluted with H2O (200 mL) and the pH was adjusted to <2 with aqueous HCl (1 M). The mixture was extracted with EtOAc (200 mL). The organic layer was washed with brine, dried over Na2SO4, then filtered and concentrated to afford 2,4-difluoro-6-hydroxybenzaldehyde (2.6 g, 82%) as a red oil. MS m/z 157.1 [M−H]−.
Step B: 2,4-Difluoro-6-hydroxybenzaldehyde (16 mmol) was combined with 1-Boc-piperazine (3.57 g, 19.2 mmol) and N,N-diisopropylethylamine (3.34 mL, 19.2 mmol) in DMSO (4 mL). The mixture was heated to 120° C. for 2 h. The mixture was purified by silica gel column chromatography (0-40% EtOAc in hexanes) to afford tert-butyl 4-(3-fluoro-4-formyl-5-hydroxyphenyl)piperazine-1-carboxylate (1.3 g, 25%) as an off white powder. 1H NMR (500 MHz, DMSO-d6): δ 11.93 (1H, s), 9.92 (1H, s), 6.08 (1H, dd, J=14.2 Hz, 2.4 Hz), 6.04 (1H, d, J=2.4 Hz), 3.59 (4H, m), 3.43 (4H, m), 1.49 (9H, s).
Step C: tert-Butyl 4-(3-fluoro-4-formyl-5-hydroxyphenyl)piperazine-1-carboxylate (65 mg, 0.2 mmol) was combined with ethyl 2-(benzo[d]thiazol-2-yl)acetate (22 mg, 0.2 mmol, prepared in Example 1, Part 1), N,N-diisopropylethylamine (35 μL, 0.2 mmol) and acetic acid (11 μL, 0.2 mmol) in EtOH (1 mL). The mixture was heated to 90° C. for 16 h. After cooling the mixture to room temperature, a precipitate was formed. The solid was collected, washed with 1:1 MeOH:H2O (1 mL) and dried under vacuum to afford tert-butyl 4-(3-(benzo[d]thiazol-2-yl)-5-fluoro-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate.
Step D: A mixture of tert-butyl 4-(3-(benzo[d]thiazol-2-yl)-5-fluoro-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate and trifluoroacetic acid (1 mL) was stirred at room temperature for 15 min, then the solvent was removed with a stream of nitrogen. The residue was partitioned in CH2Cl2 (5 mL) and aqueous K2CO3 (1 M, 5 mL). The organic layer was collected through a hydrophobic frit and concentrated to afford the title compound (38 mg, 50%) as a yellow powder: m.p. 256-260° C.; MS m/z 382.2 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.89 (1H, s), 8.15 (1H, d, J=7.8 Hz), 8.05 (1H, d, J=7.3 Hz), 7.55 (1H, m), 7.44 (1H, m), 7.02 (1H, dd, J=13.9 Hz, 2.1 Hz), 6.82 (1H, s), 3.45-3.41 (4H, m), 2.82-2.78 (4H, m), 2.46 (1H, s br).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 6 by substituting the appropriate starting materials, reagents and reaction conditions.
A mixture of ethyl 4-chloroacetoacetate (5.4 mL, 40 mmol) and 5-methylpyridin-2-amine (5.18 g, 48 mmol) in EtOH (100 mL) was heated at 70° C. for 6 h. The mixture was partitioned in EtOAc (300 mL) and an aqueous saturated NaHCO3 solution (300 mL). The organic layer was washed with brine, dried over MgSO4, filtered and concentrated. The residue was purified by silica gel column chromatography (70% EtOAc in hexanes) to give the title compound (1.6 g, 19%) as a brown oil. 1H NMR (500 MHz, DMSO-d6): δ 8.31 (1H, s), 7.74 (1H, s), 7.39 (1H, d, J=9.2 Hz), 7.08 (1H, d, J=9.2 Hz), 4.10 (2H, q, J=7.1 Hz), 3.75 (2H, s), 2.27 (3H, s), 1.20 (3H, t, J=7.1 Hz).
Step A: Following the procedure in Example 6, Step C, tert-butyl 4-(3-fluoro-4-formyl-5-hydroxyphenyl)piperazine-1-carboxylate (65 mg, 0.2 mmol), ethyl 2-(6-methylimidazo[1,2-a]pyridin-2-yl)acetate (22 mg, 0.2 mmol), N,N-diisopropylethylamine (35 μL, 0.2 mmol) and acetic acid (11 μL, 0.2 mmol) in EtOH (1 mL) gave tert-butyl 4-(5-fluoro-3-(6-methylimidazo[1,2-a]pyridin-2-yl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate.
Step B: Following the procedure in Example 6, Step D, tert-butyl 4-(5-fluoro-3-(6-methylimidazo[1,2-a]pyridin-2-yl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate and trifluoroacetic acid (1 mL) gave the title compound (18 mg, 24%) as a yellow powder: m.p. 265-270° C.; MS m/z 379.2 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.60 (1H, s), 8.42 (2H, m), 7.49 (1H, d, J=9.1 Hz), 7.16 (1H, dd, J=9.3 Hz, 1.6 Hz), 6.93 (1H, dd, J=11.6 Hz, 2.2 Hz), 6.75 (1H, d, J=1.9 Hz), 3.33-3.31 (4H, m), 2.82-2.80 (4H, m), 2.28 (3H, s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 7 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: tert-Butyl 4-(3-fluoro-4-formyl-5-hydroxyphenyl)piperazine-1-carboxylate (65 mg, 0.2 mmol, prepared in Example 6, Step B) was combined with 2-(3,5-difluorophenyl)acetic acid (55 mg, 0.2 mmol), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (57 mg, 0.3 mmol) and N,N-diisopropylethylamine (70 μL, 0.4 mmol) in DMF (1 mL). The mixture was heated to 60° C. for 1 h. After cooling to room temperature, the mixture was filtered. The solid was washed with MeOH:H2O (1:1) and dried under vacuum to afford tert-butyl 4-(3-(3,5-difluorophenyl)-5-fluoro-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate.
Step B: A mixture of tert-Butyl 4-(3-(3,5-difluorophenyl)-5-fluoro-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate and trifluoroacetic acid (1 mL) was stirred at room temperature for 15 min, then the solvent was removed with a stream of nitrogen. The residue was partitioned in CH2Cl2 (5 mL) and aqueous K2CO3 (1 M, 5 mL). The organic layer was collected through a hydrophobic frit and concentrated to afford the title compound (24 mg, 33%) as a yellow powder: m.p. 193-198° C.; MS m/z 361.3 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.11 (1H, s), 7.44 (2H, m), 7.16 (1H, tt, J=9.3 Hz, 2.4 Hz), 6.82 (1H, dd, J=13.8 Hz, 2.2 Hz), 6.65 (1H, d, J=2.4 Hz), 3.26 (4H, m), 2.73 (4H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 8 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: 3-Hydroxybenzaldehyde (6.1 g, 50 mmol) was combined with dimethylamine (37.5 mL of 2M solution in THF, 75 mmol) in 1,2-dichloroethane (200 mL). Sodium triacetoxyborohydride (15.9 g, 75 mmol) was added slowly at room temperature. Acetic acid (2.86 mL, 50 mmol) was added to the mixture. The mixture was stirred at room temperature for 16 h. To the reaction mixture was added an aqueous saturated NaHCO3 solution (100 mL). The organic layer was removed, dried over Na2SO4, then filtered and concentrated to afford crude 3-((dimethylamino)methyl)phenol (˜30 mmol, 60%).
Step B: The crude material (˜30 mmol) from Step A was dissolved in CH3CN (300 mL) and triethylamine (21 mL, 150 mmol). To the solution was added anhydrous magnesium chloride (5.7 g, 60 mmol) and paraformaldehyde (9.0 g, 300 mmol). The mixture was stirred vigorously at 60° C. for 16 h, then diluted with aqueous sodium potassium tartrate (0.1 M, 600 mL). The mixture was extracted three times with CH2Cl2 (300 mL). The combined organics were washed with brine, dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography (0-10% MeOH in CH2Cl2) to afford 4-((dimethylamino)methyl)-2-hydroxybenzaldehyde (1.7 g, 32%) as a yellow powder. MS m/z 180.1 [M+H]+.
Step C: A mixture of 4-((dimethylamino)methyl)-2-hydroxybenzaldehyde (0.5 mmol), ethyl 2-(4-chlorobenzo[d]thiazol-2-yl)acetate (128 mg, 0.5 mmol, prepared as in Example 2, Part 1), piperidine (40 μL, 0.4 mmol) and acetic acid (12 μL, 0.2 mmol) in EtOH (3 mL) was heated at reflux for 16 h. After cooling the mixture to room temperature, a precipitate formed. The solid was collected by vacuum filtration, washed with 1:1 EtOH:H2O (1 mL) and dried under vacuum to afford the title compound (184 mg, 99%) as a yellow powder: m.p. 179-182° C.; MS m/z 371.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 9.20 (1H, s), 8.18 (1H, d, J=8.0 Hz), 8.10 (1H, d, J=8.0 Hz), 7.69 (1H, d, J=7.7 Hz), 7.48 (2H, m), 7.43 (1H, d, J=8.0 Hz), 3.57 (2H, s), 2.21 (6H, s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 9 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: Following the procedure in Example 6, Step A, 3-(hydroxymethyl)phenol (6.2 g, 50 mmol), triethylamine (35 mL, 250 mmol), anhydrous magnesium chloride (9.5 g, 100 mmol) and paraformaldehyde (15 g, 500 mmol) in CH3CN (500 mL) afforded 2-hydroxy-4-(hydroxymethyl)benzaldehyde (2.2 g, 29%). MS m/z 151.1 [M−H]−.
Step B: Following the procedure in Example 9, Step C, 2-hydroxy-4-(hydroxymethyl)benzaldehyde (608 mg, 4.0 mmol), ethyl 2-(6-methylimidazo[1,2-a]pyridin-2-yl)acetate (872 mg, 4.0 mmol, prepared in Example 7, Part 1), piperidine (0.4 mL, 4.0 mmol) and acetic acid (0.24 mL, 4.0 mmol) in EtOH (4 mL) afforded 7-(hydroxymethyl)-3-(6-methylimidazo[1,2-a]pyridin-2-yl)-2H-chromen-2-one (980 mg, 80%). MS m/z 307.2 [M+H]+.
Step C: 7-(Hydroxymethyl)-3-(6-methylimidazo[1,2-a]pyridin-2-yl)-2H-chromen-2-one (900 mg, 2.9 mmol) was combined with N,N-diisopropylethylamine (1.0 mL, 6 mmol) in CH2Cl2 (15 mL). The mixture was cooled to 0° C., before adding methanesulfonyl chloride (0.28 mL, 3.6 mmol) via syringe. The mixture stirred for 1 h at 0° C., then the solvent was removed from the mixture. The residue was suspended in MeOH (5 mL) and filtered. The collected material was washed with MeOH and dried under vacuum to afford (3-(6-methylimidazo[1,2-a]pyridin-2-yl)-2-oxo-2H-chromen-7-yl)methyl methanesulfonate (1.05 g, 92%) as a tan powder. 1H NMR (500 MHz, DMSO-d6): δ 8.86 (1H, s), 8.56 (1H, s), 8.46 (1H, s), 7.99 (1H, d, J=7.9 Hz), 7.56 (1H, s), 7.51 (1H, d, J=9.1 Hz), 7.47 (1H, d, J=8.0 Hz), 7.20 (1H, d, 9.3 Hz), 5.41 (2H, s), 3.32 (3H, s), 2.30 (3H, s).
Step D: (3-(6-Methylimidazo[1,2-a]pyridin-2-yl)-2-oxo-2H-chromen-7-yl)methyl methanesulfonate (77 mg, 0.2 mmol) was combined with 2-(methylamino)ethanol (75 mg, 1.0 mmol) in DMF (2 mL). The mixture was stirred at room temperature for 1 h. To the mixture was added H2O (0.25 mL) to produce a precipitate. The solid was collected by vacuum filtration, washed with MeOH:H2O (1:1) and dried under vacuum to afford the title compound (65 mg, 90%) as an off white powder: m.p. 166-169° C.; MS m/z 364.3 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.83 (1H, s), 8.53 (1H, s), 8.46 (1H, s), 7.87 (1H, d, J=8.0 Hz), 7.50 (1H, d, J=9.4 Hz), 7.43 (1H, s), 7.37 (1H, d, J=7.9 Hz), 7.19 (1H, d, J=9.2 Hz), 4.47 (1H, t, J=5.4 Hz), 3.64 (2H, s), 3.54 (2H, q, J=5.5 Hz), 2.47 (2H, t, J=6.3 Hz), 2.29 (3H, s), 2.21 (3H, s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 10 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: 4-(3-Hydroxyphenyl)piperidine (1.7 g, 10 mmol) was added to a mixture of CH3CN (20 mL) and di-tert-butyl dicarbonate (2.4 g, 11 mmol). The mixture was stirred for 1 h at room temperature, then triethylamine (7 mL, 50 mmol), anhydrous magnesium chloride (1.9 g, 20 mmol) and paraformaldehyde (3.0 g, 100 mmol) were added. The mixture was stirred vigorously at 60° C. for 2 h, then diluted with H2O (100 mL). Aqueous HCl (1N) was added to adjust the pH of the mixture to ˜2. The mixture was extracted with EtOAc (100 mL). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography (0-5% MeOH in CH2Cl2) to afford tert-butyl 4-(4-formyl-3-hydroxyphenyl)piperidine-1-carboxylate (1.44 g, 47%) as a white powder. MS m/z 304.2 [M−H]−.
Step B: Following the procedure in Example 9, Step C, tert-butyl 4-(4-formyl-3-hydroxyphenyl)piperidine-1-carboxylate (61 mg, 0.2 mmol), ethyl 2-(4-chlorobenzo[d]thiazol-2-yl)acetate (50 mg, 0.2 mmol, prepared according to Example 2, Part 1), piperidine (10 μL, 0.1 mmol) and acetic acid (6 μL, 0.1 mmol) in EtOH (1 mL) afforded tert-butyl 4-(3-(4-chlorobenzo[d]thiazol-2-yl)-2-oxo-2H-chromen-7-yl)piperidine-1-carboxylate.
Step C: tert-Butyl 4-(3-(4-chlorobenzo[d]thiazol-2-yl)-2-oxo-2H-chromen-7-yl)piperidine-1-carboxylate was suspended in 4N HCl in 1,4-dioxane (1 mL). The mixture was stirred for 1 h, then the solvent was removed to afford the title compound (73 mg, 92%) as a yellow powder: m.p. 339-341° C.; MS m/z 397.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 9.21 (1H, s), 8.19 (1H, d, J=8.0), 8.14 (1H, d, J=8.1), 7.70 (1H, d, J=7.7 Hz), 7.49 (1H, t, J=7.8 Hz), 7.43 (1H, s), 7.40 (1H, d, J=8.2 Hz) 3.41 (2H, m), 3.01-3.07 (3H, m), 2.03 (2H, m), 1.92 (2H, m).
Step A: 4-Formyl-3-hydroxybenzoic acid (830 mg, 5 mmol) was combined with 1-methylpiperazine (0.61 mL, 5.5 mmol), triethylamine (0.77 mL, 5.5 mmol) and (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (2.86 g, 5.5 mmol) in CH2Cl2 (10 mL). The mixture was stirred at room temperature for 3 h, then concentrated and purified by silica gel column chromatography (0-5% MeOH in CH2Cl2) to afford 2-hydroxy-4-(4-methylpiperazine-1-carbonyl)benzaldehyde (1.24 g, 100%). MS m/z 249.1 [M+H]+.
Step B: Following the procedure in Example 9, Step C, 2-hydroxy-4-(4-methylpiperazine-1-carbonyl)benzaldehyde (50 mg, 0.2 mmol), ethyl 2-(4-chlorobenzo[d]thiazol-2-yl)acetate (50 mg, 0.2 mmol, prepared according to Example 2, Part 1), piperidine (20 μL, 0.2 mmol) and acetic acid (12 μL, 0.2 mmol) in EtOH (1 mL) afforded the title compound (60 mg, 68%) as a yellow powder: m.p. 230-235° C.; MS m/z 440.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 9.26 (1H, s), 8.24 (1H, d, J=8.0 Hz), 8.21 (1H, d, J=8.0 Hz), 7.72 (1H, d, J=7.6 Hz), 7.59 (1H, s), 7.51 (1H, t, J=7.9 Hz), 7.48 (1H, d, J=7.9 Hz), 3.65 (2H, m), 3.34 (2H, m), 2.42 (2H, m), 2.31 (2H, m), 2.22 (3H, s).
Step A: 3-Hydroxyphenylacetic acid (2.13 g, 14 mmol) was combined with isopropylamine (3.6 mL, 42 mmol) in THF (20 mL). The solution was cooled to 0° C. before adding propylphosphonic anhydride (9.8 mL, ˜50% in DMF, 16 mmol). The solution stirred at room temperature for 16 h. The mixture was partitioned in H2O (300 mL) and EtOAc (300 mL). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography (50% EtOAc in hexanes) to afford 2-(3-hydroxyphenyl)-N-isopropylacetamide (1.9 g, 70%) as a white powder. MS m/z 194.1 [M+H]+.
Step B: 2-(3-Hydroxyphenyl)-N-isopropylacetamide (1.9 g, 10 mmol) was dissolved in THF (20 mL). Lithium aluminum hydride (10 mL, 1 M in THF, 10 mmol) was added to the solution. The mixture was heated to 60° C. for 2 h with stirring. The excess reagent was quenched by the slow addition of H2O. After vigorous stirring for 1 h, the mixture was filtered through Celite. The filtrate was concentrated to afford crude 3-(2-(isopropylamino)ethyl)phenol, which was used without further purification.
Step C: 3-(2-(Isopropylamino)ethyl)phenol (716 mg, 4 mmol) was combined with di-tert-butyl dicarbonate (872 mg, 4 mmol) in CH2Cl2 (10 mL). The mixture was stirred at room temperature for 16 h, then concentrated and purified by silica gel column chromatography (50% EtOAc in hexanes) to afford tert-butyl 3-hydroxyphenethyl(isopropyl)carbamate (650 mg, 23%) as a white powder.
Step D: Following the procedure in Example 6, Step A, tert-butyl 3-hydroxyphenethyl(isopropyl)carbamate (650 mg, 2.3 mmol), triethylamine (1.6 mL, 11.5 mmol), anhydrous magnesium chloride (437 mg, 4.6 mmol) and paraformaldehyde (690 mg, 23 mmol) in CH3CN (8 mL) afforded tert-butyl 4-formyl-3-hydroxyphenethyl(isopropyl)carbamate (520 mg, 73%). MS m/z 306.1 [M−H]−.
Step E: Following the procedure in Example 9, Step C, tert-butyl 4-formyl-3-hydroxyphenethyl(isopropyl)carbamate (50 mg, 0.16 mmol), ethyl 2-(4-chlorobenzo[d]thiazol-2-yl)acetate (50 mg, 0.2 mmol, prepared according to Example 2, Part 1), piperidine (20 μL, 0.2 mmol) and acetic acid (12 μL, 0.2 mmol) in EtOH (1 mL) afforded tert-butyl 2-(3-(4-chlorobenzo[d]thiazol-2-yl)-2-oxo-2H-chromen-7-yl)ethyl(isopropyl)carbamate.
Step F: A mixture of 2-(3-(4-chlorobenzo[d]thiazol-2-yl)-2-oxo-2H-chromen-7-yl)ethyl(isopropyl)carbamate (0.16 mmol) and trifluoroacetic acid (1 mL) was stirred at room temperature for 15 min, then the solvent was removed with a stream of nitrogen. The residue was partitioned in CH2Cl2 (5 mL) and aqueous K2CO3 (1 M, 5 mL). The organic layer was collected through a hydrophobic frit and concentrated to afford the title compound (42 mg, 66%) as a yellow powder: m.p. 179-182° C.; MS m/z 399.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 9.23 (1H, s), 8.22 (1H, d, J=7.8 Hz), 8.10 (1H, d, J=8.0 Hz), 7.73 (1H, d, J=7.7 Hz), 7.52 (1H, t, J=7.8 Hz), 7.50 (1H, s), 7.42 (1H, d, J=8.0 Hz), 2.89 (4H, m), 2.79 (1H, m), 1.02 (6H, d, J=6.2 Hz).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 13 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: 2,4-Dihydroxybenzaldehyde (1.38 g, 10 mmol) was dissolved in CH2Cl2 (20 mL) and cooled to 0° C. To the mixture was added pyridine (0.81 mL, 10 mmol), followed by phosgene (5.0 mL, 20% in toluene, 10 mmol). The mixture was stirred for 5 min at 0° C. A solution of 1-Boc-piperazine (1.86 g, 10 mmol) and triethylamine (1.4 mL, 10 mmol) in CH2Cl2 (5 mL) was added to the mixture at 0° C. After 5 min, the mixture was washed with an aqueous saturated NaHCO3 solution. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography (20% EtOAc in hexanes) to afford 1-tert-butyl 4-(4-formyl-3-hydroxyphenyl) piperazine-1,4-dicarboxylate (480 mg, 14%). MS m/z 349.3 [M−H]−.
Step B: Following the procedure in Example 9, Step C, 1-tert-butyl 4-(4-formyl-3-hydroxyphenyl) piperazine-1,4-dicarboxylate (70 mg, 0.2 mmol), ethyl 2-(4-chlorobenzo[d]thiazol-2-yl)acetate (50 mg, 0.2 mmol, prepared according to Example 2, Part 1), piperidine (20 μL, 0.2 mmol) and acetic acid (12 μL, 0.2 mmol) in EtOH (1 mL) afforded 1-tert-butyl 4-(3-(4-chlorobenzo[d]thiazol-2-yl)-2-oxo-2H-chromen-7-yl) piperazine-1,4-dicarboxylate.
Step C: A mixture of 1-tert-butyl 4-(3-(4-chlorobenzo[d]thiazol-2-yl)-2-oxo-2H-chromen-7-yl) piperazine-1,4-dicarboxylate (0.2 mmol) and trifluoroacetic acid (1 mL) was stirred at room temperature for 15 min, then the solvent was removed with a stream of nitrogen. The residue was partitioned in CH2Cl2 (5 mL) and aqueous K2CO3 (1 M, 5 mL). The organic layer was collected through a hydrophobic frit and concentrated to afford the title compound (62 mg, 70%) as an off white powder: m.p. 236-239° C.; MS m/z 442.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 9.23 (1H, s), 8.21-8.18 (2H, m), 7.70 (1H, d, J=7.72 Hz), 7.49 (1H, t, J=7.9 Hz), 7.46 (1H, d, J=2.1 Hz), 7.31 (1H, dd, J=8.5 Hz, 2.1 Hz), 3.55 (2H, m), 3.39 (2H, m), 2.76 (4H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 14 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: To a solution of 3-hydroxyacetophenone (2.72 g, 20 mmol) in MeOH (10 mL) was added sodium borohydride (380 mg, 10 mmol). After stirring at room temperature for 2 h, the reaction mixture was acidified to pH<7 with aqueous HCl (1 N). MeOH was removed by rotoevaporation under reduced pressure. The mixture was partitioned in water and EtOAc. The organic layer was washed with water, dried over MgSO4, filtered and concentrated under reduced pressure to provide 3-(1-hydroxyethyl)phenol (2.15 g, 78%). MS m/z 137.1 [M−H]−.
Step B: To a mixture of 3-(1-hydroxyethyl)phenol (1.38 g, 10 mmol), magnesium chloride (1.94 g, 20.4 mmol) and triethylamine (7 mL, 50 mmol) in CH3CN (5 mL) was added paraformaldehyde (3 g, 100 mmol) at room temperature. The reaction mixture was heated at 60° C. overnight, then the solvent was removed by rotoevaporation under reduced pressure. The residual mixture was acidified to pH ˜2 with aqueous HCl (1 N). The aqueous mixture was extracted with EtOAc and the organic layer was concentrated. The residue was purified by silica gel column chromatography (0-20% EtOAc in CH2Cl2) to provide 2-hydroxy-4-(1-hydroxyethyl)benzaldehyde (734 mg, 44%).
Step C: To a mixture of 2-hydroxy-4-(1-hydroxyethyl)benzaldehyde (568 mg, 3.4 mmol), piperidine (674 μL, 6.8 mmol) and acetic acid (194 μL, 3.4 mmol) in EtOH (2 mL) was added ethyl 2-(benzo[d]thiazol-2-yl)acetate (800 mg, 4.1 mmol, prepared in Example 1, Part 1). The mixture was heated at 60° C. overnight. After cooling to room temperature, diethyl ether was added to the mixture to produce a precipitate. The solid was collected by filtration, washed with water and dried under vacuum to give 3-(benzo[d]thiazol-2-yl)-7-(1-hydroxyethyl)-2H-chromen-2-one (493 mg, 45%). MS m/z 324.1 [M+H]+.
Step D: To a mixture of 3-(benzo[d]thiazol-2-yl)-7-(1-hydroxyethyl)-2H-chromen-2-one (323 mg, 1 mmol) and triphenylphosphine (525 mg, 2 mmol) in CH2Cl2 (2 mL) was added N-bromosuccinimide (456 mg, 2.6 mmol) at 0° C. The reaction mixture was stirred at room temperature for 3 h. Diethyl ether was added to the mixture to produce a precipitate. The precipitate was collected by vacuum filtration, washed with water and a saturated aqueous NaHCO3 solution, and dried to give 3-(benzo[d]thiazol-2-yl)-7-(1-bromoethyl)-2H-chromen-2-one (193 mg, 50%). MS m/z 386.1, 388.1 [M+H]+.
Step E: To a solution of 3-(benzo[d]thiazol-2-yl)-7-(1-bromoethyl)-2H-chromen-2-one (40 mg, 0.10 mmol) in CH3CN (0.8 mL) was added dimethylamine (16 mg, 0.36 mmol). The reaction mixture was heated at 45° C. for 2 h. Diethyl ether was added to the mixture to produce a precipitate. The solid was collected by vacuum filtration, washed with water and an aqueous saturated NaHCO3 solution, then dried to afford the title compound (14 mg, 27%) as a yellow solid: m.p. 130-133° C.; MS m/z 351.2 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 9.25 (1H, s), 8.20 (1H, d, J=8.1 Hz), 8.09 (1H, d, J=8.1 Hz), 8.03 (1H, d, J=7.9 Hz), 7.59 (1H, t, J=7.6 Hz), 7.51-7.43 (3H, m), 3.46 (1H, q, J=6.7 Hz), 2.15 (6H, s), 1.32 (3H, d, J=6.7 Hz).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 15 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of tert-butyl 4-(4-formyl-3-hydroxyphenyl)piperazine-1-carboxylate (6.5 g, 21.2 mmol, prepared in Example 1, Part 2), ethyl cyanoacetate (2.87 mL, 29.6 mmol), piperidine (2.6 mL, 26 mmol), AcOH (1.6 mL, 29.3 mmol) and CH3CN (50 mL) was heated at 80° C. for 1 h. The reaction mixture was diluted with H2O and extracted with CH2Cl2. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography (10% EtOAc in CH2Cl2), followed by trituration with hexane/EtOAc (1:1), yielding tert-butyl 4-(3-cyano-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (5.05 g, 67%) as a yellow solid. 1H NMR (500 MHz, CDCl3): δ 8.05 (1H, s), 7.41 (1H, d, J=8.5 Hz), 6.84 (1H, dd, J=8.5 Hz, 2.5 Hz), 6.66 (1H, d, J=2.5 Hz), 3.65 (4H, m), 3.51 (4H, m), 1.52 (9H, s).
Step B: A mixture of tert-butyl 4-(3-cyano-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (400 mg, 1.13 mmol), MeOH (2 mL), CH2Cl2 (2 mL), and NH2OH (50% aqueous solution, 200 μL, 3.2 mmol) was stirred at room temperature for 8 h. The reaction mixture was concentrated with a stream of nitrogen until the total volume was halved. The reaction mixture was diluted with MeOH (40 mL) and H2O (5 mL), generating a precipitate. The precipitate was collected by vacuum filtration and dried, affording tert-butyl 4-(3-(N′-hydroxycarbamimidoyl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (386 mg, 88%) as a tan solid. MS m/z 389.2 [M+H]+.
Step C: tert-Butyl 4-(3-(N′-hydroxycarbamimidoyl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (190 mg, 0.49 mmol) was suspended in CH2Cl2 (1.5 mL) and triethylamine (85 μL, 0.6 mmol). Acetyl chloride (40 μL, 0.54 mmol) was added to the mixture. After 10 min, the mixture was diluted in CH2Cl2 and washed with aqueous HCl, followed by an aqueous saturated NaHCO3 solution. The organic layer was dried over MgSO4, filtered and concentrated under vacuum. The residue was suspended in toluene (1.5 mL) and heated at 100° C. for 30 h, then the solvent was removed with a stream of nitrogen. The residue was purified by silica gel column chromatography (10% EtOAc in CH2Cl2), followed by trituration with 2:1 hexane/acetone, yielding tert-butyl 4-(2-oxo-3-(5-phenyl-1,2,4-oxadiazol-3-yl)-2H-chromen-7-yl)piperazine-1-carboxylate (187 mg, 50%) as a yellow solid. MS m/z 475.2 [M+H]+.
Step D: tert-Butyl 4-(2-oxo-3-(5-phenyl-1,2,4-oxadiazol-3-yl)-2H-chromen-7-yl)piperazine-1-carboxylate (107 mg, 0.26 mmol) was stirred in a solution of CH2Cl2 (2.5 mL) and trifluoroacetic acid (1.0 mL) for 15 min. The reaction mixture was partitioned in CH2Cl2 and aqueous K2CO3. The organic layer was concentrated under vacuum. The residue was triturated with 2:1 hexane/acetone, yielding the title compound (116 mg, 81%) as a yellow solid: m.p. 214-221° C.; MS m/z 375.2 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.77 (1H, s), 8.18 (2H, m), 7.75 (2H, m), 7.68 (2H, m), 7.04 (1H, dd, J=9 Hz, 2 Hz), 6.87 (1H, d, J=2 Hz), 3.38 (4H, m), 2.82 (4H, m).
Step A: A mixture of tert-butyl 4-(4-formyl-3-hydroxyphenyl)piperazine-1-carboxylate (2.9 g, 9.5 mmol, prepared in Example 1, Part 2), ethyl acetoacetate (1.28 mL, 11.8 mmol), AcOH (725 μL, 13.3 mmol), piperidine (1.16 mL, 11.8 mmol), and CH3CN (23 mL) were heated at 80° C. for 2 h. The reaction mixture was partitioned between EtOAc and H2O. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography (10% EtOAc in CH2Cl2), followed by ether trituration, yielding tert-butyl 4-(3-acetyl-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (3.15 g, 89%) as a yellow solid. 1H NMR (500 MHz, CDCl3): δ 8.47 (1H, s), 7.49 (1H, d, J=9 Hz), 6.83 (1H, dd, J=9 Hz, 2.5 Hz), 6.67 (1H, d, J=2.5 Hz), 3.64 (4H, m), 3.47 (4H, m), 2.72 (3H, s), 1.52 (9H, s).
Step B: A mixture of tert-butyl 4-(3-acetyl-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (300 mg, 0.81 mmol), dimethylformamide dimethyl acetal (900 μL, 7.5 mmol) and pyrrolidine (150 μL, 1.83 mmol) was heated at 55° C. for 1 h, then the solvent was removed with a stream of nitrogen. Hydrazine (70 μL, 2.2 mmol) and AcOH (900 μL) were added. The mixture was stirred at room temperature for 45 min, then partitioned between EtOAc and H2O. The organic layer was dried over MgSO4, then filtered and concentrated under vacuum. The residue was purified by silica gel column chromatography (30% EtOAc in CH2Cl2), followed by trituration with 2:1 hexane/acetone, yielding tert-butyl 4-(2-oxo-3-(1H-pyrazol-3-yl)-2H-chromen-7-yl)piperazine-1-carboxylate (180 mg, 56%) as a yellow solid. MS m/z 397.2 [M+H]+.
Step C: A solution of tert-butyl 4-(2-oxo-3-(1H-pyrazol-3-yl)-2H-chromen-7-yl)piperazine-1-carboxylate (180 mg, 0.45 mmol) in CH2Cl2 (2.5 mL) and trifluoroacetic acid (1.0 mL) was stirred at room temperature for 15 min. The reaction mixture was partitioned between CH2Cl2 and aqueous K2CO3. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. Trituration of the residue with acetone yielded the title compound (100 mg, 75%) as a yellow solid: m.p. 224-228° C.; MS m/z 297.2 [M+H]+; 1H NMR (500 MHz, DMSO-d6:D2O, 100° C.): δ 8.31 (1H, s), 7.63 (1H, br s), 7.54 (1H, d, J=9 Hz), 6.94 (1H, dd, J=9 Hz, 2 Hz), 6.82 (1H, d, J=2 Hz), 6.77 (1H, d, J=2 Hz), 3.32 (4H, t, J=5 Hz), 2.88 (4H, t, J=5 Hz).
Step A: A mixture of tert-butyl 4-(2-oxo-3-(1H-pyrazol-3-yl)-2H-chromen-7-yl)piperazine-1-carboxylate (300 mg, 0.76 mmol, prepared in Example 17, Step B), Cs2CO3 (515 mg, 1.58 mmol), iodomethane (93 μL, 1.5 mmol), and DMF (2.0 mL) was stirred at 5° C. for 22 h. The reaction mixture was partitioned between EtOAc and H2O. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography (15% EtOAc in CH2Cl2), followed by trituration with ether to give tert-butyl 4-(3-(1-methyl-1H-pyrazol-3-yl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (215 mg, 69%) as a yellow solid. 1H NMR (500 MHz, CDCl3): δ 8.32 (1H, s), 7.41 (2H, m), 7.05 (1H, d, J=2 Hz), 6.83 (1H, dd, J=8.5 Hz, 2.5 Hz), 6.74 (1H, d, J=2 Hz), 3.97 (3H, s), 3.62 (4H, m), 3.33 (4H, m), 1.50 (9H, s).
Step B: A solution of tert-butyl 4-(3-(1-methyl-1H-pyrazol-3-yl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (215 mg, 0.52 mmol) in CH2Cl2 (2.5 mL) and trifluoroacetic acid (1.0 mL) was stirred at room temperature for 1 h. The reaction mixture was partitioned between CH2Cl2 and aqueous K2CO3. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The residue was triturated with 1:1 hexane/acetone affording the title compound (142 mg, 92%) as a yellow solid: m.p. 224-228° C.; MS m/z 311.1 [M+H]+; 1H NMR (500 MHz, CDCl3): δ 8.32 (1H, s), 7.42 (2H, m), 7.06 (1H, d, J=2 Hz), 6.85 (1H, dd, J=9 Hz, 2.5 Hz), 6.77 (1H, d, J=2 Hz), 3.99 (3H, s), 3.34 (4H, m), 3.06 (4H, m).
Step A: A mixture of tert-butyl 4-(2-oxo-3-(1H-pyrazol-3-yl)-2H-chromen-7-yl)piperazine-1-carboxylate (250 mg, 0.63 mmol, prepared in Example 17, Step B), Cs2CO3 (650 mg, 1.98 mmol), copper(I) iodide (14 mg, 0.073 mmol), iodobenzene (110 μL, 0.97 mmol), and DMF (1.6 mL) was heated at 100° C. for 24 h. The reaction mixture was partitioned between EtOAc and H2O. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography (5% EtOAc in CH2Cl2), followed by ether trituration to yield tert-butyl 4-(2-oxo-3-(1-phenyl-1H-pyrazol-3-yl)-2H-chromen-7-yl)piperazine-1-carboxylate (129 mg, 43%) as a yellow solid. 1H NMR (500 MHz, CDCl3): δ 8.52 (1H, s), 7.97 (1H, d, J=2.5 Hz), 7.77 (2H, d, J=8 Hz), 7.49 (3H, m), 7.32 (2H, m), 6.86 (1H, dd, J=8.5 Hz, 2.5 Hz), 6.77 (1H, d, J=2.5 Hz), 3.62 (4H, m), 3.36 (4H, m), 1.50 (9H, s).
Step B: A solution of tert-butyl 4-(2-oxo-3-(1-phenyl-1H-pyrazol-3-yl)-2H-chromen-7-yl)piperazine-1-carboxylate (127 mg, 0.27 mmol) in CH2Cl2 (2.5 mL) and trifluoroacetic acid (1.0 mL) was stirred at room temperature for 1 h. The reaction mixture was partitioned between CH2Cl2 and aqueous K2CO3. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The residue was triturated with 2:1 hexane/acetone to afford 3-(1-phenyl-1H-pyrazol-3-yl)-7-(piperazin-1-yl)-2H-chromen-2-one (85 mg, 84%) as a yellow solid. MS m/z 373.3 [M+H]+.
Step C: 3-(1-phenyl-1H-pyrazol-3-yl)-7-(piperazin-1-yl)-2H-chromen-2-one (55 mg, 0.15 mmol) was combined with aqueous formaldehyde (37%, 200 uL, 2.15 mmol) and sodium triacetoxyborohydride (110 mg, 0.52 mmol) in 1,2-dichloroethane (0.5 mL). The mixture was stirred 20 min at room temperature, and then quenched by the addition of an aqueous saturated NaHCO3 solution. The mixture was extracted with CH2Cl2. The organic layer was, dried over NaSO4, filtered, concentrated and purified by silica gel column chromatography (10% MeOH in CH2Cl2) to give the title compound (34 mg, 58%) as a yellow solid: m.p. 152-159° C.; MS m/z 387.3 [M+H]+; 1H NMR (500 MHz, CDCl3): δ 8.51 (1H, s), 7.97 (1H, d, J=2.5 Hz), 7.77 (2H, d, J=7.5 Hz), 7.47 (3H, m), 7.32 (2H, m), 6.86 (1H, dd, J=8.5 Hz), 6.77 (1H, d, J=2 Hz), 3.45 (4H, m), 2.66 (4H, br s), 2.43 (3H, s).
Step A: A mixture of tert-butyl 4-(4-formyl-3-hydroxyphenyl)piperazine-1-carboxylate (3.0 g, 9.8 mmol, prepared in Example 1, Part 2), ethyl 3-oxopentanoate (1.62 mL, 11.3 mmol), AcOH (650 μL, 12 mmol), piperidine (1.1 mL, 11.3 mmol), and CH3CN (24 mL) were heated at 80° C. for 4 h. The reaction mixture was partitioned between CH2Cl2 and H2O. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography (10% EtOAc in CH2Cl2), followed by ether trituration, yielding tert-butyl 4-(2-oxo-3-propionyl-2H-chromen-7-yl)piperazine-1-carboxylate (3.6 g, 95%) as a yellow solid. 1H NMR (500 MHz, CDCl3): δ 8.49 (1H, s), 7.50 (1H, d, J=8.5 Hz), 6.83 (1H, dd, J=8.5 Hz, 2.5 Hz), 6.67 (1H, d, J=2 Hz), 3.63 (4H, m), 3.47 (4H, m), 3.16 (2H, q, J=7 Hz), 1.52 (9H, s), 1.19 (3H, t, J=7 Hz).
Step B: A mixture of tert-butyl 4-(2-oxo-3-propionyl-2H-chromen-7-yl)piperazine-1-carboxylate (3.3 g, 8.55 mmol), dimethylformamide dimethyl acetal (10 mL, 830 mmol) and pyrrolidine (1.65 mL, 20.1 mmol) was heated at 60° C. for 3 h, then the solvent was removed under vacuum. The reaction mixture was dissolved in AcOH (10 mL) and cooled to 0° C. Hydrazine (820 μL, 26 mmol) was added dropwise (mild exotherm). After the addition was complete, the mixture was stirred at room temperature for 10 min. The reaction mixture was partitioned between CH2Cl2 and aqueous K2CO3. The organic layer was dried over MgSO4, then filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography (50% EtOAc in CH2Cl2), followed by trituration with 2:1 hexane/acetone, yielding tert-butyl 4-(3-(4-methyl-1H-pyrazol-3-yl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (1.01 g, 29%) as a yellow solid. 1H NMR (500 MHz, CDCl3): δ 7.95 (1H, s), 7.48 (1H, s), 7.44 (1H, d, J=9 Hz), 6.87 (1H, dd, J=8.5 Hz, 2.5 Hz), 6.74 (1H, d, J=2.5 Hz), 3.63 (4H, m), 3.38 (4H, m), 2.36 (3H, s), 1.50 (9H, s).
Step C: A solution of tert-butyl 4-(3-(4-methyl-1H-pyrazol-3-yl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (250 mg, 0.61 mmol) in CH2Cl2 (2.5 mL) and trifluoroacetic acid (1.0 mL) was stirred at room temperature for 15 min. The reaction mixture was partitioned between CH2Cl2 and aqueous K2CO3. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The residue was triturated with 1:1 hexane/acetone yielding the title compound (155 mg, 82%) as a yellow solid: m.p. 175-200° C. (decomposition range); MS m/z 311.2 [M+H]+; 1H NMR (500 MHz, DMSO-d6:D2O, 100° C.): δ 7.91 (1H, s), 7.54 (1H, d, J=9 Hz), 7.41 (1H, br s), 6.95 (1H, d, J=9 Hz), 6.80 (1H, d, J=2.5 Hz), 3.35 (4H, m), 2.92 (4H, m), 2.09 (3H, br s).
Step A: Hydrogen sulfide gas (H2S) was bubbled into a solution of ethyl cyanoacetate (4.7 mL, 44.3 mmol) in pyridine/triethylamine (500 mL, 1:1 v/v) until it became saturated. The mixture was heated at 60° C. for 18 h, then the solvent was removed under vacuum. The residue was partitioned between EtOAc and aqueous HCl. The organic layer was dried over MgSO4, then filtered and concentrated under vacuum. The resulting oil was filtered to remove solid impurities. Ethyl 3-amino-3-thioxopropanoate (6.25 g, 96%) was obtained as an orange oil. 1H NMR (500 MHz, CDCl3): δ 8.92 (1H, br s), 7.75 (1H, br s), 4.21 (2H, q, J=7 Hz), 3.82 (2H, s), 1.29 (3H, t, J=7 Hz).
Step B: A solution of ethyl 3-amino-3-thioxopropanoate (2.0 g, 13.6 mmol) and chloroacetone (1.2 mL, 15.0 mmol) in DMF (230 mL) was heated at 105° C. for 15 h. The reaction mixture was partitioned between EtOAc and H2O. The organic layer was dried over MgSO4, filtered and concentrated under vacuum. The residue was purified by silica gel column chromatography (CH2Cl2) yielding ethyl 2-(4-methylthiazol-2-yl)acetate (1.22 g, 48%) as a red oil. 1H NMR (500 MHz, CDCl3): δ 6.86 (1H, s), 4.24 (2H, q, J=7 Hz), 4.03 (2H, s), 2.44 (3H, s), 1.29 (3H, t, J=7 Hz).
Step C: A mixture of ethyl 2-(4-methylthiazol-2-yl)acetate (650 mg, 3.5 mmol), 4-fluoro-2-hydroxybenzaldehyde (490 mg, 3.5 mmol), piperidine (15 μL, 0.15 mmol), AcOH (15 μL, 0.27 mmol) and CH3CN (5 mL) was heated at 80° C. for 24 h. The reaction mixture was partitioned between CH2Cl2 and aqueous K2CO3. The organic layer was dried over MgSO4, filtered and concentrated under vacuum. The residue was triturated with 7:3 hexane/CH2Cl2 yielding 7-fluoro-3-(4-methylthiazol-2-yl)-2H-chromen-2-one (642 mg, 70%) as a yellow solid. 1H NMR (500 MHz, CDCl3): δ 8.85 (1H, s), 7.69 (1H, dd, J=8.5 Hz, 6 Hz), 7.15 (3H, m), 2.57 (3H, s).
Step D: A mixture of 7-fluoro-3-(4-methylthiazol-2-yl)-2H-chromen-2-one (100 mg, 0.38 mmol), (S)-2-methylpiperazine (46 mg, 0.46 mmol) and DMSO (600 μL) was heated at 80° C. for 15 h. The reaction mixture was diluted in an aqueous saturated NaHCO3 solution and filtered. The collected material was purified by silica gel column chromatography (10% MeOH in CH2Cl2), followed by trituration with 1:1 hexane/acetone to yield the title compound (103 mg, 79%) as a yellow solid: m.p. 194-199° C.; MS m/z 342.2 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.80 (1H, s), 7.75 (1H, d, J=9 Hz), 7.32 (1H, m), 7.06 (1H, dd, J=9 Hz, 2.5 Hz), 6.91 (1H, d, J=2.5 Hz), 3.88 (2H, t, J=11 Hz), 2.96 (1H, d, J=12 Hz), 2.81 (1H, td, J=12 Hz, 3 Hz), 2.72 (2H, m), 2.45 (4H, m), 2.36 (1H, br s), 1.04 (3H, d, J=6.5 Hz).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 21 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 4-fluoro-2-hydroxybenzaldehyde (10 g, 71.4 mmol), acetic anhydride (34 mL, 360 mmol), and triethylamine (11 mL, 79 mmol) was heated at 145° C. for 2 d. The reaction mixture was diluted in aqueous NH4OH (500 mL) and filtered. The collected material was dried, yielding 7-fluoro-2H-chromen-2-one (10.3 g, 88%) as a brown solid. 1H NMR (500 MHz. CDCl3): δ 7.69 (1H, d, J=9.5 Hz), 7.48 (1H, dd, J=8.5 Hz, 6 Hz), 7.07 (1H, dd, J=8.5 Hz, 2.5 Hz), 7.03 (1H, td, J=8.5 Hz, 2.5 Hz), 6.38 (1H, d, J=9.5 Hz).
Step B: A mixture of 7-fluoro-2H-chromen-2-one (4.33 g, 26.4 mmol), [bis(trifluoroacetoxy)iodo]benzene (18.15 g, 42.2 mmol), iodine (10.7 g, 42.2 mmol), pyridine (4.2 mL, 53 mmol), and CHCl3 (25 mL) was heated at 65° C. for 15 h. The reaction mixture was partitioned between aqueous NaHSO3 and CH2Cl2. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography (50% CH2Cl2 in hexanes, then CH2Cl2) yielding 7-fluoro-3-iodo-2H-chromen-2-one (5.6 g, 73%) as a tan solid. 1H NMR (500 MHz, CDCl3): δ 8.36 (1H, s), 7.46 (1H, dd, J=9 Hz, 6 Hz), 7.07 (2H, m).
Step C: A mixture of 7-fluoro-3-iodo-2H-chromen-2-one (5.6 g, 19.3 mmol), hexabutylditin (13.45 g, 23.2 mmol), bis(triphenylphosphine)palladium(II) dichloride (540 mg, 0.77 mmol) and 1,4-dioxane (55 mL) was heated at 80° C. for 15 h. The reaction mixture was diluted in EtOAc and filtered. The filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography (20-40% CH2Cl2 in hexanes) yielding 7-fluoro-3-(tributylstannyl)-2H-chromen-2-one (6.79 g, 77%) as a colorless oil.
Step D: A mixture of 7-fluoro-3-(tributylstannyl)-2H-chromen-2-one (1.15 g, 2.53 mmol), 4-iodoimidazole (600 mg, 3.1 mmol), bis(triphenylphosphine)palladium(II) dichloride (285 mg, 0.41 mmol), copper(I) iodide (115 mg, 0.60 mmol), and 1,4-dioxane (7 mL) was heated at 85° C. for 2 d. The reaction mixture was partitioned between NH4OH and CH2Cl2. The organic layer was concentrated under vacuum. The residue was purified by silica gel column chromatography (30% MeOH in CH2Cl2). The product was triturated with CH2Cl2, yielding 7-fluoro-3-(1H-imidazol-4-yl)-2H-chromen-2-one (298 mg, 51%) as a yellow solid. MS m/z 231.1 [M+H]+.
Step E: A mixture of 7-fluoro-3-(1H-imidazol-4-yl)-2H-chromen-2-one (90 mg, 0.39 mmol), iodobenzene (70 μL, 0.62 mmol), CuI (60 mg, 0.32 mmol), trans-1,2-bis(methylamino)cyclohexane (23 μL, 0.15 mmol), Cs2CO3 (585 mg, 1.79 mmol) and DMF (0.9 mL) was heated at 50° C. for 45 min. The reaction mixture was diluted in H2O and filtered. The solid material was partitioned between aqueous NH4OH and CH2Cl2. The organic layer was concentrated under vacuum. The residue was purified by silica gel chromatography (10% EtOAc in CH2Cl2), yielding 7-fluoro-3-(1-phenyl-1H-imidazol-4-yl)-2H-chromen-2-one (52 mg, 43%) as a white solid. 1H NMR (500 MHz, CDCl3): δ 8.58 (1H, s), 8.27 (1H, d, J=1.5 Hz), 7.94 (1H, d, J=1.5 Hz), 7.60 (1H, dd, J=8.5 Hz, 6 Hz), 7.52 (4H, m), 7.42 (1H, m), 7.11 (1H, dd, J=9 Hz, 2.5 Hz), 7.06 (1H, td, J=8 Hz, 2.5 Hz).
Step F: A mixture of 7-fluoro-3-(1-phenyl-1H-imidazol-4-yl)-2H-chromen-2-one (50 mg, 0.16 mmol), cis-2,6-dimethylpiperazine (29 mg, 0.25 mmol) and DMSO (300 μL) were heated at 100° C. for 15 h. The reaction mixture was diluted in an aqueous saturated NaHCO3 solution and filtered. The solid material was purified by silica gel column chromatography (5% MeOH in CH2Cl2), yielding the title compound (52 mg, 81%) as a yellow solid: m.p. 260-264° C.; MS m/z 401.3 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.50 (1H, s), 8.41 (1H, d, J=1 Hz), 8.14 (1H, d, J=1 Hz), 7.71 (2H, d, J=8.5 Hz), 7.64 (1H, d, J=9 Hz), 7.55 (2H, t, J=8 Hz), 7.40 (1H, t, J=7.5 Hz), 7.01 (1H, dd, J=9 Hz, 2 Hz), 6.89 (1H, d, J=2 Hz), 3.82 (2H, dd, J=12.5 Hz, 2 Hz), 2.80 (2H, m), 2.31 (2H, t, J=11.5 Hz), 2.25 (1H, br s), 1.04 (6H, d, J=6.5 Hz).
Step A: A mixture of 7-fluoro-3-(1H-imidazol-4-yl)-2H-chromen-2-one (75 mg, 0.32 mmol, prepared in Example 22, Step D), 2-iodopyridine (55 μL, 0.5 mmol), copper(I) iodide (27 mg, 0.14 mmol), trans-1,2-bis(methylamino)cyclohexane (13 μL, 0.08 mmol), Cs2CO3 (330 mg, 1.01 mmol) and DMF (750 μL) was heated at 50° C. for 30 min. The reaction mixture was diluted with H2O and filtered. The solid material was partitioned between aqueous NH4OH and CH2Cl2. The organic layer was concentrated under vacuum. The residue was purified by silica gel column chromatography (10% acetone in CH2Cl2), followed by trituration with 1:1 CH2Cl2/hexane, yielding 7-fluoro-3-(1-(pyridin-2-yl)-1H-imidazol-4-yl)-2H-chromen-2-one (62 mg, 63%) as a white solid. 1H NMR (500 MHz, CDCl3): δ 8.61 (1H, s), 8.56 (1H, d, J=1 Hz), 8.52 (1H, m), 8.50 (1H, d, J=1 Hz), 7.88 (1H, m), 7.60 (1H, dd, J=8.5 Hz, 6 Hz), 7.48 (1H, d, J=8.5 Hz), 7.29 (1H, m), 7.11 (1H, dd, J=9 Hz, 2.5 Hz), 7.06 (1H, td, J=8.5 Hz, 2.5 Hz).
Step B: Following the procedure from Example 22, Step F, 7-fluoro-3-(1-(pyridin-2-yl)-1H-imidazol-4-yl)-2H-chromen-2-one (40 mg, 0.13 mmol), cis-2,6-dimethylpiperazine (23 mg, 0.2 mmol), and DMSO (300 μL) yielded the title compound (46 mg, 88%): m.p. 201-206° C.; MS m/z 402.3 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.71 (1H, d, J=1 Hz), 8.55 (1H, m), 8.52 (1H, s), 8.45 (1H, d, J=1 Hz), 8.02 (1H, m), 7.92 (1H, d, J=8.5 Hz), 7.64 (1H, d, J=9 Hz), 7.41 (1H, dd, J=6.5 Hz, 5 Hz), 7.02 (1H, dd, J=9 Hz, 2 Hz), 6.88 (1H, d, J=2.5 Hz), 3.82 (2H, d, J=11.5 Hz), 2.80 (2H, m), 2.32 (2H, t, J=11.5 Hz), 2.28 (1H, br s), 1.04 (6H, d, J=6.5 Hz).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 23 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: Into a suspension of 7-hydroxycoumarin (16.2 g, 100 mmol) in pyridine (16.3 mL, 200 mmol) and CH2Cl2 (250 mL) at 0° C. was added dropwise a solution of triflic anhydride (20.2 mL, 120 mmol) in CH2Cl2 (50 mL). The mixture warmed to room temperature over 30 min. The mixture was washed with dilute aqueous HCl, water, brine, and then dried over NaSO4 and concentrated to give a solid 2-oxo-2H-chromen-7-yl trifluoromethanesulfonate (28.5 g, 97%) as a tan solid. MS m/z 295.0 [M+H]+.
Step B: A mixture of palladium(II) acetate (0.228 g, 1.02 mmol), 2,2′-bis(diphenylphosphino)-1,1′-binaphthalene (1.27 g, 2.04 mmol) and Cs2CO3 (8.3 g, 25.5 mmol) in toluene (75 mL) was stirred under Argon at 110° C. for 15 min until a dark red color formed. The mixture was cooled to room temperature, upon which 2-oxo-2H-chromen-7-yl trifluoromethanesulfonate (5.0 g, 17 mmol) and 1-Boc-piperazine (3.8 g, 20.4 mmol) were added. The mixture was stirred at 110° C. for 24 h. The mixture was partitioned in EtOAc and water. The organic layer was dried over NaSO4, filtered, concentrated and purified by silica gel column chromatography (0-15% EtOAc in CH2Cl2) to give tert-butyl 4-(2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (2.5 g, 45%) as a yellow solid. MS m/z 331.2 [M+H]+.
Step C: Into a mixture of tert-butyl 4-(2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (2.5 g, 7.58 mmol) and sodium acetate (1.86 g, 22.7 mmol) in acetic acid (30 mL) at room temperature was added bromine (0.4 mL, 7.95 mmol) dropwise. The mixture was stirred at room temperature for 1 h. Water was added to produce a precipitate. The solid was collected by vacuum filtration, washed with water, dried and purified by silica gel column chromatography (0-25% EtOAc in CH2Cl2) to give tert-butyl 4-(3-bromo-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (1.8 g, 58%) as a yellow solid. MS m/z 409.1 [M+H]+, 411.1 [M+2+H]+.
Step D: A mixture of tert-butyl 4-(3-bromo-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (80 mg, 0.2 mmol), 2-aminopyridine (26 mg, 0.28 mmol), bis(dibenzylideneacetone)palladium(0) (3.7 mg, 0.004 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (5.1 mg, 0.0088 mmol) and Cs2CO3 (91 mg, 0.28 mmol) in 1,4-dioxane (1.0 mL) was stirred at 100° C. overnight under Argon, then the solvent was removed. The residue was purified by silica gel column chromatography (0-10% EtOAc in CH2Cl2) to give tert-butyl 4-(2-oxo-3-(pyridin-2-ylamino)-2H-chromen-7-yl)piperazine-1-carboxylate (82 mg, 71%) as a yellow solid. MS m/z 423.2 [M+H]+.
Step E: tert-Butyl 4-(2-oxo-3-(pyridin-2-ylamino)-2H-chromen-7-yl)piperazine-1-carboxylate (71 mg, 0.168 mmol) was dissolved in trifluoroacetic acid (2.0 mL). The mixture was stirred for 15 min at room temperature, then the solvent was removed with a stream of nitrogen. The residue was partitioned in CH2Cl2 and aqueous K2CO3. The organic layer was dried over NaSO4, filtered and concentrated to give the title compound (41 mg, 76%) as a yellow solid: m.p. 191-194° C.; MS m/z 323.2 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.78 (1H, s), 8.77 (1H, s), 8.69 (1H, s), 8.24 (1H, dd, J=5.1 Hz, 1.3 Hz), 7.62-7.57 (1H, m), 7.44 (1H, d, J=8.8 Hz), 7.27 (1H, d, J=8.5 Hz), 6.95 (1H, dd, J=8.8 Hz, 2.2 Hz), 6.85-6.80 (2H, m), 3.20-3.13 (4H, m), 2.86-2.78 (4H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 24 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: To a solution of t-butyl 4-(3-acetyl-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (1.42 g, 3.8 mmol, prepared in Example 17, Step A) in 1,4-dioxane (8 mL) was added N,N-dimethylformamide dimethylacetal (6 mL, 44.7 mmol). The mixture was heated at 100° C. for 3 h. The reaction mixture was concentrated under reduced pressure. The residue was triturated with ether-hexane (1:1), producing a precipitate. The solid was collected by vacuum filtration, washed with ether-hexane and dried under nitrogen, affording (E)-tert-butyl 4-(3-(3-(dimethylamino)acryloyl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (1.5 g, 92%) as an orange powder. MS m/z 428.4 [M+H]+.
Step B: To a solution of (E)-t-butyl 4-(3-(3-(dimethylamino)acryloyl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (171 mg, 0.40 mmol) and acetamidine hydrochloride (151 mg, 1.6 mmol) in CH3CN (2 mL) was added K2CO3 (110 mg, 0.80 mmol). The mixture was heated to 100° C. for 16 h. After cooling to room temperature, water (10 mL) was added to the mixture, producing a precipitate. The precipitate was collected by vacuum filtration, washed with water and dried under nitrogen to afford t-butyl-4-(3-(2-methylpyrimidin-4-yl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (148 mg, 88%). MS m/z 423.3 [M+H]+.
Step C: To a suspension of t-butyl-4-(3-(2-methylpyrimidin-4-yl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (182 mg, 0.42 mmol) in CH2Cl2 (1 mL) was added 4N HCl in 1,4-dioxane (1 mL). The mixture was stirred for 2 h at room temperature. The suspension was diluted with ether (10 mL) and filtered. The solid was washed with ether and dried under nitrogen to afford the title compound (140 mg, 91%) as a yellow solid: m.p. 200° C. (decomp.); MS m/z 323.2 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 9.13 (2H, br), 9.05 (1H, s), 8.78 (1H, d, J=5.4 Hz), 8.22 (1H, d, J=5.4 Hz), 7.88 (1H, d, J=8.8 Hz), 7.12 (1H, dd, J=8.8 Hz, 2.5 Hz), 7.03 (1H, d, J=2.2 Hz), 3.73 (4H, m), 3.24 (4H, m), 2.71 (3H, s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 25 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: To a pressure vessel were added, 4-fluoro-2-hydroxybenzaldehyde (0.5 g, 3.6 mmol), 2-(3,4-dimethoxyphenyl)acetic acid (1.4 g, 7.2 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1.5 g, 7.9 mmol), diisopropylethylamine (2.3 mL, 14.3 mmol) and CH2Cl2 (10 mL). The mixture was stirred at 60° C. for 1 h, then quenched with an aqueous saturated NaHCO3 solution (50 mL) and extracted with EtOAc three times. The combined extracts were dried over Na2SO4 and concentrated under vacuum. The residue was purified by silica gel column chromatography (0-5% EtOAc in CH2Cl2) to give 3-(3,4-dimethoxyphenyl)-7-fluoro-2H-chromen-2-one (1.0 g, 95%). MS m/z 301.0 [M+H]+.
Step B: A mixture of 3-(3,4-dimethoxyphenyl)-7-fluoro-2H-chromen-2-one (40 mg, 0.13 mmol), piperazine (34 mg, 0.40 mmol) and DMSO (0.3 mL) was stirred at 80° C. overnight. After cooling to room temperature, the mixture was diluted with water (5 mL) to produce a precipitate. The precipitate was collected by filtration, washed with water and ethyl ether, and dried to give the title compound (14 mg, 29%) as yellow powder: m.p. 168-170° C.; MS m/z 367.2 [M+H]+; 1H NMR (500 MHz, CDCl3): δ 7.69 (1H, s), 7.38 (1H, d, J=8.8 Hz), 7.31 (1H, d, J=1.9 Hz), 7.25 (1H, d, J=2.2 Hz), 6.93 (1H, d, J=8.5 Hz), 6.85 (1H, dd, J=8.8 Hz, 2.5 Hz), 6.77 (1H, d, J=2.5 Hz), 3.95 (3H, s), 3.93 (3H, s), 3.36-3.32 (4H, m), 3.10-3.05 (4H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 26 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: To a suspension of tert-butyl 4-(3-(6-fluoropyridin-2-yl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (90 mg, 0.21 mmol, prepared according to Example 26) in isopropanol (1 mL) was added NaH (19 mg, 60% in mineral oil, 0.48 mmol). The mixture was stirred at 90° C. for 2 h, diluted with water and extracted with dichloromethane. The organic layer was concentrated and purified by silica gel column chromatography (0-10% MeOH in CH2Cl2) to give tert-butyl 4-(3-(6-isopropoxypyridin-2-yl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (50 mg, 51%). MS 466.3 m/z [M+H]+.
Step B: tert-Butyl 4-(3-(6-isopropoxypyridin-2-yl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (50 mg, 0.11 mmol) was stirred with 50% TFA in CH2Cl2 (1.0 mL) at room temperature overnight. Aqueous K2CO3 (2M solution) was added to the mixture, until the aqueous layer became basic, pH ˜9. The organic layer was separated and the aqueous layer was extracted with CH2Cl2. The combined organics were dried over Na2SO2 and concentrated to provide the title compound (37 mg, 82%) as a yellow powder: m.p. 177-180° C.; MS 366.3 m/z [M+H]+; 1H NMR (500 MHz, CDCl3): δ 8.67 (1H, s), 8.06 (1H, dd, J=7.6 Hz, 0.6 Hz), 7.63 (1H, dd, J=8.2 Hz, 7.6 Hz), 7.49 (1H, d, J=8.8 Hz), 6.86 (1H, dd, J=8.7 Hz, 2.4 Hz), 6.75 (1H, d, J=2.2 Hz), 6.65 (1H, dd, J=8.2 Hz, 0.6 Hz), 5.43 (1H, t, J=6.3 Hz), 3.41-3.31 (4H, m), 3.10-3.00 (4H, m), 1.42 (6H, d, J=6.3 Hz).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 27 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of tert-butyl 4-(3-(6-fluoropyridin-2-yl)-2-oxo-2H-chromen-7-yl)piperazine-1-carboxylate (90 mg, 0.21 mmol, prepared according to Example 26) and pyrrolidine (1 mL) was stirred at 80° C. for 2 h. The mixture was diluted with water (10 mL) and extracted with dichloromethane. The organic layer was concentrated and purified by silica gel column chromatography (0-10% MeOH in CH2Cl2) to give tert-butyl 4-(2-oxo-3-(6-(pyrrolidin-1-yl)pyridin-2-yl)-2H-chromen-7-yl)piperazine-1-carboxylate (56 mg, 50%). MS 477.0 m/z [M+H]+.
Step B: tert-Butyl 4-(2-oxo-3-(6-(pyrrolidin-1-yl)pyridin-2-yl)-2H-chromen-7-yl)piperazine-1-carboxylate was stirred with 50% TFA in CH2Cl2 (1.0 mL) at room temperature overnight. Aqueous K2CO3 (2M solution) was added to the mixture, until the aqueous layer became basic, pH ˜9. The organic layer was separated and the aqueous layer was extracted with CH2Cl2. The combined organics were dried over Na2SO2 and concentrated to provide the title compound (63 mg, 80%) as yellow powder: m.p. 190-192° C.; MS 377.3 m/z [M+H]+; 1H NMR (500 MHz, CDCl3): δ 8.72 (1H, s), 7.73 (1H, d, J=7.3 Hz), 7.54-7.44 (2H, m), 6.84 (1H, dd, J=8.8 Hz, 2.5 Hz), 6.75 (1H, d, J=2.2 Hz), 6.40-6.33 (1H, m), 3.60-3.50 (4H, m), 3.33 (4H, dd, J=6.2 Hz, 4.3 Hz), 3.10-3.01 (4H, m), 2.04 (4H, dt, J=6.5 Hz, 3.4 Hz).
Step A: A mixture of 7-fluoro-3-(6-fluoropyridin-2-yl)-2H-chromen-2-one (260 mg, 1.0 mmol, prepared according to Example 26) and NaSMe (105 mg, 1.5 mmol) in DMF (2 mL) was stirred at room temperature for 1 h. The mixture was diluted with water (10 mL) to produce a precipitate. The precipitate was collected by filtration, washed with water and CH2Cl2, and dried to give 7-fluoro-3-(6-(methylthio)pyridin-2-yl)-2H-chromen-2-one (100 mg, 35%). MS 288.3 m/z [M+H]+.
Step B: A mixture of 7-fluoro-3-(6-(methylthio)pyridin-2-yl)-2H-chromen-2-one (50 mg, 0.17 mmol), piperazine (44 mg, 0.51 mmol) and DMSO (0.5 mL) was stirred at 80° C. overnight. After cooling to room temperature, the mixture was diluted with water (5 mL) to produce a precipitate. The precipitate was collected by filtration, washed with water and ethyl ether, and dried to give the title compound (18 mg, 30%): m.p. 180-183° C.; MS m/z 354.3 [M+H]+; 1H NMR (500 MHz, CDCl3): δ 8.69 (1H, s), 7.84 (1H, d, J=7.3 Hz), 7.59 (1H, dd, J=8.5 Hz, 7.6 Hz), 7.53 (1H, d, J=8.8 Hz), 7.17-7.13 (2H, m), 6.71 (1H, s), 3.63-3.61 (4H, m), 3.09-3.03 (4H, m), 2.58-2.54 (3H, m).
Step A: A mixture of ethyl 2-(2-ethoxy-2-oxoethyl)pyrazolo[1,5-a]pyridine-3-carboxylate (1.2 g, 4.3 mmol, prepared from 1-aminopyridinium iodide and diethyl 3-oxopentanedioate according to the procedure in Japanese Patent 62-267285, 1986), NaOH (3 N, 8.6 mL) and THF (10 mL) was heated at 60° C. for 15 h. The mixture was cooled to room temperature and washed with EtOAc. The aqueous phase was acidified with aqueous HCl (6 N) to pH 3, producing a precipitate. The precipitate was collected by vacuum filtration, washed with water and dried to give 2-(carboxymethyl)pyrazolo[1,5-a]pyridine-3-carboxylic acid (0.63 g, 66%) as a white solid. MS m/z 221.1 [M+H]+.
Step B: To a suspension of 2-(carboxymethyl)pyrazolo[1,5-a]pyridine-3-carboxylic acid (0.63 g, 2.9 mmol) in water (5 mL) was added conc. H2SO4 (5 mL). The clear solution was heated at 80° C. for 15 h. The solution was cooled to room temperature. Aqueous NaOH (1 N) was added to the solution until pH 2-3 was reached. A precipitate formed. The precipitate was collected by vacuum filtration, washed with water and dried to give 2-(pyrazolo[1,5-a]pyridin-2-yl)acetic acid (0.435 g, 86%) as a white solid. MS m/z 177.1 [M+H]+.
Step C: A mixture of 2-(pyrazolo[1,5-a]pyridin-2-yl)acetic acid (0.435 g, 2.47 mmol)), 4-fluoro-2-hydroxybenzaldehyde (0.363 g, 2.59 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (0.57 g, 2.96 mmol), 4-(dimethylamino)pyridine (61 mg, 0.5 mmol) and triethylamine (0.7 mL, 5.0 mmol) in CH2Cl2 (8 mL) was heated at 50° C. After 1 h, the mixture was concentrated. The residue was suspended in CH3CN, collected by vacuum filtration, washed with CH3CN and dried to give 7-fluoro-3-(pyrazolo[1,5-a]pyridin-2-yl)-2H-chromen-2-one (0.66 g, 95%) as a yellow solid. MS m/z 281.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.83 (1H, s), 8.71 (1H, dd J=6.9 Hz, 1.0 Hz), 8.03 (1H, dd, J=8.7 Hz, 6.4 Hz), 7.78 (1H, d, J=8.8 Hz), 7.48 (1H, dd, J=9.6 Hz, 2.4 Hz), 7.34-7.24 (1H, m), 7.30 (1H, s), 7.26 (1H, m), 6.98 (1H, td, J=6.9 Hz, 1.4 Hz).
Step D: A mixture of 7-fluoro-3-(pyrazolo[1,5-a]pyridin-2-yl)-2H-chromen-2-one (56 mg, 0.2 mmol), piperazine (52 mg, 0.6 mmol) and N,N-diisopropylethylamine (52 μL, 0.3 mmol) in DMSO (0.5 mL) was heated at 120° C. for 7 h. Upon cooling to room temperature, a precipitate formed. The precipitate was collected by vacuum filtration, washed with CH3CN and dried to give the title compound (60 mg, 87%) as a yellow solid: m.p. 236-238° C.; MS m/z 347.3 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.67 (1H, dd, J=7.0 Hz, 2.3 Hz), 8.5 (1H, s), 7.73 (1H, d, J=8.9 Hz), 7.69 (1H, d, J=8.9 Hz), 7.25-7.20 (2H, m), 7.01 (1H, dd, J=8.9 Hz, 2.4 Hz), 6.92 (1H, td, J=6.8 Hz, 1.4 Hz), 6.86 (1H, d, J=2.3 Hz), 3.32 (4H, m), 2.82 (4H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 30 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 5-chloropyridin-2-amine (2.57 g, 20 mmol) and ethyl 4-chloro-3-oxobutanoate (3.95 g, 24 mmol) in EtOH (20 mL) was heated at 90° C. for 15 h, then the solvent was removed. The residue was suspended in CH3CN, collected by vacuum filtration, washed with CH3CN and dried to give ethyl 2-(6-chloroimidazo[1,2-a]pyridin-2-yl)acetate (4.14 g, 86%) as a white solid. MS m/z 239.1 [M+H]+.
Step B: To a solution of ethyl 2-(6-chloroimidazo[1,2-a]pyridin-2-yl)acetate (2.38 g, 10 mmol) in THF was added aqueous NaOH (3 N, 6.6 mL, 20 mmol). After stirring at room temperature for 3 h, the mixture was concentrated. The residual mixture was acidified with aqueous HCl (6 N) to pH 3. A precipitate formed. The precipitate was collected by vacuum filtration, washed with water and dried, yielding 2-(6-chloroimidazo[1,2-a]pyridin-2-yl)acetic acid (1.66 g, 79%) as a white solid. MS m/z 211.1 [M+H]+.
Step C: Following the procedure in Example 30, Step C, 2-(6-chloroimidazo[1,2-a]pyridin-2-yl)acetic acid (0.386 g, 2.5 mmol), 1-(4-fluoro-2-hydroxyphenyl)-ethanone (0.525 g, 2.5 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (0.623 g, 3.25 mmol), 4-(dimethylamino)pyridine (92 mg, 0.75 mmol) and triethylamine (0.91 mL, 7.5 mmol) in CH2Cl2 (4 mL) gave 3-(6-chloroimidazo[1,2-a]pyridin-2-yl)-7-fluoro-4-methyl-2H-chromen-2-one (0.426 g, 52%) as an off-white solid. MS m/z 329.1 [M+H]+.
Step D: Following the procedure in Example 30, Step D, 3-(6-chloroimidazo[1,2-a]pyridin-2-yl)-7-fluoro-4-methyl-2H-chromen-2-one (82 mg, 0.25 mmol), piperazine (65 mg, 0.75 mmol), DIEA (52 μL, 0.3 mmol) in DMSO (0.5 mL) gave the title compound (64 mg, 65%) as a yellow solid: m.p. 224-227° C.; MS m/z 395.2 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.90 (1H, d, J=2.1 Hz), 8.27 (1H, s), 7.69 (1H, d, J=9.1 Hz), 7.63 (1H, d, J=9.6 Hz), 7.30 (1H, dd, J=9.6 Hz, 2.1 Hz), 7.01 (1H, dd, J=9.1 Hz, 2.4 Hz), 6.82 (1H, d, J=2.5 Hz), 3.28 (4H, m), 2.82 (4H, m), 2.66 (3H, s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 31 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: To a stirred solution of 7-fluorocoumarin (3.04 g, 18.5 mmol, prepared in Example 22, Step A) in chloroform (20 mL), at room temperature, was added dropwise, bromine (11.9 g, 3.81 mL, 74 mmol). After the addition, the mixture was stirred at room temperature for an additional 2 hours, then cooled in an ice-water bath and diluted with dichloromethane (100 mL). Triethylamine (22.4 g, 30.7 mL, 222 mmol) was added carefully while stirring. The mixture was stirred at room temperature for an additional 2 h after the addition. The precipitate present in the mixture was removed by filtration and washed with CH2Cl2 (3×15 mL). The combined filtrate was concentrated and purified by silica gel column chromatography (CH2Cl2), yielding 3-bromo-7-fluoro-2H-chromen-2-one (4.07 g, 90%) as white solid. MS m/z 242.4, 244.4 [M+H]+; 1H NMR (500 MHz, CDCl3): δ 8.09 (1H, s), 7.47 (1H, dd, J=8.7 Hz, 5.8 Hz), 7.12-7.03 (2H, m).
Step B: A reaction tube, equipped with an open-top cap and a septum was charged with 3-bromo-7-fluoro-2H-chromen-2-one (0.49 g, 2.0 mmol), (2-(trifluoromethyl)pyridin-3-yl)boronic acid (0.42 g, 2.2 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (0.082 g, 0.1 mmol) and CH3CN (6.0 mL). After purging three times with nitrogen, aqueous K2CO3 (2.0 mL, 2.0M, 4.0 mmol) was added and the mixture was stirred at 50° C. overnight, then the solvent was removed. The residue was suspended in CH2Cl2 and filtered. The filtrate was concentrated and purified by silica gel column chromatography (0-30% EtOAc in CH2Cl2) to give 7-fluoro-3-(2-(trifluoromethyl)pyridin-3-yl)-2H-chromen-2-one (0.21 g, 34%). MS m/z 310.2 [M+H]+.
Step C: A mixture of 7-fluoro-3-(2-(trifluoromethyl)pyridin-3-yl)-2H-chromen-2-one (93 mg, 0.3 mmol) and piperazine (52 mg, 0.6 mmol) in DMSO (0.6 mL) was stirred at 80° C. for 24 h. After cooling to room temperature, the mixture was diluted with water (5 mL) to produce a precipitate. The solid was collected by filtration, washed with water and ethyl ether, and dried to give the title compound (100 mg, 89%) as yellow powder: m.p. 197-200° C.; MS m/z 376.2 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.71 (1H, s), 8.53 (1H, d, J=2.5 Hz), 8.14 (1H, t, J=7.9 Hz), 7.83 (1H, d, J=8.5 Hz), 7.75 (1H, d, J=9.1 Hz), 7.02 (1H, dd, J=9.0 Hz, 2.4 Hz), 6.85 (1H, d, J=2.2 Hz), 3.40-3.33 (4H, m), 2.85-2.78 (4H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 32 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 3-bromo-7-fluorocoumarin (122 mg, 0.5 mmol, prepared in Example 32, Step A), 2-methoxy-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (235 mg, 1.0 mmol), copper(I) chloride (50 mg, 0.5 mmol), Cs2CO3 (652 mg, 2.0 mmol), palladium(II) acetate (5.6 mg, 0.025 mmol), 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl (41 mg, 0.1 mmol) and DMF (2.0 mL) were stirred under an Argon atmosphere at 60° C. for 2 h. After cooling to room temperature, the mixture was diluted with water (10 mL) to produce a precipitate. The solid was washed with water, dried, and purified with silica gel column chromatography (0-10% EtOAc in CH2Cl2) to give 7-fluoro-3-(6-methoxypyridin-2-yl)-2H-chromen-2-one (52 mg, 38%). MS m/z 272.2 [M+H]+.
Step B: A mixture of 7-fluoro-3-(6-methoxypyridin-2-yl)-2H-chromen-2-one (52 mg, 0.19 mmol), piperazine (50 mg, 0.57 mmol) and DMSO was stirred at 80° C. overnight. After cooling to room temperature, the mixture was diluted with water (5 mL) to produce a precipitate. The precipitate was collected by filtration, dried and purified by silica gel column chromatography (0-20% MeOH in CH2Cl2) to give the title compound (20 mg, 31%) as yellow powder: m.p. 162-165° C.; MS m/z 338.2 [M+H]+. 1H NMR (500 MHz, DMSO-d6): δ 8.83 (1H, s), 7.96 (1H, dd, J=7.6 Hz, 0.9 Hz), 7.75 (1H, dd, J=8.2, 7.6 Hz), 7.69 (1H, d, J=8.8 Hz), 7.02 (1H, dd, J=9.0, 2.4 Hz), 6.85 (1H, d, J=2.2 Hz), 6.77 (1H, dd, J=8.2 Hz, 0.6 Hz), 3.98 (3H, s), 3.35-3.28 (4H, m), 2.86-2.78 (4H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 33 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 6-bromo-2-methylimidazo[1,2-a]pyridine (0.79 g, 3.75 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.14 g, 4.49 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (0.15 g, 0.19 mmol), potassium acetate (1.1 g, 11.5 mmol) in 1,4-dioxane (7.5 mL) was stirred at 80° C. overnight under Argon. The mixture was diluted with THF (20 mL) and filtered. The filtrate was evaporated to give a dark solid residue, which was used without further purification (MS m/z 177.0 [M+H]+). The residue was combined with 3-bromo-7-fluoro-2H-chromen-2-one (0.73 g, 3.0 mmol, prepared in Example 32, Step A), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (0.245 g, 0.3 mmol) and aqueous K2CO3 (2.0 M×4.5 mL, 9.0 mmol) in CH3CN (9.0 mL). The mixture was stirred at 60° C. overnight under Argon, then diluted with water and filtered. The solid was dissolved in CH2Cl2 (10% methanol), dried over Na2SO4, filtered, concentrated and purified with by silica gel chromatography (0-10% MeOH in CH2Cl2) to give 7-fluoro-3-(2-methylimidazo[1,2-a]pyridin-6-yl)-2H-chromen-2-one (0.67 g, 76%). MS m/z 295.0 [M+H]+.
Step B: A mixture of 7-fluoro-3-(2-methylimidazo[1,2-a]pyridin-6-yl)-2H-chromen-2-one (90 mg, 0.31 mmol), (S)-octahydropyrrolo[1,2-a]pyrazine (50 mg, 0.40 mmol), K2CO3 (125 mg, 0.92 mmol) in DMSO (0.6 mL) was stirred at 100° C. overnight. The mixture was diluted with an aqueous saturated NaHCO3 solution and filtered. The solid was dried and purified by silica gel chromatography (0-10% MeOH in CH2Cl2) to give the title compound (56 mg, 46%) as a yellow solid: m.p. 231-233° C.; MS m/z 401.5 [M+H]+; 1H NMR (500 MHz, CDCl3): δ 8.86 (1H, dd, J=1.7, 0.8 Hz), 7.83 (1H, s), 7.49-7.57 (1H, m), 7.35-7.44 (3H, m), 6.87 (1H, d, J=8.8 Hz), 6.77 (1H, d, J=2.2 Hz), 3.94 (1H, dd, J=12.0, 1.6 Hz), 3.80 (1H, d, J=12.6 Hz), 3.24-3.06 (3H, m), 2.76 (1H, t, J=11.0 Hz), 2.47 (3H, d, J=0.6 Hz), 2.45-2.35 (1H, m), 2.30-2.10 (2H, m), 1.99-1.87 (2H, m), 1.86-1.77 (1H, m), 1.61-1.48 (1H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 34 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: Following the procedure in Example 34, Step A, 6-bromo-2-methylbenzo[d]thiazole (0.47 g, 2.1 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (0.63 g, 2.5 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (84 mg, 0.1 mmol) in dioxane (4.0 mL) followed by reaction of the intermediate formed with 3-bromo-7-fluoro-2H-chromen-2-one (0.45 g, 1.85 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (0.16 g, 0.2 mmol), aqueous K2CO3 (2.0 M×3.0 mL, 6.0 mmol) in CH3CN (6.0 mL) yielded 7-fluoro-3-(2-methylbenzo[d]thiazol-6-yl)-2H-chromen-2-one (144 mg, 25%). MS m/z 312.0 [M+H]+.
Step B: A mixture of 7-fluoro-3-(2-methylbenzo[d]thiazol-6-yl)-2H-chromen-2-one (34 mg, 0.11 mmol), 1-methylpiperazine (22 mg, 0.22 mmol), triethylamine (49 mg, 0.49 mmol) in DMSO (0.25 mL) was stirred at 110° C. overnight. The mixture was diluted with an aqueous saturated NaHCO3 solution and filtered. The solid was dried and purified by silica gel chromatography (0-10% MeOH in CH2Cl2) to give the title compound (41 mg, 95%) as a yellow solid: m.p. 215-217° C.; MS m/z 392.4 [M+H]+; 1H NMR (500 MHz, CDCl3): δ 8.28 (1H, d, J=1.8 Hz), 7.98 (1H, dd, J=8.5, 0.6 Hz), 7.80 (1H, s), 7.73 (1H, dd, J=8.5, 1.6 Hz), 7.40 (1H, d, J=8.8 Hz), 6.86 (1H, dd, J=8.8, 2.5 Hz), 6.78 (1H, d, J=2.2 Hz), 3.42 (4H, br. s.), 2.86 (3H, s), 2.62 (4H, s), 2.40 (3H, s)
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 35 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A solution of 3-fluoropyridin-2-amine (1.0 g, 8.92 mmol) was dissolved in CH3CN (300 mL) at 0° C. N-Bromosuccinimide (800 mg, 4.5 mmol) was added to the solution. The reaction mixture was stirred at 0° C. for 20 min, then at room temperature for 20 min. The mixture was cooled to 0° C. Additional N-bromosuccinimide (800 mg, 4.5 mmol) was added. The mixture warmed to room temperature over 40 minutes. An aqueous NaHSO3 solution was added to the mixture to quench excess reagent, then the solvent was removed under vacuum. The residue was dissolved in EtOAc, then washed with aqueous K2CO3. The organic layer was dried over MgSO4, then filtered and concentrated under vacuum. Trituration of the residue with 2:1 hexanes:ether yielded 5-bromo-3-fluoropyridin-2-amine (1.18 g, 69%) as a white solid. 1H NMR (500 MHz, CDCl3): δ 7.93 (1H, d, J=2 Hz), 7.37 (1H, dd, J=9.5 Hz, 2 Hz), 4.66 (2H, br s), 2.77 (3H, s).
Step B: A solution of dimethylzinc (15 mL, 1.2 M in toluene, 18 mmol) was added to a mixture of 5-bromo-3-fluoropyridin-2-amine (1.48 g, 7.75 mmol) and [1,1′-bis(diphenylphosphino)-ferrocene]dichloropalladium(II) complex with dichloromethane (150 mg, 0.18 mmol) in 1,4-dioxane (30 mL). The mixture was heated at 95° C. for 2 h. The reaction mixture was cooled to room temperature and quenched with MeOH. The mixture was diluted with aqueous saturated NH4OH and extracted with EtOAc. The organic layer was dried over MgSO4, filtered and concentrated under vacuum. The residue was purified by silica gel column chromatography (20% acetone in CH2Cl2), followed by trituration with hexane to give 3-fluoro-5-methylpyridin-2-amine (668 mg, 68%) as a tan solid. 1H NMR (500 MHz, CDCl3): δ 7.69 (1H, s), 7.06 (1H, dd, J=11.5 Hz, 1.5 Hz), 4.43 (2H, br s), 2.21 (3H, s).
Step A: Into a mixture of 4-fluoro-2-hydroxybenzaldehyde (1.4 g, 10 mmol) and ethyl 3-oxobutanoate (1.3 g, 10 mmol) was added a few drops of piperidine. The mixture was stirred at room temperature for 10 min. A precipitate formed and was collected by vacuum filtration. The solid was washed with ethanol and aqueous HCl (1 N), filtered and dried to give 3-acetyl-7-fluoro-2H-chromen-2-one (1.96 g, 95%) as a pale yellow solid. 1H NMR (500 MHz, CDCl3): δ 8.51 (1H, s), 7.68 (1H, m), 7.13-7.07 (2H, m), 2.73 (3H, s).
Step B: Into a solution of 3-acetyl-7-fluoro-2H-chromen-2-one (1.96 g, 9.5 mmol) in CHCl3 (20 mL) was added dropwise a solution of bromine (1.6 g, 10 mmol) in CHCl3 (10 mL). The mixture was stirred at room temperature for 1 h and filtered. The solid was washed with CHCl3 and dried to give the title compound (1.96 g, 72%) as a pale yellow solid. 1H NMR (500 MHz, CDCl3): δ 8.63 (1H, s), 7.72 (1H, m), 7.17-7.10 (2H, m), 4.73 (2H, s).
Step A: A mixture of 3-(2-bromoacetyl)-7-fluoro-2H-chromen-2-one (500 mg, 1.75 mmol), 3-fluoro-5-methylpyridin-2-amine (240 mg, 1.9 mmol) and EtOH (3 mL) was heated at 95° C. for 18 h. The reaction mixture was partitioned between CH2Cl2 and aqueous K2CO3. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The residue was triturated with 1:1 hexane/acetone, yielding 7-fluoro-3-(8-fluoro-6-methylimidazo[1,2-a]pyridin-2-yl)-2H-chromen-2-one (412 mg, 75%) as an orange solid. 1H NMR (500 MHz, CDCl3): δ 8.85 (1H, s), 8.50 (1H, d, J=2.5 Hz), 7.77 (1H, m), 7.63 (1H, dd, J=9 Hz, 6 Hz), 7.11 (1H, dd, J=9 Hz, 2 Hz), 7.07 (1H, td, J=8.5 Hz, 2.5 Hz), 6.80 (1H, d, J=6 Hz), 2.34 (3H, s).
Step B: A mixture of 7-fluoro-3-(8-fluoro-6-methylimidazo[1,2-a]pyridin-2-yl)-2H-chromen-2-one (120 mg, 0.38 mmol), (S)-2-methylpiperazine (75 mg, 0.75 mmol) and DMSO (900 μL) was heated at 80° C. for 15 h. The mixture was diluted with an aqueous saturated NaHCO3 solution, causing the product to precipitate from solution. The mixture was filtered. The solid material was purified by silica gel column chromatography (10% MeOH in CH2Cl2), yielding the title compound (133 mg, 89%) as a yellow solid: m.p. 250-255° C.; MS m/z 393.3 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.73 (1H, s), 8.52 (1H, d, J=3 Hz), 8.31 (1H, s), 7.72 (1H, d, J=8.5 Hz), 7.09 (1H, d, J=12 Hz), 7.02 (1H, dd, J=9 Hz, 2 Hz), 6.88 (1H, d, J=2 Hz), 3.81 (2H, m), 2.96 (1H, m), 2.73 (3H, m), 2.39 (1H, t, J=11 Hz), 2.31 (1H, br s), 2.28 (3H, s), 1.04 (3H, d, J=6 Hz).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 36 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 4-bromo-2-hydroxybenzaldehyde (5.0 g, 24.8 mmol), piperidine (150 μL, 1.5 mmol), ethyl acetoacetate (3.15 mL, 25 mmol) and CH3CN (2.0 mL) was heated at 80° C. for 1 h. The reaction mixture was partitioned between CH2Cl2 and aqueous HCl (1 M). The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The residue was triturated with MeOH, yielding 3-acetyl-7-bromo-2H-chromen-2-one (5.45 g, 82%) as a yellow solid. 1H NMR (500 MHz, CDCl3): δ 8.46 (1H, s), 7.56 (1H, d, J=1.5 Hz), 7.51 (1H, d, J=8 Hz), 7.48 (1H, dd, J=8 Hz, 1.5 Hz), 2.72 (3H, s).
Step B: A solution of Br2 (1.1 mL, 21.4 mmol) in CHCl3 (25 mL) was added dropwise to a solution of 3-acetyl-7-bromo-2H-chromen-2-one (5.4 g, 20.2 mmol) in CHCl3 (90 mL) over a period of 90 min. The mixture was filtered. The solid material was washed with CHCl3, yielding 7-bromo-3-(2-bromoacetyl)-2H-chromen-2-one (5.6 g, 80%) as a light pink solid. 1H NMR (500 MHz, CDCl3): δ 8.58 (1H, s), 7.60 (1H, d, J=1.5 Hz), 7.55 (1H, d, J=8.5 Hz), 7.52 (1H, dd, J=8.5 Hz, 1.5 Hz), 4.72 (2H, s).
Step C: A mixture of 7-bromo-3-(2-bromoacetyl)-2H-chromen-2-one (100 mg, 0.29 mmol), 3,5-difluoropyridin-2-amine (48 mg, 0.37 mmol) and CHCl3 (500 μL) was heated at 80° C. for 25 h. The reaction mixture was partitioned between CH2Cl2 and an aqueous saturated NaHCO3 solution. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The residue was purified by silica gel column chromatography (CH2Cl2), yielding 7-bromo-3-(8-fluoro-6-methylimidazo[1,2-a]pyridin-2-yl)-2H-chromen-2-one (96 mg, 88%) as a white solid. 1H NMR (500 MHz, CDCl3): δ 8.88 (1H, s), 8.64 (1H, d, J=3 Hz), 8.00 (1H, m), 7.59 (1H, d, J=1.5 Hz), 7.53 (1H, d, J=8.5 Hz), 7.47 (1H, dd, J=8.5 Hz, 1.5 Hz), 6.99 (1H, m).
Step D: A mixture of 7-bromo-3-(8-fluoro-6-methylimidazo[1,2-a]pyridin-2-yl)-2H-chromen-2-one (40 mg, 0.11 mmol), (2-biphenyl)-di-t-butylphosphine (6 mg, 0.02 mmol), Pd2(dba)3 (6 mg, 0.0066 mmol), Cs2CO3 (55 mg, 0.17 mmol), 1-methylhomopiperazine (24 μL, 0.18 mmol), and 1,2-dimethoxyethane (450 μL) was heated at 80° C. for 90 min. The reaction mixture was then diluted in CH2Cl2 and filtered. The filtrate was concentrated. The residue was purified by silica gel column chromatography (5-10% MeOH in CH2Cl2), followed by trituration with 3:1 hexane/CH2Cl2, yielding the title compound (24 mg, 53%) as a yellow solid: m.p. 255-260° C.; MS m/z 411.2 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.79 (1H, m), 8.73 (1H, s), 8.63 (1H, d, J=3 Hz), 7.70 (1H, d, J=9 Hz), 7.55 (1H, m), 6.84 (1H, dd, J=9 Hz, 2.5 Hz), 6.67 (1H, d, J=2 Hz), 3.65 (2H, t, J=5 Hz), 3.57 (2H, t, J=5.5 Hz), 2.64 (2H, t, J=5 Hz), 2.46 (2H, t, J=5.5 Hz), 2.27 (3H, s), 1.92 (2H, pentet, J=5.5 Hz).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 37 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 3-(2-bromoacetyl)-7-fluoro-2H-chromen-2-one (0.285 g, 1.0 mmol, prepared in Example 36, Part 2) and 2-aminopyrimidine (0.19 g, 2.0 mmol) in EtOH (2.0 mL) was stirred at 95° C. overnight. The mixture was diluted with water and filtered. The solid was washed with water and dried to afford 7-fluoro-3-(imidazo[1,2-a]pyrimidin-2-yl)-2H-chromen-2-one hydrobromide (0.28 g, 78%) as a pale yellow solid. MS m/z 282.1 [M+H]+.
Step B: A mixture of 7-fluoro-3-(imidazo[1,2-a]pyrimidin-2-yl)-2H-chromen-2-one hydrobromide (50 mg, 0.18 mmol) and piperazine (61 mg, 0.71 mmol) in DMSO (0.5 mL) was stirred at 110° C. for 2 h. The mixture was diluted with an aqueous saturated NaHCO3 solution and filtered. The solid was washed with water and dried to afford the title compound (40 mg, 64%) as a yellow solid: m.p. 286° C. (decomp.); MS m/z 348.2 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.78 (1H, dd, J=6.8 Hz, 2.2 Hz), 8.55 (1H, s), 8.46 (1H, dd, J=4.2 Hz, 2.2 Hz), 8.40 (1H, s), 7.52 (1H, d, J=8.8 Hz), 6.95-6.91 (2H, m), 6.76 (1H, d, J=2.2 Hz), 3.33-3.28 (4H, m), 2.91-2.86 (4H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 38 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 2-amino-5-bromopyrimidine (2.75 g, 15.8 mmol) and di-tert-butyl dicarbonate (7.58 g, 34.8 mmol) in pyridine (30 mL) was stirred at 70° C. overnight, then the solvent was removed. The residue was partitioned between EtOAc and aqueous HCl (1 N). The aqueous layer was extracted with EtOAc. The combined organics were dried over NaSO4, then filtered and concentrated to give 2-[bis(tert-butoxycarbonyl)amino]-5-bromopyrimidine (5.5 g, 93%) as a white solid. MS m/z 398.2 [M+Na]+.
Step B: A mixture of 2-[bis(tert-butoxycarbonyl)amino]-5-bromopyrimidine (3.0 g, 8.0 mmol), dimethylzinc (1.2 M×8.0 mL, 9.6 mmol) and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (130 mg, 0.16 mmol) in 1,4-dioxane (30 mL) was stirred at 110° C. for 16 h under Argon. The mixture was cooled to room temperature, diluted with ethyl acetate and washed with saturated NH4Cl, water and brine. The organic layer was dried over NaSO4, concentrated and purified by silica gel column chromatography (0-35% EtOAc in hexanes) to give a white solid, which was dissolved in trifluoroacetic acid (5.0 mL). After 5 min, the solvent was removed and the residue was partitioned between ethyl acetate and an aqueous saturated NaHCO3 solution. The organic layer was dried over NaSO4, filtered and concentrated to give the title compound (0.7 g, 80%) as a white solid. MS m/z 110.1 [M+H]+.
Step A: Following the procedure in Example 36, Part 3, 3-(2-bromoacetyl)-7-fluoro-2H-chromen-2-one (0.855 g, 3.0 mmol) and 5-methylpyrimidin-2-amine (0.327 g, 3.0 mmol) in EtOH (6.0 mL) gave 7-fluoro-3-(6-methylimidazo[1,2-a]pyrimidin-2-yl)-2H-chromen-2-one hydrobromide (0.37 g, 42%) as a pale yellow solid. MS m/z 296.2 [M+H]+.
Step B: Following the procedure in Example 36, Part 3, 7-fluoro-3-(6-methylimidazo[1,2-a]pyrimidin-2-yl)-2H-chromen-2-one hydrobromide (80 mg, 0.21 mmol) and N-methyl piperizine (93 mg, 1.08 mmol) in DMSO (0.5 mL) gave the title compound (66 mg, 84%) as a yellow solid: m.p.>300° C.; MS m/z 362.2 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.83 (1H, dd, J=2.4 Hz, 1.1 Hz), 8.75 (1H, s), 8.44 (1H, d, J=2.2 Hz), 8.39 (1H, s), 7.71 (1H, d, J=8.8 Hz), 7.02 (1H, dd, J=8.8 Hz, 2.2 Hz), 6.86 (1H, d, J=2.2 Hz), 3.32-3.28 (4H, m), 2.86-2.75 (4H, m), 2.30 (3H, s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 39 by substituting the appropriate starting materials, reagents and reaction conditions.
2-Chloro-5-fluoropyrimidine (1.34 g, 10 mmol) was stirred with ammonium hydroxide (30%, 15 mL) at 100° C. in a sealed tube overnight. The mixture was cooled to room temperature and filtered. The solid was washed with water and dried to give the title compound (0.95 g, 80%) as a white solid.
Step A: Following the procedure in Example 36, Part 3, 3-(2-bromoacetyl)-7-fluoro-2H-chromen-2-one (1.32 g, 4.1 mmol) and 5-fluoropyrimidin-2-amine (0.465 g, 4.1 mmol) in EtOH (12.0 mL) gave 7-fluoro-3-(6-fluoroimidazo[1,2-a]pyrimidin-2-yl)-2H-chromen-2-one (0.85 g, 70%) as a pale yellow solid. MS m/z 300.1 [M+H]+.
Step B: Following the procedure in Example 36, Part 3, 7-fluoro-3-(6-fluoroimidazo[1,2-a]pyrimidin-2-yl)-2H-chromen-2-one (70 mg, 0.23 mmol) and N-methyl piperizine (46 mg, 0.46 mmol) in DMSO (0.5 mL) gave the title compound (55 mg, 63%) as a yellow solid: m.p. 275-280° C.; MS m/z 380.8 [M+H]+; 1H NMR (500 MHz, methanol-d4): δ 8.95 (1H, dd, J=3.8 Hz, 2.8 Hz), 8.65 (1H, s), 8.61 (1H, d, J=2.8 Hz), 8.53 (1H, s), 7.62 (1H, d, J=8.8 Hz), 7.02 (1H, dd, J=8.7 Hz, 2.4 Hz), 6.86 (1H, d, J=2.2 Hz), 3.51 (4H, br s), 2.81 (4H, br s), 2.50 (3H, br s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 40 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 3-(2-bromoacetyl)-7-fluoro-2H-chromen-2-one (2.85 g, 10 mmol, prepared in Example 36, Part 2) and 2-aminothiazole (1.0 g, 10 mmol) in EtOH (20 mL) was stirred at 95° C. for 6 h. After cooling to room temperature, ethyl acetate was added, causing a precipitate to form. The mixture was filtered. The solid was washed with ethyl acetate and dried, affording 6-(7-fluoro-2-oxo-2H-chromen-3-yl)imidazo[2,1-b]thiazole hydrobromide salt (1.82 g, 64%) as a tan solid. MS m/z 287.1 [M+H]+.
Step B: A mixture of 6-(7-fluoro-2-oxo-2H-chromen-3-yl)imidazo[2,1-b]thiazole hydrobromide salt (286 mg, 1.0 mmol) and 1-methylpiperazine (1.0 mL, 3.0 mmol) in DMSO (1.5 mL) was stirred at 110° C. for 2 h. The mixture was cooled to room temperature and diluted with water, producing a precipitate. The precipitate was collected by vacuum filtration, washed with water, dried and purified by silica gel column chromatography (0-10% MeOH in CH2Cl2) to give the title compound (185 mg, 51%) as a yellow solid: m.p. 256-258° C.; MS m/z 367.2 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.53 (1H, s), 8.31 (1H, s), 7.94 (1H, d, J=4.4 Hz), 7.64 (1H, d, J=8.8 Hz), 7.26 (1H, d, J=4.4 Hz), 7.02 (1H, dd, J=8.8 Hz, 2.5 Hz), 6.87 (1H, d, J=2.2 Hz), 3.45-3.23 (4H, m), 2.47-2.39 (4H, m), 2.22 (3H, s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 41 by substituting the appropriate starting materials, reagents and reaction conditions.
Into a mixture of butyraldehyde (10.8 g, 0.15 mol) and urea (22.8 g, 0.3 mol) in CHCl3 (75 mL) at 0° C. was added sulfuryl chloride (13.5 mL, 0.166 mol) dropwise. The mixture was warmed to room temperature and stirred for 1 h, then the solvent was removed. EtOH (200 mL) was added to the residue, then the mixture was heated at reflux overnight and the solvent was removed. The residue was suspended in water (200 mL) and collected by vacuum filtration to give the title compound (9.5 g, 40%) as a light brown solid. MS m/z 129.1 [M+H]+.
Step A: Following the procedure in Example 41, Step A, 3-(2-bromoacetyl)-7-fluoro-2H-chromen-2-one (0.76 g, 2.7 mmol) and 5-ethylthiazol-2-amine (0.35 g, 2.7 mmol) in EtOH (20 mL) gave 3-(2-ethylimidazo[2,1-b]thiazol-6-yl)-7-fluoro-2H-chromen-2-one hydrobromide (0.55 g, 66%) as a tan solid. MS m/z 315.2 [M+H]+.
Step B: Following the procedure in Example 41, Step B, 3-(2-ethylimidazo[2,1-b]thiazol-6-yl)-7-fluoro-2H-chromen-2-one hydrobromide (42 mg, 0.13 mmol) and 2,6-cis-dimethylpiperizine (30 mg, 0.26 mmol) in DMSO (0.25 mL) gave the title compound (3.8 mg, 7%) as a yellow solid: m.p. 251-253° C.; MS m/z 409.4 [M+H]+; 1H NMR (500 MHz, methanol-d4) δ 8.32 (1H, s), 8.21 (1H, s), 7.56-7.48 (2H, m), 7.00 (1H, dd, J=9.0 Hz, 2.4 Hz), 6.82 (1H, d, J=2.2 Hz), 3.82 (2H, dd, J=12.5 Hz, 2.4 Hz), 3.00-2.89 (2H, m), 2.82 (2H, qd, J=7.5, 1.4 Hz), 2.43 (2H, dd, J=12.5 Hz, 10.9 Hz), 1.34 (3H, t, J=7.4 Hz), 1.18 (6H, d, J=6.6 Hz).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 42 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 3-(2-bromoacetyl)-7-fluoro-2H-chromen-2-one (0.684 g, 2.4 mmol, prepared in Example 36, Part 2) and 3,5-dimethylpyrazin-2-amine (0.246 g, 2.0 mmol) in CH3CN (10 mL) was stirred at 120° C. in a sealed tube for 20 min. The mixture was cooled to room temperature and diluted with Et2O to produce a precipitate. The solid was collected by vacuum filtration, washed with Et2O and dried to give 3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-7-fluoro-2H-chromen-2-one hydrobromide (0.7 g, 90%) as a tan solid. MS m/z 310.1 [M+H]+.
Step B: 3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-7-fluoro-2H-chromen-2-one hydrobromide (100 mg, 0.25 mmol) was stirred with (R)-2-methylpiperazine (52 mg, 0.52 mmol) in DMSO (0.5 mL) with K2CO3 (0.14 g, 1.0 mmol) at 120° C. for 2 h. The mixture was cooled to room temperature and diluted with water to produce a precipitate. The solid was collected by vacuum filtration and purified by silica gel chromatography (10% MeOH in CH2Cl2) to give the title compound (64 mg, 64%) as a yellow solid. MS m/z 390.2 [M+H]+; 1H NMR (500 MHz, CDCl3): δ 8.74 (1H, s), 8.45 (1H, s), 7.77 (1H, s), 7.51 (1H, d, J=8.8 Hz), 6.88 (1H, dd, J=8.8 Hz, 2.5 Hz), 6.77 (1H, d, J=2.5 Hz), 3.77-3.67 (2H, m), 3.21-3.14 (2H, m), 3.06-2.92 (3H, m), 2.91 (3H, s), 2.64-2.56 (1H, m), 2.48 (3H, s), 1.20 (3H, d, J=6.3 Hz).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 43 by substituting the appropriate starting materials, reagents and reaction conditions.
Into a solution of 5-methylpyrazin-2-amine (0.51 g, 4.72 mmol) and ferrocene (0.263 g, 1.42 mmol) in DMSO (12 mL) was added sulfuric acid (12 mL) and a solution of CF3I in DMSO (2.4 M, 5.9 mL, 14.2 mmol). Aqueous hydrogen peroxide (30%, 0.94 mL) was added dropwise to the mixture. After stirring for 30 min at room temperature, excess reagent was quenched with ice water. The mixture was diluted with water and extracted with EtOAc. The organic layer was dried over NaSO4, filtered, concentrated and purified by silica gel column chromatography (0-20% EtOAc in CH2Cl2) to give the title compound (86 mg, 8%) as a white solid. MS m/z 219.1 [M+H]+.
Step A: Following the procedure in Example 43, Step A, 3-(2-bromoacetyl)-7-fluoro-2H-chromen-2-one (138 g, 0.49 mmol) and 5-methyl-3-(trifluoromethyl)pyrazin-2-amine (86 g, 0.49 mmol) in CH3CN (1.0 mL) gave 7-fluoro-3-(6-methyl-8-(trifluoromethyl)imidazo[1,2-a]pyrazin-2-yl)-2H-chromen-2-one hydrobromide (44 mg, 20%) as a tan solid.
Step B: Following the procedure in Example 43, Step B, 7-fluoro-3-(6-methyl-8-(trifluoromethyl)imidazo[1,2-a]pyrazin-2-yl)-2H-chromen-2-one hydrobromide (44 mg, 0.10 mmol) and 1,4-diazepane (44 mg, 0.44 mmol) in DMSO (0.25 mL) gave the title compound (43 mg, 99%) as a yellow solid: m.p.>300° C.; MS m/z 444.2 [M+H]+; 1H NMR (500 MHz, CDCl3): δ 8.79 (1H, s), 8.59 (1H, s), 8.09 (1H, s), 7.50 (1H, d, J=9.1 Hz), 6.69 (1H, dd, J=8.8 Hz, 2.2 Hz), 6.57 (1H, d, J=1.9 Hz), 3.70-3.61 (4H, m), 3.10-3.01 (2H, m), 2.88-2.83 (2H, m), 2.55 (3H, s), 2.03-1.90 (2H, m).
A mixture of 3-methylpyrazin-2-amine (109 mg, 1.0 mmol) and N-chlorosuccinimide (136 mg, 1.0 mmol) in CH2Cl2 (6.0 mL) was stirred at room temperature overnight. The mixture was washed with aqueous K2CO3 (2.0 M, 6.0 mL). The organic layer was dried over NaSO4, filtered, concentrated and purified by silica gel column chromatography (0-35% EtOAc in hexanes) to give the title compound (136 mg, 80%) as a white solid. MS m/z 144.0 [M+H]+.
Step A: Following the procedure in Example 43, Step A, 3-(2-bromoacetyl)-7-fluoro-2H-chromen-2-one (0.233 g, 0.81 mmol) and 5-chloro-3-methylpyrazin-2-amine (0.117 g, 0.81 mmol) in CH3CN (3.0 mL) gave 3-(6-chloro-8-methylimidazo[1,2-a]pyrazin-2-yl)-7-fluoro-2H-chromen-2-one hydrobromide (0.18 g, 67%) as a tan solid. MS m/z 330.1 [M+H]+.
Step B: Following the procedure in Example 43, Step B, 3-(6-chloro-8-methylimidazo[1,2-a]pyrazin-2-yl)-7-fluoro-2H-chromen-2-one hydrobromide (67 mg, 0.2 mmol), N-methyl homopiperizine (28 mg, 0.24 mmol) and triethylamine (100 mg, 1.0 mmol) in DMSO (0.5 mL) gave the title compound (70 mg, 83%) as a yellow solid: m.p. 212-218° C.; MS m/z 424 [M+H]+; 1H NMR (500 MHz, methanol-d4) δ 8.71 (1H, s), 8.55 (1H, s), 8.42 (1H, d, J=0.9 Hz), 7.56 (1H, d, J=8.8 Hz), 6.84 (1H, dd, J=8.8 Hz, 2.5 Hz), 6.67 (1H, d, J=2.2 Hz), 3.86-3.77 (2H, m), 3.65 (2H, t, J=6.3 Hz), 3.16 (2H, d, J=1.6 Hz), 3.04 (2H, br s), 2.89-2.82 (3H, m), 2.69 (3H, s), 2.27-2.14 (2H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 45 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 3-chloropyrazin-2-amine (1.29 g, 10 mmol) and sodium methanethiolate (1.05 g, 15 mmol) in DMF (10 mL) and EtOH (10 mL) was stirred at 85° C. for 2 h, and then concentrated. The mixture was diluted with water and filtered. The filtrate was extracted with ethyl acetate. The organic layer was washed with water, dried over Na2SO4, filtered and concentrated. The residue was combined with the material collected from filtration, affording the desired product 3-(methylthio)pyrazin-2-amine (1.33 g, 94%) as a white solid. MS m/z 142.1 [M+H]+.
Step B: A mixture of 3-(2-bromoacetyl)-7-fluoro-2H-chromen-2-one (2.85 g, 10 mmol, prepared in Example 36, Part 2) and 3-(methylthio)pyrazin-2-amine (1.5 g, 10 mmol) in CH3CN (40 mL) was stirred at 110° C. overnight. The mixture was cooled to room temperature and diluted with ethyl acetate to generate a precipitate. The solid was collected by vacuum filtration, washed with ethyl acetate and dried, yielding 7-fluoro-3-(8-(methylthio)imidazo[1,2-a]pyrazin-2-yl)-2H-chromen-2-one hydrobromide salt (2.15 g, 66%) as a tan solid. MS m/z 328.1 [M+H]+.
Step C: A mixture of 7-fluoro-3-(8-(methylthio)imidazo[1,2-a]pyrazin-2-yl)-2H-chromen-2-one hydrobromide (100 mg, 0.24 mmol) and piperazine (60 mg, 0.6 mmol) in DMSO (0.5 mL) was stirred at 120° C. for 5 h. The mixture was cooled to room temperature and diluted with water to produce a precipitate. The solid was collected by vacuum filtration, washed with water, dried and purified with silica gel column chromatography (5-10% MeOH in CH2Cl2) to give the title compound (52 mg, 55%) as a yellow solid. MS m/z 394.3 [M+H]+; 1H NMR (500 MHz, CDCl3): δ 8.81 (1H, s), 8.50 (1H, s), 7.81 (1H, d, J=4.4 Hz), 7.70 (1H, d, J=4.7 Hz), 7.52 (1H, d, J=8.8 Hz), 6.88 (1H, dd, J=8.8 Hz, 2.5 Hz), 6.77 (1H, d, J=2.2 Hz), 3.50 (1H, s), 3.38-3.30 (4H, m), 3.09-3.01 (4H, m), 2.71 (3H, s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 46 by substituting the appropriate starting materials, reagents and reaction conditions.
Into a solution of 7-(4-methylpiperazin-1-yl)-3-(7-(methylthio)imidazo[1,2-c]pyrimidin-2-yl)-2H-chromen-2-one (30 mg, 0.076 mmol) in dimethylacetamide (2.0 mL) at 88° C. was added a large excess of Raney Nickle. The mixture was stirred until gas evolution ceased (˜10 min). The mixture was diluted with MeOH and filtered through Celite. The filtrate was concentrated under a stream of nitrogen. The residue was purified with silica gel column chromatography (5-10% MeOH in CH2Cl2) to give the title compound (32 mg, 55%) as a yellow solid: m.p. 258-260° C.; MS m/z 348.2 [M+H]+; 1H NMR (500 MHz, CDCl3): δ 9.07 (1H, s), 8.75 (1H, s), 8.60 (1H, d, J=0.6 Hz), 8.09 (1H, dd, J=4.6 Hz, 1.4 Hz), 7.88 (1H, d, J=4.4 Hz), 7.50 (1H, d, J=8.8 Hz), 6.88 (1H, dd, J=8.8 Hz, 2.2 Hz), 6.78 (1H, d, J=2.5 Hz), 3.36 (4H, dd, J=6.1 Hz, 4.3 Hz), 3.05 (4H, dd, J=6.1 Hz, 4.3 Hz)
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 47 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 7-bromo-3-(2-bromoacetyl)-2H-chromen-2-one (2.0 g, 5.78 mmol, prepared in Example 37, Step B), 2-amino-3,5-dimethylpyrazine (825 mg, 6.71 mmol) and CH3CN (22 mL) was heated at 90° C. for 4 h. The addition of an aqueous saturated NaHCO3 solution to the mixture resulted in the formation of a precipitate. The precipitate was collected by vacuum filtration and triturated with 1:1 hexane/acetone, yielding 7-bromo-3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2H-chromen-2-one (1.9 g, 88%) as an orange solid. 1H NMR (500 MHz, DMSO-d6): δ 8.81 (1H, s), 8.61 (1H, s), 8.31 (1H, s), 7.93 (1H, d, J=8 Hz), 7.76 (1H, d, J=1.5 Hz), 7.58 (1H, dd, J=8 Hz, 1.5 Hz), 2.76 (3H, s), 2.37 (3H, s).
Step B: A mixture of 7-bromo-3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2H-chromen-2-one (150 mg, 0.40 mmol), (2-biphenyl)-di-t-butylphosphine (10 mg, 0.033 mmol), Pd2(dba)3 (10 mg, 0.011 mmol), Cs2CO3 (170 mg, 0.52 mmol), (S)-1-Boc-3-aminopyrrolidine (105 μL, 0.60 mmol) and 1,2-dimethoxyethane (1.4 mL) was heated at 80° C. for 4 h. The reaction mixture was diluted with CH2Cl2 and filtered. The filtrate was concentrated. The residue was purified by silica gel column chromatography (30-50% acetone in CH2Cl2), followed by trituration with 1:1 hexane/acetone, yielding (S)-tert-butyl 3-(3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-ylamino)pyrrolidine-1-carboxylate (109 mg, 57%) as a yellow solid. MS m/z 476.3 [M+H]+.
Step C: A mixture of (S)-tert-butyl 3-(3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-ylamino)pyrrolidine-1-carboxylate (105 mg, 0.22 mmol) was stirred in a solution of trifluoroacetic acid (1.0 mL) in CH2Cl2 (4.0 mL) for 15 min. The reaction mixture was poured into dilute aqueous NaOH. The mixture was extracted with CH2Cl2 (EtOH added to improve the solubility). The organic layer was collected and concentrated under reduced pressure, yielding the title compound (70 mg, 85%) as a yellow solid: m.p. 132° C. (decomp.); MS m/z 376.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.70 (1H, s), 8.50 (1H, s), 8.32 (1H, s), 7.68 (1H, d, J=9 Hz), 7.06 (1H, d, J=6.5 Hz), 6.68 (1H, dd, J=9 Hz, 2 Hz), 6.55 (1H, d, J=2 Hz), 4.12 (1H, m), 3.37 (1H, dd, J=12 Hz, 6 Hz), 3.18 (1H, m), 3.11 (1H, m), 2.94 (1H, dd, J=12 Hz, 4 Hz), 2.76 (3H, s), 2.37 (3H, s), 2.22 (1H, m), 1.82 (1H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 48 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of (S)-tert-butyl 3-(3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-ylamino)pyrrolidine-1-carboxylate (255 mg, 0.54 mmol, prepared in Example 48, Step B), NaH (60% mineral oil suspension, 32 mg, 0.80 mmol) and DMF (2.5 mL) were stirred at room temperature for 10 min. Iodomethane (34 μL, 0.54 mmol) was added to the mixture. After stirring the mixture for 10 min, additional iodomethane (17 μL, 0.27 mmol) was added. After stirring an additional 10 min, water was slowly added to the reaction mixture to quench any remaining NaH. The addition of H2O (15 mL) to the reaction mixture caused a precipitate to form. The precipitate was collected by vacuum filtration and purified by silica gel column chromatography (20-30% acetone in CH2Cl2), followed by ether trituration, yielding (S)-tert-butyl 3-((3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-yl)(methyl)amino)pyrrolidine-1-carboxylate (84 mg, 32%) as a yellow solid. MS m/z 490.2 [M+H]+.
Step B: A mixture of (S)-tert-butyl 3-((3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-yl)(methyl)amino)pyrrolidine-1-carboxylate (80 mg, 0.16 mmol) was stirred in a solution of trifluoroacetic acid (1.0 mL) in CH2Cl2 (4.0 mL) for 2 h. The reaction mixture was poured into dilute aqueous NaOH. The mixture was extracted with a mixture of CH2Cl2 and EtOH. The organic layer was collected and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (10% 9:1 MeOH:NH4OH in CH2Cl2), yielding the title compound (42 mg, 67%) as a yellow solid: m.p. 192-202° C.; MS m/z 390.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.70 (1H, s), 8.49 (1H, s), 8.31 (1H, s), 7.71 (1H, d, J=9 Hz), 6.91 (1H, dd, J=9 Hz, 2.5 Hz), 6.72 (1H, d, J=2 Hz), 4.53 (1H, m), 3.04 (1H, dd, J=11 Hz, 8 Hz), 2.96 (1H, m), 2.93 (3H, s), 2.77 (2H, m), 2.75 (3H, s), 2.37 (3H, s), 2.06 (1H, m), 1.66 (1H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 49 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 7-bromo-3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2H-chromen-2-one (1.1 g, 2.97 mmol, prepared in Example 48, Step A), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate (1.12 g, 3.62 mmol), K2CO3 (1.24 g, 9.0 mmol), [1,1′-bis(diphenylphosphino)-ferrocene]dichloropalladium(II) complex with dichloromethane (200 mg, 0.24 mmol) and CH3CN (8 mL) was heated at 80° C. for 4 h. The reaction mixture was partitioned between CH2Cl2 and H2O. The organic layer was concentrated under vacuum. The residue was purified by silica gel column chromatography (30-50% acetone in CH2Cl2), followed by trituation with acetone, yielding tert-butyl 4-(3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-yl)-5,6-dihydropyridine-1(2H)-carboxylate (1.17 g, 83%) as a light yellow solid. 1H NMR (500 MHz, DMSO-d6): δ 8.85 (1H, s), 8.62 (1H, s), 8.34 (1H, s), 7.94 (1H, d, J=8.5 Hz), 7.52 (1H, d, J=8.5 Hz), 7.50 (1H, s), 6.47 (1H, br s), 4.07 (2H, br s), 3.58 (2H, t, J=5 Hz), 2.77 (3H, s), 2.53 (2H, br s), 2.38 (3H, s), 1.45 (9H, s).
Step B: A solution of tert-butyl 4-(3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-yl)-5,6-dihydropyridine-1(2H)-carboxylate (300 mg, 0.63 mmol) in trifluoroacetic acid (1.0 mL) and CH2Cl2 (4 mL) was stirred at room temperature for 30 min. The reaction mixture was poured into dilute aqueous NaOH. The mixture was extracted with CH2Cl2 (EtOH added to improve the solubility). The organic layer was collected and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (30% MeOH in CH2Cl2, followed by 10-20% 9:1 MeOH:NH4OH in CH2Cl2) yielding the title compound (183 mg, 77%) as a light tan solid: m.p. 205-211° C.; MS m/z 373.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.83 (1H, s), 8.61 (1H, s), 8.33 (1H, s), 7.91 (1H, d, J=8.5 Hz), 7.50 (1H, dd, J=8 Hz, 1.5 Hz), 7.45 (1H, s), 6.52 (1H, m), 3.42 (2H, m), 2.94 (2H, t, J=6 Hz), 2.77 (3H, s), 2.40 (2H, m), 2.37 (3H, s), 2.28 (1H, br s).
A solution of 3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-7-(1,2,3,6-tetrahydropyridin-4-yl)-2H-chromen-2-one (133 mg, 0.36 mmol, prepared in Example 50) in 1,2-dichloroethane:MeOH (20 mL, 1:1) was stirred in the presence of 10% palladium on carbon (Pd/C, 107 mg) under hydrogen (40 psi). After 4 h, the reaction mixture was filtered through Celite. The filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography (10% 9:1 MeOH:NH4OH in CH2Cl2), followed by trituration with 1:1 hexanes/CH2Cl2, yielding the title compound (76 mg, 56%) as an off-white solid: m.p. 224-229° C.; MS m/z 375.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.84 (1H, s), 8.61 (1H, s), 8.34 (1H, s), 7.90 (1H, d, J=8 Hz), 7.30 (2H, m), 3.05 (2H, d, J=12 Hz), 2.77 (3H, s), 2.73 (1H, tt, J=12 Hz, 3.5 Hz), 2.59 (2H, td, J=12 Hz, 2.5 Hz), 2.38 (3H, s), 2.20 (1H, br s), 1.73 (2H, d, J=12 Hz), 1.54 (2H, qd, J=12 Hz, 3.5 Hz).
Step A: A mixture of 3-(2-bromoacetyl)-7-fluoro-2H-chromen-2-one (0.285 g, 1.0 mmol, prepared in Example 36, Part 2) and 6-chloropyridazin-3-amine (0.13 g, 1.0 mmol) in EtOH (2.0 mL) was stirred at 95° C. for 3 h. The mixture was cooled to room temperature and diluted with water to produce a precipitate. The solid was collected by vacuum filtration, washed with water and dried to give 3-(6-chloroimidazo[1,2-b]pyridazin-2-yl)-7-fluoro-2H-chromen-2-one hydrobromide (0.26 g, 82%) as a tan solid. MS m/z 316.1 [M+H]+.
Step B: A mixture of 3-(6-chloroimidazo[1,2-b]pyridazin-2-yl)-7-fluoro-2H-chromen-2-one hydrobromide (95 mg, 0.3 mmol), 1-methylpiperazine (75 mg, 0.75 mmol) in DMSO (0.5 mL) was stirred at 95° C. for 2 h. The mixture was cooled to room temperature and diluted with water to produce a precipitate. The solid was collected by vacuum filtration, washed with water, dried and purified with silica gel column chromatography (5-10% MeOH in CH2Cl2) to give 3-(6-chloroimidazo[1,2-b]pyridazin-2-yl)-7-(4-methylpiperazin-1-yl)-2H-chromen-2-one (56 mg, 50%) as a yellow solid. MS m/z 396.2 [M+H]+.
Step C: A suspension of 3-(6-chloroimidazo[1,2-b]pyridazin-2-yl)-7-(4-methylpiperazin-1-yl)-2H-chromen-2-one (50 mg, 0.13 mmol) in a mixed solvent of CH2Cl2 (2.0 mL) and MeOH (5.0 mL) was stirred with 10% Pd/C (20 mg) under hydrogen (1 atm) for 4 h. The mixture was filtered through Celite. The filtrate was concentrated. The residue was suspended in water, collected by vacuum filtration and dried to give the title compound (30 mg, 66%) as a yellow solid: m.p. 284-285° C.; MS m/z 362.3 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.78 (1H, s), 8.63 (1H, s), 8.52 (1H, dd, J=4.4 Hz, 1.6 Hz), 8.11 (1H, d, J=9.5 Hz), 7.74 (1H, d, J=8.2 Hz), 7.28 (1H, dd, J=9.5 Hz, 4.4 Hz), 7.06 (1H, d, J=9.2 Hz), 6.95 (1H, s), 3.53-3.32 (4H, m), 2.48 (3H, s), 2.38-2.15 (4H, m).
Step A: A mixture of 3-chloro-6-methylpyridazine (516 mg, 4.0 mmol), NH4OH (30%, 3 mL) and copper(II) sulfate pentahydrate (26 mg, 0.2 mmol) was stirred at 120° C. for 40 h. The mixture was cooled to room temperature and partitioned between EtOAc and brine. The aqueous layer was extracted with EtOAc five times. The combined organics were dried over NaSO4, filtered, concentrated and purified by silica gel column chromatography (0-10% MeOH in CH2Cl2) to give 6-methylpyridazin-3-amine (160 mg, 37%) as a white solid. MS m/z 109.9 [M+H]+.
Step B: A mixture of 3-(2-bromoacetyl)-7-fluoro-2H-chromen-2-one (900 mg, 3.0 mmol, prepared in Example 36, Part 2) and 6-methylpyridazin-3-amine (330 mg, 3.0 mmol) was stirred in CH3CN (6.0 mL) at 100° C. for 5 h, then the solvent was removed. The residue was purified by silica gel column chromatography (5% MeOH in CH2Cl2) to give 7-fluoro-3-(6-methylimidazo[1,2-b]pyridazin-2-yl)-2H-chromen-2-one (814 mg, 92%) as a tan solid. MS m/z 296.0 [M+H]+.
Step C: A mixture of 7-fluoro-3-(6-methylimidazo[1,2-b]pyridazin-2-yl)-2H-chromen-2-one (100 mg, 0.34 mmol) and cis-2,6-dimethylpiperazine (155 mg, 1.36 mmol) in DMSO (0.5 mL) was stirred at 100° C. overnight. The mixture was diluted with water to produce a precipitate. The solid was collected by vacuum filtration, dried under vacuum and purified by silica gel column chromatography (5% MeOH in CH2Cl2) to give the title compound (75 mg, 58%) as a yellow solid: m.p. 225-227° C.; MS m/z 390.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.64 (1H, s), 8.52 (1H, s), 7.66 (1H, d, J=9.4 Hz), 7.35 (1H, d, J=8.8 Hz), 6.81 (1H, d, J=9.4 Hz), 6.73 (1H, dd, J=8.8 Hz, 2.2 Hz), 6.66 (1H, d, J=2.2 Hz), 3.68-3.56 (2H, m), 3.12-2.96 (2H, m), 2.71-2.54 (2H, m), 2.46 (3H, s), 1.30-1.10 (6H, br s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 53 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: Following the procedure in Example 46, Step A, 6-chloropyrimidin-4-amine (1.3 g, 10 mmol) and sodium methanethiolate (1.05 g, 15 mmol) in DMF (10 mL) afforded 6-(methylthio)pyrimidin-4-amine (1.06 g, 75%). MS m/z 142.1 [M+H]+.
Step B: A mixture of 3-(2-bromoacetyl)-7-fluoro-2H-chromen-2-one (2.5 g, 8.86 mmol, prepared in Example 36, Part 2) and 6-(methylthio)pyrimidin-4-amine (1.0 g, 7.1 mmol) in CH3CN (35 mL) was stirred at 120° C. overnight. The mixture was cooled to room temperature and diluted with EtOAc to produce a precipitate. The solid was collected by vacuum filtration, washed with EtOAc and dried under vacuum to afford 7-fluoro-3-(7-(methylthio)imidazo[1,2-c]pyrimidin-2-yl)-2H-chromen-2-one hydrobromide salt (2.8 g, 97%) as a tan solid. MS m/z 328.1 [M+H]+.
Step C: A mixture of 7-fluoro-3-(7-(methylthio)imidazo[1,2-c]pyrimidin-2-yl)-2H-chromen-2-one hydrobromide (100 mg, 0.24 mmol) and 1-methylpiperazine (60 mg, 0.6 mmol) in DMSO (0.5 mL) was stirred at 120° C. for 5 h. After cooling to room temperature, the mixture was diluted with water to produce a precipitate. The solid was collected by vacuum filtration, dried under vacuum and purified by silica gel column chromatography (5% MeOH in CH2Cl2) to give 7-(4-methylpiperazin-1-yl)-3-(7-(methylthio)imidazo[1,2-c]pyrimidin-2-yl)-2H-chromen-2-one (68 mg, 69%) as a yellow solid. MS m/z 408.2 [M+H]+.
Step D: Following the procedure in Example 47, 7-(4-methylpiperazin-1-yl)-3-(7-(methylthio)imidazo[1,2-c]pyrimidin-2-yl)-2H-chromen-2-one (66 mg, 0.16 mmol) and excess Raney Ni in dimethylacetamide (2.0 mL) yielded the title compound (32 mg, 55%) as a yellow solid: m.p. 253-255° C.; MS m/z 362.3 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 9.03 (1H, s), 8.71 (1H, s), 8.56 (1H, s), 7.95 (1H, d, J=6.3 Hz), 7.54-7.44 (2H, m), 6.88 (1H, dd, J=8.8 Hz, 2.2 Hz), 6.79 (1H, d, J=2.2 Hz), 3.48 (4H, br s), 2.67 (4H, br s), 2.44 (3H, br s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 54 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 3-acetyl-7-fluoro-2H-chromen-2-one (600 mg, 2.0 mmol, prepared in Example 36, Part 2) and 5-methyl-1,3,4-thiadiazol-2-amine (241 mg, 2.0 mmol) in EtOH (6.0 mL) was stirred at 95° C. overnight in a sealed tube. The mixture was cooled to room temperature and diluted with an aqueous saturated NaHCO3 solution to produce a precipitate. The solid was collected by vacuum filtration, washed with water and dried under vacuum to give 7-fluoro-3-(2-methylimidazo[2,1-b][1,3,4]thiadiazol-6-yl)-2H-chromen-2-one (1.2 g, 62%) as a tan solid. MS m/z 303.2 [M+H]+.
Step B: A mixture of 7-fluoro-3-(2-methylimidazo[2,1-b][1,3,4]thiadiazol-6-yl)-2H-chromen-2-one (76 mg, 0.25 mmol), piperazine (64 mg, 0.75 mmol), K2CO3 (104 mg, 0.75 mmol) in DMSO (0.5 mL) was stirred at 120° C. overnight. After cooling to room temperature, the mixture was diluted with water to produce a precipitate. The solid was collected by vacuum filtration, dried under vacuum and purified by silica gel column chromatography (10% MeOH in CH2Cl2) to afford the title compound (23 mg, 25%) as a yellow solid: m.p. 288-290° C.; MS m/z 368.2 [M+H]+; 1H NMR (500 MHz, MeOD-d4): δ 8.43 (1H, s), 8.40 (1H, s), 7.54 (1H, d, J=8.8 Hz), 7.01 (1H, dd, J=8.8 Hz, 2.5 Hz), 6.84 (1H, d, J=2.2 Hz), 3.39-3.35 (4H, m), 3.00-2.95 (4H, m), 2.74 (3H, s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 55 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 2,4-difluoro-6-hydroxybenzaldehyde (8 g, 50.6 mmol), ethyl acetoacetate (6.4 mL, 50.7 mmol) and piperidine (240 μL, 2.43 mmol) was heated at 50° C. for 15 h. The reaction mixture was cooled to room temperature, and then suspended in ether. The mixture was filtered. The solid material was purified by silica gel column chromatography (50-70% CH2Cl2 in hexanes), yielding 3-acetyl-5,7-difluoro-2H-chromen-2-one (4.45 g, 39%) as an off-white solid. 1H NMR (500 MHz, CDCl3): δ 8.69 (1H, s), 6.93 (1H, dt, J=9 Hz, 2 Hz), 6.84 (1H, td, J=9 Hz, 2 Hz), 2.72 (3H, s).
Step B: A mixture of 3-acetyl-5,7-difluoro-2H-chromen-2-one (4.25 g, 19.0 mmol), tetrabutylammonium tribromide (9.85 g, 20.4 mmol) and THF (77 mL) was stirred at room temperature for 3 h, then the solvent was removed under vacuum. The residue was triturated with 1:1 hexane/CH2Cl2, yielding the title compound (3.31 g, 57%) as an off-white solid: MS m/z [303.0, 305.0][M+H]+; 1H NMR (500 MHz, CDCl3): δ 8.80 (1H, s), 6.96 (1H, m), 6.87 (1H, td, J=8.5 Hz, 2.5 Hz), 4.69 (2H, s).
Step A: A mixture of 3-(2-bromoacetyl)-5,7-difluoro-2H-chromen-2-one (160 mg, 0.53 mmol), 4-trifluoromethylpyridin-2-amine (100 mg, 0.62 mmol), and EtOH (1 mL) was heated at 80° C. for 1 h. The reaction mixture was partitioned between CH2Cl2 and an aqueous saturated NaHCO3 solution. The organic layer was dried over MgSO4, filtered, and was concentrated under vacuum. The residue was purified by silica gel column chromatography (CH2Cl2), followed by trituration with 2:1 hexane/acetone, yielding 5,7-difluoro-3-(7-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)-2H-chromen-2-one (92 mg, 47%) as a white solid. 1H NMR (500 MHz, CDCl3): δ 8.98 (1H, s), 8.64 (1H, s), 8.27 (1H, d, J=7.5 Hz), 7.95 (1H, s), 7.01 (1H, dd, J=7 Hz, 1.5 Hz), 6.96 (1H, d, J=9 Hz), 6.86 (1H, td, J=9 Hz, 2.5 Hz).
Step B: A mixture of 5,7-difluoro-3-(7-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)-2H-chromen-2-one (60 mg, 0.16 mmol), cis-2,6-dimethylpiperazine (27 mg, 0.24 mmol) and DMSO (300 μL) was heated at 80° C. for 15 h. The addition of an aqueous saturated NaHCO3 solution resulted in the formation of a precipitate. The precipitate was collected by vacuum filtration and purified by silica gel column chromatography (5% MeOH in CH2Cl2), yielding the title compound (58 mg, 79%) as a yellow solid: m.p. 210-219° C.; MS m/z 461.3 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.85 (1H, d, J=7 Hz), 8.72 (1H, s), 8.65 (1H, s), 8.08 (1H, s), 7.20 (1H, dd, J=7 Hz, 2 Hz), 6.96 (1H, dd, J=9 Hz, 2 Hz), 6.78 (1H, d, J=1.5 Hz), 3.88 (2H, d, J=12 Hz), 2.76 (2H, m), 2.37 (2H, t, J=11 Hz), 2.31 (1H, br s), 1.04 (6H, d, J=6.5 Hz).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 56 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 3-(2-bromoacetyl)-7-fluoro-2H-chromen-2-one (570 mg, 2 mmol, prepared in Example 36, Part 2) and 2-methylpyridine (186 mg, 2 mmol) in anhydrous acetone (2 mL) was heated to 50° C. for 16 h in a sealed tube. After cooling to room temperature, the reaction mixture was filtered. The collected material was washed with a small amount of CH3CN to provide 1-(2-(7-fluoro-2-oxo-2H-chromen-3-yl)-2-oxoethyl)-2-methyl-pyridinium bromide (590 mg, 78%) as a light brown solid. MS m/z 298.1 [M+H]+.
Step B: 1-(2-(7-Fluoro-2-oxo-2H-chromen-3-yl)-2-oxoethyl)-2-methyl-pyridinium bromide (590 mg, 1.6 mmol) was suspended in CH3CN (10 mL) and triethylamine (2.5 mL). The mixture was heated at reflux for 2 h. After cooling to room temperature, the mixture was filtered. The collected material was washed with CH3CN to provide 7-fluoro-3-(indolizin-2-yl)-2H-chromen-2-one (360 mg, 83%) as a yellow solid. MS m/z 280.2 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.54 (1H, s), 8.33 (1H, dd, J=7.0 Hz, 1 Hz), 8.29 (1H, d, J=1 Hz), 7.85 (1H, m), 7.45-7.42 (2H, m), 7.30 (1H, td, J=8.5 Hz, 2.5 Hz), 6.94 (1H, s), 6.74 (1H, m), 6.55 (1H, td, J=7.0 Hz, 1.5 Hz).
Step C: A mixture of 7-fluoro-3-(indolizin-2-yl)-2H-chromen-2-one (60 mg, 0.21 mmol) and piperazine (36 mg, 0.42 mmol) in anhydrous DMSO (0.3 mL) was heated to 60° C. for 16 h in a sealed tube. The mixture was purified by column chromatography on basic alumina (0-10% MeOH in CH2Cl2) to provide the title compound (39 mg, 52%) as a brown solid: m.p. 186-188° C.; MS m/z 346.4 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.35 (1H, s), 8.29 (1H, dd, J=7.0 Hz, 1 Hz), 8.22 (1H, d, J=2.5 Hz), 7.55 (1H, d, J=8.5 Hz), 7.38 (1H, d, J=8.5 Hz), 7.0 (1H, dd, J=8.5 Hz, 2.5 Hz), 6.88 (1H, s), 6.84 (1H, d, J=2.5 Hz), 6.70 (1H, m), 6.52 (1H, m), 3.29-3.27 (4H, m), 2.85-2.84 (4H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 57 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 3-(2-bromoacetyl)-7-fluoro-2H-chromen-2-one (1.0 g, 3.5 mmol) and 2,5-dimethylpyrazine (750 mg, 7.0 mmol, prepared in Example 36, Part 2) in anhydrous CH3CN (4 mL) was heated to 60° C. for 3 d in a sealed tube. After cooling to room temperature, the reaction mixture was filtered. The collected material was washed with a small amount of CH3CN to provide crude 1-(2-(7-fluoro-2-oxo-2H-chromen-3-yl)-2-oxoethyl)-2,5-dimethylpyrazin-1-ium bromide (1.8 g) as a light brown solid. MS m/z 313.3 [M+H]+.
Step B: The crude intermediate from Step A was suspended in CH3CN (20 mL) and triethylamine (5 mL). The mixture was heated to reflux for 2 h, then cooled and filtered. The collected material was washed with a small amount of CH3CN to provide 7-fluoro-3-(indolizin-2-yl)-2H-chromen-2-one (850 mg, 83% over 2 steps) as an orange solid. MS m/z 295.3 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.82 (1H, s), 8.62 (1H, s), 8.34 (1H, s), 8.16 (1H, s), 7.86 (1H, m), 7.47 (1H, d, J=2.5 Hz), 7.34-7.31 (2H, m), 2.37 (3H, s).
Step C: Following the procedure in Example 57, Step C, 7-fluoro-3-(3-methylpyrrolo[1,2-a]pyrazin-7-yl)-2H-chromen-2-one (50 mg, 0.17 mmol) and piperazine (30 mg, 0.34 mmol) in DMSO (0.5 mL) yielded the title compound (21 mg, 34%) as a yellow solid: m.p. 220° C. (dec.); MS m/z 361.4 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.77 (1H, s), 8.45 (1H, s), 8.29 (1H, s), 8.14 (1H, s), 7.58 (1H, d, J=8.5 Hz), 7.28 (1H, s), 7.04 (1H, dd, J=9 Hz, 2.5 Hz), 6.90 (1H, d, J=2.5 Hz), 3.40-3.36 (4H, m), 2.99-2.97 (4H, m), 2.38 (3H, s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 58 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 4-chloro-2-methylpyrimidine (512 mg, 4 mmol) and sodium methanethiolate (280 mg, 4 mmol) in anhydrous CH3CN (4 mL) was heated to 60° C. for 16 h. After cooling to room temperature, the mixture was filtered. The collected material was washed with CH2Cl2. The combined filtrate was concentrated to provide crude 2-methyl-4-(methylthio)pyrimidine, which was used in the following step without further purification.
Step B: A mixture of the crude product from Step A and 3-(2-bromoacetyl)-7-fluoro-2H-chromen-2-one (855 mg, 3.0 mmol, prepared in Example 36, Part 2) in anhydrous CH3CN (4 mL) was heated to 60° C. for 3 days in a sealed tube. The mixture was cooled to room temperature and filtered. The collected material was washed with a small amount of CH3CN to provide crude 1-(2-(7-fluoro-2-oxo-2H-chromen-3-yl)-2-oxoethyl)-2-methyl-4-(methylthio)pyrimidin-1-ium bromide (822 mg) as a light brown solid, which was used in the following step without further purification.
Step C: The crude product from Step B was suspended in CH3CN (10 mL) and triethylamine (2.5 mL). The mixture was heated to reflux for 2 h, then cooled and filtered. The collected material was washed with CH3CN, affording 7-fluoro-3-(2-(methylthio)pyrrolo[1,2-a]pyrimidin-7-yl)-2H-chromen-2-one (1.0 g, 77% over three steps) as a brown solid. MS m/z 327.1 [M+H]+.
Step D: To a solution of 7-fluoro-3-(2-(methylthio)pyrrolo[1,2-a]pyrimidin-7-yl)-2H-chromen-2-one (100 mg, 0.31 mmol) in dimethylacetamide (3 mL) was carefully added Raney Ni (slurry in H2O, ˜50 mg) at 60° C. After 15 min, additional Raney Ni was added in small portions until UPLC/MS indicated complete conversion. After cooling to room temperature, the reaction mixture was filtered through Celite. The filter cake was washed with CH2Cl2. The filtrate was concentrated, providing 7-fluoro-3-(pyrrolo[1,2-a]pyrimidin-7-yl)-2H-chromen-2-one (60 mg, 69% yield) as a brown gummy oil. MS m/z 281.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.75 (1H, m), 8.65 (1H, s), 8.26 (1H, d, J=1 Hz), 8.13 (1H, m), 7.86 (1H, m), 7.45 (1H, dd, J=9.5 Hz, 2.5 Hz), 7.31 (1H, td, J=8.5 Hz, 2.5 Hz), 7.07 (1H, s), 6.70 (1H, m).
Step E: Following the procedure in Example 57, Step C, 7-fluoro-3-(pyrrolo[1,2-a]pyrimidin-7-yl)-2H-chromen-2-one (60 mg, 0.21 mmol) and piperazine (36 mg, 0.42 mmol) in DMSO (0.5 mL) yielded the title compound (28 mg, 38%) as a yellow solid: m.p. 235-238° C.; MS m/z 347.2 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.70 (1H, dd, J=2 Hz, 1 Hz), 8.47 (1H, s), 8.20 (1H, d, J=1.5 Hz), 8.09 (1H, dd, J=3.5 Hz, 1.5 Hz), 7.57 (1H, d, J=8.5 Hz), 7.04-7.01 (2H, m), 6.86 (1H, d, J=2.5 Hz), 6.67-6.65 (1H, m), 3.31-3.29 (4H, m), 2.87-2.85 (4H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 59 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of hexane-2,5-dione (6 mL, 51 mmol) and hydrazine monohydrate (2.5 mL, 51 mmol) in ethanol (50 mL) was brought to reflux for 3 h, then the solvent was removed under reduced pressure. The residue was combined with 10% Pd/C (1.1 g) in anhydrous benzene (200 mL). The reaction mixture was heated at reflux overnight, then cooled to room temperature and filtered through a pad of Celite. The filtrate was concentrated and purified by silica gel column chromatography (6% MeOH in CH2Cl2) to provide 3,6-dimethylpyridazine (3.1 g, 56%) as a light brown oil. 1H NMR (500 MHz, CDCl3): δ 7.23 (2H, s), 2.69 (6H, s).
Step B: A mixture of 3,6-dimethylpyridazine (81 mg, 0.75 mmol) and 3-(2-bromoacetyl)-7-fluoro-2H-chromen-2-one (143 mg, 0.5 mmol, prepared in Example 36, Part 2) in anhydrous CH3CN (1 mL) was stirred at room temperature for 5 d in a sealed tube to afford 1-(2-(7-fluoro-2-oxo-2H-chromen-3-yl)-2-oxoethyl)-3,6-dimethylpyridazin-1-ium bromide as a crude mixture in CH3CN.
Step C: The crude reaction mixture from Step B was diluted with anhydrous CH3CN (2 mL) and triethylamine (1 mL). The mixture was heated at reflux for 2 h, then cooled and filtered. The collected material was washed with CH3CN, affording 7-fluoro-3-(2-methylpyrrolo[1,2-b]pyridazin-6-yl)-2H-chromen-2-one (103 mg, 70%) as a brown solid. MS m/z 295.0 [M+H]+.
Step D: Following the procedure in Example 57, Step C, 7-Fluoro-3-(2-methylpyrrolo[1,2-b]pyridazin-6-yl)-2H-chromen-2-one (40 mg, 0.13 mmol) and piperazine (22 mg, 0.26 mmol) in DMSO (0.5 mL) yielded the title compound (24 mg, 48%) as a light brown solid: m.p. 213-216° C.; MS m/z 361.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.23 (1H, s), 8.15 (1H, d, J=1.5 Hz), 7.68 (1H, d, J=9 Hz), 7.39 (1H, d, J=9 Hz), 6.87-6.83 (2H, m), 6.74 (1H, d, J=2 Hz), 6.43 (1H, d, J=9 Hz), 3.27-3.22 (4H, m), 2.84-2.83 (4, m), 2.33 (3H, s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 60 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: To an oven dry round bottom flask were added thieno-5-pyridine (0.6 g, 4.4 mmol), triisopropyl borate (1.38 mL, 6.8 mmol) and THF (16 mL). The mixture was cooled to −78° C. under nitrogen before the addition of LDA (3.6 mL, 1.5 M, 5.2 mmol) with stirring. After 1 h the reaction mixture was poured onto ice water (20 mL) and acidified to pH 2 with 2 N HCl. The precipitate was collected by filtration, washed with water and dried to provide thieno-5-pyridine-2-boronic acid (0.7 g, 88%). MS m/z 135.0 [M+H]+.
Step B: A mixture of thieno-5-pyridine-2-boronic acid (0.59 g, 3.29 mmol), 3-bromo-7-fluoro-2H-chromen-2-one (1.0 g, 4.12 mmol, prepared in Example 32, Step A), tris(dibenzylideneacetone)dipalladium(0) (0.19 g, 0.21 mmol), tri-tert-butylphosphonium tetrafluoroborate (0.14 g, 0.49 mmol) and potassium fluoride (1.21 g, 20.57 mmol) in THF (10 mL) was stirred at 50° C. under Argon for 15 h. The mixture was then diluted with 10% MeOH in CH2Cl2 (100 mL) and filtered through Celite. The filtrate was concentrated. The residue was washed with CH2Cl2 and dried to give 7-fluoro-3-(thieno[3,2-c]pyridin-2-yl)-2H-chromen-2-one (0.43 g, 43%). MS m/z 298.0 [M+H]+.
Step C: A mixture of 7-fluoro-3-(thieno[3,2-c]pyridin-2-yl)-2H-chromen-2-one (75 mg, 0.25 mmol), (2S,6R)-2,6-dimethylpiperazine (57 mg, 0.50 mmol) and DMSO (1.0 mL) was stirred at 90° C. overnight. The mixture was cooled to room temperature and diluted with water (10 mL) to produce a precipitate. The precipitate was collected by filtration, washed with water and ethyl ether, and then dried to give the title compound (20 mg, 21%): m.p. 163-165° C.; MS 392.3 m/z [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 9.11 (1H, d, J=1.0 Hz), 8.60 (1H, s), 8.39 (1H, d, J=5.4 Hz), 8.19 (1H, s), 8.03 (1H, d, J=5.7 Hz), 7.62 (1H, d, J=8.8 Hz), 7.07 (1H, dd, J=8.8 Hz, 2.2 Hz), 6.91 (1H, d, J=2.5 Hz), 3.93-3.85 (2H, m), 2.84-2.74 (2H, m), 2.41-2.33 (2H, m), 1.04 (6H, d, J=6.3 Hz).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 61 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: 2,6-Dimethylpyrimidin-4-ol (5.0 g, 40 mmol) was dissolved in aqueous NaOH solution (50 mL, 1 M, 50 mmol). To the solution was added iodine (10.2 g, 40 mmol). The mixture was gradually heated to 80° C. and stirred for 2 h. After cooling the mixture to room temperature, acetic acid was added to adjust the pH ˜6. A precipitate formed and was collected by filtration. The solid was washed with water and dried to give 5-iodo-2,6-dimethylpyrimidin-4-ol (6.51 g, 65%). MS m/z 251.2 [M+H]+.
Step B: 5-Iodo-2,6-dimethylpyrimidin-4-ol (4.6 g, 18.4 mmol) was combined with phosphorus oxychloride (15 mL). The mixture was stirred at 110° C. for 2 h, then the solvent was removed under vacuum. The residue was dissolved in CH2Cl2 and washed with an aqueous saturated NaHCO3 solution and brine. The organic layer was concentrated and purified by silica gel column chromatography (0-10% EtOAc in CH2Cl2) to give the title compound (3.86 g, 78%) as colorless oil that solidified on standing. MS m/z 269.2 [M+H]+.
Step A: A mixture of 3-bromo-7-fluorocoumarin (1.82 g, 7.5 mmol, prepared in Example 38, Step A), ethynyltrimethylsilane (0.88 g, 9.0 mmol), copper(I) iodide (0.071 g, 0.38 mmol), bis(triphenylphosphine)palladium(II) dichloride (0.26 g, 0.38 mmol), triethylamine (1.52 g, 15.0 mmol) and CH3CN (15 mL) was stirred under an Argon atmosphere at room temperature for 12 h, then the solvent was removed. The residue was purified by silica gel column chromatography (0-50% EtOAc in hexanes) to give 7-fluoro-3-((trimethylsilyl)ethynyl)-2H-chromen-2-one as white solid, used directly for the next step. MS m/z 261.2 [M+H]+.
Step B: The intermediate obtained in Step A was dissolved in MeOH (50 mL) and cooled in an ice-water bath. K2CO3 (1.55 g, 11.25 mmol) was added and the mixture was stirred at 0° C. for 1.5 h. Saturated aqueous NH4Cl (200 mL) was added to produce a precipitate. The precipitate was collected, washed with water, dried and purified by silica gel column chromatography (0-10% EtOAc in CH2Cl2) to give 3-ethynyl-7-fluoro-2H-chromen-2-one (1.14 g, 81% two steps) as white needles. MS m/z 189.2 [M+H]+.
Step C: A mixture of 3-ethynyl-7-fluoro-2H-chromen-2-one (376 mg, 2.0 mmol), 4-chloro-5-iodo-2,6-dimethylpyrimidine (590 mg, 2.2 mmol), copper(I) iodide (19 mg, 0.1 mmol), bis(triphenylphosphine)palladium(II) dichloride (70 mg, 0.1 mmol), triethylamine (404 mg, 4.0 mmol) and CH3CN (4.0 mL) was stirred at 50° C. under an Argon atmosphere overnight, then the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (5% MeOH in CH2Cl2) to give 3-((4-chloro-2,6-dimethylpyrimidin-5-yl)ethynyl)-7-fluoro-2H-chromen-2-one (92 mg, 14%). MS m/z 329.3 [M+H]+.
Step D: 3-((4-Chloro-2,6-dimethylpyrimidin-5-yl)ethynyl)-7-fluoro-2H-chromen-2-one (92 mg, 0.28 mmol) was stirred with sodium hydrosulfide (47 mg, 0.84 mmol) in EtOH (2.0 mL) at 80° C. for 1 h. The mixture was cooled to room temperature and diluted with water (8 mL) to produce a precipitate. The precipitate was collected, washed with water and dried to give 3-(2,4-dimethylthieno[2,3-d]pyrimidin-6-yl)-7-fluoro-2H-chromen-2-one as white powder (66 mg, 73%). MS m/z 327.3 [M+H]+.
Step E: 3-(2,4-Dimethylthieno[2,3-d]pyrimidin-6-yl)-7-fluoro-2H-chromen-2-one (66 mg, 0.2 mmol), piperazine (43 mg, 0.5 mmol) in DMSO (0.5 mL) was stirred at 80° C. for 6 h. The mixture was cooled to room temperature and diluted with water (6 mL) to produce a precipitate. The precipitate was collected, washed with water and dried to give the title compound (75 mg, 96%) as yellow powder: m.p. 275° C. (decomp.); MS m/z 393.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.65 (1H, s), 8.15 (1H, s), 7.60 (1H, d, J=8.83 Hz), 7.05 (1H, dd, J=9.0 Hz, 2.4 Hz), 6.89 (1H, d, J=2.5 Hz), 3.38-3.33 (4H, m), 2.84-2.79 (4H, m), 2.74 (3H, s), 2.66 (3H, s).
Step A: 6-Methylpyridin-3-ol (0.5 g, 4.58 mmol) was dissolved in aqueous NaOH (4.5 mL, 1 M, 4.5 mmol). To the solution was added iodine (1.28 g, 5.2 mmol) and the mixture was stirred at room temperature overnight. The mixture was neutralized with aqueous HCl (2 M) to pH ˜7. A white precipitate formed and was collected by filtration. The solid was washed with water and dried to give 2-iodo-6-methylpyridin-3-ol (0.8 g, 50%). MS m/z 236.0 [M+H]+.
Step B: A mixture of 2-iodo-6-methylpyridin-3-ol (325 mg, 1.38 mmol) and 3-ethynyl-7-fluoro-2H-chromen-2-one (200 mg, 1.06 mmol, prepared in Example 62, Part 2), bis(triphenylphosphine)palladium(II) dichloride (37 mg, 0.05 mmol), copper(I) iodide (10 mg, 0.05 mmol), triethylamine (0.25 mL, 2.12 mmol) in CH3CN (3.0 mL) was stirred under an Argon atmosphere at 40° C. for 4 h. The mixture was diluted with water (50 mL) to produce a precipitate. The precipitate was collected, washed with ethyl ether and dried to give 3-(5-methylfuro[3,2-b]pyridin-2-yl)-7-(piperazin-1-yl)-2H-chromen-2-one (142 mg, 48%). MS m/z 297.2 [M+H]+.
Step C: A mixture of 3-(5-Methylfuro[3,2-b]pyridin-2-yl)-7-(piperazin-1-yl)-2H-chromen-2-one (100 mg, 0.33 mmol), 1-methylpiperazine (67 mg, 0.67 mmol) in DMSO (1 mL) was stirred at 90° C. for 2 h. The mixture was cooled to room temperature and diluted with water (10 mL) to produce a precipitate. The precipitate was collected by filtration, washed with water and dried to give the title compound (99 mg, 80%) as a yellow solid: m.p. 178-180° C.; MS 376.0 m/z [M+H]+; 1H NMR (500 MHz, CDCl3): δ 8.22 (1H, s), 7.64 (1H, d, J=0.95 Hz), 7.54 (1H, dd, J=8.5 Hz, 1.0 Hz), 7.39 (1H, d, J=8.8 Hz), 7.00 (1H, d, J=8.5 Hz), 6.80 (1H, dd, J=8.8 Hz, 2.5 Hz), 6.68 (1H, d, J=2.2 Hz), 3.38-3.33 (4H, m), 2.59 (3H, s), 2.54-2.47 (4H, m), 2.30 (3H, s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 63 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: 2,6-Dimethylpyridin-3-ol (1.0 g, 8.1 mmol) was dissolved in aqueous NaOH (4.05 mL, 2 M, 8.1 mmol). To the solution was added iodine (2.62 g, 10.3 mmol) at room temperature. The mixture was stirred at 50° C. for 2 h, then neutralized (pH ˜7) with aqueous HCl (2 N). Excess reagent was quenched with sodium thiosulfate, then the solvent was removed under vacuum. The residue was suspended in 10% MeOH in CH2Cl2 (100 mL) and filtered. The filtrate was concentrated to give 4-iodo-2,6-dimethylpyridin-3-ol (0.96 g, 50%). MS m/z 250.0 [M+H]+.
Step B: A mixture of 4-iodo-2,6-dimethylpyridin-3-ol (343 mg, 1.38 mmol), 3-ethynyl-7-fluoro-2H-chromen-2-one (200 mg, 1.06 mmol, prepared in Example 62, Step B), bis(triphenylphosphine)palladium(II) dichloride (37 mg, 0.05 mmol), copper(I) iodide (10 mg, 0.05 mmol), triethylamine (0.25 mL, 2.12 mmol) in DMF (3.0 mL) was stirred under an Argon atmosphere at 40° C. overnight. The mixture was diluted with water (50 mL) to produce a precipitate. The precipitate was collected to give 3-(5,7-dimethylfuro[2,3-c]pyridin-2-yl)-7-fluoro-2H-chromen-2-one (185 mg, 60%). MS m/z 311.2 [M+H]+.
Step C: A mixture of 3-(5,7-dimethylfuro[2,3-c]pyridin-2-yl)-7-fluoro-2H-chromen-2-one (100 mg, 0.32 mmol), piperazine (58 mg, 0.67 mmol) in DMSO (1.0 mL) was stirred at 90° C. for 2 h. The mixture was cooled to room temperature and diluted with water (10 mL) to produce a precipitate. The precipitate was collected by filtration, washed with water and dried to give the title compound (89 mg, 74%) as yellow powder: m.p. 218-220° C.; MS 376.0 m/z [M+H]+; 1H NMR (500 MHz, CDCl3): δ 8.36 (1H, s), 7.56-7.49 (2H, m), 7.21 (1H, s), 6.92-6.87 (1H, m), 6.77-6.74 (1H, m), 3.39 (4H, m, J=10.4 Hz), 3.09-3.03 (4H, m), 2.79 (3H, s), 2.61 (3H, s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 64 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: Diisopropyl azodicarboxylate (5.5 mL, 28 mmol) was added dropwise to a mixture of 7-hydroxycoumarin (4.6 g, 28 mmol), (R)-tert-butyl 3-hydroxypyrrolidine-1-carboxylate (6.0 g, 31 mmol), triphenylphosphine (7.4 g, 28 mmol) and triethylamine (3.9 mL, 28 mmol) in THF (28 mL) at 0° C. The mixture was stirred at room temperature overnight. The solids were removed by filtration and washed with cold THF. The solid was dissolved in EtOAc. The solution was washed with aqueous HCl (0.5 N), dried over NaSO4, then filtered and concentrated to give (S)-tert-butyl 3-(2-oxo-2H-chromen-7-yloxy)pyrrolidine-1-carboxylate (4.85 g, 52%) as a pale yellow solid. MS m/z 232.2 [M-Boc+H]+.
Step B: Into a mixture of (S)-tert-butyl 3-(2-oxo-2H-chromen-7-yloxy)pyrrolidine-1-carboxylate (1.5 g, 3.6 mmol) and sodium acetate (1.0 g, 12.8 mmol) in acetic acid (11.0 mL) was added bromine (0.186 mL, 3.6 mmol) dropwise at room temperature. The mixture was stirred at room temperature overnight, then the solvent was removed and the residue was purified by silica gel column chromatography (0-60% EtOAc in hexanes) to afford (S)-tert-butyl 3-(3-bromo-2-oxo-2H-chromen-7-yloxy)pyrrolidine-1-carboxylate (2.9 g, 80%) as a pale yellow solid. MS m/z 312.1 [M-Boc+H]+.
Step C: A mixture of (S)-tert-butyl 3-(3-bromo-2-oxo-2H-chromen-7-yloxy)pyrrolidine-1-carboxylate (1.2 g, 2.9 mmol), tributyl(1-ethoxyvinyl)stannane (1.2 g, 3.2 mmol), copper(I) iodide (0.13 g, 0.7 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.34 g, 0.29 mmol) in 1,4-dioxane (30 mL) was stirred at 100° C. for 2 h under Argon. The mixture was partitioned between EtOAc and water. The organic layer was washed with brine, dried over NaSO4, filtered and concentrated. The residue was purified by silica gel column chromatography (0-35% EtOAc in CH2Cl2) to afford (S)-tert-butyl 3-(3-(1-ethoxyvinyl)-2-oxo-2H-chromen-7-yloxy)pyrrolidine-1-carboxylate (1.84 g, 65%) as a pale yellow solid. MS m/z 402.3 [M+H]+.
Step D: Into a solution of (S)-tert-butyl 3-(3-(1-ethoxyvinyl)-2-oxo-2H-chromen-7-yloxy)pyrrolidine-1-carboxylate (0.78 g, 1.84 mmol) in THF (10 mL) and water (1 mL) was added N-bromosuccinimide (0.344 g, 1.93 mmol) portionwise. The mixture was stirred at room temperature for 10 min then partitioned between EtOAc and an aqueous saturated NaHCO3 solution. The organic layer was washed with brine, dried over NaSO4, filtered and concentrated. The residue was purified by silica gel column chromatography (0-30% EtOAc in CH2Cl2) to afford (S)-tert-butyl 3-(3-(2-bromoacetyl)-2-oxo-2H-chromen-7-yloxy)pyrrolidine-1-carboxylate (0.77 g, 90%) as a pale yellow solid. MS m/z 454.1 [M+H]+.
Step E: A mixture of (S)-tert-butyl 3-(3-(2-bromoacetyl)-2-oxo-2H-chromen-7-yloxy)pyrrolidine-1-carboxylate (100 mg, 0.21 mmol) and 2-aminopyrimidine (20 mg, 0.21 mmol) in EtOH (0.6 mL) was stirred at 95° C. for 10 h in a sealed tube. The mixture was cooled to room temperature and diluted with an aqueous saturated NaHCO3 solution (2.0 mL) to produce a precipitate. The solid was collected by vacuum filtration, washed with water and dried to give (S)-tert-butyl 3-(3-(2-bromoacetyl)-2-oxo-2H-chromen-7-yloxy)pyrrolidine-1-carboxylate, which was dissolved in a solution of 4 N HCl in dioxane (1.0 mL, 4.0 mmol). The mixture was stirred for 1 h at room temperature, then the solvent was removed. The residue was suspended in acetone, collected by vacuum filtration and dried to give the title compound as the hydrochloride salt (61 mg, 70%) as an off white solid: m.p. 240-250° C.; MS m/z 349.2 [M+H]+; 1H NMR (500 MHz, MeOD-d4): δ 9.27 (1H, dd, J=1.6, 6.8 Hz), 9.07 (1H, dd, J=1.6, 4.4 Hz), 8.83 (1H, s), 8.64 (1H, s), 7.83 (1H, d, J=8.2 Hz), 7.67 (1H, dd, J=4.4, 7.0 Hz), 7.17-7.12 (2H, m), 5.45-5.40 (1H, m), 3.76-3.46 (4H, m), 2.49-2.35 (2H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 65 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 2,4-dihydroxybenzaldehyde (141 mg, 1.0 mmol), ethyl 2-(benzo[d]oxazol-2-yl)acetate (195 mg, 0.95 mmol, prepared according to Example 1, Part 1), piperidine (86 mg μL, 1.0 mmol) and acetic acid (305 mg, 5.0 mmol) in CH3CN (1.0 mL) was stirred at 120° C. overnight, then the solvent was removed. The residue was suspended in water, collected by vacuum filtration, washed with CH3CN and dried to yield 3-(benzo[d]oxazol-2-yl)-7-hydroxy-2H-chromen-2-one (211 mg, 76%) as a gray solid. MS m/z 280.1 [M+H]+.
Step B: Following the procedure in Example 65, Step A, 3-(benzo[d]oxazol-2-yl)-7-hydroxy-2H-chromen-2-one (280 mg, 1.0 mmol), tert-Butyl 4-hydroxypiperidine-1-carboxylate (227 mg, 1.1 mmol), diisopropyl azodicarboxylate (0.20 mL, 1.0 mmol), triphenylphosphine (0.27 g, 1.0 mmol), triethylamine (0.14 mL, 1.0 mmol) in THF (1.0 mL) yielded tert-Butyl 4-(3-(benzo[d]oxazol-2-yl)-2-oxo-2H-chromen-7-yloxy)piperidine-1-carboxylate (303 mg, 66%) as an off white solid. MS m/z 463.2 [M+H]+.
Step C: tert-Butyl 4-(3-(benzo[d]oxazol-2-yl)-2-oxo-2H-chromen-7-yloxy)piperidine-1-carboxylate was dissolved in CH2Cl2 (2.0 mL) and trifluoroacetic acid (1.0 mL). The mixture was stirred at room temperature for 0.5 h, then the solvent was removed. The residue was suspended in aqueous K2CO3 (2.0 M, 5.0 mL), collected by vacuum filtration, then washed with water, and dried to give the title compound (158 mg, 67%) as a gray solid: m.p. 198-201° C.; MS m/z 363.2 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.90 (1H, s), 7.81-7.76 (2H, m), 7.70 (1H, d, J=7.5 Hz), 7.49-7.39 (2H, m), 7.08-7.03 (2H, m), 4.77-4.69 (1H, m), 3.16-3.08 (2H, m), 2.86-2.78 (2H, m), 2.14-2.06 (2H, m), 1.81-1.69 (2H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 66 by substituting the appropriate starting materials, reagents and reaction conditions.
A mixture of (S)-3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-7-(3-methylpiperazin-1-yl)-2H-chromen-2-one (250 mg, 0.64 mmol, analogously prepared according to the procedure of Example 43), acetaldehyde (71 μL, 1.29 mmol), sodium triacetoxyborohydride (409 mg, 1.93 mmol) in CH2Cl2 (10% MeOH) (10 mL) was stirred at room temperature overnight. The reaction was quenched by the addition of an aqueous saturated NaHCO3 solution. The mixture was extracted with CH2Cl2 (10% MeOH). The organic layer was dried over NaSO4, filtered, concentrated and purified by silica gel column chromatography (10% MeOH in CH2Cl2) to give the title compound (192 mg, 72%) as a yellow solid: m.p. 208-209° C.; MS m/z 418.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.70 (1H, s), 8.50 (1H, s), 8.30 (1H, d, J=0.9 Hz), 7.73 (1H, d, J=8.8 Hz), 7.02 (1H, s), 6.88 (1H, d, J=1.9 Hz), 3.73 (2H, br. s.), 3.11-2.97 (1H, m), 2.91-2.82 (1H, m), 2.74 (5H, m), 2.48-2.40 (1H, m), 2.35-2.21 (5H, m), 1.06 (3H, d, J=6.3 Hz), 0.98 (3H, t)
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 67 by substituting the appropriate starting materials, reagents and reaction conditions.
A mixture of 3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-7-(piperidin-4-yl)-2H-chromen-2-one (50 mg, 0.13 mmol, prepared in Example 51), Cs2CO3 (150 mg, 0.46 mmol), 2-iodopropane (20 μL, 0.20 mmol) and DMF (300 μL) was heated at 60° C. for 3 h. The reaction mixture was diluted with H2O, causing a precipitate to form. The mixture was filtered. The solid material was purified by silica gel column chromatography (5% 10:1 MeOH:NH4OH in CH2Cl2), followed by trituration with 2:1 hexane:CH2Cl2, yielding the title compound as a tan solid: m.p. 206-211° C.; MS m/z 417.5 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.85 (1H, s), 8.62 (1H, s), 8.34 (1H, s), 7.90 (1H, d, J=8 Hz), 7.34 (1H, s), 7.31 (1H, dd, J=8 Hz, 1.5 Hz), 2.91 (2H, d, J=11 Hz), 2.77 (3H, s), 2.73 (1H, m), 2.62 (1H, m), 2.38 (3H, s), 2.24 (2H, t, J=11 Hz), 1.81 (2H, d, J=12 Hz), 1.67 (2H, qd, J=12 Hz, 3.5 Hz), 1.01 (6H, d, J=6.5 Hz).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 68 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 2-oxo-2H-chromen-7-yl trifluoromethanesulfonate (2.94 g, 10 mmol, prepared in Example 24, Step A), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate (3.7 g, 12 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (0.81 g, 1.0 mmol) and K2CO3 (4.14 g, 30 mmol) in CH3CN (40 mL) was stirred at 80° C. for 4 h. The mixture was partitioned in water and EtOAc. The organic layer was dried over Na2SO4, then concentrated and chromatographed on silica gel, eluting with 0-10% EtOAc in CH2Cl2 to give tert-butyl 4-(2-oxo-2H-chromen-7-yl)-5,6-dihydropyridine-1(2H)-carboxylate (3.4 g, 100%). MS m/z 328.2 [M+H]+.
Step B: A solution of tert-butyl 4-(2-oxo-2H-chromen-7-yl)-5,6-dihydropyridine-1(2H)-carboxylate (3.4 g, 10 mmol) in CH2Cl2 (50 mL) and EtOAc (100 mL) was stirred with 5% Pd/C (0.35 g) under H2 (1 atm) at room temperature for 3 h. The mixture was filtered. The filtrate was concentrated to give tert-butyl 4-(2-oxo-2H-chromen-7-yl)piperidine-1-carboxylate (3.4 g, 100%). MS m/z 330.2 [M+H]+.
Step C: Bromine (2.1 g, 13 mmol) was added dropwise at room temperature into a solution of tert-butyl 4-(2-oxo-2H-chromen-7-yl)piperidine-1-carboxylate (3.4 g, 10 mmol) and sodium acetate (2.46 g, 30 mmol) in acetic acid (15 mL). After stirring 5 h at room temperature, the mixture was diluted with water and filtered. The solid was dissolved in dichloromethane and washed with an aqueous saturated NaHCO3 solution. The organic layer was dried over Na2SO4, concentrated and chromatographed on silica gel, eluting with 0-10% EtOAc in CH2Cl2 to give tert-butyl 4-(3-bromo-2-oxo-2H-chromen-7-yl)piperidine-1-carboxylate (0.9 g, 22%).
Step D: A mixture of tert-butyl 4-(3-bromo-2-oxo-2H-chromen-7-yl)piperidine-1-carboxylate (0.5 g, 1.23 mmol), (2-methylimidazo[1,2-a]pyridin-6-yl)boronic acid (300 mg, 1.7 mmol, prepared in Example 34, Step A), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (136 mg, 0.166 mmol) and K2CO3 (2.5 mL of a 2.0 M aqueous solution, 5.0 mmol) in 1,4-dioxane (4 mL) was heated at 88° C. for 2 h. The mixture was partitioned in EtOAc and water. The organic layer was concentrated and chromatographed on silica gel, eluting with 20-100% EtOAc in CH2Cl2 to provide tert-butyl 4-(3-(2-methylimidazo[1,2-a]pyridin-6-yl)-2-oxo-2H-chromen-7-yl)piperidine-1-carboxylate (0.4 g, 70%). MS m/z 460.4 [M+H]+.
Step E: A solution of tert-butyl 4-(3-(2-methylimidazo[1,2-a]pyridin-6-yl)-2-oxo-2H-chromen-7-yl)piperidine-1-carboxylate (0.4 g, 0.87 mmol) in CH2Cl2 (2.0 mL) and TFA (2.0 mL) was stirred at room temperature for 15 min. The mixture was concentrated. The residue was partitioned in CH2Cl2 and aqueous K2CO3. The organic layer was concentrated, and then triturated with acetone to provide the title compound (0.25 g, 80%) as a gray powder. MS m/z 360.3 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 9.01 (1H, m), 8.37 (1H, s), 7.80 (1H, s), 7.71 (1H, d, J=7.9 Hz), 7.54 (2H, m), 7.31 (2H, m), 3.04 (2H, m), 2.74 (1H, m), 2.61 (2H, m), 2.34 (3H, d, J=0.6 Hz), 1.73 (2H, m), 1.57 (2H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 69 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: A mixture of 6-chloropyridazin-3-amine (2.6 g, 20 mmol) and 1-bromo-2,2-dimethoxypropane (4.0 g, 22 mmol) in CH3CN (20 mL) was stirred at 100° C. overnight. The mixture was concentrated and chromatographed on silica gel, eluting with 20-50% EtOAc in CH2Cl2 to give 6-chloro-2-methylimidazo[1,2-b]pyridazine (0.53 g, 16%). MS m/z 168.1 [M+H]+.
Step B: Lithium bis(trimethylsilyl)amide (8.25 mL, 1 M in toluene, 8.25 mmol) was added to a mixture of 6-chloro-2-methylimidazo[1,2-b]pyridazine (0.46 g, 2.75 mmol), t-butyl acetate (0.55 mL, 4.12 mmol) and chloro[2-(di-tert-butylphosphino)-2′,4′,6′-triisopropyl-1,1′-biphenyl][2-(2-aminoethyl)phenyl)]palladium(II) (47 mg, 0.069 mmol). The mixture was stirred for 5 min at room temperature, and then washed with water. The organic layer was concentrated and chromatographed on silica gel, eluting with 0-35% EtOAc in CH2Cl2 to give tert-butyl 2-(2-methylimidazo[1,2-b]pyridazin-6-yl)acetate (0.12 g, 18%).
Step C: A mixture of 4-bromo-2-hydroxybenzaldehyde (0.12 g, 0.6 mmol), tert-butyl 2-(2-methylimidazo[1,2-b]pyridazin-6-yl)acetate (0.12 g, 0.49 mmol), piperidine (0.145 mL, 1.47 mmol) and acetic acid (42 μL, 0.74 mmol) in ethanol (3.0 mL) was stirred at 120° C. overnight. The mixture was diluted with water and filtered, affording 7-bromo-3-(2-methylimidazo[1,2-b]pyridazin-6-yl)-2H-chromen-2-one.
Step D: 7-Bromo-3-(2-methylimidazo[1,2-b]pyridazin-6-yl)-2H-chromen-2-one was mixed with tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate (0.56 g, 0.6 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (40 mg, 0.049 mmol) and aqueous 2 M K2CO3 (1.0 mL, 2.0 mmol) in CH3CN (2.0 mL). The mixture was stirred at 80° C. for 3 h, then diluted into EtOAc and washed with water. The organic layer was chromatographed on silica gel, eluting with 0-50% EtOAc in CH2Cl2 to give tert-butyl 4-(3-(3-(1-(tert-butoxycarbonyl)-1,2,3,6-tetrahydropyridin-4-yl)-2-methylimidazo[1,2-b]pyridazin-6-yl)-2-oxo-2H-chromen-7-yl)-5,6-dihydropyridine-1(2H)-carboxylate (120 mg, 38%). MS m/z 640.6 [M+H]+.
Step E: The compound from Step D was dissolved in 4 N HCl in dioxane (2.0 mL). The mixture was stirred at room temperature for 15 min, then concentrated, treated with aqueous sodium bicarbonate, and filtered. The solid was washed with water and dried to afford the title compound (15 mg, 18%). MS m/z 440.5 [M+H]+; 1H NMR (500 MHz, DMSO-d6) δ 9.26 (1H, s), 8.08 (1H, s), 8.02 (1H, m), 7.81 (1H, m), 7.54 (1H, m), 7.48 (1H, m), 6.70 (1H, m), 6.55 (1H, m), 3.45 (4H, m), 2.90 (4H, m), 2.39 (3H, s), 2.36-2.18 (4H, m).
Step A: A mixture of 3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-7-iodo-2H-chromen-2-one (0.21 g, 0.5 mmol, prepared according to Example 48, Step A), (R)-tert-butyl 2-(hydroxymethyl)pyrrolidine-1-carboxylate (0.145 g, 0.72 mmol), CuI (9.5 mg, 0.05 mmol), 3,4,7,8-tetramethyl-1,10-phenanthroline (24 mg, 0.1 mmol) and Cs2CO3 (0.33 g, 1.0 mmol) in toluene (1.5 mL) and dioxane (1.0 mL) was stirred at 110° C. for 16 h. The mixture was concentrated and chromatographed on silica gel, eluting with 0-10% MeOH in CH2Cl2 to give a crude mixture containing (R)-tert-butyl 2-(((3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-yl)oxy)methyl)pyrrolidine-1-carboxylate (0.21 g). MS m/z 491.2 [M+H]+.
Step B: The mixture from Step A was treated with 4 N HCl in dioxane (2.0 mL). After 1 h, the mixture was concentrated, then dissolved in methanol (7N in NH3). The solvent was removed and the residue was chromatographed on silica gel, eluting with 0-15% MeOH in CH2Cl2 to give the title compound (25 mg, 13%) as a light yellow powder: m.p. 172-174° C.; MS m/z 391.3 [M+H]+; 1H NMR (500 MHz, CD3OD) δ 8.71 (1H, s), 8.46 (1H, s), 8.04 (1H, d, J=0.9 Hz), 7.61 (1H, d, J=8.8 Hz), 6.98 (1H, m), 6.90 (1H, d, J=2.2 Hz), 4.08 (1H, m), 3.99 (1H, m), 3.55 (1H, m), 2.98 (2H, m), 2.83 (3H, s), 2.42 (3H, d, J=0.9 Hz), 2.02 (1H, m), 1.96-1.76 (2H), 1.64 (1H, m).
Step A: Into a solution of 2-(4-bromo-2-(methoxymethoxy)phenyl)-1,3-dioxolane (1.45 g, 5.0 mmol) in THF (20 mL) at −78° C. was added BuLi (3.75 mL of a 1.6 M solution in hexane, 6.0 mmol). The mixture was stirred at −78° C. for 30 min, then benzyl 4-oxopiperidine-1-carboxylate (1.75 g, 7.5 mmol) was added to the mixture in one portion. The temperature of the mixture was allowed to rise to room temperature slowly. The mixture was stirred at room temperature for 2 h, then the reaction was quenched with saturated NH4Cl solution. The mixture was partitioned in EtOAc and water. The organic layer was washed with brine, dried over Na2SO4, then concentrated and chromatographed on silica gel, eluting with 0-65% EtOAc in CH2Cl2 to give benzyl 4-(4-(1,3-dioxolan-2-yl)-3-(methoxymethoxy)phenyl)-4-hydroxypiperidine-1-carboxylate (1.2 g, 54%).
Step B: Into a solution of benzyl 4-(4-(1,3-dioxolan-2-yl)-3-(methoxymethoxy)phenyl)-4-hydroxypiperidine-1-carboxylate (1.2 g, 2.7 mmol) in CH2Cl2 (6.0 mL) at −78° C. was added diethylaminosulfur trifluoride (0.49 mL, 3.0 mmol) dropwise. The temperature of the mixture was allowed to rise to room temperature. The mixture was stirred at room temperature for an additional hour before it was quenched by with saturated Na2CO3 solution. The organic layer was concentrated and chromatographed on silica gel, eluting with 0-20% EtOAc in CH2Cl2 to give benzyl 4-fluoro-4-(4-formyl-3-(methoxymethoxy)phenyl)piperidine-1-carboxylate (0.83 g, 76%). 1H NMR (500 MHz, acetone-d6) δ 10.47 (1H, d, J=0.6 Hz), 7.78 (1H, dd, J=8.2, 0.6 Hz), 7.30-7.44 (6H, m), 7.18-7.22 (1H, m), 5.43 (2H, s), 5.15 (2H, s), 4.20 (2H, m), 3.52 (3H, s), 3.33-3.02 (2H, m), 2.22-2.06 (2H, m), 1.95 (2H, m).
Step C: Into a mixture of benzyl 4-fluoro-4-(4-formyl-3-(methoxymethoxy)phenyl)piperidine-1-carboxylate (0.83 g, 2.1 mmol) in ethanol (2.0 mL) and water (2.0 mL) was added concentrated HCl (12 N, 2.0 mL, 24 mmol). The mixture was stirred at 50° C. for 3 h. The mixture was partitioned in EtOAc and water. The organic layer was washed with brine, dried over Na2SO4, and concentrated to give benzyl 4-fluoro-4-(4-formyl-3-hydroxyphenyl)piperidine-1-carboxylate as a pure product (0.75 g, 100%). MS m/z 356.2 [M−H]−.
Step D: A mixture of benzyl 4-fluoro-4-(4-formyl-3-hydroxyphenyl)piperidine-1-carboxylate (0.75 g, 2.1 mmol), ethyl 2-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)acetate (0.54 g, 2.3 mmol), piperidine (1.5 μL, 0.042 mmol), acetic acid (0.5 mL) in ethanol (2.0 mL) was stirred at 120° C. for 2 h. The mixture was concentrated and chromatographed on silica gel, eluting with 30-100% EtOAc in CH2Cl2 to give benzyl 4-(3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-yl)-4-fluoropiperidine-1-carboxylate (0.35 g, 47%). MS m/z 527.3 [M+H]+.
Step E: A mixture of benzyl 4-(3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-yl)-4-fluoropiperidine-1-carboxylate (0.17 g, 0.32 mmol) and 10% Pd/C (17 mg) in methanol and dichloromethane (3:1, 10 mL) was stirred overnight under H2 (1 atm). The mixture was filtered through Celite, concentrated and chromatographed on silica gel, eluting with 10% MeOH in CH2Cl2 to give 3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-7-(4-fluoropiperidin-4-yl)-2H-chromen-2-one (75 mg, 60%). MS m/z 393.3 [M+H]+.
Step F: 3-(6,8-Dimethylimidazo[1,2-a]pyrazin-2-yl)-7-(4-fluoropiperidin-4-yl)-2H-chromen-2-one (75 mg, 60%) was dissolved in CH3OH:CH2Cl2 10:1 (2.0 mL). Acetaldehyde (0.1 mL of a 6.5 M solution in isopropanol, 0.65 mmol) was added to the solution, followed by solid sodium triacetoxyborohydride (85 mg, 0.4 mmol). The mixture was stirred at room temperature for 30 min, then quenched with aqueous K2CO3. The organic layer was concentrated and chromatographed on silica gel, eluting with 0-10% MeOH in CH2Cl2 to give the title compound (34 mg, 40%) as a gray solid: m.p. 162-164° C.; MS m/z 421.3 [M+H]+; 1H NMR (500 MHz, CD3OD) δ 8.79 (1H, s), 8.53 (1H, s), 8.09 (1H, d, J=0.6 Hz), 7.76 (1H, m), 7.42 (2H, m), 3.03 (2H, m), 2.84 (3H, s), 2.63 (2H, d, J=7.3 Hz), 2.54 (2H, br s), 2.43 (3H, d, J=0.9 Hz), 2.23 (2H, m), 2.05 (2H, br s), 1.20 (3H, t, J=7.3 Hz).
Step A: Into a stirred solution of 1H-pyrazole (6.95 g, 0.1 mmol) and sodium hydroxide (16 g, 0.4 mol) in water (400 mL0 was added bromine (48 g, 0.3 mol) dropwise over 1 h. The mixture was stirred for 1 h, and then filtered. The cake was washed with water and dried to give 3,4,5-tribromo-1H-pyrazole (25.2 g, 81%). MS m/z 302.9 [M+H]+.
Step B: Into a mixture of 3,4,5-tribromo-1H-pyrazole (1.54 g, 5.1 mmol) and K2CO3 (2.1 g, 15.3 mmol) in acetone (20 mL) was added chloroacetone (0.52 g, 5.6 mmol) dropwise. The mixture was stirred at room temperature for 2 h, then partitioned between water and CH2Cl2. The organic layer was dried over Na2SO4 and concentrated to give 1-(3,4,5-tribromo-1H-pyrazol-1-yl)propan-2-one (1.80 g, 99%). MS m/z 361.0 [M+H]+.
Step C: A mixture of 1-(3,4,5-tribromo-1H-pyrazol-1-yl)propan-2-one (3.6 g, 10.0 mmol), tributyl(1-ethoxyvinyl)stannane (3.5 mL, 10.0 mmol) and bis(triphenylphosphine)palladium(II) dichloride (0.35 g, 0.5 mmol) in 1,4-dioxane (25 mL) was stirred at 100° C. overnight. The reaction was cooled to room temperature, and then filtered through Celite. The filtrate was concentrated and partitioned between THF (100 mL) and aqueous 1 N HCl (100 mL). The mixture was stirred at 60° C. for 1 h. The organic layer was separated. The aqueous layer was extracted with ethyl acetate (50 mL×3). The combined organic layers were dried over Na2SO4 and concentrated. The residue was chromatographed on silica gel, eluting with CH2Cl2 to give 1-(5-acetyl-3,4-dibromo-1H-pyrazol-1-yl)propan-2-one (1.1 g, 34%). MS m/z 325.0 [M+H]+.
Step D: A mixture of 1-(5-acetyl-3,4-dibromo-1H-pyrazol-1-yl)propan-2-one (3.24 g, 10 mmol) and ammonium acetate (7.7 g, 100 mmol) in acetic acid (10 mL) was stirred at 120° C. for 30 min. The mixture was cooled to room temperature and diluted with water (150 mL). The mixture was stirred for 20 min and filtered. The solid was dried and chromatographed on silica gel, eluting with CH2Cl2 to give 2,3-dibromo-4,6-dimethylpyrazolo[1,5-a]pyrazine (1.55 g, 50%). MS m/z 305.9 [M+H]+.
Step E: Into a solution of 2,3-dibromo-4,6-dimethylpyrazolo[1,5-a]pyrazine (1.55 g, 5.1 mmol) in THF (50 mL) at 0° C. was added a solution of i-PrMgCl (2.7 mL of a 2.0 M solution in THF, 5.4 mmol). The mixture was stirred at 0° C. for 30 min and excess reagent was quenched with the addition of methanol (25 mL). The mixture was partitioned between EtOAc and water. The organic layer was washed with brine, dried over Na2SO4, concentrated and chromatographed on silica gel, eluting with 0-35% EtOAc in hexanes to provide 2-bromo-4,6-dimethylpyrazolo[1,5-a]pyrazine (0.95 g, 80%). MS m/z 228.1 [M+H]+.
Step F: A mixture of 2-bromo-4,6-dimethylpyrazolo[1,5-a]pyrazine (0.226 mg, 1.0 mmol), hexamethyldistannane (0.397 g, 1.2 mmol) and tetrakis(triphenylphosphine) palladium(0) (0.116 g, 0.1 mmol) in toluene (3.0 mL) was stirred at 100° C. overnight under Argon. The mixture was cooled to room temperature and chromatographed on silica gel, eluting with 0-45% EtOAc in hexanes to give 4,6-dimethyl-2-(trimethylstannyl)pyrazolo[1,5-a]pyrazine (0.212 g, 70%) as a white solid. MS m/z 312.2 [M+H]+; 1H NMR (500 MHz, acetone-d6) δ 8.29 (1H, s), 6.95 (1H, s), 2.66 (3H, s), 2.42 (3H, d, J=0.9 Hz), 0.36 (9H, s).
Step A: A mixture of tert-butyl 4-(3-bromo-2-oxo-2H-chromen-7-yl)piperidine-1-carboxylate (110 mg, 0.24 mmol, prepared in Example 69, Step C), 4,6-dimethyl-2-(trimethylstannyl)pyrazolo[1,5-a]pyrazine (75 mg, 0.24 mmol), tetrakis(triphenylphosphine) palladium(0) (28 mg, 0.024 mmol) in a 0.5 M solution of LiCl in THF (1.44 mL, 0.72 mmol) was stirred at 100° C. for 20 h, then the solvent was removed. The residue was chromatographed on silica gel, eluting with 0-60% EtOAc in CH2Cl2 to give tert-butyl 4-(3-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-yl)piperidine-1-carboxylate (98 mg, 86%). MS m/z 475.3 [M+H]+.
Step B: Following the procedure in Example 69, Step E, tert-butyl 4-(3-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-yl)piperidine-1-carboxylate (98 mg, 0.21 mmol) and TFA (0.4 mL) in dichloromethane (0.4 mL) provided 3-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-7-(piperidin-4-yl)-2H-chromen-2-one (80 mg, 100%). MS m/z 375.3 [M+H]+.
Step C: Following the procedure in Example 67, 3-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-7-(piperidin-4-yl)-2H-chromen-2-one (61 mg, 0.1 mmol), acetaldehyde (30 μL of a 6.5 M solution in isopropanol, 0.2 mmol) and NaBH(OAc)3 (64 mg, 0.3 mmol) provided the title compound (36 mg, 90%) as a gray solid. MS m/z 403.3 [M+H]+; 1H NMR (500 MHz, CD3OD) δ 8.69 (1H, s), 8.23 (1H, s), 7.68 (1H, d, J=8.5 Hz), 7.61 (1H, s), 7.30 (2H, dd, J=3.8 Hz, 2.8 Hz), 3.41 (2H, br), 2.89 (3H, d, J=7.3 Hz), 2.75 (3H, s), 2.66 (2H, m), 2.48 (3H, s), 2.07 (2H, br), 1.95 (2H, m), 1.29 (3H, t, J=7.3 Hz).
Step A: Following the procedure in Example 37, Step A, 2,4-dihydroxybenzaldehyde (1.4 g, 10 mmol), ethyl acetoacetate (1.28 mL, 10 mmol), piperidine (1.0 mL, 10 mmol) and acetic acid (3 mL, 50 mmol) in CH3CN (20 mL) provided 3-acetyl-7-hydroxy-2H-chromen-2-one (1.77 g, 82%). MS m/z 203.1 [M−H]−.
Step B: Diisopropyl azodicarboxylate (2.0 mL, 10 mmol) was added dropwise into a mixture of 3-acetyl-7-hydroxy-2H-chromen-2-one (2.15 g, 10 mmol), tert-butyl 2-hydroxyethylcarbamate (1.9 mL, 12 mmol), triphenylphosphine (2.62 g, 10 mmol) and triethylamine (1.4 mL, 10 mmol) in THF (10 mL) at 0° C. The mixture warmed to room temperature and stirred 48 h. The mixture was filtered. The solid material was washed with ether and hexane to give tert-butyl 2-(3-acetyl-2-oxo-2H-chromen-7-yloxy)ethylcarbamate (1.81 g, 50%). MS m/z 348.2 [M+H]+.
Step C: Bromine (26 μL, 0.5 mmol) in CH2Cl2 (0.5 mL) was added to a mixture of tert-butyl 2-(3-acetyl-2-oxo-2H-chromen-7-yloxy)ethylcarbamate (183 mg, 0.5 mmol), CHCl3 (3.0 mL) and EtOH (1.0 mL). The mixture was stirred at room temperature for 4 h, then the solvent was removed from the mixture. The residue was chromatographed on silica gel, eluting with 0-15% MeOH in CH2Cl2 to give tert-butyl 2-(3-(2-bromoacetyl)-2-oxo-2H-chromen-7-yloxy)ethylcarbamate (0.108 g, 50%). MS m/z 428.1 [M+H]+.
Step D: Following the procedure in Example 43, Step A, tert-butyl 2-(3-(2-bromoacetyl)-2-oxo-2H-chromen-7-yloxy)ethylcarbamate (108 mg, 0.25 mmol) and 3,5-dimethylpyrazin-2-amine (32 mg, 0.25 mmol) in CH3CN (2.0 mL) provided tert-butyl 2-(3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-yloxy)ethylcarbamate (88 mg, 80%). MS m/z 451.3 [M+H]+.
Step E: Following the procedure in Example 69, Step E, tert-butyl 2-(3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-yloxy)ethylcarbamate (88 mg, 0.20 mmol) and TFA (0.3 mL) in CH2Cl2 (0.6 mL) provided 7-(2-aminoethoxy)-3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2H-chromen-2-one (66 mg, 95%). MS m/z 351.2 [M+H]+.
Step F: Following the procedure in Example 67, 7-(2-aminoethoxy)-3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2H-chromen-2-one (35 mg, 0.1 mmol), formaldehyde (37% aqueous, 0.1 mL) and NaBH(OAc)3 (64 mg, 0.3 mmol) provided the title compound (25 mg, 67%) as a pale yellow solid: m.p. 251-253° C.; MS m/z 379.3 [M+H]+; 1H NMR (500 MHz, CD3OD) δ 8.79 (1H, s), 8.51 (1H, s), 8.09 (1H, m), 7.69 (1H, d, J=8.5 Hz), 7.05 (1H, m), 6.99 (1H, m), 4.30 (2H, s), 3.12 (2H, br s), 2.86 (3H, s), 2.59 (6H, s), 2.45 (3H, s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 74 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: Into a mixture of 3-acetyl-7-hydroxy-2H-chromen-2-one (2.3 g, 10 mmol), ethylene glycol (1.7 mL, 30 mmol) and triphenylphosphine (2.88 g, 11 mmol) in THF (10 mL) was added triethylamine (1.53 mL, 11 mmol). The mixture was cooled to 0° C. Diisopropyl azodicarboxylate (2.2 mL, 11 mmol) was added dropwise to the mixture. The mixture was stirred at room temperature overnight, after which the solvent was removed. The residue was chromatographed on silica gel, eluting with 0-45% EtOAc in CH2Cl2. The resulting material was chromatographed on silica gel a second time, eluting with 10-100% EtOAc in hexanes to provide 3-acetyl-7-(2-hydroxyethoxy)-2H-chromen-2-one (0.50 g, 20%). MS m/z 249.1 [M+H]+.
Step B: Following the procedure of Example 74, Step C, 3-acetyl-7-(2-hydroxyethoxy)-2H-chromen-2-one (0.5 g, 2.0 mmol) and bromine (0.105 mL, 2.0 mmol) in CHCl3 (18 mL) and EtOH (2.0 mL) provided 3-(2-bromoacetyl)-7-(2-hydroxyethoxy)-2H-chromen-2-one (0.26 g, 40%). MS m/z 329.0 [M+H]+.
Step C: Following the procedure in Example 43, Step A, 3-(2-bromoacetyl)-7-(2-hydroxyethoxy)-2H-chromen-2-one (0.26 g, 0.8 mmol) and 3,5-dimethylpyrazin-2-amine (0.10 g, 0.8 mmol) in CH3CN (2.0 mL) provided 3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-7-(2-hydroxyethoxy)-2H-chromen-2-one (0.32 g, 88%). MS m/z 352.2 [M+H]+.
Step D: Methanesulfonyl chloride (0.065 mL, 0.81 mmol) in CH2Cl2 (1.5 mL) was added dropwise into a solution of 3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-7-(2-hydroxyethoxy)-2H-chromen-2-one (0.29 g, 0.66 mmol) and triethylamine (0.28 mL, 1.98 mmol) in CH2Cl2 (1.5 mL) at 0° C. The mixture was stirred at 0° C. for 20 min, and then at room temperature for 30 min, then the solvent was removed. The residue was triturated with methanol, and then filtered to give 2-(3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-yloxy)ethyl methanesulfonate (0.18 g, 63%). MS m/z 430.2 [M+H]+.
Step E: A mixture of 2-(3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-yloxy)ethyl methanesulfonate (61 mg, 0.14 mmol) and pyrrolidine (0.07 mL, 0.85 mmol) in DMF (0.3 mL) was stirred at 60° C. for 30 min. The mixture was chromatographed on silica gel, eluting with 0-10% MeOH in CH2Cl2 to give the title compound (25 mg, 40%) as a pale yellow solid: m.p. 201-203° C.; MS m/z 405.2 [M+H]+; 1H NMR (500 MHz, CD3OD) δ 8.83 (1H, m), 8.55 (1H, m), 8.11 (1H, m), 7.74 (1H, m), 7.07 (2H, m), 4.46 (2H, m), 3.75 (2H, m), 3.70 (2H, m), 3.23 (2H, m), 2.88 (3H, s), 2.46 (3H, s), 2.22 (2H, m), 2.08 (2H, m).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 75 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: Methyl 2-(triphenylphosphoranylidene)acetate (1.84 g, 5.5 mmol) was dissolved in CHCl3 (10 mL) and cooled to 0° C. N-Bromosuccinimide (980 mg, 5.5 mmol) was added to the solution. The mixture warmed to room temperature and stirred 20 min. The mixture was concentrated. 4-Bromosalicaldehyde (1.0 g, 5.0 mmol) and diphenyl ether (10 mL) were added to the residue. The mixture was heated at 220° C. for 3 h. The mixture was loaded directly onto silica gel, eluting with 0-30% EtOAc in hexanes to afford 3,7-dibromo-2H-chromen-2-one (650 mg, 43%) as a white solid. 1H NMR (500 MHz, DMSO-d6): δ 8.62 (1H, s), 7.78 (1H, d, J=1.8 Hz), 7.65 (1H, d, J=8.3 Hz), 7.60 (1H, dd, J=8.3 Hz, 1.8 Hz).
Step B: 3,7-Dibromo-2H-chromen-2-one (200 mg, 0.66 mmol) was combined with t-Boc-piperazine (186 mg, 1.0 mmol) and triethylamine (0.18 mL, 1.3 mmol) in DMSO (1 mL). The mixture was heated at 100° C. for 30 min. The mixture was partitioned in CH2Cl2 (5 mL) and H2O (5 mL). The organic layer was loaded directly onto silica gel, eluting with 0-50% EtOAc in hexanes to afford tert-butyl 4-(7-bromo-2-oxo-2H-chromen-3-yl)piperazine-1-carboxylate (80 mg, 30%) as a white solid. 1H NMR (500 MHz, DMSO-d6): δ 7.72 (1H, s), 7.52 (1H, d, J=8.4 Hz), 7.43 (1H, dd, J=8.4 Hz, 1.6 Hz), 7.31 (1H, d, J=1.6 Hz), 3.81 (4H, m), 3.55 (4H, m), 1.49 (9H, s).
Step C: tert-Butyl 4-(7-bromo-2-oxo-2H-chromen-3-yl)piperazine-1-carboxylate (80 mg, 0.2 mmol) was combined with (2-methylimidazo[1,2-a]pyridin-6-yl)boronic acid (70 mg, 0.4 mmol, prepared in Example 34, Step A) and tetrakis(triphenylphosphine)palladium(0) (23 mg, 0.02 mmol) in CH3CN (1 mL). Aqueous K2CO3 (1 mL, 1 M) was added to the mixture. The mixture was heated under nitrogen at 80° C. for 16 h. The organic layer was removed, concentrated and chromatographed on silica gel, eluting with 0-8% MeOH in CH2Cl2 to afford tert-butyl 4-(7-(2-methylimidazo[1,2-a]pyridin-6-yl)-2-oxo-2H-chromen-3-yl)piperazine-1-carboxylate (57 mg, 62%) as a white powder.
Step D: tert-Butyl 4-(7-(2-methylimidazo[1,2-a]pyridin-6-yl)-2-oxo-2H-chromen-3-yl)piperazine-1-carboxylate (57 mg, 0.12 mmol) was dissolved in TFA (1 mL). The mixture was stirred for 15 min at room temperature, then the solvent was removed with a nitrogen stream. The residue was partitioned in CH2Cl2 and aqueous K2CO3. The organic layer was collected and concentrated to afford the title compound (24 mg, 50%) as a white powder: m.p. 174-178° C.; MS m/z 361.3 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.99 (1H, s), 8.07 (1H, s), 7.89 (1H, d, J=8.1 Hz), 7.76 (1H, s), 7.74 (1H, d, J=8.4 Hz), 7.68 (1H, d, J=9.2 Hz), 7.59 (1H, d, J=9.4 Hz), 7.47 (1H, s), 3.67 (1H, br), 3.35 (4H, m), 2.81 (4H, m), 2.37 (3H, s).
Step A: Following the procedure in Example 6, Step A, 3-(hydroxymethyl)phenol (1.24 g, 100 mmol), triethylamine (70 mL, 500 mmol), anhydrous magnesium chloride (19.0 g, 200 mmol) and paraformaldehyde (30 g, 1000 mmol) in CH3CN (400 mL) afforded 2-hydroxy-4-(hydroxymethyl)benzaldehyde (4.5 g, 29%). MS m/z 151.1 [M−H]−.
Step B: 2-Hydroxy-4-(hydroxymethyl)benzaldehyde (3.04 g, 20 mmol) was combined with ethyl acetoacetate (2.55 mL, 20 mmol) and piperidine (0.2 mL, 2.0 mmol) in CH3CN (10 mL). The mixture was heated at 80° C. for 1 h. The mixture was concentrated and chromatographed on silica gel, eluting with 0-50% EtOAc in hexanes to afford 3-acetyl-7-(hydroxymethyl)-2H-chromen-2-one (1.88 g, 43%) as a tan powder. 1H NMR (500 MHz, DMSO-d6): δ 8.66 (1H, s), 7.90 (1H, d, J=7.7 Hz), 7.36 (2H, m), 5.55 (1H, t, J=5.8 Hz), 4.65 (2H, d, J=5.8 Hz), 2.59 (3H, s).
Step C: 3-Acetyl-7-(hydroxymethyl)-2H-chromen-2-one (1.88 g, 8.3 mmol) was suspended in CHCl3 (8 mL). Bromine (0.43 mL, 8.3 mmol) was added dropwise at room temperature, then the mixture was stirred at room temperature for 30 min. The solid material in the mixture was collected, washed with CHCl3 and dried to afford 3-(2-bromoacetyl)-7-(hydroxymethyl)-2H-chromen-2-one (2.1 g, 85%) as a tan powder. MS m/z 297.1, 299.1 [M+H]+.
Step D: 3-(2-Bromoacetyl)-7-(hydroxymethyl)-2H-chromen-2-one (2.1 g, 7.0 mmol) was combined with 3,5-dimethylpyrazin-2-amine (940 mg, 7.7 mmol) in CH3CN (10 mL). The mixture was heated at 80° C. for 16 h. The mixture was cooled to room temperature and filtered. The solid material was washed with CH3CN and dried to afford 3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-7-(hydroxymethyl)-2H-chromen-2-one hydrobromide (1.0 g, 35%) as a tan powder. MS m/z 322.3 [M+H]+. 1H NMR (500 MHz, DMSO-d6): δ 8.91 (1H, s), 8.73 (1H, s), 8.45 (1H, s), 7.96 (1H, d, J=8.1 Hz), 7.41 (1H, s), 7.37 (1H, d, J=8.1 Hz), 4.65 (2H, s), 2.83 (3H, s), 2.42 (3H, s).
Step E: 3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-7-(hydroxymethyl)-2H-chromen-2-one hydrobromide (1.0 g, 2.5 mmol) was combined with diisopropylethylamine (1.3 mL, 7.5 mmol) in CH2Cl2 (10 mL). To the mixture was added methanesulfonyl chloride (0.39 mL, 5 mmol) at 0° C. The mixture was stirred 30 min at 0° C., then loaded directly onto silica gel, eluting with 0-30% EtOAc in CH2Cl2 to afford (3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-yl)methyl methanesulfonate (900 mg, 90%) as a white powder. MS m/z 400.3 [M+H]+. 1H NMR (500 MHz, DMSO-d6): δ 8.90 (1H, s), 8.65 (1H, s), 8.36 (1H, s), 8.06 (1H, d, J=8.1 Hz), 7.57 (1H, s), 7.48 (1H, d, J=8.1 Hz), 5.41 (2H, s), 2.78 (3H, s), 2.39 (3H, s).
Step F: (3-(6,8-Dimethylimidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-yl)methyl methanesulfonate (60 mg, 0.15 mmol) was suspended in THF:DMF (1:1, 1 mL). Dimethylamine (0.75 mmol, 1 M in THF) was added to the mixture. The mixture was stirred at room temperature for 1 h, then loaded directly onto silica gel and eluted with 0-10% MeOH (3% NH3) in CH2Cl2 to afford the title compound (31 mg, 59%) as an off-white powder: m.p. 192-196° C.; MS m/z 349.3 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.88 (1H, s), 8.64 (1H, s), 8.36 (1H, s), 7.95 (1H, d, J=8.1 Hz), 7.38 (1H, s), 7.35 (1H, d, J=8.1 Hz), 3.53 (2H, s), 2.78 (3H, s), 2.38 (3H, s), 2.20 (6H, s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 77 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: 7-Hydroxycoumarin (4.86 g, 30 mmol) was dissolved in DMF (60 mL). Sodium hydride (1.8 g, 45 mmol, 60% dispersion in mineral oil) was added to the solution. After 5 min of vigorous stirring, N,N-bis(trifluoromethylsulfonyl)aniline (11.8 g, 33 mmol) was added to the mixture. The mixture was stirred vigorously for 30 min at room temperature, then ice water (120 mL) was added. The mixture was allowed to stand for 30 min, then filtered to remove the solid product. The solid was washed with water and dried, affording 2-oxo-2H-chromen-7-yl trifluoromethanesulfonate (6.9 g, 78%) as a white crystalline solid. MS m/z 295.2 [M+H]+.
Step B: 2-Oxo-2H-chromen-7-yl trifluoromethanesulfonate (2.94 g, 10 mmol) was combined with 3-butyne-1-ol (1.13 mL, 15 mmol), copper(I) iodide (380 mg, 2.0 mmol), tetrakis(triphenylphosphine)palladium(0) (1.16 g, 1.0 mmol) and triethylamine (2.78 mL, 20 mmol) in DMF (30 mL). The mixture was stirred at 60° C. for 2 h, then the solvent was removed by rotary evaporation. The residue was chromatographed on silica gel, eluting with 0-20% EtOAc in CH2Cl2 to afford 7-(4-hydroxybut-1-yn-1-yl)-2H-chromen-2-one (2.1 g, quant.) as a tan powder. MS m/z 215.2 [M+H]+.
Step C: 7-(4-Hydroxybut-1-yn-1-yl)-2H-chromen-2-one (1.72 g, 8 mmol) was suspended in MeOH (20 mL) with 10% Pd/C (300 mg). The mixture was stirred vigorously under H2 (1 atm) for 16 h, then the catalyst was removed by vacuum filtration. The filtrate was concentrated to afford 7-(4-hydroxybutyl)-2H-chromen-2-one (1.67 g, 96%) as a colorless oil. MS m/z 219.2 [M+H]+.
Step D: 7-(4-Hydroxybutyl)-2H-chromen-2-one (1.67 g, 7.6 mmol) was dissolved in AcOH (20 mL). Sodium acetate (1.87 g, 22.8 mmol) and bromine (1.18 mL, 22.8 mmol) were added sequentially. The mixture was stirred at 40° C. for 2 h., then the solvent was removed by rotary evaporation. MeOH (10 mL) and triethylamine (1 mL) were added to the residue and the mixture was stirred at 70° C. for 10 min. The solvent was removed by rotary evaporation, then the residue was partitioned in water and CH2Cl2. The organic layer was collected, concentrated and chromatographed on silica gel, eluting with 20-80% EtOAc in hexanes to afford 3-bromo-7-(4-hydroxybutyl)-2H-chromen-2-one (1.2 g, 53%) as a white powder. MS m/z 297.1, 299.1 [M+H]+.
Step E: 3-Bromo-7-(4-hydroxybutyl)-2H-chromen-2-one (1.2 g, 4 mmol) was combined with tributyl(1-ethoxyvinyl)stannane (1.8 g, 5 mmol), copper(I) iodide (190 mg, 1 mmol) and tetrakis(triphenylphosphine)palladium(0) (462 mg, 0.4 mmol) in 1,4-dioxane (20 mL). The mixture was stirred at 60° C. for 4 h, then the solvent was removed by rotary evaporation. The residue was chromatographed on silica gel, eluting with 20-50% EtOAc in hexanes to afford 3-(1-ethoxyvinyl)-7-(4-hydroxybutyl)-2H-chromen-2-one (860 mg, 75%) a colorless oil. MS m/z 261.2 [M+H]+ (hydrolysis during UPLC/MS analysis provides the mass of 3-acetyl-7-(4-hydroxybutyl)-2H-chromen-2-one).
Step F: 3-(1-Ethoxyvinyl)-7-(4-hydroxybutyl)-2H-chromen-2-one (860 mg, 3.0 mmol) was dissolved in THF (12 mL) and H2O (3 mL). N-Bromosuccinimide (587 mg, 3.3 mmol) was added to the mixture. After 10 min, THF was removed with a nitrogen stream. The residue was suspended in H2O (10 mL) and filtered. The collected solid was washed with H2O and dried affording 3-(2-bromoacetyl)-7-(4-hydroxybutyl)-2H-chromen-2-one (1.0 g, 99%) as a tan solid. MS m/z 339.1, 341.1 [M+H]+.
Step G: Following the procedure in Example 77, Step D, 3-(2-bromoacetyl)-7-(4-hydroxybutyl)-2H-chromen-2-one (1.0 g, 3.0 mmol), 3,5-dimethylpyrazin-2-amine (403 mg, 3.3 mmol) in CH3CN (10 mL) yielded 3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-7-(4-hydroxybutyl)-2H-chromen-2-one hydrobromide (880 mg, 66%) as a tan powder. MS m/z 364.2 [M+H]+.
Step H: Following the procedure in Example 77, Step E, 3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-7-(4-hydroxybutyl)-2H-chromen-2-one hydrobromide, diisopropylethylamine (1.3 mL, 7.5 mmol) and methanesulfonyl chloride (0.39 mL, 5 mmol) in CH2Cl2 (10 mL) yielded 4-(3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-yl)butyl methanesulfonate (705 mg, 80%) as a pale yellow solid. MS m/z 442.2 [M+H]+.
Step I: Following the procedure in Example 77, Step F, 4-(3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-yl)butyl methanesulfonate (36 mg, 0.08 mmol), dimethylamine (2 mmol, 2 M in THF) in DMF (1 mL) yielded the title compound (27 mg, 86%) as an off white powder: m.p. 190-193° C.; MS m/z 391.5 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.86 (1H, s), 8.62 (1H, s), 8.35 (1H, s), 7.90 (1H, d, J=7.9 Hz), 7.32 (1H, s), 7.27 (1H, d, J=7.9 Hz), 2.78 (3H, s), 2.73 (2H, t, J=6.9 Hz), 2.38 (3H, s), 2.22 (2H, t, J=6.9 Hz), 2.10 (6H, s), 1.64 (2H, p, J=7.5 Hz), 1.43 (2H, p, J=7.5 Hz).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 78 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: Following the procedure in Example 78, Step B, 2-oxo-2H-chromen-7-yl trifluoromethanesulfonate (4.2 g, 14.3 mmol, prepared in Example 78, Step A) propargyl alcohol (1.13 mL, 21.4 mmol), copper(I) iodide (266 mg, 1.4 mmol), tetrakis(triphenylphosphine) palladium(0) (1.62 g, 1.4 mmol) and triethylamine (2.98 mL, 21.5 mmol) in DMF (30 mL) yielded 7-(3-hydroxyprop-1-yn-1-yl)-2H-chromen-2-one (1.5 g, 52%) as a tan powder. MS m/z 201.1 [M+H]+.
Step B: Following the procedure in Example 78, Step C, 7-(3-hydroxyprop-1-yn-1-yl)-2H-chromen-2-one (1.5 g, 8 mmol), 10% Pd/C (200 mg) in MeOH (20 mL) yielded 7-(3-hydroxypropyl)-2H-chromen-2-one (1.5 g, quant.) as a white powder. MS m/z 205.2 [M+H]+.
Step C: Following the procedure in Example 78, Step D, 7-(3-hydroxypropyl)-2H-chromen-2-one (1.5 g, 7.3 mmol), sodium acetate (1.74 g, 21.2 mmol) and bromine (1.1 mL, 21.2 mmol) in AcOH (20 mL) yielded 3-bromo-7-(3-hydroxypropyl)-2H-chromen-2-one (595 mg, 29%) as a white powder. MS m/z 283.1, 285.1 [M+H]+.
Step D: 3-bromo-7-(3-hydroxypropyl)-2H-chromen-2-one (142 mg, 0.5 mmol) was combined with (2-methylimidazo[1,2-a]pyridin-6-yl)boronic acid (132 mg, 0.75 mmol, prepared in Example 34, Step A) and tetrakis(triphenylphosphine)palladium(0) (58 mg, 0.05 mmol) in CH3CN (2 mL). Aqueous K2CO3 (2 mL, 1 M) was added to the mixture. The mixture was heated under nitrogen at 80° C. for 2 h. The organic layer was removed, concentrated and chromatographed on silica gel, eluting with 0-8% MeOH in CH2Cl2 to afford 7-(3-hydroxypropyl)-3-(2-methylimidazo[1,2-a]pyridin-6-yl)-2H-chromen-2-one (65 mg, 39%) as a tan powder. MS m/z 335.2 [M+H]+.
Step F: Following the procedure in Example 77, Step E, 7-(3-hydroxypropyl)-3-(2-methylimidazo[1,2-a]pyridin-6-yl)-2H-chromen-2-one (65 mg, 0.2 mmol), diisopropylethylamine (0.15 mL, 0.8 mmol) and methanesulfonyl chloride (30 μL, 0.4 mmol) in CH2Cl2 (2 mL) yielded 3-(3-(2-methylimidazo[1,2-a]pyridin-6-yl)-2-oxo-2H-chromen-7-yl)propyl methanesulfonate (35 mg, 47%) as a tan powder. MS m/z 413.3 [M+H]+.
Step G: Following the procedure in Example 77, Step F, 3-(3-(2-methylimidazo[1,2-a]pyridin-6-yl)-2-oxo-2H-chromen-7-yl)propyl methanesulfonate (35 mg, 0.08 mmol), dimethylamine (2 mmol, 2 M in THF) in THF (1 mL) yielded the title compound (22 mg, 76%) as an off white powder: m.p. 176-178° C.; MS m/z 362.3 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 9.01 (1H, s), 8.38 (1H, s), 7.80 (1H, s), 7.70 (1H, d, J=7.9 Hz), 7.54 (2H, m), 7.34 (1H, s), 7.28 (1H, d, J=7.9 Hz), 2.73 (2H, t, J=7.1 Hz), 2.36 (3H, s), 2.22 (2H, t, J=7.1 Hz), 2.13 (6H, s), 1.76 (2H, p, J=7.3 Hz).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 79 by substituting the appropriate starting materials, reagents and reaction conditions.
3-(3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-yl)propyl methanesulfonate (300 mg, 0.70 mmol, prepared according to Example 77) was combined with sodium azide (57 mg, 0.88 mmol) in DMF (5 mL). The mixture was heated at 70° C. for 2 h. Triphenylphosphine (367 mg, 1.4 mmol) and H2O (63 μL, 3.5 mmol) were added to the solution. The mixture was stirred for an additional 1 h at 70° C., then loaded directly onto silica gel, eluting with 9.7:0.3:90 MeOH:NH3:CH2Cl2 to afford the title compound (160 mg, 66%) as an off white powder: m.p. 212-216° C.; MS m/z 349.3 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.83 (1H, s), 8.60 (1H, s), 8.33 (1H, s), 7.88 (1H, d, J=7.9 Hz), 7.32 (1H, s), 7.26 (1H, d, J=7.9 Hz), 2.77 (3H, s), 2.75 (2H, t, J=6.9 Hz), 2.57 (2H, t, J=6.9 Hz), 2.37 (3H, s), 1.70 (2H, p, J=7.5 Hz).
7-(3-aminopropyl)-3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2H-chromen-2-one (40 mg, 0.11 mmol, prepared in Example 80) was suspended in 1,2-dichloroethane (1 mL) and EtOH (0.5 mL). To the mixture was added benzaldehyde (122 μL, 1.2 mmol) and sodium triacetoxyborohydride (47 mg, 0.22 mmol). The mixture was stirred at room temperature for 16 h, then loaded directly onto silica gel, eluting with 2-8% MeOH (3% NH3) in CH2Cl2 to afford the title compound (28 mg, 65%) as a tan powder: m.p. 143-147° C.; MS m/z 439.3 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.85 (1H, s), 8.62 (1H, s), 8.35 (1H, s), 7.88 (1H, d, J=7.9 Hz), 7.34-7.19 (7H), 3.69 (2H, s), 2.78 (3H, s), 2.75 (2H, m), 2.52 (2H, m), 2.38 (3H, s), 1.79 (2H, p, J=6.7 Hz).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 81 by substituting the appropriate starting materials, reagents and reaction conditions.
A mixture of 7-bromo-3-(6,8-dimethylimidazo[1,2-a]pyrazin-2-yl)-2H-chromen-2-one (74.4 mg, 0.2 mmol, prepared in Example 48, Step A), 3-(dimethylamino)azetidine dihydrochloride (69.2 mg, 0.4 mmol), bis(dibenzylideneacetone)palladium(0) (11.5 mg, 0.02 mmol), RuPhos (9.3 mg, 0.02 mmol), BrettPhos (10.7 mg, 0.02 mmol) and Cs2CO3 (228.1 mg, 0.7 mmol) in toluene (0.2 mL) and t-butanol (0.2 mL) was heated at 100° C. for 5 h. The solvent was removed by rotary evaporation, then the residue was suspended in diethyl ether and filtered. The solid was washed thoroughly with water and dried to afford the title compound (59.5 mg, 76%) as a yellow solid: m.p. 246-250° C.; MS m/z 390.3 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.72 (1H, s), 8.50 (1H, s), 8.32 (1H, s), 7.74 (1H, d, J=8.6 Hz), 6.47 (1H, dd, J=8.6 Hz, 2.2 Hz), 6.35 (1H, d, J=1.9 Hz), 4.05 (2H, m), 3.78 (2H, m), 3.27 (1H, m), 2.75 (3H, s), 2.37 (3H, s), 2.15 (6H, s).
Step A: 6-[3-(6,8-Dimethyl-imidazo[1,2-a]pyrazin-2-yl)-2-oxo-2H-chromen-7-yl]-2,6-diaza-spiro[3.3]heptane-2-carboxylic acid tert-butyl ester (49 mg, 0.100 mmol, prepared according to Example 43) was stirred in CH2Cl2 (5 mL) with trifluoroacetic acid (1.25 mL) at room temperature for 2 h, then the solvent was removed in vacuo. The residue was partitioned in CH2Cl2/MeOH (9/1) and an aqueous saturated NaHCO3 solution (1 M, 5 mL). The organic phase was dried over Na2SO4 and purified by silica gel chromatography (CH2Cl2/MeOH 95/5, 1% aq. NH3) to give the title compound (33 mg, 85%) as a yellow solid. MS m/z 388.3 [M+H]+. 1H NMR (300 MHz, CDCl3): δ 8.91 (1H, d, J=1.2 Hz), 8.30 (1H, s), 7.91 (1H, d), 7.53 (1H, d), 7.48 (1H, d), 6.48 (1H, dd), 6.40 (1H, d), 4.14 (4H, s), 4.06 (4H, s), 2.36 (3H, s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 83 by substituting the appropriate starting materials, reagents and reaction conditions.
Step A: 3-fluoropyridin-2-amine (5.0 g, 45 mmol) was combined with N-bromosuccinimide (8.0 g, 45 mmol) in CH3CN. The mixture was stirred at room temperature for 30 min. Chloroacetone (4.3 mL, 54 mmol) was added to the mixture, which was heated at 100° C., allowing CH3CN to evaporate. After 1 h, the temperature was further raised to 120° C. for 2 h. The mixture solidified upon cooling. The solid material was dissolved in H2O (50 mL) and an aqueous saturated NaHCO3 solution (100 mL) was added. A precipitate formed, and was collected by vacuum filtration. The solid material was washed with H2O and vacuum dried. The material was chromatographed on silica gel (0-30% EtOAc in CH2Cl2), providing the title compound as a tan powder (4.65 g, 45%). MS m/z 229.2 [M+H]+.
Step B: A mixture of 6-bromo-8-fluoro-2-methyl-imidazo[1,2-a]pyridine (500 mg, 2.18 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (665 mg, 2.62 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloro-palladium(II) complex with dichloromethane (89.1 mg, 0.109 mmol), and potassium acetate (643 mg, 6.55 mmol) in 1,4-dioxane (4.4 mL) was stirred at 80° C. overnight under Argon. The mixture was diluted with THF (12 mL) and filtered. The filtrate was concentrated to give a dark solid residue, which was used without further purification. The residue was combined with 3-bromo-7-fluoro-2H-chromen-2-one (250 mg, 1.03 mmol, prepared in Example 32, step A), [1,1′-bis(diphenylphosphino)-ferrocene]dichloropalladium(II) complex with dichloromethane (84 mg, 0.101 mmol) and aqueous K2CO3 (2.0 M×1.55 mL, 3.09 mmol) in CH3CN (3.5 mL). The mixture was stirred at 60° C. for 5 h under Argon, then cooled to room temperature, diluted with water and filtered. The solid was dissolved in CH2Cl2 (10% methanol), dried over Na2SO4, filtered, concentrated and purified by silica gel chromatography (0-5% MeOH in CH2Cl2) to give 7-fluoro-3-(8-fluoro-2-methyl-imidazo[1,2-a]pyridin-6-yl)-chromen-2-one (286 mg, 89%) as a brown solid.
Step C: A mixture of 7-fluoro-3-(8-fluoro-2-methyl-imidazo[1,2-a]pyridin-6-yl)-chromen-2-one (87 mg, 0.279 mmol), triethyl amine (0.14 mL, 1.00 mmol) and tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate hemioxalate (158 mg, 0.325 mmol) in DMSO (1 mL) was stirred at 100° C. for 20 h. The mixture was diluted with an aqueous saturated NaHCO3 solution and filtered. The solid was dried and purified by silica gel chromatography (0-10% MeOH in CH2Cl2) to give 6-[3-(8-Fluoro-2-methyl-imidazo[1,2-a]pyridin-6-yl)-2-oxo-2H-chromen-7-yl]-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester as a yellow solid.
Step D: 6-[3-(8-Fluoro-2-methyl-imidazo[1,2-a]pyridin-6-yl)-2-oxo-2H-chromen-7-yl]-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester (136 mg, 0.27 mmol) was stirred in CH2Cl2 (3 mL) with trifluoroacetic acid (0.5 mL) at room temperature for 2 h, then the solvent was removed in vacuo. The residue was partitioned in CH2Cl2/MeOH (9/1) and an aqueous NaHCO3 solution (1 M, 5 mL). The organic phase was dried over Na2SO4, concentrated and purified by silica gel chromatography (CH2Cl2/MeOH 80/20, 1% aq. NH3) to give the title product (32 mg, 29%) as a yellow solid. MS m/z 391.7 [M+H]+. 1H NMR (300 MHz, CDCl3): δ 8.71 (1H, s), 8.42 (1H, s), 7.75 (1H, s), 7.45 (1H, d, J=8.7 Hz), 6.36 (1H, dd, J=8.7 Hz, J=2.1 Hz), 6.28 (1H, d, J=2.1 Hz), 4.10 (4H, s), 3.48 (4H, s), 2.90 (3H, s), 2.17 (3H, s).
As shown in Table 1 below, additional compounds disclosed herein may be prepared according to Example 84 by substituting the appropriate starting materials, reagents and reaction conditions.
Table 1 provides isolated compounds of a free base form of a compound of Formula (I) that may be prepared according to the procedures of the indicated Example by substituting the appropriate starting materials, reagents and reaction conditions. The preparation of any salt, isotopologue, stereoisomer, racemate, enantiomer, diastereomer or tautomer from a free base form of a compound of Formula (I) is also contemplated and further included within the scope of the description herein. Where a free base form of the compound was not isolated from the salt form, a person of ordinary skill in the art could be expected to perform the required reactions to prepare and isolate the free base form of the compound.
The term “Cpd” represents Compound number, the term “Ex” represents “Example Number” (wherein * indicates that the corresponding Example for the Compound is provided above), the term “M.P.” represents “Melting Point (° C.),” the term “MS” represents “Mass Spectroscopy Peak(s) m/z [M+H]+/−”, the term “D” represents “Decomposition/Decomposed,” the term “DR” represents “Decomposition Range,” the term “S” represents “Softens,” the term “ND” indicates that the value was “Not Determined” and the term “NI” indicates that the compound was “Not Isolated.”
or a salt, isotopologue, stereoisomer, racemate, enantiomer, diastereomer or tautomer thereof.
Table 2 further provides certain isolated compounds of a salt form of a compound of Formula (I) that may be prepared according to the procedures of the indicated Example by using the appropriate reactants, reagents and reaction conditions. The preparation of any free base, isotopologue, stereoisomer, racemate, enantiomer, diastereomer or tautomer from a salt form of a compound of Formula (I) is also contemplated and further included within the scope of the description herein. Where a free base form of the compound was not isolated from the salt form, a person of ordinary skill in the art could be expected to perform the required reactions to prepare and isolate the free base form of the compound.
The term “Cpd” represents Compound number, the term “Ex” represents “Example Number” (wherein * indicates that the corresponding Example for the Compound is provided above), the term “M.P.” represents “Melting Point (° C.),” the term “MS” represents “Mass Spectroscopy Peak(s) m/z [M+H]+/−,” the term “D” represents “Decomposition/Decomposed,” the term “DR” represents “Decomposition Range,” the term “S” represents “Softens” and the term “ND” indicates that the value was “Not Determined.”
or a free base, isotopologue, stereoisomer, racemate, enantiomer, diastereomer or tautomer thereof.
To describe in more detail and assist in understanding the present description, the following non-limiting biological examples are offered to more fully illustrate the scope of the description and are not to be construed as specifically limiting the scope thereof. Such variations of the present description that may be now known or later developed, which would be within the purview of one skilled in the art to ascertain, are considered to fall within the scope of the present description and as hereinafter claimed. These examples illustrate the testing of certain compounds described herein in vitro and/or in vivo and demonstrate the usefulness of the compounds for treating of SMA by enhancing the inclusion of exon 7 of SMN2 into mRNA transcribed from the SMN2 gene. Compounds of Formula (I) enhance inclusion of exon 7 of SMN2 into mRNA transcribed from the SMN2 gene and increase levels of Smn protein produced from the SMN2 gene, and thus can be used to treat SMA in a human subject in need thereof.
SMN2 Minigene Construct
DNA corresponding to a region of the SMN2 gene starting from the 5′ end of exon 6 (ATAATTCCCCC) (SEQ ID NO. 14) and ending at nucleic acid residue 23 of exon 8 (CAGCAC) (SEQ ID NO. 15) was amplified by PCR using the following primers:
The 5′ end of each primer was designed to add a BamHI restriction endonuclease recognition site at both the 5′ end of exon 6 (GGATCC) (SEQ ID NO. 18) and the 3′ end after the 23th nucleotide of exon 8. Using the BamHI restriction endonuclease recognition sites, the PCR fragment was cloned into a derivative of the original pcDNA 3.1/Hygro vector which was modified as disclosed in United States Patent Publication US2005/0048549.
New UTRs were added to the modified vector using the HindIII site and the BamHI restriction sites comprising a 5′DEG UTR: 5′-TAGCTTCTTACCCGTACTCCACCGTTGGCAGCACGATCGCACGTCCCACGT GAACCATTGGTAAACCCTG-3′ (SEQ ID NO. 19) was cloned into the modified pcDNA3.1/Hygro vector together with a start codon upstream of the BamHI restriction site; and
a 3′DEG UTR: 5′-ATCGAAAGTACAGGACTAGCCTTCCTAGCAACCGCGGGCTGGGAGTCTGA GACATCACTCAAGATATATGCTCGGTAACGTATGCTCTAGCCATCTAACTATTCCCT ATGTCTTATAGGG-3′ (SEQ ID NO. 20) was cloned into the modified pcDNA3.1/Hygro vector using the NotI restriction endonuclease recognition site and the XhoI restriction endonuclease recognition site with a stop codon immediately downstream of the NotI restriction site. In addition, a firefly luciferase gene lacking its start codon was cloned into the vector using the BamHI and NotI restriction sites.
The resulting minigene comprises, in 5′ to 3′ order: the 5′-DEG UTR, the start codon, six additional nucleotides forming a BamHI restriction site, the nucleic acid residues of exon 6, the nucleic acid residues of intron 6 of SMN2, the nucleic acid residues of exon 7 of SMN2, the nucleic acid residues of intron 7 of SMN2, and the first 23 nucleic acid residues of exon 8 of SMN2, an additional six nucleotides forming a BamHI restriction site and the firefly luciferase gene lacking the start codon.
A single adenine residue was inserted after nucleotide 48 of exon 7 of SMN2 by site-directed mutagenesis. This minigene construct is referred to as SMN2-A.
SMN2 transcripts derived from minigenes containing exon 6 through 8 and the intervening introns recapitulate the splicing of their endogenous pre-mRNAs (Lorson et al, Proc. Natl. Acad. Sci. U.S.A., 1999, 96 (11), 6307). An SMN2-alternative splicing reporter construct which contains exons 6 through 8 and the intervening introns followed by a luciferase reporter gene was generated. Salient features of this construct are the lack of the start codon in the luciferase gene, inactivation of the termination codon (in the open reading frame that encodes the SMN protein) of exon 7 by insertion of a nucleotide after nucleic acid 48 of exon 7 and addition of a start codon (ATG) immediately upstream of exon 6. A single adenine (SMN2-A) was inserted after nucleic residue 48 of exon 7.
The SMN2 minigene was designed such that the luciferase reporter is in frame with the ATG start codon immediately upstream of exon 6 when exon 7 is present in the mRNA and the luciferase reporter is out of frame with the ATG start codon immediately upstream of exon 6 if exon 7 of SMN2 is removed during splicing of the pre-mRNA. In addition, in the absence of exon 7, the open reading frame that starts from the ATG start codon immediately upstream of exon 6 contains a stop codon in the fragment of exon 8 of SMN. Thus, in the presence of compounds that increase the inclusion of exon 7 of SMN2 into mRNA transcribed from the SMN2 gene, more transcripts containing exon 7 and more functional reporter are produced. A schematic illustration of this description can be found in
The DNA sequence of the minigene from the SMN2-A construct SEQ ID NO. 21 is provided in
SMN2 Minigene mRNA Splicing RT-qPCR Assay in Cultured Cells
The reverse transcription-quantitative PCR-based (RT-qPCR) assay is used to quantify the level of the full length SMN2 minigene mRNA containing SMN2 exon 7 in a HEK293H cell line stably transfected with said minigene and treated with a test compound.
Materials
Protocol. HEK293H cells stably transfected with the SMN2-A minigene construct described above (10,000 cells/well) are seeded in 200 μL of cell culture medium (DMEM plus 10% FBS, with 200 μg/mL hygromycin) in 96-well flat-bottom plates and the plate is immediately swirled to ensure proper dispersal of cells, forming an even monolayer of cells. Cells are allowed to attach for at least 4-6 hours. Test compounds are serially diluted 3.16-fold in 100% DMSO to generate a 7-point concentration curve. A solution of test compound (1 μL, 200× in DMSO) is added to each cell-containing well and the plate is incubated for 24 hours in a cell culture incubator (37° C., 5% CO2, 100% relative humidity). 2 replicates are prepared for each test compound concentration. The cells are then lysed in Cells-To-Ct lysis buffer and the lysate is stored at −80° C.
Full length SMN2-A minigene and GAPDH mRNA are quantified using the following primers and probes provided in Table 3. Primer SMN Forward A (SEQ ID NO. 1) hybridizes to a nucleotide sequence in exon 7 (nucleotide 22 to nucleotide 40), primer SMN Reverse A (SEQ ID NO. 2) hybridizes to a nucleotide sequence in the coding sequence of Firefly luciferase, SMN Probe A (SEQ ID NO. 3) hybridizes to a nucleotide sequence in exon 7 (nucleotide 50 to nucleotide 54) and exon 8 (nucleotide 1 to nucleotide 21). The combination of these three oligonucleotides detects only SMN1 or SMN2 minigenes (RT-qPCR) and will not detect endogenous SMN1 or SMN2 genes.
1Primers and probes designed by PTC Therapeutics, Inc.;
2Commercially available from Life Technologies, Inc. (formerly Invitrogen).
The SMN forward and reverse primers are used at final concentrations of 0.4 μM. The SMN probe is used at a final concentration of 0.15 μM. The GAPDH primers are used at final concentrations of 0.2 μM and the probe at 0.15 μM.
The SMN2-minigene GAPDH mix (15 μL total volume) is prepared by combining 7.5 μL of 2×RT-PCR buffer, 0.4 μL of 25×RT-PCR enzyme mix, 0.75 μL of 20×GAPDH primer-probe mix, 4.0075 μL of water, 2 μL of 10-fold diluted cell lysate, 0.06 μL of 100 μM SMN forward primer, 0.06 μL of 100 μM SMN reverse primer, and 0.225 μL of 100 μM SMN probe.
PCR is carried out at the following temperatures for the indicated time: Step 1: 48° C. (15 min); Step 2: 95° C. (10 min); Step 3: 95° C. (15 sec); Step 4: 60° C. (1 min); then repeat Steps 3 and 4 for a total of 40 cycles.
Each reaction mixture contains both SMN2-A minigene and GAPDH primers/probe sets (multiplex design), allowing simultaneous measurement of the levels of two transcripts.
Two SMN spliced products are generated from the SMN2 minigene. The first spliced product containing exon 7, corresponding to full length SMN2 mRNA, is called SMN2mini FL. The second one lacking exon 7 is called SMN2mini Δ7.
The increase of SMN2mini FL mRNA relative to that in cells treated with vehicle control is determined from real-time PCR data using a modified ΔΔCt method (as described in Livak and Schmittgen, Methods, 2001, 25:402-8). The amplification efficiency E is calculated from the slope of the amplification curve for SMN2mini FL and GAPDH individually. The abundances of SMN2mini FL and GAPDH are then calculated as (1+E)−Ct, where Ct is the threshold value for each amplicon. The abundance of SMN2mini FL is normalized to GAPDH abundance. The normalized SMN2mini FL abundance from test compound-treated samples is then divided by normalized SMN2mini FL abundance from vehicle-treated cells to determine the level of SMN2 FL mRNA relative to vehicle control.
Results. As seen in
For compounds of Formula (I) or a form thereof disclosed herein, Table 4 provides the EC1.5x for production of full length SMN2 mRNA that was obtained from the 7-point concentration data generated for each test compound according to the procedure of Biological Example 2. The term “EC1.5x for production of full length SMN2 mRNA” is defined as that concentration of test compound that is effective in increasing the amount of full length SMN2 mRNA to a level 1.5-fold greater relative to that in vehicle-treated cells. An EC1.5x for production of full length SMN2 mRNA between >3 μM and ≦30 μM is indicated by one star (*), an EC1.5x between >1 μM and ≦3 μM is indicated by two stars (**), an EC1.5x between >0.3 μM and ≦1 μM is indicated by three stars (***), an EC1.5x between >0.1 μM and ≦0.3 μM is indicated by four stars (****) and an EC1.5x≦0.1 μM is indicated by five stars (*****).
Endogenous SMN2 mRNA RT-qPCR Splicing Assay in Cultured Cells
The reverse transcription-quantitative PCR-based (RT-qPCR) assay is used to quantify the levels of the full length and Δ7 SMN2 mRNAs in primary cells and cell lines containing the SMN2 gene treated with a test compound.
Materials
Protocol. GM03813 SMA patient cells (5,000 cells/well) are seeded in 200 μL, of cell culture medium (DMEM plus 10% FBS) in 96-well flat-bottom plates and the plate is immediately swirled to ensure proper dispersal of cells, forming an even monolayer of cells. Cells are allowed to attach for at least 4-6 hrs. Test compounds are serially diluted 3.16-fold in 100% DMSO to generate a 7-point concentration curve. A solution of test compound (1 μL, 200× in DMSO) is added to each test well and 1 μL, DMSO is added to each control well. The plate is incubated for 24 hrs in a cell culture incubator (37° C., 5% CO2, 100% relative humidity). The cells are then lysed in Cells-To-Ct lysis buffer and the lysate is stored at −80° C.
SMN2-specific spliced products and GAPDH mRNA are identified using the following primers and probes in Table 5. Primer SMN FL Forward B (SEQ ID NO. 7) hybridizes to a nucleotide sequence in exon 7 (nucleotide 32 to nucleotide 54) and exon 8 (nucleotide 1 to nucleotide 4), primer SMN Δ7 Forward B (SEQ ID NO. 8) hybridizes to a nucleotide sequence in exon 6 (nucleotide 87 to nucleotide 111) and exon 8 (nucleotide 1 to nucleotide 3), primer SMN Reverse B (SEQ ID NO. 9) hybridizes to a nucleotide sequence in exon 8 (nucleotide 39 to nucleotide 62), probe SMN Probe B (SEQ ID NO. 10) hybridizes to a nucleotide sequence in exon 8 (nucleotide 7 to nucleotide 36). These primers and probe hybridize to nucleotide sequences common to human SMN1 and SMN2 mRNAs. Since the SMA patient cells used in Example 3 contain only the SMN2 gene, RT-qPCR can quantify only SMN2 full-length and Δ7 mRNA.
1Primers and probes designed by PTC Therapeutics, Inc.;
2Commercially available from Life Technologies, Inc. (formerly Invitrogen).
The SMN forward and reverse primers are used at final concentrations of 0.4 μM. The SMN probe is used at a final concentration of 0.15 μM. GAPDH primers are used at final concentrations of 0.1 μM and the probe at 0.075 μM. TaqMan gene expression assays were conducted at 20× concentrations with the GAPDH primers and Vic labeled probe provided as a 20× mixture. The One-Step RT-PCR kit was used as the Real-Time PCR Mix.
The SMN-GAPDH mix (10 μL total volume) is prepared by combining 5 μL of 2×RT-PCR buffer, 0.4 μL of 25×RT-PCR enzyme mix, 0.25 μL of 20×GAPDH primer-probe mix, 1.755 μL water, 2.5 μL of cell lysate, 0.04 μL of 100 μM SMN FL or SMN Δ7 forward primer, 0.04 μL of 100 μM SMN reverse primer, and 0.015 μL of 100 μM probe.
PCR is carried out at the following temperatures for indicated time: Step 1: 48° C. (15 min); Step 2: 95° C. (10 min); Step 3: 95° C. (15 sec); Step 4: 60° C. (1 min); then, repeat Steps 3 and 4 for a total of 40 cycles.
Each reaction mixture contains either SMN2 FL and GAPDH or SMN2 Δ7 and GAPDH primers/probe sets (multiplex design), allowing simultaneous measurement of the levels of two transcripts.
The endogenous SMN2 gene gives rise to two alternatively spliced mRNAs. The full length SMN2 mRNA contains exon 7 and termed SMN2 FL. The truncated mRNA lacks exon 7 and termed SMN2 Δ7.
The increase of SMN2 FL and decrease in SMN2 Δ7 mRNAs relative to those in cells treated with vehicle control are determined from real-time PCR data using a modified ΔΔCt method (as described in Livak and Schmittgen, Methods, 2001, 25:402-8). The amplification efficiency E is calculated from the slope of the amplification curve for SMN2 FL, SMN2 Δ7, and GAPDH individually. The abundances of SMN2 FL, SMN2 Δ7, and GAPDH are then calculated as (1+E)−Ct, where Ct is the threshold value for each amplicon. The abundances of SMN2 FL and SMN2 Δ7 are normalized to GAPDH abundance. The normalized SMN2 FL and SMN2 Δ7 abundances from test compound-treated samples are then divided by normalized SMN2 FL and SMN2 Δ7 abundances, respectively, from vehicle-treated cells to determine the levels of SMN2 FL and SMN2 Δ7 mRNAs relative to vehicle control.
Results. As seen in
Endogenous SMN2 mRNA End-Point Semi-Quantitative RT-PCR Splicing Assay in Cultured Cells
The endpoint reverse transcription-PCR splicing assay is used to visualize and quantify the levels of the full length and Δ7 SMN2 mRNAs in primary cells and cell lines containing the SMN2 gene treated with a test compound.
Materials
Protocol. GM03813 SMA patient cells (5,000 cells/well) are seeded in 200 μL of cell culture medium (DMEM plus 10% FBS) in 96-well flat-bottom plates and the plate is immediately swirled to ensure proper dispersal of cells, forming an even monolayer of cells. Cells are allowed to attach for at least 4-6 hrs. Test compounds are serially diluted 3.16-fold in 100% DMSO to generate a 7-point concentration curve. A solution of test compound (1 μL, 200× in DMSO) is added to each test well and 1 μL DMSO is added to each control well. The plate is incubated for 24 hrs in a cell culture incubator (37° C., 5% CO2, 100% relative humidity). The cells are then lysed in Cells-To-Ct lysis buffer and the lysate is stored at −80° C.
SMN FL and Δ7 mRNAs are identified using the following primers in Table 6. These primers hybridize to a nucleotide sequence in exon 6 (SMN Forward C, SEQ ID NO. 11) (nucleotide 43 to nucleotide 63) and exon 8 (SMN Reverse C, SEQ ID NO. 12) (nucleotide 51 to nucleotide 73) common to human SMN1 and SMN2 mRNAs. Since the SMA patient cells used in Example 4 contain only the SMN2 gene, RT-PCR can visualize and quantify only SMN2 full-length and SMN2 Δ7 mRNAs.
1Primers designed by PTC Therapeutics, Inc.
To synthesize cDNA, 5 μL of lysate, 4 μL of 5× iScript reaction mix, 1 μL of reverse transcriptase, and 10 μL of water are combined and incubated 5 min at 25° C. followed by 30 min at 42° C., followed by 5 min at 85° C. cDNA solution is stored at −20° C.
To perform endpoint PCR, 5 μL of cDNA, 0.2 μL of 100 μM forward primer, 0.2 μL of 100 μM reverse primer, and 22.5 μL of polymerase super mix are combined in a 96 well semiskirted PCR plate. PCR is carried out at the following temperatures for indicated time: Step 1: 94° C. (2 min), Step 2: 94° C. (30 sec), Step 3: 55° C. (30 sec), Step 4: 68° C. (1 min), then repeat Steps 2 to 4 for a total of 33 cycles, then hold at 4° C.
10 μL of each PCR sample is electrophoretically separated on a 2% agarose E-gel for 14 minutes stained with ds DNA staining reagents (e.g., ethidium bromide) and visualized using a gel imager.
Results. As seen in
SMN2 mRNA RT-qPCR Splicing Assay in Animal Tissues
The reverse transcription-quantitative PCR-based (RT-qPCR) assay is used to quantify the levels of the full length and Δ7 SMN2 mRNAs in tissues from mice treated with test compound.
Materials
Protocol. C/C-allele SMA mice are treated by oral gavage two times per day for 10 days with test compounds re-suspended in 0.5% HPMC and 0.1% Tween-80. Tissue samples were collected and snap frozen for RNA purification.
Tissue samples (20-40 mg) are homogenized in QIAzol Lysis Reagent for 2 minutes at 20 Hz in the TissueLyser II using one stainless steel bead. After addition of chloroform, the homogenate is separated into aqueous and organic phases by centrifugation. RNA partitioned to the upper, aqueous phase is extracted and ethanol is added to provide appropriate binding conditions. The sample is then applied to the RNeasy spin column from the RNeasy Mini Kit, where total RNA binds to the membrane. The RNA is eluted in RNase-free water then stored at −20° C. and subsequently analyzed using the TaqMan RT-qPCR on the 7900HT Thermocycler. Total RNA is diluted ten fold and 2.5 μL of the diluted sample is added to the TaqMan RT-qPCR mixture.
SMN2 spliced products are identified using the following primers and probes in Table 7. Primer SMN FL Forward B (SEQ ID NO. 7) hybridizes to a nucleotide sequence in exons 7 and 8, primer SMN Δ7 Forward B (SEQ ID NO. 8) hybridizes to a nucleotide sequence in exons 6 and 8, primer SMN Reverse B (SEQ ID NO. 9) hybridizes to a nucleotide sequence in exon 8, probe SMN Probe B (SEQ ID NO. 10) hybridizes to a nucleotide sequence in exon 8. These primers and probe hybridize to nucleotide sequences common to human SMN1 and SMN2 mRNAs. Since the SMA patient cells used in Example 5 contain only the SMN2 gene, RT-qPCR can quantify only SMN2 full-length and Δ7 mRNAs.
1Primers and probes designed by PTC Therapeutics, Inc.
The SMN forward and reverse primers are used at final concentrations of 0.4 μM. The SMN probe is used at a final concentration of 0.15 μM. The SMN-GAPDH Mix (10 μL total volume) is prepared by combining 5 μL of 2×RT-PCR buffer, 0.4 μL of 25×RT-PCR enzyme mix, 0.5 μL of 20×GAPDH primer-probe mix, 1.505 μL of water, 2.5 μL of RNA solution, 0.04 μL of 100 μM forward primer, 0.04 μL of 100 μM reverse primer, and 0.015 μL of 100 μM SMN probe.
Each PCR cycle was carried out at the following temperatures for indicated time: Step 1: 48° C. (15 min); Step 2: 95° C. (10 min); Step 3: 95° C. (15 sec); Step 4: 60° C. (1 min); then, repeat Steps 3 and 4 for a total of 40 cycles.
Each reaction mixture contains either SMN2 FL and mGAPDH or SMN2 Δ7 and mGAPDH primers/probe sets (multiplex design), allowing simultaneous measurement of the levels of two transcripts.
The increase of SMN2 FL and decrease in SMN2 Δ7 mRNAs relative to those in tissues from animals treated with vehicle control are determined from real-time PCR data using a modified ΔΔCt method (as described in Livak and Schmittgen, Methods, 2001, 25:402-8). The amplification efficiency E is calculated from the slope of the amplification curve for SMN2 FL, SMN2 Δ7, and GAPDH individually. The abundances of SMN2 FL, SMN2 Δ7, and GAPDH are then calculated as (1+E)−Ct, where Ct is the threshold value for each amplicon. The abundances of SMN2 FL and SMN2 Δ7 are normalized to GAPDH abundance. The normalized SMN2 FL and SMN2 Δ7 abundances from test compound-treated samples are then divided by normalized SMN2 FL and SMN2 Δ7 abundances, respectively, from vehicle-treated cells to determine the levels of SMN2 FL and SMN2 Δ7 mRNAs relative to vehicle control.
Results. As seen in
Endogenous SMN2 mRNA End-Point Semi-Quantitative RT-PCR Splicing Assay in Animal Tissues
The endpoint reverse transcription-PCR (RT-PCR) splicing assay is used to quantify the levels of the full length and Δ7 SMN2 mRNAs in tissues from mice treated with test compound.
Materials
Protocol. C/C-allele SMA mice are treated by oral gavage two times per day for 10 days with test compounds in 0.5% HPMC and 0.1% Tween-80. Tissue samples are collected and snap frozen for RNA purification.
Tissue samples (20-40 mg) are homogenized in QIAzol Lysis Reagent for 2 minutes at 20 Hz in the TissueLyser II using one stainless steel bead. After addition of chloroform, the homogenate is separated into aqueous and organic phases by centrifugation. RNA partitioned to the upper, aqueous phase is extracted and ethanol is added to provide appropriate binding conditions. The sample is then applied to the RNeasy spin column from the RNeasy Mini Kit, where total RNA binds to the membrane. The RNA is eluted in RNase-free water then stored at −20° C.
SMN2 spliced products are identified using the following amplification primers in Table 8. These primers hybridize to a nucleotide sequence in exon 6 (SMN Forward D, SEQ ID NO. 13) (nucleotide 22 to nucleotide 46) and exon 8 (SMN Reverse C, SEQ ID NO. 12) common to human SMN1 and SMN2 mRNAs.
1Primers designed by PTC Therapeutics, Inc.
To synthesize cDNA, combine 1 μL of RNA solution (25-50 ng), 4 μL of 5× iScript reaction mix, 1 μL of reverse transcriptase, and 10 μL of water are combined and incubates 25° C. for 5 min followed by 42° C. for 30 min followed by 85° C. for 5 min. cDNA solution is stored at −20° C.
To perform endpoint PCR, 5 μL of cDNA, 0.2 μL of 100 μM forward primer, 0.2 μL of 100 μM reverse primer, and 22.5 μL of polymerase super mix are combined in a 96 well semiskirted PCR plate. PCR is carried out at the following temperatures for indicated time: Step 1: 94° C. (2 min), Step 2: 94° C. (30 sec), Step 3: 55° C. (30 sec), Step 4: 68° C. (1 min), then repeat Steps 2 to 4 for a total of 33 cycles, then hold at 4° C.
10 μL of each PCR sample is electrophoretically separated on a 2% agarose E-gel for 14 minutes, stained with ds DNA staining reagents (e.g., ethidium bromide) and visualized using a gel imager.
Results. As seen in
Smn Protein Assay in Cultured Cells
The SMN HTRF (homogeneous time resolved fluorescence) assay is used to quantify the level of Smn protein in SMA patient fibroblast cells treated with test compounds. The results of the assay are shown in Table 9.
Materials
Protocol. Cells are thawed and cultured in DMEM-10% FBS for 72 hours. Cells are trypsinized, counted and re-suspended to a concentration of 25,000 cells/mL in DMEM-10% FBS. The cell suspensions are plated at 5,000 cells per well in a 96 well microtiter plate and incubated for 3 to 5 hours. To provide a control signal, three (3) wells in the 96 well plate do not receive cells and, thus, serve as Blank control wells. Test compounds are serially diluted 3.16-fold in 100% DMSO to generate a 7-point concentration curve. 1 μL of test compound solution is transferred to cell-containing wells and cells are incubated for 48 hours in a cell culture incubator (37° C., 5% CO2, 100% relative humidity). Triplicate samples are set up for each test compound concentration. After 48 hours, the supernatant is removed from the wells and 25 μL of the RIPA lysis buffer, containing protease inhibitors, is added to the wells and incubated with shaking at room temperature for 1 hour. 25 μL of the diluent is added and then 35 μL of the resulting lysate is transferred to a 384-well plate, where each well contains 5 μL of the antibody solution (1:100 dilution of anti-SMN d2 and anti-SMN kryptate in SMN reconstitution buffer). The plate is centrifuged for 1 minute to bring the solution to the bottom of the wells, then incubated overnight at room temperature. Fluorescence for each well of the plate at 665 nm and 620 nm is measured on an EnVision multilabel plate reader (Perkin-Elmer).
The normalized fluorescence signal is calculated for each sample, Blank and vehicle control well by dividing the signal at 665 nm by the signal at 620 nm. Normalizing the signal accounts for possible fluorescence quenching due to the matrix effect of the lysate. The ΔF value (a measurement of Smn protein abundance) for each sample well is calculated by subtracting the normalized average fluorescence for the Blank control wells from the normalized fluorescence for each sample well and then dividing this difference by the normalized average fluorescence for the Blank control wells. The resulting ΔF value for each sample well represents the Smn protein abundance from test compound-treated samples. The ΔF value for each sample well is divided by the ΔF value for the vehicle control wells to calculate the fold increase in Smn protein abundance relative to the vehicle control.
Results. As seen in
For compounds of Formula (I) or a form thereof disclosed herein, Table 9 provides the EC1.5x for Smn protein expression that was obtained from the 7-point concentration data generated for each test compound according to the procedure of Biological Example 7. The term “EC1.5x for Smn protein expression” is defined as that concentration of test compound that is effective in producing 1.5 times the amount of Smn protein in a SMA patient fibroblast cell compared to the amount produced from the DMSO vehicle control. An EC1.5x for Smn protein expression between >3 μM and ≦10 μM is indicated by one star (*), an EC1.5x between >1 and ≦3 μM is indicated by two stars (**), an EC1.5x between >0.3 μM and ≦1 μM is indicated by three stars (***) and an EC1.5x≦0.3 μM is indicated by four stars (****).
For compounds of Formula (I) or a form thereof disclosed herein, Table 10 provides the maximum fold (Fold) increase of Smn protein that was obtained from the 7-point concentration data generated for each test compound according to the procedure of Biological Example 7. A maximum fold increase of ≦1.2 is indicated by one star (*), a fold increase between >1.2 and ≦1.35 is indicated by two stars (**), a fold increase between >1.35 and ≦1.5 is indicated by three stars (***), a fold increase between >1.5 and ≦1.65 is indicated by four stars (****) and a fold increase >1.65 is indicated by five stars (*****).
Gems Count (Smn-dependent Nuclear Speckle Count) Assay
The level of Smn protein directly correlates with the amount of nuclear foci, also known as gems, produced upon staining the cell with a fluorescently labeled anti-Smn antibody (Liu and Dreyfuss, EMBO J., 1996, 15:3555). Gems are multi-protein complexes whose formation is nucleated by the Smn protein and the gems count assay is used to evaluate the level of Smn protein in the cell. As described herein, the gems count assay is used to quantify the level of Smn protein in SMA patient fibroblast cells treated with a test compound.
Materials
Protocol: Cells are thawed and incubated in DMEM-10% FBS for 72 hours, then trypsinized, counted and resuspended to 100,000 cells/mL in DMEM-10% FBS. 2 mL of the cell suspension is plated in a 6-well cell culture plate with a sterile cover slip and incubated for 3 to 5 hours. Test compounds are serially diluted 3.16-fold in 100% DMSO to generate a 7-point dilution curve. 10 μL of test compound solution is added to each cell-containing well and incubated for 48 hours in a cell culture incubator (37° C., 5% CO2, 100% relative humidity). Duplicates are set up for each test compound concentration. Cells containing DMSO at a final concentration of 0.5% are used as controls.
Cell culture medium is aspirated from the wells containing cover slips and gently washed three times with cold PBS. The cells are fixed by incubation for 20 minutes at room temperature while in paraformaldehyde. The cells are then washed two times with cold PBS followed by incubation for 5 minutes at room temperature with 0.05% Triton X-100 in PBS to permeabilize the cells. After the fixed cells are washed three times with cold PBS, they are blocked with 10% FBS for 1 hour. 60 μL of primary antibody diluted 1:1000 in blocking buffer is added and the mixture is incubated for one hour at room temperature. The cells are washed three times with PBS and 60 μL of secondary antibody diluted 1:5000 in blocking buffer is added, then the mixture is incubated for one hour at room temperature. The cover slips are mounted onto the slides with the aid of mounting medium and allowed to dry overnight. Nail polish is applied to the sides of the cover slip and the slides are stored, protected from light. A Zeiss Axovert 135 with a 63× Plan-Apochromat, NA=1.4 objective is used for immunofluorescence detection and counting. The number of gems is counted per ≧150 nuclei and % activation is calculated using DMSO and 10 nM bortezomib as controls. For each test compound, the cells are examined at all wavelengths to identify test compounds with inherent fluorescence.
Results. As seen in
Smn Protein Assay in Human Motor Neurons
Smn immunofluorescent confocal microscopy is used to quantify the level of Smn protein in human motor neurons treated with test compounds.
Protocol. Human motor neurons derived from SMA iPS cells (Ebert et al., Nature, 2009, 457:2770; and, Rubin et al., BMC Biology, 2011, 9:42) are treated with test compound at various concentrations for 72 hours. The level of Smn protein in the cell nucleus is quantified using Smn immunostaining and confocal fluorescence microscopy essentially as described in Makhortova et al., Nature Chemical Biology, 2011, 7:544. The level of Smn protein in compound-treated samples is normalized to that in vehicle-treated samples and plotted as a function of the compound concentration.
Results. As seen in
Smn Protein Assay in Animal Tissues
This Smn protein assay compares tissues from test compound treated mice with those from DMSO vehicle treated mice to determine the increase in levels of Smn protein produced from the human SMN2 gene.
Materials
Protocol. The tissue samples in Safe-Lock tubes are weighed and the volume of RIPA buffer containing the protease inhibitor cocktail is added based on the weight to volume ratios for each type of tissue: Brain (50 mg/mL), Muscle (50 mg/mL) and Spinal Cord (25 mg/mL).
Tissues are homogenized using the TissueLyzer by bead milling. 5 mm stainless steel beads are added to the sample and shaken vigorously for 5 minutes at 30 Hz in the TissueLyzer. The samples are then centrifuged for 20 minutes at 14,000×g in a microcentrifuge and the homogenates transferred to the PCR plate. The homogenates are diluted in RIPA buffer to approximately 1 mg/mL for HTRF and approximately 0.5 mg/mL for total protein measurement using the BCA protein assay. For the SMN HTRF assay, 35 μL of the tissue homogenate is transferred to a 384-well plate containing 5 μL of the antibody solution (1:100 dilution of each of the anti-SMNd2 and anti-SMN Kryptate in reconstitution buffer). To provide a control signal, three (3) wells in the plate contain only RIPA Lysis Buffer and, thus, serve as Blank control wells. The plate is centrifuged for 1 minute to bring the solution to the bottom of the wells and then incubated overnight at room temperature. Fluorescence for each well of the plate at 665 nm and 620 nm is measured on an EnVision multilabel plate reader (Perkin-Elmer). The total protein in the tissue homogenate is measured using the BCA assay according to the manufacturer's protocol.
The normalized fluorescence signal is calculated for each sample, Blank and vehicle control well by dividing the signal at 665 nm by the signal at 620 nm. Normalizing the signal accounts for possible fluorescence quenching due to the matrix effect of the tissue homogenate. The ΔF value (a measurement of Smn protein abundance) for each tissue sample well is calculated by subtracting the normalized average fluorescence for the Blank control wells from the normalized fluorescence for each tissue sample well and then dividing this difference by the normalized average fluorescence for the Blank control wells. The ΔF value for each tissue sample well is divided by the total protein quantity (determined using the BCA assay) for that tissue sample. The change in Smn protein abundance for each tissue sample relative to the vehicle control is calculated as the percent difference in the ΔF value of the tissue sample in the presence of the test compound and the averaged ΔF value of the vehicle control signal divided by the averaged ΔF value of the vehicle control signal.
Smn Protein Assay in Tissues of Adult C/C-Allele SMA Mice
The tissues for use in the assay for Smn protein in adult C/C-allele SMA mice are prepared as described in Example 10. The assay assesses whether treatment of C/C-allele SMA mice with a test compound for 10 days increases levels of Smn protein produced from the SMN2 gene.
Materials
Protocol. C/C-allele SMA mice are dosed twice a day orally (in 0.5% hydroxypropylmethyl cellulose (HPMC) with 0.1% Tween-80) with a test compound at 10 mg/kg for 10 days. Age-matched heterozygous mice are dosed with vehicle for use as a control. Tissues are collected for analysis of protein levels according to Example 10.
Results. As seen in
Smn Protein in Tissues of Neonatal Δ7 SMA Mice
The assay for Smn protein in neonatal SMA mice tissues is used to determine whether treatment with a test compound increases Smn protein levels produced from the SMN2 gene.
Materials
Protocol. SMA Δ7 homozygous knockout mice are dosed once a day (QD) intraperitoneally (IP) with a test compound or vehicle (100% DMSO) from postnatal day (PND) 3 to day 9. Tissues are collected for analysis of protein levels according to Example 10.
Results. As seen in
Body Weight of Neonatal Δ7 SMA Mice
The change in body weight of neonatal SMA mice is used to determine whether treatment with a test compound improves body weight.
Materials
Protocol. SMA Δ7 homozygous knockout mice are dosed intraperitoneally (IP) with test compound or vehicle (100% DMSO) once per day (QD) from postnatal day (PND) 3 until the dose regimen is switched to an oral dose twice per day (BID) in 0.5% hydroxypropylmethyl cellulose (HPMC) with 0.1% Tween-80 at a dose 3.16-fold higher than the dose used for IP. Body weights of SMA Δ7 mice treated with test compound or vehicle and age matched heterozygous mice are recorded every day.
Results. As seen in
Righting Reflex in Neonatal Δ7 SMA Mice
The functional change in righting reflex of neonatal SMA mice is used to determine whether treatment with a test compound improves righting reflex.
Materials
Protocol. SMA Δ7 homozygous knockout mice are dosed intraperitoneally (IP) with test compound or vehicle (100% DMSO) once per day (QD) from postnatal day (PND) 3 until the dose regimen is switched to an oral dose twice per day (BID) in 0.5% hydroxypropylmethyl cellulose (HPMC) with 0.1% Tween-80 at a dose 3.16-fold higher than the dose used for IP. The righting reflex time is measured as the time taken by a mouse to flip over onto its feet after being laid on its back. Righting reflex is measured five times for each mouse (allowing a maximal time of 30 sec for each try) with 5 minutes between each measurement. The righting reflex time for SMA Δ7 homozygous knockout mice treated with test compound or vehicle and age-matched heterozygous mice is measured on PND 10, 14 and 18 and plotted.
Results. As seen in
Survival of Neonatal Δ7 SMA Mice
The change in the number of surviving mice over time is used to determine whether treatment with a test compound improves survival.
Materials
Protocol. SMA Δ7 homozygous knockout mice are dosed intraperitoneally (IP) with test compound or vehicle (100% DMSO) once per day (QD) from postnatal day (PND) 3 until the dose regimen is switched to an oral dose twice per day (BID) in 0.5% hydroxypropylmethyl cellulose (HPMC) with 0.1% Tween-80 at a dose 3.16-fold higher than the dose used for IP. The number of surviving mice in each group is recorded every day and plotted as a percent of total number of mice. Tissues of SMA Δ7 and age-matched heterozygous mice are collected for the measurement of Smn protein levels and processed as detailed in Example 10. The total protein normalized Smn protein levels measured in the tissues are plotted as a percent of those in the age-matched heterozygous mice tissues, with the heterozygous levels represented as 100 percent. The level of Smn protein in the test compound treated mice tissue relative to that in heterozygous mice tissue is indicated as a percent value above each bar in the graph.
Results. As seen in
Results. As seen in
Without regard to whether a document cited herein was specifically and individually indicated as being incorporated by reference, all documents referred to herein are incorporated by reference into the present application for any and all purposes to the same extent as if each individual reference was fully set forth herein.
Although certain embodiments have been described in detail above, those having ordinary skill in the art will clearly understand that many modifications are possible in the embodiments without departing from the teachings thereof. All such modifications are intended to be encompassed within the claims as described herein.
This application is a U.S. national stage application of International Patent Application No. PCT/US2012/071899, filed Dec. 28, 2012, which claims the benefit of U.S. Provisional Application No. 61/582,064, Dec. 30, 2011, each of which is incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2012/071899 | 12/28/2012 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2013/101974 | 7/4/2013 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 3242177 | Carl-wolfgang et al. | Mar 1966 | A |
| 3311636 | Moffett | Mar 1967 | A |
| 3646052 | Otto et al. | Feb 1972 | A |
| 3681397 | Knupfer et al. | Aug 1972 | A |
| 4141900 | Schlapfer | Feb 1979 | A |
| 4284787 | Knupfer et al. | Aug 1981 | A |
| 4404389 | Vamvakaris et al. | Sep 1983 | A |
| 4426530 | Berneth et al. | Jan 1984 | A |
| 5194393 | Hugl et al. | Mar 1993 | A |
| 7569337 | Auberson | Aug 2009 | B2 |
| 20060205741 | Zhang et al. | Sep 2006 | A1 |
| 20070208033 | Yamazaki et al. | Sep 2007 | A1 |
| 20100004233 | Iikura et al. | Jan 2010 | A1 |
| 20100035279 | Gubernator et al. | Feb 2010 | A1 |
| 20110086833 | Paushkin et al. | Apr 2011 | A1 |
| Number | Date | Country |
|---|---|---|
| 1448431 | Oct 2003 | CN |
| 1448431 | Oct 2003 | CN |
| 1020636 | Dec 1957 | DE |
| 2950291 | Dec 1979 | DE |
| WO 2009124800 | Oct 2009 | DE |
| 0030703 | Jun 1981 | EP |
| 0448241 | Sep 1991 | EP |
| 2361680 | Mar 1978 | FR |
| 991202 | May 1965 | GB |
| S4891129 | Nov 1973 | JP |
| S56140990 | Nov 1981 | JP |
| 7-3179 | Jan 1995 | JP |
| 0700317 | Jun 1995 | JP |
| WO 03074519 | Sep 2003 | WO |
| WO 2009151546 | May 2009 | WO |
| WO 2010019236 | Aug 2009 | WO |
| WO 2009124800 | Oct 2009 | WO |
| WO 2010049044 | May 2010 | WO |
| Entry |
|---|
| Sreenivasulu, Proceedings—Indian Academy of Sciences, Section A (1974), 79(1), 41-7. |
| Sreenivasulu, Proceedings—Indian Academy of Sciences, Section A (1973), 78(4), 159-68. |
| Steyer Applied Physics (Berlin) (1975), 7(2), 113-22. |
| Czerney, Journal fuer Praktische Chemie (Leipzig) (1982), 324(2), 255-66. |
| U.S. Appl. No. 14/373,937, filed Jul. 23, 2014, Chen et al. |
| U.S. Appl. No. 14/377,531, filed Aug. 8, 2014, Qi et al. |
| U.S. Appl. No. 14/380,385, filed Aug. 22, 2014, Lee et al. |
| U.S. Appl. No. 14/386,524, filed Sep. 19, 2014, Yang et al. |
| Le et al., (2005) “SMND7, the major product of the centromeric survival motor neuron (SMN2) gene, extends survival in mice with spinal muscular atrophy and associates with full-length SMN,” Human Molecular Genetics, vol. 14(6) pp. 845-857 (2005). |
| Passini et al., (2001) “Antisense Oligonucleotides Delivered to the Mouse CNS Ameliorate Symptoms of Severe Spinal Muscular Atrophy,” Sci Transl Med., vol. 3(72) (2001). |
| Hua et al., (2012) “Peripheral SMN restoration is essential for long-term rescue of a severe SMA mouse model,” Nature, vol. 478(7367): pp. 123-126 (2012). |
| Coady et al., (2010) “Trans-splicing-mediated improvement in a severe mouse model of spinal muscular atrophy,” J Neurosci., vol. 30(1), pp. 126-130 (2010). |
| Greene et al, (1991) Protective Groups in Organic Synthesis (1991), Wiley, New York. |
| Higuchi and W. Stella, (1987) “Pro-drugs as Novel Delivery Systems,” vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press (1987). |
| Liu et al., (1996) “A novel nuclear structure containing the survival of motor neurons protein,” EMBO J., vol. 15(14), pp. 3555-3565 (1996). |
| PCT International Search Report Issued Apr. 18, 2013 in connection with PCT/US2012/071899. |
| PCT International Preliminary Report Issued Jul. 10, 2014 in connection with PCT/US2012/071899. |
| Czerney et al., “Heterocyclisch Substituierte Cumarine Aus Beta-Chloropropeniminiumsalzen Heterocyclic Substituted Coumarines From Beta-Chloropropeniminium Salts,” Journal Fuer Praktische Chemie, Wiley VCH, Weinheim, DE, vol. 324, No. 2, Jan. 1, 1982, pp. 255-266, XP009045598 (English abstract included). |
| English translation of Chinese Office Action for Chinese Patent Application Serial No. 201280071056.5 (PCT/US2012/071899) (This Office Action cites references G01, H01-H04 and I02 listed here.). |
| “Medicinal Chemistry”, Chief editor is Qidong You, Chemical Industry Press, Jul. 2008, pp. 27-29. |
| Number | Date | Country | |
|---|---|---|---|
| 20150119380 A1 | Apr 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61582064 | Dec 2011 | US |
