Patent Application
20250066389
More than 90% of human genes produce multiple mature transcripts via alternative splicing. This process is essential for generating different transcripts in different cell and tissue types, during the developmental process, and in response to internal and external signals. Alternative splicing are prevalent not only for protein-coding genes but also for most other kinds of genes including microRNA genes and long noncoding genes. Splicing is carried out by the spliceosome. Small nuclear RNAs (snRNAs) are key components of the spliceosome. The major spliceosome comprises the U1, U2, U4. U5, and U6 snRNAs, and it catalyzes the removal of ˜95% of human introns, while the remaining introns (called the U12-type of introns) are removed by the minor spliceosome, comprising the U11, U12, U4atac, U5, and U6atac snRNAs. These snRNAs are in complex with their respective protein partners to form the functional unit of small nuclear ribonucleoproteins (snRNPs).
Splicing is a highly regulated process, with the regulation exerted by both cis-elements and trans-factors. The cis-elements that are recognized by the snRNAs include the 5′-splice site, 3˜-splice site, and the branchpoint, each of these associating with a sequence motif that is recognized by a component of the spliceosome. In addition, there are intronic splicing enhancers (ISE), intronic splicing silencer (ISS), exonic splicing enhancer (ESE), and exonic splicing enhancer (ESS), which are recognized by a myriad of trans-factors commonly known as RNA-binding proteins (RBPs). Some of these RBPs directly bind to the cis-elements in a sequencing-specific way, while other RBPs recognize RNA structures (e.g., RNA duplex or unpaired loop region), yet others function via protein-protein interaction. There are ˜1600 RBPs annotated in the human genome, and they are expressed in a cell-type-specific manner and form an extensive regulatory network for splicing regulation.
Dysregulation of splicing is implicated in roughly half of human diseases. Some diseases are caused by mutations in the spliceosome components or RBPs, while others by mutations in the cis-elements such as splice sites, branchpoint, or the various splicing enhancers and silencers. Although current approaches to treating these diseases, such as CRISPR-based genome editing, virus-aided gene therapy, or a variety of oligonucleotide-based technologies, continue to improve, they still suffer major technical and clinical challenges. In particular, oligonucleotide-based therapeutics show unfavorable pharmacokinetics, can not be orally administered, and can not be delivered effectively to many tissues, especially the brain. Small-molecule drugs have excellent pharmacokinetics, effective delivery, and bioavailability, and have only recently become available for modulating RNA splicing. Yet, the currently molecules come from a few limited chemical series. Thus, there is a great need to develop additional small molecule splicing modulators (SMSMs).
Almost 50 inherited disorders in humans result from an increase in the number of copies of single repeats in genomic DNA. These DNA repeats appear to be predisposed to such expansion because they have unusual structural characteristics, which disrupt cellular replication, repair, and recombination machinery. The presence of expanding DNA repeats alters gene expression in human cells, leading to disease.
One of these inherited disorders is Huntington's disease (HD). HD is a deadly neurodegenerative disorder with no cure associated with cognitive impairment, dementia, and loss of motor coordination. It is characterized by the progressive and hereditary increase in the length of the CAG trinucleotide repeats that encode a stretch of polyglutamine, in the Huntington gene (HTT) coding region. These repeats can increase in number from one generation to the next. The normal allele of the HTT gene contains fewer than 36 CAG repeats, while the mutant allele contains more than 36 repeats. Most HD patients carry one normal allele and one mutant allele that causes the disease. Functionally, the aberrant accumulation of CAG repeats is believed to confer a toxic gain of function on the mutant HD protein, causing it to aggregate, form protein deposits (ie, inclusion bodies), and induce cell death. The severity of the disease generally reflects the extent of repeat expansion in the mutant HTT protein.
Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are associated with long repeats of polyCUG and polyCCUG in the 3′-UTR and intron 1 regions of the transcription of myotonic dystrophy protein kinase (DMPK) and protein 9 of zinc finger (ZNF9), respectively. While normal individuals have up to 30 CTG repeats, DMI patients have a higher number of repeats ranging from 50 to thousands. The severity of the disease and the age of onset correlate with the number of repetitions. Adult-onset patients show milder symptoms and have fewer than 100 repeats, juvenile-onset DM1 patients have up to 500 repeats, and congenital cases typically have around 1,000 CTG repeats. Expanded transcripts containing CUG repeats form a secondary structure, accumulate in the nucleus as nuclear foci, and sequester RNA-binding proteins (RNA-BP).
Besides the extra copies of repeats inherited at birth, for many repeat expansion diseases, repeats are highly unstable and their repeat numbers continue to expand throughout the life time of patients. This repeat instability has been shown experimentally to be mediated by proteins in DNA mismatch repair (MMR) processes including PMS1, MLH1, MSH3. Human genetics data from genome-wide association studies has indicated that variants in MMR proteins are associated with clinically relevant HD symptomatology including age at motor onset, rate of progression and somatic instability. Knocking down and knocking out MMR genes have been shown to stall or slow the somatic repeat expansion in various preclinical models of repeat expansion diseases. Thus, there is a need for splicing modulators of MMR genes as potential therapeutic agents to treat a variety of repeat expansion diseases.
Here we describe a series of novel small molecule splicing modulators (SMSMs), which can be used to treat a wide variety of diseases, including neurodegenerative and repeat expansion diseases. These SMSMs target regions of a primary RNA transcript that are cis-elements, such as splice sites, branch points, splicing enhancers, or splicing silencers. These regions may contain unpaired nucleotides in an RNA duplex, called bulges. The bulges may be naturally occurring or caused by diseases. When the SMSMs come into contact with the RNA transcript, it may be bound by the spliceosome or the other trans-factors, most notably RNA-binding proteins (RBPs). The SMSMs reported herein may cause an alteration in the sequence or abundance of the mature transcript, which may, in turn, cause a difference in the sequence or abundance of the functional protein should the transcript be protein-coding or the sequence or abundance of the functional RNA should the transcript be non-coding.
In some aspects, the present disclosure provides, inter alia, a compound of Formula (I):
or a pharmaceutically acceptable salt thereof, wherein:
In some aspects, the present disclosure provides a compound obtainable by, or obtained by, a method for preparing a compound as described here (e.g., a method comprising one or more steps described in herein).
In some aspects, the present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, and a pharmaceutically acceptable diluent or carrier.
In some aspects, the present disclosure provides an intermediate as described herein, being suitable for use in a method for preparing a compound as described herein (e.g., the intermediate is selected from the intermediates described herein).
In some aspects, the present disclosure provides a method of treating or preventing a disease or disorder disclosed herein in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt, solvate, or prodrug thereof, or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a method of treating a disease or disorder disclosed herein in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt, solvate, or prodrug thereof, or a pharmaceutical composition of the present disclosure.
In some aspects, the present disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt, solvate, or prodrug thereof for use in treating or preventing a disease or disorder disclosed herein.
In some aspects, the present disclosure provides a compound of the present disclosure or a pharmaceutically acceptable salt, solvate, or prodrug thereof for use in treating a disease or disorder disclosed herein.
In some aspects, the present disclosure provides use of a compound of the present disclosure or a pharmaceutically acceptable salt, solvate, or prodrug thereof for treating or preventing a disease or disorder disclosed herein.
In some aspects, the present disclosure provides use of a compound of the present disclosure or a pharmaceutically acceptable salt, solvate, or prodrug thereof for treating a disease or disorder disclosed herein.
In some aspects, the present disclosure provides use of a compound of the present disclosure or a pharmaceutically acceptable salt, solvate, or prodrug thereof in the manufacture of a medicament for treating or preventing a disease or disorder disclosed herein.
In some aspects, the present disclosure provides use of a compound of the present disclosure or a pharmaceutically acceptable salt, solvate, or prodrug thereof in the manufacture of a medicament for treating a disease or disorder disclosed herein.
In some aspects, the present disclosure provides a method of preparing a compound of the present disclosure.
In some aspects, the present disclosure provides a method of preparing a compound, comprising one or more steps described herein.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
Other features and advantages of the disclosure will be apparent from the following detailed description and claims.
Compounds described herein are generally designed to treat diseases and disorders disclosed herein.
Unless otherwise stated, the following terms used in the specification and claims have the following meanings set out below.
As used herein, “alkyl”, “C1, C2, C3, C4, C5, C6, or C7 alkyl” or “C1-C7 alkyl” is intended to include C1, C2, C3, C4, C5, C6, or C7 straight chain (linear) saturated aliphatic hydrocarbon groups and C3, C4, C5, C6, or C7 branched saturated aliphatic hydrocarbon groups. For example, C1-C7 alkyl is intended to include C1, C2, C3, C4, C5, C6, and C7 alkyl groups. Examples of alkyl include, moieties having from one to six carbon atoms, such as, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, or n-hexyl. In some embodiments, a straight chain or branched alkyl has six or fewer carbon atoms (e. g., C1-C6 for straight chain, C3-C6 for branched chain), and in another embodiment, a straight chain or branched alkyl has four or fewer carbon atoms.
As used herein, “alkenyl” is intended to include straight-chain or branched hydrocarbon groups having from 2 to 6 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds (“C2-C6alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C2-C6alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like.
As used herein, “alkynyl” is intended to include straight-chain or branched hydrocarbon groups having from 2 to 6 carbon atoms, one or more carbon-carbon triple bonds, and optionally one or more double bonds (“C2-C6 alkynyl”). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C2-C4alkynyl groups include, without limitation, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like.
As used herein, the term “optionally substituted alkyl” refers to unsubstituted alkyl or alkyl having designated substituents replacing one or more hydrogen atoms on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulphydryl, alkylthio, arylthio, thiocarboxylate, sulphates, alkylsulphinyl, sulphonato, sulphamoyl, sulphonamido, nitro, trifluoromethyl, cyano, azido, heterocycloalkyl, alkylaryl, or an aromatic or heteroaromatic moiety.
Other optionally substituted moieties (such as optionally substituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl) include both the unsubstituted moieties and the moieties having one or more of the designated substituents. For example, substituted heterocycloalkyl includes those substituted with one or more alkyl groups, such as 2,2,6,6-tetramethyl-piperidinyl and 2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridinyl.
As used herein, “heteroalkyl”, “C1, C2, C3, C4, C5, C6, C7, or C8 heteroalkyl” or “C1-C8 heteroalkyl” is intended to include C1, C2, C3, C4, C5, C6, C7, or C8 straight chain (linear) saturated aliphatic hydrocarbon groups and C3, C4, C5, C6, C7, or C8 branched saturated aliphatic hydrocarbon groups, in which at least one of the carbons has been replaced with N. O, or S. The heteroatom will be bonded to any required hydrogens to complete the heteroatom's valence (e.g., a CH2 may be replaced with an “O” or a “NH”, a CH may be replaced with an N, etc.)). Such substituents can include, for example, —O—CH(CH3)2, —CH2—N(CH3)—CH2CH2OCH3, —S—CH2CH2—O—CH2CH3, and so forth.
As used herein, “heteroalkylene” is a bivalent heteroalkyl group with two open valences. Such substituents can include, for example, —CH2—O—CH2—, —O—CH2CH(CH)—NH—CH2—, —CH2—O—CH2CH2—S—CH2—, etc.
As used herein, the term “alkoxy” refers to the group —OR where R is alkyl. Particular alkoxy groups are methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, and 1,2-dimethylbutoxy. Particular alkoxy groups are lower alkoxy, i.e., with between 1 and 6 carbon atoms.
As used herein, the term “cycloalkyl” refers to a saturated or partially unsaturated hydrocarbon monocyclic or polycyclic (e.g., fused, bridged, or spiro rings) system having 3 to 30 carbon atoms (e.g., C3-C12, C3-C10, or C3-C8). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, 1,2,3,4-tetrahydronaphthalenyl, and adamantyl.
In the case of polycyclic cycloalkyl, only one of the rings in the cycloalkyl needs to be non-aromatic.
As used herein, the term “heterocycloalkyl” or “heterocyclyl” refers to a saturated or partially unsaturated 3-8 membered monocyclic, 7-12 membered bicyclic (fused, bridged, or spiro rings), or 11-14 membered tricyclic ring system (fused, bridged, or spiro rings) having one or more heteroatoms (such as O, N, S, P, or Se), e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or e.g., 1, 2, 3, 4, 5, or 6 heteroatoms, independently selected from the group consisting of nitrogen, oxygen and sulphur, unless specified otherwise. Examples of heterocycloalkyl groups include, but are not limited to, piperidinyl, piperazinyl, pyrrolidinyl, dioxanyl, tetrahydrofuranyl, isoindolinyl, indolinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl, oxiranyl, azetidinyl, oxetanyl, thietanyl, 1,2,3,6-tetrahydropyridinyl, tetrahydropyranyl, dihydropyranyl, pyranyl, morpholinyl, tetrahydrothiopyranyl, 1,4-diazepanyl, 1,4-oxazepanyl, 2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, 2-oxa-6-azaspiro[3.3]heptanyl, 2,6-diazaspiro[3.3]heptanyl, 1,4-dioxa-8-azaspiro[4.5]decanyl, 1,4-dioxaspiro[4.5]decanyl, 1-oxaspiro[4.5]decanyl, I-azaspiro[4.5]decanyl, 3′H-spiro[cyclohexane-1,1′-isobenzofuran]-yl, 7′H-spiro[cyclohexane-1,5′-furo[3,4-b]pyridin]-yl, 3′H-spiro[cyclohexane-1,1′-furo[3,4-c]pyridin]-yl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[3.1.0]hexan-3-yl, 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazolyl, 3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidinyl, 4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridinyl, 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidinyl, 2-azaspiro[3.3]heptanyl, 2-methyl-2-azaspiro[3.3]heptanyl, 2-azaspiro[3.5]nonanyl, 2-methyl-2-azaspiro[3.5]nonanyl, 2-azaspiro[4.5]decanyl, 2-methyl-2-azaspiro[4.5]decanyl, 2-oxa-azaspiro[3.4]octanyl, 2-oxa-azaspiro[3.4]octan-6-yl, and the like. In the case of multicyclic heterocycloalkyl, only one of the rings in the heterocycloalkyl needs to be non-aromatic (e.g., 4,5,6,7-tetrahydrobenzo[c]isoxazolyl).
As used herein, the term “cycloalkyloxy” refers to a —O-cycloalkyl group in which cycloalkyl is as defined herein. Preferably the cycloalkyloxy is a C1-C6cycloalkyloxy. Examples include, but are not limited to, cyclopropanoxy and cyclobutanoxy.
As used herein, the term “aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system. Examples of aryl groups include, but are not limited to, phenyl, naphthyl and the like. Conveniently, an aryl is phenyl.
As used herein, the term “heteroaryl” is intended to include a stable 5-, 6-, or 7-membered monocyclic or 7-, 8-, 9-, 10-, 11- or 12-membered bicyclic aromatic heterocyclic ring which consists of carbon atoms and one or more heteroatoms, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or e.g., 1, 2, 3, 4, 5, or 6 heteroatoms, independently selected from the group consisting of nitrogen, oxygen and sulphur. The nitrogen atom may be substituted or unsubstituted (i.e., N or NR wherein R is H or other substituents, as defined). The nitrogen and sulphur heteroatoms may optionally be oxidised (i.e., N→O and S(O)p, where p=1 or 2).
It is to be noted that total number of S and O atoms in the aromatic heterocycle is not more than 1. Examples of heteroaryl groups include pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like. Heteroaryl groups can also be fused or bridged with alicyclic or heterocyclic rings, which are not aromatic so as to form a multicyclic system (e.g., 4,5,6,7-tetrahydrobenzo[c]isoxazolyl).
Furthermore, the terms “aryl” and “heteroaryl” include multicyclic aryl and heteroaryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, quinoline, isoquinoline, naphthridine, indole, benzofuran, purine, benzofuran, deazapurine, indolizine.
The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring can be substituted atone or more ring positions (e.g., the ring-forming carbon or heteroatom such as N) with such substituents as described above, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulphydryl, alkylthio, arylthio, thiocarboxylate, sulphates, alkylsulphinyl, sulphonato, sulphamoyl, sulphonamido, nitro, trifluoromethyl, cyano, azido, heterocycloalkyl, alkylaryl, or an aromatic or heteroaromatic moiety. Aryl and heteroaryl groups can also be fused or bridged with alicyclic or heterocyclic rings, which are not aromatic so as to form a multicyclic system (e.g., tetralin, methylenedioxyphenyl such as benzo[d][1,3]dioxole-5-yl).
As used herein, the term “substituted,” means that any one or more hydrogen atoms on the designated atom is replaced with a selection from the indicated groups, provided that the designated atom's normal valency is not exceeded, and that the substitution results in a stable compound. When a substituent is oxo or keto (i.e., ═O), then 2 hydrogen atoms on the atom are replaced. Keto substituents are not present on aromatic moieties. Ring double bonds, as used herein, are double bonds that are formed between two adjacent ring atoms (e.g., C═C, C═N or N═N). “Stable compound” and “stable structure” are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
When a bond to a substituent is shown to cross a bond connecting two atoms in a ring, then such substituent may be bonded to any atom in the ring. When a substituent is listed without indicating the atom via which such substituent is bonded to the rest of the compound of a given formula, then such substituent may be bonded via any atom in such formula. Combinations of substituents and/or variables are permissible, but only if such combinations result in stable compounds.
When any variable (e.g., R) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-2 R moieties, then the group may optionally be substituted with up to two R moieties and R at each occurrence is selected independently from the definition of R. Also, combinations of substituents and/or variables are permissible, but only if such combinations result in stable compounds.
As used herein, the term “hydroxy” or “hydroxyl” includes groups with an —OH or —O−.
As used herein, the term “cyano” refers to the group —CN.
As used herein, the term “halo” or “halogen” refers to fluoro, chloro, bromo and iodo.
As used herein, the term “haloalkyl” refers to a branched or unbranched alkyl substituted with one or more halogens. For example, a C1-7haloalkyl is an alkyl group of from one to seven carbons wherein at least one H is substituted by a halogen. Examples of haloalkyl include but are not limited to CFH2, CF2H, CF3, CH2CF3, CF2CF3, C(F)(CH3)2, CH2CH2Br, CH(I)CH2F, and CH2Cl.
As used herein, the term “optionally substituted haloalkyl” refers to unsubstituted haloalkyl having designated substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulphydryl, alkylthio, arylthio, thiocarboxylate, sulphates, alkylsulphinyl, sulphonato, sulphamoyl, sulphonamido, nitro, trifluoromethyl, cyano, azido, heterocycloalkyl, alkylaryl, or an aromatic or heteroaromatic moiety.
As used herein, the expressions “one or more of A, B, or C,” “one or more A, B, or C,” “one or more of A. B, and C,” “one or more A, B, and C,” “selected from the group consisting of A, B, and C”, “selected from A, B, and C”, and the like are used interchangeably and all refer to a selection from a group consisting of A, B, and/or C, i.e., one or more As, one or more Bs, one or more Cs, or any combination thereof, unless indicated otherwise.
It is to be understood that the present disclosure provides methods for the synthesis of the compounds of any of the Formulae described herein. The present disclosure also provides detailed methods for the synthesis of various disclosed compounds of the present disclosure according to the following schemes as well as those shown in the Examples.
It is to be understood that, throughout the description, where compositions are described as having, including, or comprising specific components, it is contemplated that compositions also consist essentially of, or consist of, the recited components. Similarly, where methods or processes are described as having, including, or comprising specific process steps, the processes also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions can be conducted simultaneously.
It is to be understood that the synthetic processes of the disclosure can tolerate a wide variety of functional groups, therefore various substituted starting materials can be used. The processes generally provide the desired final compound at or near the end of the overall process, although it may be desirable in certain instances to further convert the compound to a pharmaceutically acceptable salt thereof.
It is to be understood that compounds of the present disclosure can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or from readily prepared intermediates, by employing standard synthetic methods and procedures either known to those skilled in the art, or which will be apparent to the skilled artisan in light of the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be obtained from the relevant scientific literature or from standard textbooks in the field. Although not limited to any one or several sources, classic texts such as Smith, M. B., March, J., March's Advanced Organic Chemistry: Reactions. Mechanisms, and Structure, 5th edition, John Wiley & Sons: New York, 2001; Greene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons: New York, 1999; R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989): L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), incorporated by reference herein, are useful and recognised reference textbooks of organic synthesis known to those in the art.
One of ordinary skill in the art will note that, during the reaction sequences and synthetic schemes described herein, the order of certain steps may be changed, such as the introduction and removal of protecting groups. One of ordinary skill in the art will recognise that certain groups may require protection from the reaction conditions via the use of protecting groups. Protecting groups may also be used to differentiate similar functional groups in molecules. A list of protecting groups and how to introduce and remove these groups can be found in Greene, T. W., Wuts. P. G. M., Protective Groups n Organic Synthesis, 3rd edition, John Wiley & Sons: New York, 1999.
It is to be understood that, unless otherwise stated, any description of a method of treatment includes use of the compounds to provide such treatment or prophylaxis as is described herein, as well as use of the compounds to prepare a medicament to treat or prevent such condition. It is to be understood that, unless otherwise stated, any description of a method of treatment includes use of the compounds to provide such treatment or prophylaxis as is described herein, as well as use of the compounds to prepare a medicament to treat such condition. The treatment includes treatment of human or non-human animals including rodents and other disease models.
As used herein, the term “subject” is interchangeable with the term “subject in need thereof”, both of which refer to a subject having a disease or having an increased risk of developing the disease. A “subject” includes a mammal. The mammal can be e.g., a human or appropriate non-human mammal, such as primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep or a pig. The subject can also be a bird or fowl. In one embodiment, the mammal is a human. A subject in need thereof can be one who has been previously diagnosed or identified as having a disease or disorder disclosed herein. A subject in need thereof can also be one who is suffering from a disease or disorder disclosed herein. Alternatively, a subject in need thereof can be one who has an increased risk of developing such disease or disorder relative to the population at large (i.e., a subject who is predisposed to developing such disorder relative to the population at large). A subject in need thereof can have a refractory or resistant a disease or disorder disclosed herein (i.e., a disease or disorder disclosed herein that does not respond or has not yet responded to treatment). The subject may be resistant at start of treatment or may become resistant during treatment. In some embodiments, the subject in need thereof received and failed all known effective therapies for a disease or disorder disclosed herein. In some embodiments, the subject in need thereof received at least one prior therapy.
As used herein, the term “treating” or “Treat” describes the management and care of a subject for the purpose of combating a disease, condition, or disorder and includes the administration of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term “treat” can also include treatment of a cell in vitro or an animal model.
It is to be appreciated that references to “treating” or “treatment” include the alleviation of established symptoms of a condition. “Treating” or “treatment” of a state, disorder or condition therefore includes: (1) delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition. (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving or attenuating the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.
It is to be understood that a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, can or may also be used to prevent a relevant disease, condition or disorder, or used to identify suitable candidates for such purposes.
As used herein, the term “preventing,” “prevent,” or “protecting against” describes reducing or eliminating the onset of the symptoms or complications of such disease, condition or disorder.
It is to be understood that one skilled in the art may refer to general reference texts for detailed descriptions of known techniques discussed herein or equivalent techniques. These texts include Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2000); Coligan et al., Current Protocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., Current Protocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., The Pharmacological Basis of Therapeutics (1975). Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th edition (1990). These texts can, of course, also be referred to in making or using an aspect of the disclosure.
It is to be understood that the present disclosure also provides pharmaceutical compositions comprising any compound described herein in combination with at least one pharmaceutically acceptable excipient or carrier.
As used herein, the term “pharmaceutical composition” is a formulation containing the compounds of the present disclosure in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler or a vial. The quantity of active ingredient (e.g., a formulation of the disclosed compound or salt, hydrate, solvate or isomer thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the subject. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational, buccal, sublingual, intrapleural, intrathecal, intranasal, and the like. Dosage forms for the topical or transdermal administration of a compound of this disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. In one embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.
As used herein, the term “pharmaceutically acceptable” refers to those compounds, anions, cations, materials, compositions, carriers, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
As used herein, the term “pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable excipient” as used in the specification and claims includes both one and more than one such excipient.
It is to be understood that a pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., ingestion), inhalation, transdermal (topical), and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulphite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
It is to be understood that a compound or pharmaceutical composition of the disclosure can be administered to a subject in many of the well-known methods currently used for chemotherapeutic treatment. For example, a compound of the disclosure may be injected into the blood stream or body cavities or taken orally or applied through the skin with patches. The dose chosen should be sufficient to constitute effective treatment but not so high as to cause unacceptable side effects. The state of the disease condition (e.g., a disease or disorder disclosed herein) and the health of the subject should preferably be closely monitored during and for a reasonable period after treatment.
As used herein, the term “therapeutically effective amount”, refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend upon the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.
A “therapeutically effective amount” means the amount of a compound that, when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.
It is to be understood that, for any compound, the therapeutically effective amount can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually rats, mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
Therapeutic/prophylactic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The dosage may vary within this range depending upon the dosage form employed, sensitivity of the subject, and the route of administration.
Dosage and administration are adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
The pharmaceutical compositions containing active compounds of the present disclosure may be manufactured in a manner that is generally known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilising processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and/or auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically. Of course, the appropriate formulation is dependent upon the route of administration chosen.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible pharmaceutically acceptable carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin, or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebuliser.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The active compounds can be prepared with pharmaceutically acceptable carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved.
In therapeutic applications, the dosages of the pharmaceutical compositions used in accordance with the disclosure vary depending on the agent, the age, weight, and clinical condition of the recipient subject, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage.
Generally, the dose should be sufficient to result in slowing, and preferably regressing, the symptoms of the disease or disorder disclosed herein and also preferably causing complete regression of the disease or disorder. An effective amount of a pharmaceutical agent is that which provides an objectively identifiable improvement as noted by the clinician or other qualified observer. Improvement in survival and growth indicates regression. As used herein, the term “dosage effective manner” refers to amount of an active compound to produce the desired biological effect in a subject or cell.
It is to be understood that the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
It is to be understood that, for the compounds of the present disclosure being capable of further forming salts, all of these forms are also contemplated within the scope of the claimed disclosure.
As used herein, the term “pharmaceutically acceptable salts” refer to derivatives of the compounds of the present disclosure wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 2-acetoxybenzoic, 2-hydroxyethane sulphonic, acetic, ascorbic, benzene sulphonic, benzoic, bicarbonic, carbonic, citric, edetic, ethane disulphonic, 1,2-ethane sulphonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, glycoloylarsanilic, hexylresorcinic, hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic, isethionic, lactic, lactobionic, lauryl sulphonic, maleic, malic, mandelic, methane sulphonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic, salicylic, stearic, subacetic, succinic, sulphamic, sulphanilic, sulphuric, tannic, tartaric, toluene sulphonic, and the commonly occurring amine acids, e.g., glycine, alanine, phenylalanine, arginine, etc.
In some embodiments, the pharmaceutically acceptable salt is a sodium salt, a potassium salt, a calcium salt, a magnesium salt, a diethylamine salt, a choline salt, a meglumine salt, a benzathine salt, a tromethamine salt, an ammonia salt, an arginine salt, or a lysine salt.
Other examples of pharmaceutically acceptable salts include hexanoic acid, cyclopentane propionic acid, pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulphonic acid, 2-naphthalenesulphonic acid, 4-toluenesulphonic acid, camphorsulphonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, muconic acid, and the like. The present disclosure also encompasses salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. In the salt form, it is understood that the ratio of the compound to the cation or anion of the salt can be 1:1, or any ratio other than 1:1, e.g., 3:1, 2:1, 1:2, or 1:3.
The compounds, or pharmaceutically acceptable salts thereof, are administered orally, nasally, transdermally, pulmonary, inhalationally, buccally, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally and parenterally. In one embodiment, the compound is administered orally. One skilled in the art will recognise the advantages of certain routes of administration.
A salt, for example, can be formed between an anion and a positively charged group (e.g., amino) on a substituted compound disclosed herein. Suitable anions include chloride, bromide, iodide, sulphate, bisulphate, sulphamate, nitrate, phosphate, citrate, methanesulphonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulphonate, and acetate (e.g., trifluoroacetate).
As used herein, the term “pharmaceutically acceptable anion” refers to an anion suitable for forming a pharmaceutically acceptable salt. Likewise, a salt can also be formed between a cation and a negatively charged group (e.g., carboxylate) on a substituted compound disclosed herein. Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as tetramethylammonium ion or diethylamine ion. The substituted compounds disclosed herein also include those salts containing quaternary nitrogen atoms.
It is to be understood that the compounds of the present disclosure, for example, the salts of the compounds, can exist in either hydrated or unhydrated (the anhydrous) form or as solvates with other solvent molecules. Nonlimiting examples of hydrates include monohydrates, dihydrates, etc. Nonlimiting examples of solvates include ethanol solvates, acetone solvates, etc.
As used herein, the term “solvate” means solvent addition forms that contain either stoichiometric or non-stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water the solvate formed is a hydrate; and if the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one or more molecules of water with one molecule of the substance in which the water retains its molecular state as H2O.
As used herein, the term “analog” refers to a chemical compound that is structurally similar to another but differs slightly in composition (as in the replacement of one atom by an atom of a different element or in the presence of a particular functional group, or the replacement of one functional group by another functional group). Thus, an analog is a compound that is similar or comparable in function and appearance, but not in structure or origin to the reference compound.
As used herein, the term “derivative” refers to compounds that have a common core structure and are substituted with various groups as described herein.
As used herein, the term “bioisostere” refers to a compound resulting from the exchange of an atom or of a group of atoms with another, broadly similar, atom or group of atoms. The objective of a bioisosteric replacement is to create a new compound with similar biological properties to the parent compound. The bioisosteric replacement may be physicochemically or topologically based. Examples of carboxylic acid bioisosteres include, but are not limited to, acyl sulphonamides, tetrazoles, sulphonates and phosphonates. See. e.g., Patani and LaVoie, Chem. Rev. 96, 3147-3176, 1996.
It is also to be understood that certain compounds of any one of the Formulae disclosed herein may exist in solvated as well as unsolvated forms such as, for example, hydrated forms. A suitable pharmaceutically acceptable solvate is, for example, a hydrate such as hemi-hydrate, a mono-hydrate, a di-hydrate or a tri-hydrate.
Reference to Formula (I) can include subformulas of Formula (I), e.g., Formulas (Ix), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), (Im), (In), (Io), (Ip), and (Iq).
Compounds of any one of the Formulae disclosed herein may exist in a number of different tautomeric forms and references to compounds of Formula (I) include all such forms. For the avoidance of doubt, where a compound can exist in one of several tautomeric forms, and only one is specifically described or shown, all others are nevertheless embraced by Formula (I). Examples of tautomeric forms include keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, and nitro/aci-nitro.
Compounds of any one of the Formulae disclosed herein containing an amine function may also form N-oxides. A reference herein to a compound of Formula (I) that contains an amine function also includes the N-oxide. Where a compound contains several amine functions, one or more than one nitrogen atom may be oxidised to form an N-oxide. Particular examples of N-oxides are the N-oxides of a tertiary amine or a nitrogen atom of a nitrogen-containing heterocycle. N-oxides can be formed by treatment of the corresponding amine with an oxidising agent such as hydrogen peroxide or a peracid (e.g. a peroxycarboxylic acid), see for example Advanced Organic Chemistry, by Jerry March, 4th Edition, Wiley Interscience, pages. More particularly, N-oxides can be made by the procedure of L. W. Deady (Syn. Comm. 1977, 7, 509-514) in which the amine compound is reacted with meta-chloroperoxybenzoic acid (mCPBA), for example, in an inert solvent such as dichloromethane.
As used herein, the term “isomerism” means compounds that have identical molecular formulae but differ in the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.” Stereoisomers that are not mirror images of one another are termed “diastereoisomers,” and stereoisomers that are non-superimposable mirror images of each other are termed “enantiomers” or sometimes optical isomers. A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is termed a “racemic mixture.”
As used herein, the term “chiral center” refers to a carbon atom bonded to four nonidentical substituents.
As used herein, the term “chiral isomer” means a compound with at least one chiral center. Compounds with more than one chiral center may exist either as an individual diastereomer or as a mixture of diastereomers, termed “diastereomeric mixture.” When one chiral center is present, a stereoisomer may be characterised by the absolute configuration (R or S) of that chiral center. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385: errata 511: Cahn et al., Angew. Chem. 1966, 78, 413; Calm and Ingold, J Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J Chem. Educ. 1964, 41, 116).
As used herein, the term “geometric isomer” means the diastereomers that owe their existence to hindered rotation about double bonds or a cycloalkyl linker (e.g., 1,3-cyclobutyl). These configurations are differentiated in their names by the prefixes cis and trans, or Z and E, which indicate that the groups are on the same or opposite side of the double bond in the molecule according to the Cahn-Ingold-Prelog rules.
It is to be understood that the compounds of the present disclosure may be depicted as different chiral isomers or geometric isomers. It is also to be understood that when compounds have chiral isomeric or geometric isomeric forms, all isomeric forms are intended to be included in the scope of the present disclosure, and the naming of the compounds does not exclude any isomeric forms, it being understood that not all isomers may have the same level of activity.
It is to be understood that the structures and other compounds discussed in this disclosure include all atropic isomers thereof. It is also to be understood that not all atropic isomers may have the same level of activity.
As used herein, the term “atropic isomers” are a type of stereoisomer in which the atoms of two isomers are arranged differently in space. Atropic isomers owe their existence to a restricted rotation caused by hindrance of rotation of large groups about a central bond.
Such atropic isomers typically exist as a mixture, however as a result of recent advances in chromatography techniques, it has been possible to separate mixtures of two atropic isomers in select cases.
As used herein, the term “tautomer” is one of two or more structural isomers that exist in equilibrium and is readily converted from one isomeric form to another. This conversion results in the formal migration of a hydrogen atom accompanied by a switch of adjacent conjugated double bonds. Tautomers exist as a mixture of a tautomeric set in solution. In solutions where tautomerisation is possible, a chemical equilibrium of the tautomers will be reached. The exact ratio of the tautomers depends on several factors, including temperature, solvent and pH. The concept of tautomers that are interconvertible by tautomerisations is called tautomerism. Of the various types of tautomerism that are possible, two are commonly observed. In keto-enol tautomerism a simultaneous shift of electrons and a hydrogen atom occurs. Ring-chain tautomerism arises as a result of the aldehyde group (—CHO) in a sugar chain molecule reacting with one of the hydroxy groups (—OH) in the same molecule to give it a cyclic (ring-shaped) form as exhibited by glucose.
It is to be understood that the compounds of the present disclosure may be depicted as different tautomers. It should also be understood that when compounds have tautomeric forms, all tautomeric forms are intended to be included in the scope of the present disclosure, and the naming of the compounds does not exclude any tautomer form. It will be understood that certain tautomers may have a higher level of activity than others.
Compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed “isomers”. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers”. Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers”. When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterised by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarised light and designated as dextrorotatory or levorotatory (i.e., as (+) or (−)-isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture”.
The compounds of this disclosure may possess one or more asymmetric centers; such compounds can therefore be produced as individual (R)- or (S)-stereoisomers or as mixtures thereof. Unless indicated otherwise, the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures, racemic or otherwise, thereof. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art (see discussion in Chapter 4 of “Advanced Organic Chemistry”, 4th edition J. March, John Wiley and Sons. New York, 2001), for example by synthesis from optically active starting materials or by resolution of a racemic form. Some of the compounds of the disclosure may have geometric isomeric centers (E- and Z-isomers).
Accordingly, the present disclosure includes those compounds of any one of the Formulae disclosed herein as defined hereinbefore when made available by organic synthesis and when made available within the human or animal body by way of cleavage of a prodrug thereof. Accordingly, the present disclosure includes those compounds of any one of the Formulae disclosed herein that are produced by organic synthetic means and also such compounds that are produced in the human or animal body by way of metabolism of a precursor compound, that is a compound of any one of the Formulae disclosed herein may be a synthetically-produced compound or a metabolically-produced compound.
The dosage regimen utilising the compounds is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the subject; the severity of the condition to be treated: the route of administration; the renal and hepatic function of the subject; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the condition. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to counter or arrest the progress of the condition.
Techniques for formulation and administration of the disclosed compounds of the disclosure can be found in Remington: the Science and Practice of Pharmacy, 19th edition, Mack Publishing Co., Easton, PA (1995). In an embodiment, the compounds described herein, and the pharmaceutically acceptable salts thereof, are used in pharmaceutical preparations in combination with a pharmaceutically acceptable carrier or diluent. Suitable pharmaceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous or organic solutions. The compounds will be present in such pharmaceutical compositions in amounts sufficient to provide the desired dosage amount in the range described herein.
All percentages and ratios used herein, unless otherwise indicated, are by weight. Other features and advantages of the present disclosure are apparent from the different examples. The provided examples illustrate different components and methodology useful in practicing the present disclosure. The examples do not limit the claimed disclosure. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present disclosure.
In the synthetic schemes described herein, compounds may be drawn with one particular configuration for simplicity. Such particular configurations are not to be construed as limiting the disclosure to one or another isomer, tautomer, regioisomer or stereoisomer, nor does it exclude mixtures of isomers, tautomers, regioisomers or stereoisomers; however, it will be understood that a given isomer, tautomer, regioisomer or stereoisomer may have a higher level of activity than another isomer, tautomer, regioisomer or stereoisomer.
All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The invention having now been described by way of written description, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples below are for purposes of illustration and not limitation of the claims that follow.
As use herein, the phrase “compound of the disclosure” refers to those compounds which are disclosed herein, both generically and specifically.
In some aspects, the present disclosure provides, inter alia, a compound of formula (I):
wherein A is optionally substituted with 1-4 R9;
In some aspects, the present disclosure provides, inter alia, a compound of formula (I):
In some aspects, the present disclosure provides, inter aha, a compound of Formula (I):
In some aspects, the present disclosure provides, inter alia, a compound of formula (Ix):
In some embodiments, 0, 1, or 2 of X, Y, and Z are N.
In some embodiments, the compound is of Formula (Ia),
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound is of Formula (Ib),
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound is of Formula (Ic),
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound is of Formula (Id),
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound is of Formula (Ie),
or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound is of Formula (If),
or a pharmaceutically acceptable salt thereof.
In some embodiments, R1, R2, R3 and R4 are each independently selected from the group consisting of H halogen, C1-C6alkyl, C1-C6haloalkyl, C1-C6alkoxy, and C1-C6cycloalkyloxy. In some embodiments, R1, R2, R3 and R4 are each independently selected from the group consisting of H, halogen, C1-C6alkoxy, and C1-C6alkyl. In some embodiments, R1, R2, R3 and R4 are each H.
In some embodiments, R5 is H.
In some embodiments, A is selected from the group consisting of
wherein A is optionally substituted with 1-3 R9.
In some embodiments, A is selected from the group consisting of
wherein A is optionally substituted with 1-3 R9.
In some embodiments, A is selected from the group consisting of
wherein A is optionally substituted with 1-3 R9.
In some embodiments, A is substituted by one R9 selected from the group consisting of halogen and C1-C6alkyl. In some embodiments, A is not substituted by R9.
In some embodiments, R6 is Me.
In some embodiments, R7 is C1-C6alkyl, C1-C6cycloalkyl, or heterocycloalkyl. In some embodiments, R7 is Me, Et, isopropyl, or cyclobutyl.
In some embodiments, A is selected from the group consisting of
In some embodiments A is selected from the group consisting of
In some embodiments, A is
In some embodiments, A is
In some embodiments, A is
In some embodiments, A is
In some embodiments, A is
In some embodiments, A is
In some embodiments, B is NR10R11, wherein
In some embodiments, R10 and R11 are taken together with the nitrogen atom to which they are attached to form a monocyclic heterocycloalkyl of 4-7 ring atoms with 1 or 2 total nitrogen ring atoms and 0 or 1 additional ring heteroatoms selected from O and S, and the heterocycloalkyl is optionally substituted with 1, 2, 3, or 4 R12.
In some embodiments, B is
In some embodiments, each R12 is independently selected from the group consisting of halogen, hydroxy, 4-7-membered monocyclic heterocycloalkyl, C1-C6heteroalkyl, and C1-C6alkyl, wherein alkyl is optionally substituted with one or more halogen, hydroxyl, methoxy. C3-C8cycloalkyl, or NH2, and heterocycloalkyl is optionally substituted with one or more halogen, hydroxyl, methoxy, or C1-C6 alkyl; and
In some embodiments, each R12 is independently selected from the group consisting of halogen, hydroxy, and C1-C6alkyl, wherein alkyl is optionally substituted with one or more halogen, hydroxyl, methoxy, C3-C8cycloalkyl, or NH2; and
In some embodiments, each R12 is independently C1-C6alkyl.
In some embodiments, B is
In some embodiments, R13 is H or unsubstituted C1-C6alkyl.
In some embodiments, each R12 is independently C1-C6alkyl.
In some embodiments, B is a bicyclic 6-14 membered heterocycloalkyl comprising 1, 2 or 3 total nitrogen ring atoms and 0 or 1 additional ring heteroatoms selected from O and S, and the heterocycloalkyl is optionally substituted with 1, 2, 3, or 4 R12.
In some embodiments, each R12 is independently C1-C6alkyl.
In some embodiments, B is NR10R11 and R11 is hydrogen, C1-7alkyl, C1-7haloalkyl, or C3-8cycloalkyl.
In some embodiments, R11 is H or C1-7alkyl, and R10 is C1-C8heteroalkyl comprising at least one nitrogen atom.
In some embodiments, R10 is —(CH2)0-3heterocycloalkyl comprising at least 1 nitrogen ring atom, each R10 optionally substituted with 1 to 6 R12.
In some embodiments, the compound is of formula (Ig)
In some embodiments, the compound is of formula (Ih)
In some embodiments, the compound is of formula (Ii)
In some embodiments, the compound is of formula (Ij)
In some embodiments, the compound is of formula (Ik)
In some embodiments, the compound is of formula (In)
In some embodiments, the compound is of formula (Io)
In some embodiments, the compound is of formula (Ip)
In some embodiments, the compound is of formula (Iq)
In some embodiments, the compound is of formula (Is)
In some embodiments, the compound is of formula (It)
In some embodiments, the compound is of formula (Iu)
In some embodiments, the compound is selected from a compound of Table 1.
In some aspects, the disclosure provides a pharmaceutical composition comprising a compound of the present disclosure, or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients.
In some aspects, the present disclosure provides use of a compound of the present disclosure or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating or preventing a disease or disorder disclosed herein.
In some aspects, the present disclosure provides a method of preparing a compound of the present disclosure.
In some aspects, the present disclosure provides a method of preparing a compound, comprising one or more steps described herein.
In some embodiments, the compound is selected from the compounds described in Table 1 and pharmaceutically acceptable salts thereof.
In some embodiments, the compound is selected from the compounds described in Table 1.
or a pharmaceutically acceptable salt thereof.
For the avoidance of doubt it is to be understood that, where in this specification a group is qualified by “described herein”, the said group encompasses the first occurring and broadest definition as well as each and all of the particular definitions for that group.
The various functional groups and substituents making up the compounds of the Formula (I) are typically chosen such that the molecular weight of the compound does not exceed 1000 daltons. More usually, the molecular weight of the compound will be less than 900, for example less than 800, or less than 750, or less than 700, or less than 650 daltons.
More conveniently, the molecular weight is less than 600 and, for example, is 550 daltons or less.
It will be understood that the compounds of any one of the Formulae disclosed herein and any pharmaceutically acceptable salts thereof, comprise stereoisomers and mixtures of stereoisomers of said compounds.
It is to be understood that the compounds of any Formula described herein include the compounds themselves, as well as their salts, and their solvates, if applicable.
The in vivo effects of a compound of any one of the Formulae disclosed herein may be exerted in part by one or more metabolites that are formed within the human or animal body after administration of a compound of any one of the Formulae disclosed herein. As stated hereinbefore, the in vivo effects of a compound of any one of the Formulae disclosed herein may also be exerted by way of metabolism of a precursor compound (a prodrug).
Suitably, the present disclosure excludes any individual compounds not possessing the biological activity defined herein.
By way of example only, provided is a scheme for preparing the small molecule splicing modulators (SMSMs) described herein.
In some embodiments, a scheme for preparing an SMSM is described herein in
In some aspects, the present disclosure provides a method of preparing a compound of the present disclosure.
In some aspects, the present disclosure provides a method of preparing a compound, comprising one or more steps as described herein.
In some aspects, the present disclosure provides a compound obtainable by, or obtained by, or directly obtained by a method for preparing a compound as described herein.
In some aspects, the present disclosure provides an intermediate as described herein, being suitable for use in a method for preparing a compound as described herein.
The compounds of the present disclosure can be prepared by any suitable technique known in the art. Particular processes for the preparation of these compounds are described further in the accompanying examples.
In the description of the synthetic methods described herein and in any referenced synthetic methods that are used to prepare the starting materials, it is to be understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment and workup procedures, can be selected by a person skilled in the art.
It is understood by one skilled in the art of organic synthesis that the functionality present on various portions of the molecule must be compatible with the reagents and reaction conditions utilized.
It will be appreciated that during the synthesis of the compounds of the disclosure in the processes defined herein, or during the synthesis of certain starting materials, it may be desirable to protect certain substituent groups to prevent their undesired reaction. The skilled chemist will appreciate when such protection is required, and how such protecting groups may be put in place, and later removed. For examples of protecting groups see one of the many general texts on the subject, for example, ‘Protective Groups in Organic Synthesis’ by Theodora Green (publisher: John Wiley & Sons). Protecting groups may be removed by any convenient method described in the literature or known to the skilled chemist as appropriate for the removal of the protecting group in question, such methods being chosen so as to effect removal of the protecting group with the minimum disturbance of groups elsewhere in the molecule. Thus, if reactants include, for example, groups such as amino, carboxy or hydroxy it may be desirable to protect the group in some of the reactions mentioned herein.
By way of example, a suitable protecting group for an amino or alkylamino group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an alkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl, or t-butoxycarbonyl group, an arylmethoxycarbonyl group, for example benzyloxycarbonyl, or an aroyl group, for example benzoyl. The deprotection conditions for the above protecting groups necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or alkoxycarbonyl group or an aroyl group may be removed by, for example, hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide. Alternatively an acyl group such as a tert-butoxycarbonyl group may be removed, for example, by treatment with a suitable acid as hydrochloric, sulphuric or phosphoric acid or trifluoroacetic acid and an arylmethoxycarbonyl group such as a benzyloxycarbonyl group may be removed, for example, by hydrogenation over a catalyst such as palladium on carbon, or by treatment with a Lewis acid for example boron tris(trifluoroacetate). A suitable alternative protecting group for a primary amino group is, for example, a phthaloyl group which may be removed by treatment with an alkylamine, for example dimethylaminopropylamine, or with hydrazine.
A suitable protecting group for a hydroxy group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an aroyl group, for example benzoyl, or an arylmethyl group, for example benzyl. The deprotection conditions for the above protecting groups will necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or an aroyl group may be removed, for example, by hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium, sodium hydroxide or ammonia. Alternatively an arylmethyl group such as a benzyl group may be removed, for example, by hydrogenation over a catalyst such as palladium on carbon.
A suitable protecting group for a carboxy group is, for example, an esterifying group, for example a methyl or an ethyl group which may be removed, for example, by hydrolysis with a base such as sodium hydroxide, or for example a tert-butyl group which may be removed, for example, by treatment with an acid, for example an organic acid such as trifluoroacetic acid, or for example a benzyl group which may be removed, for example, by hydrogenation over a catalyst such as palladium on carbon.
Once a compound of Formula (I) has been synthesised by any one of the processes defined herein, the processes may then further comprise the additional steps of: (i) removing any protecting groups present; (ii) converting the compound of Formula (I) into another compound of Formula (I); and/or (iii) forming a pharmaceutically acceptable salt, hydrate or solvate thereof.
The resultant compounds of Formula (I) can be isolated and purified using techniques well known in the art.
Conveniently, the reaction of the compounds is carried out in the presence of a suitable solvent, which is preferably inert under the respective reaction conditions. Examples of suitable solvents comprise but are not limited to hydrocarbons, such as hexane, petroleum ether, benzene, toluene or xylene; chlorinated hydrocarbons, such as trichlorethylene, 1,2-dichloroethane, tetrachloromethane, chloroform or dichloromethane; alcohols, such as methanol, ethanol, isopropanol, n-propanol, n-butanol or tert-butanol; ethers, such as diethyl ether, diisopropyl ether, tetrahydrofuran (THF), 2-methyltetrahydrofuran, cyclopentylmethyl ether (CPME), methyl tert-butyl ether (MTBE) or dioxane; glycol ethers, such as ethylene glycol monomethyl or monoethyl ether or ethylene glycol dimethyl ether (diglyme); ketones, such as acetone, methylisobutylketone (MIBK) or butanone; amides, such as acetamide, dimethylacetamide, dimethylformamide (DMF) or N-methylpyrrolidinone (NMP); nitriles, such as acetonitrile; sulphoxides, such as dimethyl sulphoxide (DMSO); nitro compounds, such as nitromethane or nitrobenzene; esters, such as ethyl acetate or methyl acetate, or mixtures of the said solvents or mixtures with water.
The reaction temperature is suitably between about −100° C. and 300° C., depending on the reaction step and the conditions used.
Reaction times are generally in the range between a fraction of a minute and several days, depending on the reactivity of the respective compounds and the respective reaction conditions. Suitable reaction times are readily determinable by methods known in the art, for example reaction monitoring. Based on the reaction temperatures given above, suitable reaction times generally lie in the range between 10 minutes and 48 hours.
Moreover, by utilising the procedures described herein, in conjunction with ordinary skills in the art, additional compounds of the present disclosure can be readily prepared. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds. As will be understood by the person skilled in the art of organic synthesis, compounds of the present disclosure are readily accessible by various synthetic routes, some of which are exemplified in the accompanying examples. The skilled person will easily recognise which kind of reagents and reactions conditions are to be used and how they are to be applied and adapted in any particular instance—wherever necessary or useful—in order to obtain the compounds of the present disclosure. Furthermore, some of the compounds of the present disclosure can readily be synthesised by reacting other compounds of the present disclosure under suitable conditions, for instance, by converting one particular functional group being present in a compound of the present disclosure, or a suitable precursor molecule thereof, into another one by applying standard synthetic methods, like reduction, oxidation, addition or substitution reactions; those methods are well known to the skilled person. Likewise, the skilled person will apply—whenever necessary or useful—synthetic protecting (or protective) groups; suitable protecting groups as well as methods for introducing and removing them are well-known to the person skilled in the art of chemical synthesis and are described, in more detail, in, e.g., P. G. M. Wuts, T. W. Greene. “Greene's Protective Groups in Organic Synthesis”, 4th edition (2006) (John Wiley & Sons).
General routes for the preparation of a compound of the application are described herein.
Compounds designed, selected and/or optimised by methods described above, once produced, can be characterised using a variety of assays known to those skilled in the art to determine whether the compounds have biological activity. For example, the molecules can be characterised by conventional assays, including but not limited to those assays described below, to determine whether they have a predicted activity, binding activity and/or binding specificity.
In some aspects, the present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure as an active ingredient. In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one compound of each of the formulae described herein, or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable carriers or excipients. In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one compound selected from Table 1.
As used herein, the term “composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
The compounds of present disclosure can be formulated for oral administration in forms such as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions. The compounds of present disclosure on can also be formulated for intravenous (bolus or in-fusion), intraperitoneal, topical, subcutaneous, intramuscular or transdermal (e.g., patch) administration, all using forms well known to those of ordinary skill in the pharmaceutical arts.
The formulation of the present disclosure may be in the form of an aqueous solution comprising an aqueous vehicle. The aqueous vehicle component may comprise water and at least one pharmaceutically acceptable excipient. Suitable acceptable excipients include those selected from the group consisting of a solubility enhancing agent, chelating agent, preservative, tonicity agent, viscosity/suspending agent, buffer, and pH modifying agent, and a mixture thereof.
Any suitable solubility enhancing agent can be used. Examples of a solubility enhancing agent include cyclodextrin, such as those selected from the group consisting of hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, randomly methylated-β-cyclodextrin, ethylated-β-cyclodextrin, triacetyl-β-cyclodextrin, peracetylated-β-cyclodextrin, carboxymethyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 2-hydroxy-3-(trimethylammonio)propyl-β-cyclodextrin, glucosyl-β-cyclodextrin, sulphated β-cyclodextrin (S-β-CD), maltosyl-β-cyclodextrin, β-cyclodextrin sulphobutyl ether, branched-β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, randomly methylated-γ-cyclodextrin, and trimethyl-γ-cyclodextrin, and mixtures thereof.
Any suitable chelating agent can be used. Examples of a suitable chelating agent include those selected from the group consisting of ethylenediaminetetraacetic acid and metal salts thereof, disodium edetate, trisodium edetate, and tetrasodium edetate, and mixtures thereof.
Any suitable preservative can be used. Examples of a preservative include those selected from the group consisting of quaternary ammonium salts such as benzalkonium halides (preferably benzalkonium chloride), chlorhexidine gluconate, benzethonium chloride, cetyl pyridinium chloride, benzyl bromide, phenylmercury nitrate, phenylmercury acetate, phenylmercury neodecanoate, merthiolate, methylparaben, propylparaben, sorbic acid, potassium sorbate, sodium benzoate, sodium propionate, ethyl p-hydroxybenzoate, propylaminopropyl biguanide, and butyl-p-hydroxybenzoate, and sorbic acid, and mixtures thereof.
The aqueous vehicle may also include a tonicity agent to adjust the tonicity (osmotic pressure). The tonicity agent can be selected from the group consisting of a glycol (such as propylene glycol, diethylene glycol, triethylene glycol), glycerol, dextrose, glycerin, mannitol, potassium chloride, and sodium chloride, and a mixture thereof.
The aqueous vehicle may also contain a viscosity/suspending agent. Suitable viscosity/suspending agents include those selected from the group consisting of cellulose derivatives, such as methyl cellulose, ethyl cellulose, hydroxyethylcellulose, polyethylene glycols (such as polyethylene glycol 300, polyethylene glycol 400), carboxymethyl cellulose, hydroxypropylmethyl cellulose, and cross-linked acrylic acid polymers (carbomers), such as polymers of acrylic acid cross-linked with polyalkenyl ethers or divinyl glycol (Carbopols −such as Carbopol 934, Carbopol 934P, Carbopol 971, Carbopol 974 and Carbopol 974P), and a mixture thereof.
In order to adjust the formulation to an acceptable pH (typically a pH range of about 5.0 to about 9.0, more preferably about 5.5 to about 8.5, particularly about 6.0 to about 8.5, about 7.0 to about 8.5, about 7.2 to about 7.7, about 7.1 to about 7.9, or about 7.5 to about 8.0), the formulation may contain a pH modifying agent. The pH modifying agent is typically a mineral acid or metal hydroxide base, selected from potassium hydroxide, sodium hydroxide, and hydrochloric acid, and mixtures thereof, and preferably sodium hydroxide and/or hydrochloric acid. These acidic and/or basic pH modifying agents are added to adjust the formulation to the target acceptable pH range. Hence it may not be necessary to use both acid and base—depending on the formulation, the addition of one of the acid or base may be sufficient to bring the mixture to the desired pH range.
The aqueous vehicle may also contain a buffering agent to stabilise the pH. When used, the buffer is selected from the group consisting of a phosphate buffer (such as sodium dihydrogen phosphate and disodium hydrogen phosphate), a borate buffer (such as boric acid, or salts thereof including disodium tetraborate), a citrate buffer (such as citric acid, or salts thereof including sodium citrate), and ε-aminocaproic acid, and mixtures thereof.
The formulation may further comprise a wetting agent. Suitable classes of wetting agents include those selected from the group consisting of polyoxypropylene-polyoxyethylene block copolymers (poloxamers), polyethoxylated ethers of castor oils, polyoxyethylenated sorbitan esters (polysorbates), polymers of oxyethylated octyl phenol (Tyloxapol), polyoxyl 40 stearate, fatty acid glycol esters, fatty acid glyceryl esters, sucrose fatty esters, and polyoxyethylene fatty esters, and mixtures thereof.
According to a further aspect of the disclosure there is provided a pharmaceutical composition which comprises a compound of the disclosure as defined hereinbefore, or a pharmaceutically acceptable salt, hydrate or solvate thereof, in association with a pharmaceutically acceptable diluent or carrier.
The compositions of the disclosure may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular, intraperitoneal or intramuscular dosing or as a suppository for rectal dosing).
The compositions of the disclosure may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents.
An effective amount of a compound of the present disclosure for use in therapy is an amount sufficient to treat or prevent a disease or disorder referred to herein, slow its progression and/or reduce the symptoms associated with the condition.
An effective amount of a compound of the present disclosure for use in therapy is an amount sufficient to treat a disease or disorder referred to herein, slow its progression and/or reduce the symptoms associated with the condition.
The size of the dose for therapeutic or prophylactic purposes of a compound of Formula (I) will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or subject and the route of administration, according to well-known principles of medicine.
Provided herein is a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound or a pharmaceutical composition described herein, wherein the disease is selected from the group consisting of Dentatorubropallidoluysian atrophy, Huntington's disease, Spinal and bulbar muscular atrophy. SCA1 (Spinocerebellar ataxia Type 1), SCA2 (Spinocerebellar ataxia Type 2), SCA3 (Spinocerebellar ataxia Type 3 or Machado-Joseph disease), SCA6 (Spinocerebellar ataxia Type 6), SCA7 (Spinocerebellar ataxia Type 7), SCA12 (Spinocerebellar ataxia Type 12), SCA17 (Spinocerebellar ataxia Type 17), FRAXA (Fragile X syndrome), FXTAS (Fragile X-associated tremor/ataxia syndrome), FRAXE (Fragile XE mental retardation), Baratela-Scott syndrome, FRDA (Friedreich's ataxia), DM1 (Myotonic dystrophy Type 1), DM2 (Myotonic dystrophy Type 2) SCA8 (Spinocerebellar ataxia Type 8), Fuchs endothelial corneal dystrophy, Desbuquois dysplasia, amyotrophic lateral sclerosis, frontotemporal dementia.
In some embodiments, the disease is Huntington's disease.
Provided herein is a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound or a pharmaceutical composition disclosed herein, wherein the disease is Huntington's disease.
In some embodiments, the disease is myotonic dystrophy 1.
Provided herein is a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound or a pharmaceutical composition disclosed herein, wherein the disease is myotonic dystrophy 1.
In some embodiments, the disease is selected from the group consisting of FRAXA (Fragile X syndrome), FXTAS (Fragile X-associated tremor/ataxia syndrome). FRAXE (Fragile XE mental retardation).
Provided herein is a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound or a pharmaceutical composition disclosed herein, wherein the disease is selected from the group consisting of FRAXA (Fragile X syndrome), FXTAS (Fragile X-associated tremor/ataxia syndrome), FRAXE (Fragile XE mental retardation).
The compounds of the disclosure or pharmaceutical compositions comprising these compounds may be administered to a subject by any convenient route of administration, whether systemically/peripherally or topically (i.e., at the site of desired action).
Routes of administration include, but are not limited to, oral (e.g. by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including. e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eye drops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol. e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intra-arterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly.
The present disclosure include the following enumerated embodiments,
1. A compound of Formula (I):
2. The compound of embodiment 1, wherein 0, 1, or 2 of X, Y, and Z are N.
3. The compound of embodiment 1 or 2, wherein the compound is of Formula (Ia),
4. The compound of embodiment 1 or 2, wherein the compound is of Formula (Ib),
5. The compound of embodiment 1 or 2, wherein the compound is of Formula (Ic),
6. The compound of embodiment 1 or 2, wherein the compound is of Formula (Id).
7. The compound of embodiment 1 or 2, wherein the compound is of Formula (Ic),
8. The compound of embodiment 1 or 2, wherein the compound is of Formula (If),
9. The compound of any one of embodiments 1-8, wherein R1, R2, R3 and R4 are each independently selected from the group consisting of H, halogen, C1-C6alkyl, C1-C6haloalkyl. C1-C6alkoxy, and C1-C6cycloalkyloxy.
10. The compound of any one of embodiments 1-8, wherein R1, R2, R3 and R4 are each independently selected from the group consisting of H, halogen, C1-C6alkoxy, and C1-C6alkyl.
11. The compound of any one of embodiments 1-8, wherein R1, R2, R3 and R4 are each H.
12. The compound of any one of embodiments 1-11, wherein R5 is H.
13. The compound of any one of embodiments 1-12, wherein A is selected from the group consisting of
14. The compound of any one of embodiments 1-13, wherein A is substituted by one R9 selected from the group consisting of halogen and C1-C6alkyl.
15. The compound of any one of embodiments 1-13, wherein A is not substituted by R9.
16. The compound of any one of embodiments 1-15, wherein R6 is Me.
17. The compound of any one of embodiments 1-16, wherein R8 is C1-C6alkyl, C1-C6cycloalkyl, or heterocycloalkyl.
18. The compound of any one of embodiments 1-17, wherein R7 is Me, Et, isopropyl, or cyclobutyl.
19. The compound according to any one of embodiments 1-18, wherein A is selected from the group consisting of
20. The compound of any one of embodiments 1-19, wherein A is
21. The compound of any one of embodiments 1-20, wherein A is
22. The compound according to any one of embodiments 1-21, wherein B is NR10R11, wherein
23. The compound of embodiment 22, wherein R10 and R11 are taken together with the nitrogen atom to which they are attached to form a monocyclic heterocycloalkyl of 4-7 ring atoms with 1 or 2 total nitrogen ring atoms and 0 or 1 additional ring heteroatoms selected from O and S. and the heterocycloalkyl is optionally substituted with 1, 2, 3, or 4 R12.
24. The compound of any one of embodiments 1-23, wherein B is
25. The compound of embodiment 24, wherein each R12 is independently selected from the group consisting of halogen, hydroxy, 4-7-membered monocyclic heterocycloalkyl, C1-C6heteroalkyl, and C1-C6alkyl, wherein alkyl is optionally substituted with one or more halogen, hydroxyl, methoxy. C3-C8cycloalkyl, or NH2, and heterocycloalkyl is optionally substituted with one or more halogen, hydroxyl, methoxy, or C1-C6alkyl; and
27. The compound of embodiment 23 or 24, wherein B is
28. The compound of embodiment 27, wherein R13 is H or unsubstituted C1-C6alkyl.
29. The compound of any one of embodiments 27 or 28, wherein each R12 is independently C1-C6alkyl.
30. The compound of embodiment 1, wherein the compound is of formula (Ig)
31. The compound of embodiment 1, wherein the compound is of formula (Ih)
32. The compound of embodiment 1, wherein the compound is of formula (Ii)
33 The compound of embodiment 1, wherein the compound is of formula (Ij)
34. The compound of embodiment 1, wherein the compound is of formula (Ik)
35. The compound of embodiment 1, wherein the compound is of formula (Im)
36. The compound of embodiment 1, wherein the compound is of formula (In)
37. The compound of embodiment 1, wherein the compound is of formula (Io)
38. The compound of embodiment 1, wherein the compound is of formula (Ip)
39. The compound of embodiment 1, wherein the compound is of formula (Iq)
40. The compound according to any one of the preceding embodiments, selected from a compound of Table 1.
41. A pharmaceutical composition comprising a compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients.
42. A compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt thereof for use as a small molecule splicing modulator.
43. A pharmaceutical composition comprising a compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, solvate, or prodrug thereof and one or more pharmaceutically acceptable excipients.
44. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of embodiments 1-42 or a pharmaceutical composition of embodiment 43, wherein the disease is selected from the group consisting of Dentatorubropallidoluysian atrophy, Huntington's disease, Spinal and bulbar muscular atrophy, SCA1 (Spinocerebellar ataxia Type 1), SCA2 (Spinocerebellar ataxia Type 2), SCA3 (Spinocerebellar ataxia Type 3 or Machado-Joseph disease), SCA6 (Spinocerebellar ataxia Type 6), SCA7 (Spinocerebellar ataxia Type 7), SCA12 (Spinocerebellar ataxia Type 12), SCA17 (Spinocerebellar ataxia Type 17), FRAXA (Fragile X syndrome), FXTAS (Fragile X-associated tremor/ataxia syndrome), FRAXE (Fragile XE mental retardation), Baratela-Scott syndrome, FRDA (Friedreich's ataxia), DM1 (Myotonic dystrophy Type 1), DM2 (Myotonic dystrophy Type 2) SCA8 (Spinocerebellar ataxia Type 8), Fuchs endothelial corneal dystrophy, Desbuquois dysplasia, amyotrophic lateral sclerosis, frontotemporal dementia.
45. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of embodiments 1-42 or a pharmaceutical composition of embodiment 43, wherein the disease is Huntington's disease.
46. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of embodiments 1-42 or a pharmaceutical composition of embodiment 43, wherein the disease is Myotonic dystrophy 1.
47. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of embodiments 1-42 or a pharmaceutical composition of embodiment 43, wherein the disease is selected from the group consisting of FRAXA (Fragile X syndrome), FXTAS (Fragile X-associated tremor/ataxia syndrome), FRAXE (Fragile XE mental retardation)
For exemplary purpose, neutral compounds of Formula (I) are synthesized and tested in the examples. It is understood that the neutral compounds of Formula (I) may be converted to the corresponding pharmaceutically acceptable salts of the compounds using routine techniques in the art (e.g., by saponification of an ester to the carboxylic acid salt, or by hydrolyzing an amide to form a corresponding carboxylic acid and then converting the carboxylic acid to a carboxylic acid salt).
Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz or 300 MHz as stated; the chemical shifts (6) are reported in parts per million (ppm). Spectra were recorded using a Bruker or Varian instrument with 8, 16 or 32 scans.
LC-MS chromatograms and spectra were recorded using an Agilent 1200 or Shimadzu LC-20 AD&MS 2020 instrument using a C-18 column such as C18 2.1×30 mm, unless otherwise stated. Injection volumes were 0.7-8.0 μl and the flow rates were typically 0.8 or 1.2 ml/min. Detection methods were diode array (DAD) or evaporative light scattering (ELSD) as well as positive ion electrospray ionisation. MS range was 100-1000 Da.
Solvents were gradients of water and acetonitrile both containing a modifier (typically 0.01-0.04%) such as trifluoroacetic acid or ammonium carbonate.
To a mixture of methyl 5-aminopyrazine-2-carboxylate (5 g, 32.7 mmol) in EtOH (50 mL) was added 1-bromopropan-2-one (9 g, 63.4 mmol). The mixture was stirred at 80° C. for 2 days. After cooling to rt, the reaction mixture was concentrated. The crude was purified by silica gel column (DCM/MeOH=10:1) to give title product (3.5 g, Y: 52%) as a grey solid. ESI-MS (M+H)+: 206.1. 1H NMR (400 MHz, DMSO-d6) δ 9.58 (d, J=0.9 Hz, 1H), 9.26 (s, 1H), 8.28 (s, 1H), 4.41 (q, J=7.1 Hz, 2H), 2.55 (s, 3H), 1.37 (t, J=7.1 Hz, 4H).
To a mixture of ethyl 2-methylimidazo[1,2-a]pyrazine-6-carboxylate (400 mg, 1.94 mmol) in THF/water (12 mL, 5:1) was added LiOH·H2O (388 mg, 9.71 mmol). The mixture was stirred at rt for 2 h. After concentration, the residue was adjusted to pH=5 with 1M HCl. The cured was purified by reverse phase column to give title product (200 mg, Y: 58%) as a grey solid. ESI-MS (M+H)+: 178.1.
To a mixture of 6-bromo-8-fluoro-2-methylimidazo[1,2-a]pyridine (1 g, 4.37 mmol) in MeOH (20 mL) was added TEA (1.3 g, 13.11 mmol) and Pd(dppf)Cl2 (322 mg, 0.44 mmol). The resulting mixture was stirred overnight at 60° C. under CO (balloon). The mixture was allowed to cooling down to room temperature and concentrated. The residue was purified by silica gel column chromatography (PE:EA=5:1) to give title product (800 mg, 88%) as a yellow solid. ESI-MS (M+H)+: 209.0 1H NMR (400 MHz, DMSO-d6) δ 9.15 (d, J=1.0 Hz, 1H), 7.97 (d, J=3.0 Hz, 1H), 7.38 (dd, J=11.5, 1.0 Hz, 1H), 3.89 (s, 3H), 2.38 (s, 3H).
To a mixture of methyl 8-fluoro-2-methylimidazo[1,2-a]pyridine-6-carboxylate (800 mg, 3.85 mmol) in THF (20 mL) was added H2O (4 mL) and LiOH·H2O (809 mg, 19.23 mmol). The reaction mixture was stirred for 2 h at room temperature. After concentration, the residue was diluted with water and the mixture was adjusted to pH to 5 with HCl (2M). The solid was formed and collected by filtration and the cake was washed with water. The solid was dried under vacuum at 55° C. to give title product (670 mg, 90%) as a white solid. ESI-MS (M+H)+: 195.0 1H NMR (400 MHz, DMSO-d6) δ 9.32 (d, J=1.1 Hz, 1H), 8.18 (d, J=1.4 Hz, 1H), 7.84 (d, J=10.8 Hz, 1H), 2.48 (d, J=0.6 Hz, 3H).
To a mixture of 6-bromo-2,8-dimethylimidazo[1,2-a]pyrazine (500 mg, 2.21 mmol) in MeOH (20 mL) was added TEA (670 mg, 6.64 mmol) and Pd(dppf)Cl2 (162 mg, 0.22 mmol). The resulting mixture was stirred at 80° C. overnight under CO. The mixture was allowed to cooling down to room temperature and concentrated. The residue was purified by silica gel column chromatography (PE:EA=5:1) to give methyl 2,8-dimethylimidazo[1,2-a]pyrazine-6-carboxylate (420 mg, 92.6%) as a yellow solid. ESI-MS (M+H)+: 206.
To a mixture of methyl 2,8-dimethylimidazo[1,2-a]pyrazine-6-carboxylate (420 mg, 2.04 mmol) in MeOH (10 mL) and H2O (1 mL) was added LiOH·H2O (150 mg, 6.12 mmol). The reaction mixture was stirred for 2 h at room temperature. The mixture was adjust pH to 5 with HCl (2M). The reaction was concentrated to give 2,8-dimethylimidazo [1,2-a]pyrazine-6-carboxylic acid (450 mg, crude, containing some inorganic salt) as a yellow solid, which was used to next step without further purification. ESI-MS (M+H)+: 192.1.
To a solution of 4-methoxypyridin-2-amine (1.00 g, 8.07 mmol) in MeCN (10 mL) was added NBS (1.45 g, 8.07 mmol) portionwise at 0° C. and stirred at 0° C. for 1 h. The reaction mixture was diluted with ethyl acetate (80 mL) and washed with water (80 mL) and brine (80 mL). The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo to give 5-bromo-4-methoxypyridin-2-amine (1.5 g, crude) as an off-white solid, which was used to next step without further purification. ESI-MS (M+H)+: 203.0.
To a solution of 5-bromo-4-methoxypyridin-2-amine (1.50 g, 7.40 mmol) in EtOH (30 mL) was added 1-bromopropan-2-one (2.00 g, 14.90 mmol). The mixture was stirred at 80° C. for 24 h. After cooling to rt, the reaction mixture was concentrated. The residue was dissolved with EA and the organic phase was stirred with sat. aqueous sodium carbamate for 30 min. The two phases were separated. The organic phase was concentrated. The crude was purified by silica gel column (DCM/MeOH=10:1) to give title product (0.8 g, Y: 47%) as a grey solid. ESI-MS (M+H)+: 241.0. 1H NMR (400 MHz, DMSO-d6) δ 8.78 (s, 1H), 7.45 (s, 1H), 6.95 (s, 1H), 3.89 (s, 3H), 2.26 (s, 3H).
To a mixture of 6-bromo-7-methoxy-2-methylimidazo[1,2-a]pyridine (0.80 g, 3.32 mmol) in MeOH (20 mL) was added TEA (1.00 g, 9.96 mmol) and Pd(dppf)Cl2 (243 mg, 0.33 mmol. The mixture was charged with CO for three times and stirred at 80° C. for 16 h. The reaction mixture was diluted with ethyl acetate (100 mL) and washed with water (50 mL) and brine (50 mL). The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo to give methyl-7-methoxy-2-methylimidazo [1,2-a]pyridine-6-carboxylate (0.5 g, crude) as a gray solid. ESI-MS (M+H)+: 221.2.
To a mixture of methyl 7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylate (250 mg, 1.14 mmol) in THF/water (6 mL, 5:1) was added LiOH·H2O (100 mg, 3.41 mmol). The mixture was stirred at it for 2 h. After concentration, the residue was adjusted to pH=5 with 1M HCL. The cured was purified by reverse phase column to give 7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (200 mg, Y: 85%) as a gray solid. ESI-MS (M+H)+: 207.1.
5-Bromo-4-methoxypyridin-2-amine (30.2 g, 148.74 mmol), 3-bromoprop-1-yne (21.23 g, 178.49 mmol) and 2-propanol (350 mL) were added to a small Schlenk flask and stirred vigorously at 80° C. overnight. After that, the mixture was allowed to cool to room temperature and the excess of solvent and propargyl bromide was removed under high vacuum. The resulting crude residue of 2-amino-5-bromo-4-methoxy-1-(prop-2-yn-1-yl)pyridin-1-ium bromide (47.0 g, 90.0% purity, 131.37 mmol, 88.3% yield) was used in the next step without purification. 1H NMR (400 MHz, DMSO-d6) δ: 8.6 (br s, 2H), 8.57 (s, 1H), 6.75 (s, 1H), 5.06 (s, 2H), 3.97 (s, 3H).
To a stirring solution of sodium hydroxide (5.89 g, 147.36 mmol, 5.89 mL, 1.01 equiv.) in deionised H2O (300 mL) was added 2-amino-5-bromo-4-methoxy-1-(prop-2-yn-1-yl)pyridin-1-ium bromide (46.98 g, 145.9 mmol) via powder addition funnel over a period of 30 minutes. Immediately upon addition, the solution phase turned yellow and yellow oil became dispersed as a distinct separate phase. The oil product was extracted with EtOAc (2×150 mL), dried over anhydrous MgSO4, filtered and concentrated under reduced pressure to afford 6-bromo-7-methoxy-2-methylimidazo[1,2-a]pyridine (22.5 g, 90.0% purity, 84.0 mmol, 57.6% yield) as a pale yellow solid. 1H NMR (400 MHz, DMSO-d6) δ: 8.76 (s, 1H), 7.43 (s, 1H), 6.94 (s, 1H), 3.87 (s, 3H), 2.55 (s, 3H).
6-Bromo-7-methoxy-2-methylimidazo[1,2-a]pyridine (30.68 g, 127.26 mmol) was dissolved in MeOH (250 mL) and [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane (2.08 g, 2.55 mmol) was added followed by triethylamine (28.33 g, 279.98 mmol). The resulting mixture was transferred into autoclave and stirred at 130° C. under 40 bar pressure of CO overnight. Then the MeOH was evaporated and the residue was partitioned between water (150 mL) and EtOAc (300 mL). The organic layer was separated, dried over Na2SO4 and evaporated under reduced pressure to give crude methyl 7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylate (19.15 g, 86.96 mmol, 68.3% yield), which was used in the next step without purification. ESI-MS (M+H)+: 221.2.
A mixture of methyl 7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylate (19.15 g, 86.96 mmol) and potassium hydroxide (7.32 g, 130.43 mmol) was stirred in methanol (150 mL) and H2O (50 mL) overnight. The reaction mixture was concentrated under reduced pressure to remove methanol, and the resulting aqueous solution was neutralized with 1 N HCl to pH=5 to precipitate the carboxylic acid. The solid 7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (14.0 g, 95.0% purity, 64.5 mmol, 74.2% yield) was isolated by filtration, washed with H2O (20 mL) and MeCN (20 mL), dried and used directly in the next step without further purification. 1H NMR (400 MHz, DMSO-d6) δ: 9.23 (s, 1H), 7.88 (s, 1H), 7.21 (s, 1H), 3.97 (s, 3H), 2.39 (s, 3H).
To a stirred mixture of tert-butyl (4-methoxypyridin-2-yl)carbamate (4.47 g, 19.93 mmol) in dry THF (100 mL) was added n-BuLi (2.5 M, 19.9 mL) under N2 atmosphere with ice-water bathing, the reaction was stirred for 1 h at 0° C. then CH3I was added by dropwise within 30 min, the mixture was stirred for 2 h at room temperature. The mixture was quenched with H2O (50 mL) and extracted with EtOAc (50 mL×3). The organic phase was washed with saturated NaCl (20 mL) and dried with Na2SO4. The mixture was concentrated under vacuum to afford crude product, the crude product was purified by silica gel column (PE/EA=1:1) to afford title product (4.3 g, Y: 86.1%) as light yellow solid. ESI-MS (M+H)+: 239.1.
To a stirred mixture of tert-butyl (4-methoxy-3-methylpyridin-2-yl)carbamate (4.0 g, 16.78 mmol) in DCM (50 mL) was added TFA (10 mL) by dropwise under N2 atmosphere at room temperature, the reaction was stirred for 4 h at room temperature. The mixture was concentrated under vacuum to afford title product TFA salt (4.1 g Y: 99%) as light yellow solid, the crude mixture was directly used for the next step without further purification. ESI-MS (M+H)+: 139.1.
To a stirred mixture of 4-methoxy-3-methylpyridin-2-amine (2.70 g, 19.55 mmol) in AcOH (20 mL) was added Br2 (15.62 g, 97.75 mmol) by dropwise at 40° C. within 30 min. the mixture was stirred for 1 h at 40° C. The mixture was quenched with H2O (10 mL) and neutralized with NH4HCO3, extracted with i-PrOH/CHCl3 (1:3, v/v, 50 mL×3). The organic phase was washed with saturated NaCl (20 mL) and dried with Na2SO4. The mixture was concentrated under vacuum to afford crude product, the crude product was purified by silica gel column (DCM/MeOH=10:1) to afford title product (2.9 g, Y: 68.3%) as white solid. ESI-MS (M+H)+: 217.1. 1H NMR (400 MHz, DMSO-d6) δ 7.98 (s, 1H), 5.97 (s, 2H), 3.71 (s, 3H), 1.99 (s, 3H).
To a stirred mixture of 5-bromo-4-methoxy-3-methylpyridin-2-amine (2.0 g, 9.21 mmol) in EtOH (10 mL) was added 1-bromopropan-2-one (2.52 g, 18.42 mmol) by portion at room temperature, the mixture was stirred for 8 h at 80° C. in a sealed tube. The mixture was quenched with H2O (10 mL) and neutralized with Na2CO3, extracted with EtOAc (50 mL×3). The organic phase was washed with saturated NaCl (20 mL) and dried with Na2SO4. After concentration, the crude product was purified by silica gel column (DCM/MeOH=10:1) to afford title product (1.3 g, Y: 55.3%) as white solid. ESI-MS (M+H)+: 255.0.
To a solution of 6-bromo-7-methoxy-2,8-dimethylimidazo[1,2-a]pyridine (500 mg, 1.96 mmol) in MeOH (10 mL) were added TEA (595 mg, 5.88 mmol) and Pd(dppf)Cl2 (143.28 mg, 0.196 mmol) at room temperature under CO atmosphere (balloon). The mixture was stirred for 4 h at 90° C. After concentration, the residue was purified by silica gel column (DCM/MeOH=10:1) to afford title product (245 mg, Y: 53.3%) as light yellow solid. ESI-MS (M+H)+: 235.1.
To a solution of compound methyl 7-methoxy-2,8-dimethylimidazo[1,2-a]pyridine-6-carboxylate (200 mg, 0.85 mmol) in THF (4.0 mL) were added H2O (1 mL) and LiOH·H2O (59 mg, 2.46 mmol). The reaction mixture was stirred at room temperature for 1 h. The organic solvent was concentrated under reduced pressure and extracted with CH2Cl2 (50 mL×3). Then the aqueous solution was acidified to pH=2 with 1M HCl. Then the aqueous solution was extracted with EA (50 mL×3). The combined organic layer was washed with water and brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give title product (100 mg, yield: 53.4%) as a yellow solid. ESI-MS (M+H)+: 221.0.
To a solution of 5-bromo-6-methoxy-1H-indazole (3.2 g, 14.1 mmol) in anhydrous THF (30 mL) was added sodium hydride (1.13 g, 28.2 mmol) and the mixture was stirred under nitrogen for 0.5 h. Then iodomethane (4 g, 28.2 mmol) was added and the resulting mixture was stirred at 55° C. for 3 h. The mixture was carefully diluted with water and extracted with EA. The organic phase was washed with brine (50 mL), dried over Na2SO4 and concentrated to dryness. The crude was purified by column chromatography (EA:PE=2:1, v/v) to give title product (780 mg, Y: 23%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.86 (s, 1H), 7.77 (s, 1H), 7.02 (s, 1H), 4.17 (s, 3H), 3.95 (s, 3H). ESI-MS (M+H)+: 240.8, MS (M+2+H)+: 242.8.
A mixture of 5-bromo-6-methoxy-2-methyl-2H-indazole (1.10 g, 4.60 mmol), TEA (1.39 g, 13.8 mmol) and Pd(dppf)Cl2 (341 mg, 0.46 mmol) in MeOH (30 mL) was purged with carbon monoxide for three times at rt. The mixture was stirred for at 85° C. under carbon monoxide overnight. The catalyst was filtered off and the filtrate was concentrated to give crude product, which was purified by column chromatography (EA:PE=4:1, v/v) to give methyl 6-methoxy-2-methyl-2H-indazole-5-carboxylate (770 mg, Y: 76%) as an orange solid. ESI-MS (M+H)+: 221.
To a mixture of methyl 6-methoxy-2-methyl-2H-indazole-5-carboxylate (770 mg, 3.21 mmol) in MeOH (10 mL) and H2O (5 mL) was added LiOH·H2O (1.28 g, 32.10 mmol). The reaction mixture was stirred for 2 h at room temperature. After concentration, the residue was diluted with water and adjusted to pH=6 with 1M HCl. The precipitate was collected by filtration and dried under vacuum to give 6-methoxy-2-methyl-2H-indazole-5-carboxylic acid (450 mg, crude) as a yellow solid, which was used to next step without further purification. ESI-MS (M+H)+: 207.1.
To a suspension of 3,6-dichloropyridazine (1.0 g, 6.75 mmol) and tert-butyl piperazine-1-carboxylate (1.3 g, 6.75 mmol) in NMP (20 mL) was added TEA (2.0 g, 20.2 mmol), and the reaction mixture was stirred for 2 h at 120° C. After cooling to rt, the mixture was diluted with water 150 mL and 150 mL of EA. The EA layer was separated, washed with brine and water, dried over Na2SO4 and concentrated to dryness. The crude was purified by silica gel column (10-50% EA in PE). The product was obtained as off-white solid (1.2 g, Y: 60%). 1H NMR (400 MHz, CDCl3) δ 6.96 (d, J=9.6 Hz, 1H), 6.77 (d, J=9.6 Hz, 1H), 3.55 (dd, J=6.3, 4.0 Hz, 4H), 3.42 (d, J=5.2 Hz, 4H), 1.48 (s, 9H).
A mixture of tert-butyl 4-(6-chloropyridazin-3-yl)piperazine-1-carboxylate (1.5 g, 5.01 mmol), diphenylmethanimine (1.4 g, 7.55 mmol), Pd2(dba)3 (289 mg, 0.25 mmol), BINAP (144 mg, 0.25 mmol) and Cs2CO3 (489 mg, 15.0 mmol) in toluene (15 mL) was purged with N2 for three times at room temperature. Then the reaction mixture was stirred at 100° C. for 16 h. After cooling to rt, the mixture was diluted with water (150 mL) and EA (150 mL). The EA layer was separated, washed with brine and water, dried over Na2SO4, concentrated to dryness. The crude was purified by silica gel column (10-50% EA in PE). The product was obtained as off-white solid (1.25 g, yield: 56%.) ESI-MS: [M+H]+ 480.1
A mixture of tert-butyl 4-(6-((diphenylmethylene)amino)pyridazin-3-yl)piperazine-1-carboxylate (1.2 g, 2.71 mmol), NH2OH·HCl (378 mg, 5.42 mmol) and NaOAc (1.1 g, 13.5 mmol) in MeOH (15 mL) was stirred for 1 h at rt. After diluting with water, the mixture was extracted with EA (30 mL×2). The combined organics was washed with brine, dried over Na2SO4. After concentration, the crude was used to next step without further purification. 1H NMR (400 MHz, CDCl3) δ 7.23 (d, J=9.5 Hz, 1H), 6.90 (d, J=9.5 Hz, 1H), 3.62 (br s, 4H), 3.59-3.55 (m, 4H), 1.49 (s, 9H).
A mixture of 5-bromo-3-fluoropyridin-2-amine (50 g; 262 mmol), 1-bromo-2,2-dimethoxypropane (57.5 g; 315 mmol) and PPTS (6.6 g; 26.2 mmol) in i-PrOH (300 mL) was stirred at 70° C. for 16 h. Next, the reaction mixture was filtered and the obtained solid was rinsed with cold i-PrOH (2×100 mL), dried in vacuo to give 6-bromo-8-fluoro-2-methylimidazo[1,2-a]pyridine as HBr salt (35 g; 113 mmol; 43% yield). ESI-MS: 230.1 (M+H)+.
A mixture of 6-bromo-8-fluoro-2-methylimidazol[1,2-a]pyridine HBr (35 g; 113 mmol), Pd(dppf)Cl2 (9.2 g; 11.3 mmol) and TEA (63 mL; 452 mmol) in MeOH (300 mL) was stirred at 50° C. in autoclave (under CO atmosphere, 10 atm.) for 14 h. Next, the reaction mixture was concentrated in vacuo and purified by column chromatography to afford the title compound (9 g; 43 mmol; 38% yield). ESI-MS: 209.2 (M+H)+.
Lithium hydroxide monohydrate (2.4 g, 100 mmol) was added to a stirred solution of methyl 8-fluoro-2-methylimidazol[1,2-a]pyridine-6-carboxylate (9 g; 43 mmol) in THF/water (100 mL/50 mL) and the obtained solution was stirred at r.t. for 14 h. Next, the reaction mixture was concentrated in vacuo, acidified to pH=6 and extracted with EtOAc. Organics were washed with water, dried with Na2SO4 and evaporated to give 8-fluoro-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (7.2 g; 37 mmol; 86% yield). ESI-MS: 195.2 (M+H)+
A mixture of 2,4-difluoro-1-nitrobenzene (100 mg, 0.63 mmol), K2CO3 (217 mg, 1.57 mmol) and tert-butyl piperazine-1-carboxylate (98 mg, 0.52 mmol) in HMPA (1 mL) was heated to 100° C. for 4 h. After cooling to rt, the reaction mixture was diluted with ethyl acetate (30 mL) and washed with water (30 mL) and brine (30 mL). The organic layer was dried over Na2SO4, concentrated to dryness. The crude was purified by pre-TLC (EA:PE=1:5, v/v) to give title product (140 mg, Y: 82.8%) as a yellow solid. ESI-MS (M+H)+: 326.1. 1H NMR (400 MHz, CDCl3) δ 8.07-8.02 (m, 1H), 6.61-6.50 (m, 2H), 3.62-3.48 (m, 4H), 3.47-3.36 (m, 4H), 1.49 (s, 9H).
A mixture of tert-butyl 4-(3-fluoro-4-nitrophenyl)piperazine-1-carboxylate (140 mg, 0.43 mmol) and Pd/C (14 mg, 10% wt/wt) in MeOH (10 mL) was purged with hydrogen for three times at rt. The mixture was stirred for 30 min at rt under hydrogen. The catalyst was filtered off and the filtrate was concentrated to give title product, which was used in next step without further purification. ESI-MS (M+H)+: 296.1.
To a stirred solution of 2-methylimidazo[1,2-a]pyrazine-6-carboxylic acid (100 mg, 0.56 mmol), tert-butyl 4-(4-amino-3-fluorophenyl)piperazine-1-carboxylate (110 mg, 0.373 mmol) and DIPEA (240 mg, 1.865 mmol) in DMF (6 mL) was added HATU (284 mg, 0.746 mmol) at rt. After addition was completed, the mixture was stirred for 2 h. The reaction mixture was treated with EA/Water (100 mL, 1:1). The organic phase was separated and the aqueous phase was extracted with EA (50 mL×1). The combined organic layers was washed with brine and dried over sodium sulfate. After concentration under reduced pressure, the crude was slurry with EA/PE (1:10, v/v, 5 mL) for 30 min at rt. Then the solid was collected by filtration and washed with EA/PE (1:10, v/v, 3 mL×3). The product was obtained (64 mg. Y: 37.9%) as a light brown solid, which was used to next step without further purification. ESI-MS (M+H)+: 455.1.
To a solution of tert-butyl 4-(3-fluoro-4-(2-methylimidazo[1,2-a]pyrazine-6-carboxamido)phenyl)piperazine-1-carboxylate (64 mg, 0.141 mmol) in DCM (5 mL) was added HCl-dioxane (4 M, 0.36 mL, 1.41 mmol) at 0° C. and the mixture was allowed to warm to rt and stirred for 1 h. After concentration, the residue was treated with DCM (10 mL) and EA (10 mL). The solid was collected by filtration and washed EA three times (3 mL×3). The solid was dried under vacuum at 55° C. to provide title product as light grey solid (35.3 mg, yield: 60%). ESI-MS (M+H)+: 355.1. 1H NMR (400 MHz, DMSO-d6) δ 10.20 (s, 1H), 9.47 (s, 3H), 9.19 (s, 1H), 8.24 (s, 1H), 7.80-7.74 (m, 1H), 7.01 (dd, J=13.9, 2.3 Hz, 1H), 6.87 (d J=8.7 Hz, 1H), 3.48-3.42 (m, 4H), 3.20 (s, 4H), 2.53 (s, 3H).
A mixture of 1-fluoro-4-nitrobenzene (500 mg, 3.55 mmol), tert-butyl piperazine-1-carboxylate (989 mg, 5.32 mmol) and triethyl amine (1.1 g, 10.65 mmol) in MeCN (10 mL) was stirred at 80° C. for 4 h. After cooling to rt, the mixture was concentrated. The crude was purified by silica gel column (PE:EA=10:1) to give title product (490 mg, Y: 45%) as a yellow solid. ESI-MS (M+H)+: 308.2.
A mixture of tert-butyl 4-(4-nitrophenyl)piperazine-1-carboxylate (490 mg, 1.6 mmol) and Pd/C (50 mg, 10% wt/wt) in MeOH (10 mL)/THF (2 mL) was purged with hydrogen for three times at rt. The mixture was stirred for 1 h at rt under hydrogen. The catalyst was filtered off and the filtrate was concentrated to give title product (400 mg. Y: 90%), which was used in next step without further purification. ESI-MS (M+H)+: 278.1. 1H NMR (400 MHz, DMSO-d6) δ 6.70 (d, J=8.8 Hz, 2H), 6.49 (d, J=8.8 Hz, 2H), 4.60 (s, 2H), 3.46-3.38 (m, 4H), 2.87-2.78 (m, 4H), 1.41 (s, 9H).
To a stirred solution of 2-methylimidazo[1,2-a]pyrazine-6-carboxylic acid (150 mg, 0.85 mmol), tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (196 mg, 0.71 mmol) and DIPEA (275 mg, 2.13 mmol) in DMF (3 mL) was added HATU (405 mg, 1.07 mmol) at it. After addition was completed, the mixture was stirred for 1 h. After diluting with water, the precipitate was collected by filtration. The solid was washed with MeCN and dried to give title product (160 mg, Y: 52%) as a yellow solid. ESI-MS (M+H)+: 437.2. 1H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 9.29 (s, 1H), 8.99 (s, 1H), 8.09 (s, 1H), 7.76 (d, J=7.7 Hz, 2H), 6.96 (d, J=7.8 Hz, 2H), 3.46 (br s, 4H), 3.07 (br s, 4H), 2.46 (s, 3H), 1.42 (s, 9H).
To a solution of tert-butyl 4-(4-(2-methylimidazo[1,2-a]pyrazine-6-carboxamido)phenyl)piperazine-1-carboxylate (80 mg, 0.18 mmol) in DCM (2 mL) was added HCl-dioxane (4 M, 0.5 mL, 2 mmol) at 0° C. and the mixture was allowed to warm to rt and stirred for 2 h. After concentration, the residue was treated with EA (5 mL). The solid was collected by filtration and washed EA three times (5 mL). The solid was dried under vacuum to provide title product as yellow solid (60 mg, yield: 90%). ESI-MS (M+H)+: 337.1. 1H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 9.47 (s, 1H), 9.37 (br s, 2H), 9.19 (s, 1H), 8.26 (s, 1H), 7.81 (d, J=9.1 Hz, 2H), 7.05 (d, J=9.1 Hz, 2H), 3.45-3.33 (m, 4H), 3.24-3.21 (m, 4H), 2.54 (s, 3H).
A mixture of 2-chloro-5-nitropyridine (1.0 g, 6.3 mmol), Et3N (1.92 g, 18.9 mmol) and tert-butyl piperazine-1-carboxylate (2.35 g, 12.6 mmol) in CH3CN (10 mL) was stirred at 100° C. for 1 h. After cooing to rt, the reaction mixture was diluted with ethyl acetate (30 mL) and washed with water once (30 mL), brine (30 mL). The organic layer was dried over Na2SO4, concentrated to dryness. The crude was purified by silica gel column (EA:PE=1:5, v/v) to give title product (1.8 g. Y: 92%) as a yellow solid. ESI-MS (M+H)+: 309.4.
A mixture of tert-butyl 4-(5-nitropyridin-2-yl)piperazine-1-carboxylate (1.0 g, 3.2 mmol) and Pd/C (100 mg, 10% wt/wt) in MeOH (10 mL) was purged with nitrogen for three times and then hydrogen for three times at rt. The mixture was stirred for 30 min at it under hydrogen. The catalyst was filtered off and the filtrate was concentrated to provide crude title product, which was used to next step without further purification. ESI-MS (M+H)+: 279.0.
To a stirred solution of 2-methylimidazo[1,2-a]pyrazine-6-carboxylic acid (76 mg, 0.43 mmol), tert-butyl 4-(5-aminopyridin-2-yl)piperazine-1-carboxylate (100 mg, 0.36 mmol) and DIPEA (138 mg, 1.07 mmol) in DMF (5 mL) was added HATU (204 mg, 0.54 mmol) at rt. The mixture was stirred at rt for 2 hrs. The reaction mixture was treated with EA/Water (50 mL, 1:1). The organic phase was separated and the aqueous phase was extracted with EA (50 mL×1). The combined organic layers was dried over sodium sulfate. After concentration, the crude was purified by silica gel column (DCM/MeOH=10:1) to provide title product (46 mg, Y: 24%) as black solid. ESI-MS (M+H)+: 438.2. 1H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H), 9.30 (s, 1H), 8.99 (s, 1H), 8.60 (s, 1H), 8.04-8.09 (m, 2H), 6.89 (d, J=8.0 Hz, 1H), 3.44 (s, 8H), 2.46 (s, 3H), 1.43 (s, 9H).
To a solution of tert-butyl 4-(5-(2-methylimidazo[1,2-a]pyrazine-6-carboxamido)pyridin-2-yl)piperazine-1-carboxylate (26 mg, 0.059 mmol) in DCM (5 mL) was added HCl-dioxane (4 M, 0.50 mL, 2 mmol) at 0° C. and warmed to rt for 1 h. After concentration, the residue was treated with DCM (5 mL×1) and EA (5 mL×1). The solid was collected by filtration and washed with EA three times (3 mL×3). The solid was dried under vacuum at 55° C. to provide title product as light grey solid (8.6 mg, yield: 39%). ESI-MS (M+H)+: 338.1. 1H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H), 9.38-9.36 (m, 3H), 9.03 (s, 1H), 8.69 (s, 1H), 8.22-8.13 (m, 2H), 7.08-7.10 (d, J=8.0 Hz, 1H), 3.69-3.64 (m, 4H), 3.19 (s, 4H), 2.47 (s, 3H).
A mixture of 2-chloro-5-nitropyridine (0.30 g, 1.90 mmol), Et3N (0.60 g, 5.93 mmol) and tert-butyl (R)-2-methylpiperazine-1-carboxylate (0.46 g, 2.30 mmol) in CH3CN (10 mL) was stirred 100° C. for 1 h. After cooling to rt, the reaction mixture was diluted with ethyl acetate (100 mL) and washed with water (30 mL), brine (30 mL). The organic layer was dried over Na2SO4, concentrated to dryness. The crude was purified by silica gel column (EA:PE=1:5, v/v) to give the title compound (500 mg, Y: 81%) as a yellow solid. ESI-MS (M-56+H)+:267.0.
A mixture of tert-butyl (R)-2-methyl-4-(5-nitropyridin-2-yl)piperazine-1-carboxylate (0.2 g, 0.62 mmol) and Pd/C (20 mg, 10% wt/wt) in MeOH (10 mL) was purged with nitrogen for three times and then hydrogen for three times at rt. The mixture was stirred for 30 min at rt under hydrogen. The solid was filtered off. The mother liquor was concentrated under vacuum to provide title product (180 mg, Y: 99%) as a purple solid, which was used to next step without further purification. ESI-MS (M+H)+: 293.4.
To a stirred solution of 2-methylimidazo[1,2-a]pyrazine-6-carboxylic acid (73 mg, 0.41 mmol), tert-butyl (R)-4-(5-aminopyridin-2-yl)-2-methylpiperazine-1-carboxylate (100 mg, 0.34 mmol) and DIPEA (133 mg, 1.03 mmol) in DMF (6 mL) was added HATU (195 mg, 0.51 mmol) at rt. The mixture was stirred at rt for 2 hrs. The reaction mixture was treated with EA/Water (80 mL, 1:1). The organic phase was separated and the aqueous phase was extracted with EA (50 mL×1). The combined organic layers was dried over sodium sulfate. After concentration under reduced pressure, the crude was slurried with EA/PE (1:10, v/v, 5 mL) for 30 min at rt. Then the solid was collected by filtration and washed with EA/PE (1:10, v/v, 3 mL×3). The product was obtained (30 mg, Y: 19.5%) as a light brown solid, which was used to next step without further purification. ESI-MS (M+H)+: 452.4.
To a solution of tert-butyl (R)-2-methyl-4-(5-(2-methylimidazo[1,2-a]pyrazine-6-carboxamido)pyridin-2-yl)piperazine-1-carboxylate (30 mg, 0.067 mmol) in DCM (5 mL) was added HCl-dioxane (4 M, 0.50 mL, 2 mmol) at 0° C. and the mixture was allowed to warm to rt and stirred for 1 h. After concentration, the residue was treated with DCM (5 mL×1) and EA (5 mL×1). The solid was collected by filtration and washed with EA three times (3 mL×3). The solid was dried under vacuum at 55° C. to provide title product as light grey solid (8.2 mg, yield: 32%). ESI-MS (M+H)+: 352.2. 1H NMR (400 MHz, CD3OD) δ 9.49 (s, 1H), 9.32 (s, 1H), 8.85 (s, 1H), 8.41 (d, J=8.0 Hz, 1H), 8.28 (s, 1H), 7.46 (d, J=8.0 Hz, 1H), 4.35-4.32 (m, 2H), 3.49-3.63 (m, 4H), 3.33-3.39 (m, 1H), 2.66 (s, 3H), 1.45 (d, J=8.0 Hz, 3H).
A mixture of 2,4-difluoro-1-nitrobenzene (318 mg, 2.0 mmol), tert-butyl (R)-2-methylpiperazine-1-carboxylate (400 mg, 2.0 mmol) and potassium carbonate (276 mg, 2.0 mmol) in HMPA (5 mL) was stirred at 60° C. for 4 h. After cooling to rt, the reaction mixture was diluted with ethyl acetate and washed with water and brine. The organic layer was dried over Na2SO4, concentrated to dryness. The crude was purified by pre-TLC (EA:PE=1:10, v/v) to give title product (200 mg, Y: 29%) as a brown oil. ESI-MS (M-56+H)+: 284.0. 1H NMR (400 MHz, CDCl3) δ 8.04 (t, J=9.1 Hz, 1H), 6.60-6.42 (m, 2H), 4.34 (br s, 1H), 3.98-3.89 (m, 1H), 3.75-3.67 (m, 1H), 3.61-3.54 (m, 1H), 3.44-3.32 (m, 2H), 3.22-3.12 (m, 1H), 1.49 (s, 9H), 1.22 (d, J=6.7 Hz, 3H).
A mixture of tert-butyl (R)-4-(3-fluoro-4-nitrophenyl)-2-methylpiperazine-1-carboxylate (220 mg, 0.65 mmol) and Pd/C (25 mg) in MeOH (3 mL) was purged with nitrogen for three times and then hydrogen for three times at rt. The mixture was stirred for 30 min at rt under hydrogen. LCMS showed the starting material was consumed completely and all of starting material was converted to desired product. The reaction was stopped and the solid was filtered off. The mother liquor was removed under vacuum. The residue title compound was used in next step without further purification. ESI-MS (M+H)+: 310.4.
To a stirred solution of tert-butyl (R)-4-(4-amino-3-fluorophenyl)-2-methylpiperazine-1-carboxylate (100 mg, 0.32 mmol) in DMF (3 mL) was added 2-methylimidazo[1,2-a]pyrazine-6-carboxylic acid (63 mg, 0.36 mmol), DIEA (120 mg, 0.97 mmol) and HATU (185 mg, 0.49 mmol). The mixture was stirred for 3 h at rt. Water was added and the precipitate was collected by filtration. The filter cake was washed with water (5 mL×3) and dried under vacuum to provide the title compound (96 mg, Y: 62%) as a yellow solid, which was used to next step without further purification. ESI-MS (M+H)+: 469.2. 1H NMR (400 MHz, CDCl3) δ 9.88 (s, 1H), 9.01-8.89 (m, 2H), 8.30 (t, J=8.9 Hz, 1H), 7.62 (s, 1H), 6.76-6.62 (m, 2H), 4.35 (br s, 1H), 3.95 (d, J=13.2 Hz, 1H), 3.53-3.45 (m, 1H), 3.41-3.21 (m, 2H), 3.01-2.94 (m, 1H), 2.82-2.73 (m, 1H), 2.57 (s, 3H), 1.49 (s, 9H), 1.30 (d J=6.7 Hz, 3H).
To a solution of tert-butyl (R)-4-(3-fluoro-4-(2-methylimidazo[1,2-a]pyrazine-6-carboxamido)phenyl)-2-methylpiperazine-1-carboxylate (100 mg, 0.213 mmol) in DCM (3 mL) was added HCl solution in 1,4-dioxane (0.55 mL, 4 mmoL, 4M) at 0° C. The mixture was stirred at it for 1.5 h. The mixture was filtered and the filtrate was concentrated under reduce pressure, the residue was washed by EA (5 mL) and PE (5 mL) to give title product (68 mg, Y:87%) as a yellow solid. ESI-MS (M+H)+: 369.1. 1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 9.42 (br s, 1H), 9.37 (d, J=1.2 Hz, 1H), 9.16 (br s, 1H), 9.08 (s, 1H), 8.15 (s, 1H), 7.81 (t, J=9.0 Hz, 1H), 7.03 (dd, J=14.0, 2.5 Hz, 1H), 6.88 (dd, J=8.9, 2.3 Hz, 1H), 3.90-3.76 (m, 2H), 3.40-3.29 (m, 2H), 3.11-3.00 (m, 2H), 2.84-2.80 (m, 1H), 2.49 (s, 3H), 1.31 (d, J=6.5 Hz, 3H).
A mixture of 2,4-difluoro-1-nitrobenzene (100 mg, 0.63 mmol), tert-butyl (1R,5S)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (133 mg, 0.63 mmol) and potassium carbonate (260 mg, 1.89 mmol) in HMPA (1 mL) was stirred at 60° C. for 2 h. After cooling to rt, the reaction mixture was diluted with ethyl acetate and washed with water and brine. The organic layer was dried over Na2SO4, concentrated to dryness. The crude was purified by pre-TLC (EA:PE=1:10, v/v) to give title product (70 mg, Y: 32%) as a yellow solid. ESI-MS (M+H)+: 352.1. 1H NMR (400 MHz, CDCl3) δ 8.04 (t, J=9.2 Hz, 1H), 6.58-6.45 (m, 2H), 4.42 (s, 2H), 3.57-3.38 (m, 2H), 3.20 (d, J=9.0 Hz, 2H), 2.08-1.98 (m, 2H), 1.81-1.75 (m, 2H), 1.48 (s, 9H).
A mixture of tert-butyl (1R,5S)-3-(3-fluoro-4-nitrophenyl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (70 mg, 0.19 mmol) and Pd/C (10 mg) in MeOH (10 mL) was purged with hydrogen for three times at rt. The mixture was stirred for 1 h at rt under hydrogen. The catalyst was filtered off and the filtrate was concentrated to give title product (60 mg, Y: 94%) as a pink solid, which was used in next step without further purification. ESI-MS (M+H)+: 322.0.
To a stirred solution of 2-methylimidazo[1,2-a]pyrazine-6-carboxylic acid (41 mg, 0.23 mmol), tert-butyl (1R,5S)-3-(4-amino-3-fluorophenyl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (50 mg, 0.16 mmol) and DIPEA (60 mg, 0.46 mmol) in DMF (2 mL) was added HATU (89 mg, 0.23 mmol) at rt. After addition was completed, the mixture was stirred for 1 h. After diluting with water, the precipitate was collected by filtration. The solid was washed with PE/EA (10:1) and dried to give title product (66 mg, Y: 74%) as a yellow solid. ESI-MS (M+H)+: 481.2.
To a solution of tert-butyl (1R,5S)-3-(3-fluoro-4-(2-methylimidazo[1,2-a]pyrazine-6-carboxamido)phenyl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (66 mg, 0.14 mmol) in DCM (10 mL) was added HCl-dioxane (4 M, 0.5 mL, 2 mmol) at 0° C. and the mixture was allowed to warm to rt and stirred for 2 h. After concentration, the residue was treated with EA. The solid was collected by filtration and washed EA. The solid was dried under vacuum to provide title product as yellow solid (16 mg, yield: 27%). ESI-MS (M+H)+: 381.0. 1H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H), 9.42-9.27 (m, 3H), 9.06 (s, 1H), 8.13 (s, 1H), 7.84-7.72 (m, 1H), 6.99-6.86 (m, 1H), 6.82-6.73 (m, 1H), 4.14 (s, 2H), 3.71-3.59 (m, 2H), 3.17-3.08 (m, 2H), 2.48 (s, 3H), 2.03-1.90 (m, 4H).
To a suspension of 5-bromo-2-nitropyridine (1 g, 4.93 mmol) and tert-butyl (S)-2-methylpiperazine-1-carboxylate (1.1 g, 5.42 mmol) in NMP (15 mL) was added TEA (1.5 g, 14.8 mmol), and the reaction mixture was stirred for 6 h at 120° C. After cooling to rt and diluting with water, the mixture was extracted with EA. The combined organics were washed with brine and water, dried over Na2SO4 and concentrated to dryness. The crude was purified by silica gel column (PE/EA=3:1). The product was obtained as yellow solid (I g, Y: 63%). ESI-MS (M+H)+: 323.0.
To a mixture of tert-butyl (S)-2-methyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate (1 g, 3.11 mmol) in MeOH (10 mL) was added Pd/C (100 mg). The reaction mixture was stirred for 1 h at rt under hydrogen atmosphere (balloon pressure). The solvent was filtered and the filter cake was washed with MeOH (50 mL). The filtrate was concentrated to give desired product as purple semi-solid (750 mg, yield: 83%), which was used to next step without further purification. ESI-MS (M+H)+: 293.1. 1H NMR (400 MHz, DMSO-d6) δ 7.60 (d, J=2.8 Hz, 1H), 7.18-7.10 (m, 1H), 6.41 (d, J=8.8 Hz, 1H), 5.45 (s, 2H), 4.18 (br s, 1H), 3.81-3.72 (m, 1H), 3.27-3.20 (m, 1H), 3.15-3.07 (m, 2H), 2.66-2.60 (m, 1H), 2.49-2.40 (m, 1H), 1.42 (s, 9H), 1.22 (d, J=6.7 Hz, 3H).
To a mixture of 8-fluoro-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (300 mg, 1.95 mmol) in DMF (8 mL) was added tert-butyl (S)-4-(6-aminopyridin-3-yl)-2-methylpiperazine-1-carboxylate (299 mg, 1.54 mmol), DIEA (399 mg, 3.09 mmol) and HATU (585 mg, 1.54 mmol). The mixture was stirred at rt for 5 h. After diluting with water, the mixture was filtered and the cake was purified by silica gel column chromatography (DCM:MeOH=20:1) to give title product (280 mg, 33%) as a yellow solid. ESI-MS (M+H)+: 469.0.
To a solution of tert-butyl (S)-4-(6-(8-fluoro-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyridin-3-yl)-2-methylpiperazine-1-carboxylate (100 mg, 0.21 mmol) in DCM (10 ml) was added TFA (1 mL) at 0° C. The mixture was stirred for 1 h at rt. After concentration, the residue was diluted with DCM (5 m) and concentrated again. The residue was purified by Prep-HPLC to give title product (31.72 mg, yield: 41%) as a red solid. ESI-MS (M+H)+: 369.1. 1H NMR (400 MHz, DMSO-d6) δ 10.91 (s, 1H), 9.25-9.24 (m, 2H), 8.85 (d, J=9.4 Hz, 1H), 8.19 (d, J=2.9 Hz, 1H), 8.07-8.02 (m, 2H), 7.89-7.83 (m, 1H), 7.57 (dd, J=9.2, 3.0 Hz, 1H), 3.87-3.78 (m, 2H), 3.45-3.42 m, 2H), 3.19-3.17 (m, 1H), 3.01-2.98 (m, 1H), 2.80-2.76 (m, 1H), 2.43 (s, 3H), 1.30 (d, J=6.5 Hz, 3H).
To a stirred solution of 8-fluoro-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (100 mg 0.0.52 mmol), tert-butyl 4-(4-amino-3-fluorophenyl)piperazine-1-carboxylate (168 mg, 0.57 mmol) and DIEA (335 mg, 2.60 mmol) in DMF (10 mL) was added HATU (396 mg, 1.04 mmol) at rt. After addition was completed, the mixture was stirred for 2 h. After concentration, the crude was purified by prep-HPLC (0.05% FA in water/CH3CN) to give title product (123 mg, yield: 51.2%) as a brown solid. ESI-MS (M+H)+: 472.4.
To a solution of tert-butyl 4-(3-fluoro-4-(8-fluoro-2-methylimidazo[1,2-a]pyridine-6-carboxamido)phenyl)piperazine-1-carboxylate (50 mg, 0.11 mmol) in EtOAc (1 mL) was added HCl-EA (3M, 1 mL), the mixture was stirred at rt for 1 h. The precipitate was filtered and lyophilized to give title product (35 mg, yield: 70%). ESI-MS (M+H)+: 372.2. 1H NMR (400 MHz, CD3OD) δ 9.26 (s, 1H), 8.30-8.17 (m, 2H), 7.61 (t, J=8.6 Hz, 1H), 6.93 (dd, J=17.9, 11.5 Hz, 2H), 3.48 (d, J=4.8 Hz, 4H), 3.39 (d, J=4.6 Hz, 4H), 2.62 (s, 3H).
To a mixture of 2,8-dimethylimidazo[1,2-a]pyrazine-6-carboxylic acid (50 mg, 0.26 mmol) in DMF (8 mL) were added tert-butyl 4-(4-amino-3-fluorophenyl)piperazine-1-carboxylate (115 mg, 0.39 mmol), DIEA (101 mg, 0.78 mmol) and HATU (149 mg, 0.39 mmol). The mixture was stirred at it for 5 h and quenched with water (10 mL). The mixture was filtered and the cake was purified by silica gel column chromatography (DCM:MeOH=20:1) to give tert-butyl 4-(4-(2,8-dimethylimidazo [1,2-a]pyrazine-6-carboxamido)-3-fluorophenyl)piperazine-1-carboxylate (50 mg, 41%) as a yellow solid. ESI-MS (M+H)+: 469.3.
To a solution of tert-butyl 4-(4-(2,8-dimethylimidazo [1,2-a]pyrazine-6-carboxamido)-3-fluorophenyl)piperazine-1-carboxylate (50 mg, 0.11 mmol) was added HCl in dioxane (5 mL) at rt. The mixture was stirred for 1 h at rt. The precipitate was filtered and lyophilized to give N-(2-fluoro-4-(piperazin-1-yl)phenyl)-2,8-dimethylimidazo [1,2-a]pyrazine-6-carboxamide hydrochloride (3 mg, yield: 7.6%) as a yellow solid. ESI-MS (M+H)+: 369.3. 1H NMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H), 9.20 (s, 1H), 8.94 (s, 2H), 8.07 (s, 1H), 7.88-7.83 (m, 1H), 7.02 (d, J=14.2 Hz, 1H), 6.87 (d, J=9.2 Hz, 1H), 3.40 (d, J=4.8 Hz, 4H), 3.23 (s, 4H), 2.82 (s, 3H), 2.46 (s, 3H).
To a stirred solution of 7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (100 mg, 0.48 mmol), tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate (140 mg, 0.49 mmol) and DIEA (190 mg, 1.46 mmol) in DMF (4 mL) was added HATU (289 mg, 0.76 mmol) at rt. After addition was completed, the mixture was stirred for 1 h. After diluting with water, the solid was collected by filtration and washed with MeCN to give product (100 mg, Y: 44.8%) as a light brown solid, which was used to next step without further purification. ESI-MS (M+H)+: 467.3. 1H NMR (400 MHz, DMSO-d6) δ 10.30 (s, 1H), 9.00 (s, 1H), 8.10-8.08 (m, 2H), 7.67 (s, 1H), 7.50 (d, J=8.9 Hz, 1H), 7.03 (s, 1H), 4.00 (s, 3H), 3.47 (br.s, 4H), 2.34 (s, 3H), 1.43 (s, 9H).
To a solution of tert-butyl 4-(6-(7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyridin-3-yl)piperazine-1-carboxylate (100 mg, 0.214 mmol) in 1,4-dioxane (1 mL) was added HCl-dioxane (4 M, 0.5 mL) at rt and stirred for 1 h. The precipitate was filtered and lyophilized to give 7-methoxy-2-methyl-N-(5-(piperazin-1-yl)pyridin-2-yl)imidazo [1,2-a]pyridine-6-carboxamide HCl salt (17 mg, yield: 21, 8%) as a yellow solid. ESI-MS (M+H)+: 367.2. 1H NMR (400 MHz, DMSO-d6) δ 10.91 (s, 1H), 9.82 (s, 2H), 9.23 (s, 1H), 8.17-8.05 (m, 2H), 8.00 (s, 1H), 7.64 (d, J=7.2 Hz, 1H), 7.36 (s, 1H), 4.07 (s, 3H), 3.48 (s, 4H), 3.20 (s, 4H), 2.45 (s, 3H).
To a stirred solution of 7-methoxy-2-methylimidazo [1,2-a]pyridine-6-carboxylic acid (100 mg, 0.48 mmol), tert-butyl 4-(4-amino-3-fluorophenyl)piperazine-1-carboxylate (130 mg, 0.49 mmol) and DIEA (190 mg, 1.46 mmol) in DMF (4 mL) was added HATU (289 mg, 0.76 mmol) at rt. After addition was completed, the mixture was stirred for 1 h. After diluting with water, the solid was collected by filtration and washed with MeCN to give title product (30 mg, Y: 12.8%) as a light brown solid, which was used to next step without further purification. ESI-MS (M+H)+: 484.3.
To a solution of tert-butyl 4-(3-fluoro-4-(7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)phenyl)piperazine-1-carboxylate (30 mg, 0.06 mmol) in 1,4-dioxane (1 mL) was added HCl-dioxane (4 M, 0.5 mL) at rt and stirred for 1 h. The precipitate was filtered and lyophilized to give N-(2-fluoro-4-(piperazin-1-yl) phenyl)-7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamide (12 mg, yield: 52.2%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H), 9.68 (s, 2H), 9.24 (s, 1H), 7.98 (s, 1H), 7.68 (t, J=8.9 Hz, 1H), 7.34 (s, 1H), 6.99 (d, J=13.8 Hz, 1H), 6.86 (d, J=7.8 Hz, 1H), 4.09 (s, 3H), 3.45 (s, 4H), 3.18 (s, 4H), 2.46 (s, 3H). ESI-MS (M+H)+: 384.2.
To a stirred solution of 7-methoxy-2,8-dimethylimidazo[1,2-a]pyridine-6-carboxylic acid (100 mg, 0.45 mmol), tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate (152 mg, 0.55 mmol) and DIEA (176 mg, 1.36 mmol) in DMF (5 mL) was added HATU (259 mg, 0.681 mmol) at rt. After addition was completed, the mixture was stirred for 2 h. The reaction mixture was treated with EA/Water (100 mL, 1:1). The organic phase was separated and the aqueous phase was extracted with EA (50 mL×1). The combined organic layers was washed with brine and dried over sodium sulfate. The mixture was purified by prep-HPLC (0.05% FA in water/MeCN) to give tert-butyl 4-(6-(7-methoxy-2,8-dimethylimidazo[1,2-a]pyridine-6-carboxamido) pyridin-3-yl)piperazine-1-carboxylate (60 mg, yield: 27.6%) as a white solid. ESI-MS (M+H)+: 481.1.
To a solution of tert-butyl tert-butyl 4-(6-(7-methoxy-2,8-dimethylimidazo[1,2-a]pyridine-6-carboxamido)pyridin-3-yl)piperazine-1-carboxylate (60 mg, 0.124 mmol) in EA (3 mL) was added HCl-EA (3 M, 2 mL) at 0° C. and the mixture was allowed to warm to rt and stirred for 1 h. After concentration, the residue purified by prep-HPLC (0.05% HCl in water/MeCN) to give title compound (5.0 mg, yield: 10.6%) as a yellow solid. 1H NMR (400 MHz, MeOD-d4) δ 9.16 (s, 1H), 8.30 (dd, J=9.5, 2.6 Hz, 1H), 8.07 (s, 1H), 8.00 (s, 1H), 7.80 (d, J=9.4 Hz, 1H), 4.07 (s, 3H), 3.69-3.61 (m, 4H), 3.48-3.40 (m, 4H), 2.59 (s, 3H), 2.57 (s, 3H). ESI-MS (M+H)+: 381.1.
To a stirred solution of 6-methoxy-2-methyl-2H-indazole-5-carboxylic acid (80 mg, 0.38 mmol), tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate (128 mg, 0.46 mmol) and DIEA (152 mg, 1.16 mmol) in DMF (6 mL) was added HATU (220 mg, 0.58 mmol) at rt. After addition was completed, the mixture was stirred for 2 h. The reaction mixture was treated with EA/Water (40 mL, 1:1). The organic phase was separated and the aqueous phase was extracted with EA (25 mL). The combined organic layers was washed with brine and dried over sodium sulfate. After concentration under reduced pressure, the crude was purificated by C18 flash (0.1% FA in water/CH3CN) to give tert-butyl 4-(6-(6-methoxy-2-methyl-2H-indazole-5-carboxamido)pyridin-3-yl)piperazine-1-carboxylate (60 mg, Y: 33.2%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 10.31 (s, 1H), 8.70 (s, 1H), 8.35 (d, J=9.1 Hz, 1H), 8.03-7.97 (m, 2H), 7.33 (dd, J=9.0, 2.9 Hz, 1H), 7.10 (s, 1H), 4.20 (s, 3H), 4.11 (s, 3H), 3.64-3.57 (m, 4H), 3.15-3.09 (m, 4H), 1.62 (s, 9H). ESI-MS (M+H)+: 467.3.
To a solution of 4-(6-(6-methoxy-2-methyl-2H-indazole-5-carboxamido)pyridin-3-yl) piperazine-1-carboxylate (60 mg, 0.129 mmol) in EA (2 mL) was added HCl-EA (3 M, 2 mL) at 0° C. and the mixture was allowed to warm to rt and stirred for 1 h. After concentration, the solid was collected by filtration and washed EA three times (3 mL×3). The solid was dried under vacuum at 55° C. to provide title product (10 mg, yield: 21.2%) as light yellow solid. 1H NMR (400 MHz, MeOD-d4) δ 8.66 (s, 1H), 8.52 (s, 1H), 8.33 (dd, J=9.5, 2.3 Hz, 1H), 8.00 (d, J=2.0 Hz, 1H), 7.77 (d, J=9.5 Hz, 1H), 7.24 (s, 1H), 4.29 (s, 3H), 4.11 (s, 3H), 3.68-3.60 (m, 4H), 3.49-3.41 (m, 4H). ESI-MS (M+H)+: 367.2.
A mixture of 2,4-difluoro-1-nitrobenzene (5 g, 21 mmol), K2CO3 (6.9 g, 50 mmol) and tert-butyl (S)-2-methylpiperazine-1-carboxylate (4.2 g, 21 mmol) in HMPA (50 ml) was heated to 100° C. for 4 h. After cooling to rt, the reaction mixture was diluted with ethyl acetate (300 mL) and washed with water (100 mL) and brine (100 mL). The organic layer was dried over Na2SO4, concentrated to dryness. The crude was purified by silica gel column (EA:PE=1:5, v/v) to give title product (4 g, Y: 80%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.98 (t, J=9.1 Hz, 1H), 6.47 (dd, J=9.5, 2.5 Hz, 1H), 6.40 (dd, J=14.8, 2.6 Hz, 1H), 4.27 (s, 1H), 3.93-3.82 (m, 1H), 3.65-3.62 (m, 1H), 3.51 (d, J=11.2 Hz, 1H), 3.34-3.24 (m, 2H), 3.14-3.05 (m, 1H), 1.46 (s, 9H), 1.15 (d, J=6.7 Hz, 3H).
A mixture of tert-butyl (S)-4-(3-fluoro-4-nitrophenyl)-2-methylpiperazine-1-carboxylate (2 g, 5.9 mmol) and Pd/C (320 mg, 10% wt/wt) in MeOH (50 mL) was purged with hydrogen for three times at it. The mixture was stirred for 30 min at rt under hydrogen. The catalyst was filtered off and the filtrate was concentrated to give title product, which was used in next step without further purification. 1H NMR (400 MHz, CDCl3) δ 6.77-6.69 (m, 11H), 6.62 (dd, J=13.4, 2.5 Hz, 1H), 6.55 (dd, J=8.5, 2.1 Hz, 1H), 4.32 (s, 1H), 3.95-3.92 (m, 1H), 3.45 (s, 2H), 3.32-3.12 (m, 3H), 2.79-2.76 (m, 1H), 2.61-2.59 (m, 1H), 1.48 (s, 9H), 1.30 (d, J=6.7 Hz, 3H). ESI-MS (M+H)+: 310.2.
To a stirred solution of 6-methoxy-2-methyl-2H-indazole-5-carboxylic acid (80 mg, 0.38 mmol), tert-butyl (S)-4-(4-amino-3-fluorophenyl)-2-methylpiperazine-1-carboxylate (181 mg, 0.58 mmol) and DIEA (253 mg, 0.76 mmol) in DMF (10 mL) was added HATU (297 mg, 0.78 mmol) at rt. After addition was completed, the mixture was stirred for 2 h. The reaction mixture was treated with EA/Water (100 mL, 1:1). The organic phase was separated and the aqueous phase was extracted with EA (50 mL×1). The combined organic layers was washed with brine and dried over sodium sulfate. After concentration under reduced pressure, the crude was slurry with EA/PE (1:10, v/v, 5 mL) for 30 min at rt. Then the solid was collected by filtration and washed with EA/PE (1:10, v/v, 3 mL×3). The product was obtained (90 mg, Y: 49%) as a light brown solid. ESI-MS (M+H)+: 498.3. 1H NMR (400 MHz, CDCl3) δ 10.21 (d, J=2.3 Hz, 1H), 8.73 (d, J=3.8 Hz, 1H), 8.41 (dd, J=11.7, 6.7 Hz, 1H), 7.98 (d, J=5.2 Hz, 1H), 7.09 (d, J=6.7 Hz, 1H), 6.72-6.56 (m, 2H), 4.35 (s, 1H), 4.20 (s, 3H), 4.09 (s, 3H), 3.95-3.91 (m, 1H), 3.46-3.42 (m, 1H), 3.33-3.30 (m, 1H), 3.29-3.21 (m, 1H), 2.94-2.90 (m, 1H), 2.79-2.71 (m, 1H), 1.49 (s, 9H), 1.30 (d J=6.7 Hz, 3H).
To a solution of tert-butyl (S)-4-(3-fluoro-4-(6-methoxy-2-methyl-2H-indazole-5-carboxamido)phenyl)-2-methylpiperazine-1-carboxylate (64 mg, 0.141 mmol) in EtOAc (1 mL) was added HCl-EA (3M, 1 mL), the mixture was stirred at rt for 1 h. The precipitate was filtered and lyophilized to give (S)—N-(2-fluoro-4-(3-methylpiperazin-1-yl) phenyl)-6-methoxy-2-methyl-2H-indazole-5-carboxamide hydrochloride (35.3 mg, yield: 60%). ESI-MS (M+H)+: 398. 1H NMR (400 MHz, MeOD-d4) δ 8.71 (s, 1H), 8.55 (s, 1H), 8.05 (t, J=8.9 Hz, 1H), 7.24 (s, 1H), 7.00-6.85 (m, 2H), 4.30 (s, 3H), 4.14 (s, 3H), 3.85-3.81 (m, 2H), 3.55-3.45 (m, 2H), 3.36-3.32 (m, 1H), 3.09-2.95 (m, 1H), 2.85-2.82 (m, 1H), 1.41 (d, J=6.6 Hz, 3H).
To a stirred solution of 6-methoxy-2-methyl-2H-indazole-5-carboxylic acid (60 mg, 0.29 mmol), tert-butyl 4-(4-amino-3-fluorophenyl)piperazine-1-carboxylate (96 mg, 0.32 mmol) and DIEA (114 mg, 0.87 mmol) in DMF (3 mL) was added HATU (150 mg, 0.39 mmol) at rt. After addition was completed, the mixture was stirred for 2 h. The crude was purified by prep-HPLC (0.05% FA in water/CH3CN) to give title product (80 mg, yield:57.1%) as a yellow solid. ESI-MS (M+H)+: 484.1 1H NMR (400 MHz, CDCl3) δ 10.24 (s, 1H), 8.73 (s, 1H), 8.42 (dd, J=11.6, 6.8 Hz, 1H), 7.99 (s, 1H), 7.10 (s, 1H), 6.75-6.68 (m, 2H), 4.20 (s, 3H), 4.09 (s, 3H), 3.60-3.57 (m, 4H), 3.14-3.09 (m, 4H), 1.49 (s, 9H).
To a solution of tert-butyl 4-(3-fluoro-4-(6-methoxy-2-methyl-2H-indazole-5-carboxamido)phenyl)piperazine-1-carboxylate (80 mg, 0.16 mmol) in EA (0.5 mL) was added HCl-EA (3 M, 0.5 mL) and the mixture was stirred for 1 h. The precipitate was filtered and lyophilized to give title product (35 mg, yield:55.5%) as a yellow solid. ESI-MS (M+H)+: 383.9. 1H NMR (400 MHz, MeOD-d4) δ 8.70 (s, 1H), 8.55 (s, 1H), 8.05 (t, J=8.8 Hz, 1H), 7.24 (s, 1H), 6.96 (dd, J=13.6, 2.6 Hz, 1H), 6.88 (dd, J=8.8, 2.4 Hz, 1H), 4.31 (s, 3H), 4.13 (s, 3H), 3.46-3.43 (m, 4H), 3.39-3.36 (m, 4H).
A mixture of 2,4-difluoro-1-nitrobenzene (722 mg, 4.54 mmol), K2CO3 (732 mg, 5.30 mmol) and tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate (300 mg, 1.51 mmol) in HMPA (10 mL) was heated to 100° C. for 4 h. After cooling to rt, the reaction mixture was diluted with ethyl acetate (60 mL) and washed with water (60 mL) and brine (60 mL). The organic layer was dried over Na2SO4, concentrated to dryness. The crude was purified by pre-TLC (EA:PE=1:5, v/v) to give title product (260 mg, Y: 50.9%) as a yellow solid. ESI-MS (M+H)+: 338.1.
A mixture of tert-butyl 6-(3-fluoro-4-nitrophenyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (460 mg, 1.36 mmol) and Pd/C (92 mg, 20% wt/wt) in MeOH (30 mL) was purged with hydrogen for three times at rt. The mixture was stirred for 30 min at rt under hydrogen. The catalyst was filtered off and the filtrate was concentrated to give title product, which was used in next step without further purification. 1H NMR (400 MHz, CDCl3) δ 6.70 (t, J=9.0 Hz, 1H), 6.19 (dd, J=12.5, 2.3 Hz, 1H), 6.11 (dd, J=8.4, 1.7 Hz, 1H), 4.07 (br.s, 4H), 3.87 (br.s, 4H), 1.44 (s, 9H).
To a stirred solution of 6-methoxy-2-methyl-2H-indazole-5-carboxylic acid (60 mg, 0.29 mmol), tert-butyl 6-(4-amino-3-fluorophenyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (80 mg, 0.26 mmol) and DIEA (114 mg, 0.87 mmol) in DMF (3 mL) was added HATU (150 mg, 0.38 mmol) at rt. After addition was completed, the mixture was stirred for 2 h. The crude was purified by prep-HPLC (0.05% FA in water/CH3CN) to give title product (50 mg, yield: 39.0%) as a yellow solid. ESI-MS (M+H)+: 496.1. 1H NMR (400 MHz, CDCl3) δ 10.15 (d, J=2.2 Hz, 1H), 8.72 (s, 1H), 8.34 (t, J=8.8 Hz, 1H), 7.99 (s, 1H), 7.09 (s, 1H), 6.24 (dd, J=9.5, 4.9 Hz, 2H), 4.20 (s, 3H), 4.10 (s, 4H), 4.08 (s, 3H), 3.96 (s, 4H), 1.45 (s, 9H).
To a solution of tert-butyl 6-(3-fluoro-4-(6-methoxy-2-methyl-2H-indazole-5-carboxamido)phenyl)-2,6-diazaspiro[3.3] heptane-2-carboxylate (50 mg, 0.10 mmol) in DCM (4 mL) was added TFA (1 mL) and the mixture was stirred for 1 h. The mixture was concentrated in vacuo, the residue was purified by prep-HPLC (0.05% FA in water/CH3CN) to give title product (10 mg, yield:25%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.89 (s, 1H), 8.78 (s, 1H), 8.43 (s, 1H), 8.33 (s, 1H), 7.82 (t, J=8.8 Hz, 1H), 7.13 (s, 1H), 6.43-6.27 (m, 2H), 4.18 (s, 4H), 4.14 (s, 3H), 3.98 (d, J=3.5 Hz, 7H). ESI-MS (M+H)+: 396.1.
A mixture of 2,4-difluoro-1-nitrobenzene (450 mg, 2.83 mmol), K2CO3 (391 mg, 2.83 mmol) and tert-butyl hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (300 mg, 1.41 mmol) in HMPA (8.00 mL) was heated at 100° C. for 4 h. After cooling to rt, the reaction mixture was diluted with ethyl acetate (100.0 mL) and washed with water (60.0 mL) and brine (30.0 mL). The organic layer was dried over Na2SO4. After concentration, the crude was purified by gel silica chromatograph (PE:EA=10:1-5:1) to give title product (270 mg, Y: 64.5%) as a yellow solid. ESI-MS (M+H-56)+: 296.1.
A mixture of tert-butyl 5-(3-fluoro-4-nitrophenyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (270 mg, 0.912 mmol) and Pd/C (50.0 mg) in EA (5.00 mL) was purged with hydrogen for three times at rt. The mixture was stirred for overnight at rt under hydrogen. The catalyst was filtered off and the filtrate was concentrated to give title product, which was used in next step without further purification. ESI-MS (M+H)+: 322.1.
To a mixture of tert-butyl 5-(4-amino-3-fluorophenyl)hexahydropyrrolo[3,4-c]pyrrole-2(0H)-carboxylate (100 mg, 0.31 mmol) in DMF (8 mL) were added 6-methoxy-2-methyl-2H-indazole-5-carboxylic acid (128 mg, 0.62 mmol), DIEA (121 mg, 0.93 mmol) and HATU (178 mg, 0.47 mmol). The mixture was stirred at rt for 2 h. After diluting with water, he mixture was filtered and the cake was purified by C18 (0.1% FA in water/CH3CN) to give title product (40 mg, 25%) as a white solid. ESI-MS (M+H)+: 510.1. 1H NMR (400 MHz, MeOD-d4) δ 8.51 (s, 1H), 8.31 (s, 1H), 7.75 (dd, J=8.8, 6.2 Hz, 1H), 7.12 (s, 1H), 6.83 (dd, J=11.1, 2.8 Hz, 1H), 6.74-6.69 (m, 1H), 4.19 (s, 3H), 4.04 (s, 3H), 3.63 (dd, J=11.3, 7.9 Hz, 2H), 3.34 (s, 2H), 3.24 (dd, J=11.3, 4.0 Hz, 2H), 3.09 (d, J=9.0 Hz, 2H), 3.00 (dd, J=8.6, 5.1 Hz, 2H), 1.43 (s, 9H).
To a solution of tert-butyl 5-(3-fluoro-4-(6-methoxy-2-methyl-2H-indazole-5-carboxamido)phenyl)hexahydropyrrolo [3,4-c]pyrrole-2(1H)-carboxylate (40 mg, 0.078 mmol) was added HCl-EA (3 M, 5 mL) at RT and the mixture stirred for 1 h at rt. The precipitate was filtered and lyophilized to give title product (24 mg, yield: 75%) as white solid. 1H NMR (400 MHz, MeOD-d4) δ 8.70 (s, 1H), 8.51 (s, 1H), 7.63 (dd, J=8.8, 6.1 Hz, 1H), 7.27 (s, 1H), 6.93 (dd, J=10.8, 2.7 Hz, 1H), 6.83 (dd, J=8.3, 2.7 Hz, 1H), 4.32 (s, 3H), 4.13 (s, 3H), 3.53-3.46 (m, 2H), 3.32 (s, 2H), 3.19-3.15 (m, 6H). ESI-MS (M+H)+: 410.2.
7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (0.229 g, 1.11 mmol) was suspended in DMF (4 mL) and ethylbis(propan-2-yl)amine (0.215 g, 1.66 mmol, 290.0 μL, 1.5 equiv.) was added followed by [(dimethylamino)(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methylidene]dimethylazanium, hexafluoro-lambda5-phosphanide (0.506 g, 1.33 mmol). The resulting mixture was stirred for 30 min at r.t. After that, tert-Butyl 4-(6-aminopyridazin-3-yl)piperazine-1-carboxylate (0.223 g, 798.34 μmol) was added in one portion and the reaction mixture was stirred overnight at r.t. The precipitate formed was filtered, washed with MeCN (2 mL), MTBE (2 mL), dried in vacuo to give pure tert-butyl 4-(6-7-methoxy-2-methylimidazo[1,2-a]pyridine-6-amidopyridazin-3-yl)piperazine-1-carboxylate (0.120 g, 256.67 μmol, 23.1% yield). ESI-MS (M+H)+: 468.4.
To a stirred solution of tert-butyl 4-(6-7-methoxy-2-methylimidazo[1,2-a]pyridine-6-amidopyridazin-3-yl)piperazine-1-carboxylate (0.050 g, 106.97 μmol) in dichloromethane (15 mL) 2,2,2-trifluoroacetic acid (0.122 g, 1.07 mmol) was added in one portion and the resulting mixture was stirred overnight at r.t. The solvents were evaporated and the residue was triturated with MTBE/MeCN (5/2 mL). The precipitate was filtered and dried under vacuo to give 7-methoxy-2-methyl-N-[6-(piperazin-1-yl)pyridazin-3-yl]imidazo[1,2-a]pyridine-6-carboxamide TFA salt (0.042 g, 101.78 μmol, quantitative) as a white solid. 1H NMR (500 MHz, DMSO-d6) δ 11.09 (s, 1H), 9.14 (s, 1H), 9.07-8.82 (br s, 2H), 8.19 (d, J 9.9 Hz, 1H), 7.90 (s, 1H), 7.51 (d, J=9.8 Hz, 1H), 7.34 (s, 1H), 4.05 (s, 3H), 3.77 (s, 4H), 3.23 (s, 4H). ESI-MS (M+H)+: 368.2.
The 7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (0.200 g 969.94 μmol) was suspended in DMF (4 mL) and ethylbis(propan-2-yl)amine (0.314 g, 2.43 mmol) was added followed by [(dimethylamino)(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methylidene]dimethylazanium, hexafluoro-lambda5-phosphanide (0.443 g, 1.17 mmol). The resulting mixture was stirred for 30 min at r.t. After that, tert-butyl 4-(5-aminopyridin-2-yl)piperazine-1-carboxylate (0.270 g, 970.99 μmol) was added in one portion and the reaction mixture was stirred overnight at r.t. The precipitate formed was filtered, washed with MeCN (2 mL), MTBE (2 mL), dried in vacuo to give pure tert-butyl 4-(5-7-methoxy-2-methylimidazo[1,2-a]pyridine-6-amidopyridin-2-yl)piperazine-1-carboxylate (0.250 g, 535.87 μmol, 55.2% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.96 (s, 1H), 8.81 (s, 1H), 8.39 (s, 1H), 7.86 (d, J 9.2 Hz, 1H), 7.55 (s, 1H), 6.91 (s, 1H), 6.84 (d, J=8.9 Hz, 1H), 3.89 (s, 3H), 3.39 (s, 8H), 2.24 (s, 3H), 1.39 (s, 9H). ESI-MS (M+H)+: 467.2.
tert-Butyl 4-(5-7-methoxy-2-methylimidazo[1,2-a]pyridine-6-amidopyridin-2-yl)piperazine-1-carboxylate (0.050 g, 107.25 μmol) was dissolved in dichloromethane (10 mL) and 2,2,2-trifluoroacetic acid (0.122 g, 1.07 mmol) was added. The resulting mixture was stirred overnight at r.t., evaporated and triturated with mixture of MTBE/MeCN (5/2 mL). The precipitate formed was filtered and dried in vacuo to give desired 7-methoxy-2-methyl-N-[6-(piperazin-1-yl)pyridin-3-yl]imidazo[1,2-a]pyridine-6-carboxamide TFA salt (0.109 g, 226.88 μmol, quantitative) as a grey solid. 1H NMR (400 MHz, DMSO-d6) δ 10.31 (s, 1H), 9.11 (s, 1H), 8.80 (s, 2H), 8.42 (s, 1H), 7.88 (m, 2H), 7.30 (s, 1H), 6.95 (s, 1H), 4.02 (s, 3H), 3.64 (s, 4H), 3.17 (s, 4H), 2.40 (s, 3H). ESI-MS (M+H)+: 367.2.
The 7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (0.200 g, 969.94 μmol) was suspended in DMF (4 mL) and ethylbis(propan-2-yl)amine (0.313 g, 2.42 mmol) was added followed by [(dimethylamino)(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methylidene]dimethylazanium, hexafluoro-lambda5-phosphanide (0.442 g, 1.16 mmol). The resulting mixture was stirred for 30 min at r.t. After that, tert-butyl 4-(5-aminopyrimidin-2-yl)piperazine-1-carboxylate (0.270 g, 969 μmol) was added in one portion and the reaction mixture was stirred overnight at r.t. The precipitate formed was filtered, washed with MeCN (2 mL), MTBE (2 mL), dried in vacuo to give pure tert-butyl 4-(5-(7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyrimidin-2-yl)piperazine-1-carboxylate (0.234 g, 500 μmol, 51.7% yield). ESI-MS (M+H)+: 468.2.
tert-buty 4-(5-(7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyrimidin-2-yl)piperazine-1-carboxylate (0.234 g, 500 μmol) was dissolved in dichloromethane (10 mL) and 2,2,2-trifluoroacetic acid (0.571 g, 5.01 mmol) was added. The resulting mixture was stirred overnight at r.t., evaporated and triturated with mixture of MTBE/MeCN (5/2 mL). The precipitate formed was filtered and dried in vacuo to give desired 7-methoxy-2-methyl-N-(2-(piperazin-1-yl)pyrimidin-5-yl)imidazo[1,2-a]pyridine-6-carboxamide TFA salt (0.215 g, 447 μmol, quantitative) as a beige solid. 1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 9.14 (s, 1H), 8.94 (s, 2H), 8.68 (d, J=1.7 Hz, 2H), 7.88 (s, 1H), 7.32 (s, 1H), 4.02 (s, 3H), 3.89 (s, 4H), 3.15 (s, 4H), 2.40 (s, 3H). ESI-MS (M+H)+: 368.2.
A mixture of 8-fluoro-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (0.5 g; 2.6 mmol). HATU (1.18 g; 3.12 mmol), tert-butyl 4-(5-aminopyridin-2-yl)piperazine-1-carboxylate (0.7 g; 2.5 mmol) and TEA (0.72 mL; 5.2 mmol) in DMF (20 mL) was stirred at r.t. for 14 h. Next, the reaction mixture was diluted with water and extracted with EtOAc. Organics were washed with water, dried with Na2SO4 and evaporated to give crude title compound (0.9 g; 2 mmol; 79% yield), which was used in the next step without further purification. ESI-MS: 455.2 (M+H)+.
tert-butyl 4-(5-(8-fluoro-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyridin-2-yl)piperazine-1-carboxylate (0.9 g; 2 mmol) was dissolved in MeOH (20 mL). Next, 10% dioxane/HCl solution was added and the obtained solution was stirred at r.t. for 10 h. The reaction mixture was evaporated in vacuo and the residue was purified by HPLC to afford the title compound as hydrochloride salt (73 mg; 19% yield). ESI-MS: 355.2 (M+H)+; H NMR (400 MHz, DMSO-d6): δ 10.18 (s, 1H), 9.05 (s, 1H), 8.41 (d, J=2.8 Hz, 1H), 7.96 (d, J=2.4 Hz, 1H), 7.86 (dd, J=9.2, 2.8 Hz, 1H), 7.61 (d, J=12 Hz, 1H), 6.83 (d, J=9.6 Hz, 1H), 3.33-3.39 (m, 4H), 2.74-2.82 (m, 4H), 2.38 (s, 3H).
To a solution of 3,6-dichloropyridazine (3.0 g, 15 mmol) in 1,4-dioxane (20 mL) were added DIEA (3.87 g, 30 mmol), tert-butyl (S)-2-methylpiperazine-1-carboxylate (2.4 g, 12 mmol). The mixture was stirred at 100° C. overnight. The reaction mixture was diluted with saturated sodium bicarbonate and extracted with EtOAc (50 mL×3), the organic layer was dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by column chromatography (PE/EA=5:1) to give title product (1.4 g, Y:36.8%) as a light yellow solid. ESI-MS (M+H)+: 313.1. 1H NMR (400 MHz, CDCl3) δ 7.22 (d, J=9.5 Hz, 1H), 6.86 (d, J=9.5 Hz, 1H), 4.36 (br s, 1H), 4.15 (d, J=11.6 Hz, 1H), 4.06-3.92 (m, 2H), 3.37 (dd, J=13.2, 4.0 Hz, 1H), 3.29-3.26 (m, 1H), 3.11-3.08 (m, 1H), 1.49 (s, 9H), 1.19 (d, J=6.7 Hz, 3H).
To a solution of tert-butyl (S)-4-(6-chloropyridazin-3-yl)-2-methylpiperazine-1-carboxylate (1.4 g, 4.5 mmol) in 1,4-dioxane (20 mL) were added Pd(OAc)2 (412 mg, 0.45 mmol), Cs2CO3 (3.0 g, 9.0 mmol), BINAP (518 mg, 0.9 mmol) and diphenylmethanimine (851 mg, 6.6 mmol), and the mixture was stirred at 80° C. overnight under nitrogen. The reaction mixture was diluted with water and extracted with EtOAc. The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by column chromatography (PE/EA=5:1) to give title product (590 mg, Y: 29.5%) as a light yellow solid. ESI-MS (M+H)+: 458.2
To a solution of tert-butyl (S)-4-(6-((diphenylmethylene)amino) pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (590 mg, 1.3 mmol) in MeOH (10 mL) was added NaOAc (320 mg, 3.9 mmol), NH2OH·HCl (449 mg, 6.5 mmol) and the mixture was stirred at rt overnight. The resultant reaction mixture was neutralized with saturated aqueous sodium hydrogen carbonate solution (25 mL), and extracted with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The crude product was washed with tert-butyl methyl ether (10 mL) to give title product (330 mg, crude) as a yellow solid. ESI-MS (M+H)+: 294.2. 1H NMR (400 MHz, CDCl3) δ 6.89 (d, J=9.6 Hz, 1H), 6.74 (d, J=9.5 Hz, 1H), 4.47-4.30 (m, 3H), 3.96-3.94 (m, 2H), 3.81-3.78 (m, 1H), 3.25-3.22 (m, 1H), 3.13-3.07 (m, 1H), 2.92-2.87 (m, 1H), 1.48 (s, 9H), 1.23 (d, J=6.7 Hz, 3H).
A solution of 6-bromo-7-methoxy-2-methylimidazo[1,2-a]pyridine (5 g, 21 mmol) in BBr3 (210 mL, 1M in DCM) was stirred at rt overnight. The reaction mixture was diluted with saturated sodium bicarbonate and concentrated, the residue was purified by column chromatography (DCM/MeOH=20:1) to give title product (2.8 g, Y: 58%) as a light yellow solid. ESI-MS (M+H)+:226.9. 1HNMR (400 MHz, CDCl3) δ 8.56 (s, 1H), 7.28 (s, 1H), 6.47 (s, 1H), 2.33 (s, 3H).
To a solution of 6-bromo-2-methylimidazo[1,2-a]pyridin-7-ol (0.8 g, 3.5 mmol) in DMF (10 mL) was added iodoethane (0.6 ml, 7.1 mmol) and K2CO3 (0.966 mg, 7 mmol). The mixture was stirred at RT overnight. The reaction mixture was diluted with water and extracted with EtOAc. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column (EA/PE=1:1) to give title product (255 mg, Y: 28.2%) as a gray solid. ESI-MS (M+H)+: 254.8/256.8. 1H NMR (400 MHz, CDCl3) δ 8.07 (s, 1H), 7.06 (s, 1H), 6.73 (s, 1H), 4.18-3.92 (m, 2H), 2.32 (d, J=0.7 Hz, 3H), 1.44 (t, J=7.0 Hz, 3H).
To a solution of 6-bromo-7-ethoxy-2-methylimidazo[1,2-a]pyridine (255 mg, 1.00 mmol) in MeOH (10 mL) was added TEA (303 mg, 3.00 mmol) and Pd(dppf)Cl2 (73.2 mg, 0.1 mmol). The mixture was charged with CO fort three times and stirred at 80° C. for 16 h. The reaction mixture was diluted with ethyl acetate (100 mL) and washed with water (50 mL) and brine (50 mL). The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo to give title product (220 mg, crude) as a gray solid. ESI-MS (M+H)+:235.0.
To a mixture of methyl 7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylate (210 mg, 0.9 mmol) in MeOH/water (12 mL, 5:1) was added LiOH·H2O (43 mg, 1.8 mmol). The mixture was stirred at rt for 2 h. After concentration, the residue was adjusted to pH=5 with 1M HCl. The residue was purified by reverse phase column to give title product (100 mg) as a gray solid. ESI-MS (M+H)+: 221.0.
To a mixture of 6-bromo-2-methylimidazo [1,2-a]pyridin-7-ol (800 mg, 3.51 mmol) in DMF (10 mL) was added NaH (280 mg, 7.02 mmol) at 0° C. the mixture was stirred at 0° C. for 0.5 h. Then 2-bromopropane (1.8 g, 10.53 mmol) was added and the reaction mixture was stirred at it overnight. The reaction mixture was diluted with water and extracted with EtOAc. The organics were washed with brine, dried with Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography (EA) to give title compound (900 mg, 95% yield), ESI-MS (M+H)+: 269.0. 1H NMR (400 MHz, DMSO-d6) δ 8.77 (s, 1H), 7.44 (s, 1H), 6.97 (s, 1H), 4.77-4.73 (m, 1H), 2.26 (s, 3H), 1.31 (d7.2 Hz, 6H).
To a mixture of 6-bromo-7-isopropoxy-2-methylimidazo [1,2-a]pyridine (400 mg, 1.48 mmol) in MeOH (20 mL) were added TEA (449 mg, 4.44 mmol) and Pd(dppf)Cl2 (108 mg, 0.15 mmol). The resulting mixture was charged with CO for three times and stirred overnight at 80° C. under CO. The mixture was allowed to cool down to room temperature and concentrated. The residue was used directly without purification. ESI-MS (M+H)+: 249.0.
To a mixture of methyl 7-isopropoxy-2-methylimidazo [1,2-a]pyridine-6-carboxylate (400 mg, 1.61 mmol) in THF (5 mL) and H2O (1 mL) was added LiOH·H2O (145 mg, 6.04 mmol). The reaction mixture was stirred for 2 h at room temperature. The mixture was exacted with DCM (15 mL). The aqueous phase was adjusted to pH to 5 with HCl (1M). The aqueous phase was concentrated to provide as a black solid and the solid was washed with DCM. The organic phase was concentrated to give title product (420 mg, crude). ESI-MS (M+H)+: 235.1
A mixture of 6-bromo-2-methylimidazo[1,2-a]pyridin-7-ol (800 mg, 3.51 mmol), bromocyclobutane (2.37 g, 17.54 mmol), KI (291 mg, 1.75 mmol) and Cs2CO3 (3.43 g, 10.52 mmol) in DMF (15 mL) was stirred at 100° C. for 16 h. The reaction mixture was diluted with water (30 mL) and extracted with EtOAc (60 mL×2). The combined organic layer was washed with brine, dried with Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography (PE/EA=2:1) to give title compound (630 mg, 64% yield) as a yellow solid. ESI-MS (M+H)+: 281.0. 1H NMR (400 MHz, CDCl3) δ 8.14 (s, 1H), 7.12 (s, 1H), 6.68 (s, 1H), 4.71 (p, J=7.1 Hz, 1H), 2.60-2.49 (m, 2H), 2.38 (s, 3H), 2.32-2.20 (m, 2H), 1.97-1.88 (m, 1H), 1.78-1.75 (m, 1H).
To a mixture of 6-bromo-7-cyclobutoxy-2-methylimidazo[1,2-a]pyridine (630 mg, 2.24 mmol) in MeOH (20 mL) were added TEA (679 mg, 6.72 mmol) and Pd(dppf)Cl2 (183 mg, 0.22 mmol). The resulting mixture was stirred overnight at 80° C. under CO. The mixture was allowed to cool down to room temperature and concentrated. The residue was used directly without purification. ESI-MS (M+H)+: 260.9
To a mixture of methyl 7-cyclobutoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylate (300 mg, 1.14 mmol) in THF (5 mL) and H2O (1 mL) was added LiOH·H2O (138 mg, 5.74 mmol). The reaction mixture was stirred for 2 h at room temperature. The mixture was exacted with DCM (15 mL). The aqueous phase was adjusted pH to 5 with HCl (1M). The aqueous phase was concentrated to provide as a black solid and the solid was washed with DCM. The organic phase was concentrated to give title product (450 mg, crude). ESI-MS (M+H)+: 247.2.
To a suspension of 2,5-dibromopyrazine (2.95 g, 12.50 mmol) and tert-butyl (S)-2-methylpiperazine-1-carboxylate (3.00 g, 15.00 mmol) in NMP (60 mL) was added DIEA (3.23 g, 25.00 mmol), the reaction mixture was stirred for 3 h at 110° C. After cooling to rt, the mixture was diluted with water 150 mL and 150 mL of EA. The EA layer was separated, washed with brine and water, dried over Na2SO4 and concentrated to dryness. The crude was purified by silica gel column (10-50% EA in PE). The product was obtained as a yellow solid (3.30 g, Y: 62%). 1H NMR (400 MHz, CDCl3) δ 8.12 (d, J=1.2 Hz, 1H), 7.84 (d, J=1.2 Hz, 1H), 4.35-4.34 (m, 1H), 4.07-4.02 (m, 1H), 3.97-3.93 (m, 2H), 3.30-3.21 (m, 2H), 3.08-3.01 (m, 1H), 1.48 (s, 9H), 1.18 (d, J=6.4 Hz, 3H).
A mixture of tert-butyl (S)-4-(5-bromopyrazin-2-yl)-2-methylpiperazine-1-carboxylate (3.30 g, 9.27 mmol), diphenylmethanimine (2.01 g, 11.12 mmol), Pd(OAc)2 (104 mg, 0.46 mmol), BINAP (577 mg, 0.93 mmol) and Cs2CO3 (6.04 g, 18.54 mmol) in 1,4-dioxane (80 mL) was purged with N2 for three times at room temperature. Then the reaction mixture was stirred at 120° C. for 48 h. After cooling to rt, the mixture was diluted with water (150 mL) and EA (150 mL). The EA layer was separated, washed with brine and water, dried over Na2SO4, concentrated to dryness. The crude was purified by silica gel column (10-50% EA in PE). The product was obtained as a yellow solid (1.50 g, yield: 36%) ESI-MS: [M+H]+ 458.2
A mixture of tert-butyl (S)-4-(5-((diphenylmethylene)amino)pyrazin-2-yl)-2-methylpiperazine-1-carboxylate (1.5 g, 3.28 mmol), NH2OH·HCl (1.13 g, 16.4 mmol) and NaOAc (807 mg, 9.84 mmol) in MeOH (30 mL) was stirred for 1 h at rt. After diluting with water, the mixture was extracted with EA (30 mL×2). The combined organics was washed with brine, dried over Na2SO4. After concentration, the crude was used to next step without further purification. 1H NMR (400 MHz, CDCl3) δ 7.68 (d, J=1.6 Hz, 1H), 7.64 (d, J=1.6 Hz, 1H), 4.39-4.32 (m, 1H), 4.05 (br s, 2H), 3.97-3.93 (m, 1H), 3.89-3.84 (m, 1H), 3.75-3.71 (m, 1H), 3.27-3.20 (m, 1H), 3.00-2.97 (m, 1H), 2.82-2.76 (m, 1H), 1.48 (s, 9H), 1.25 (d, J=6.8 Hz, 3H).
To a mixture of 7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (100 mg, 0.45 mmol) in DMF (4 mL) were added HATU (343 mg, 0.9 mmol), DIEA (291 mg 0.9 mmol), tert-butyl 4-(6-aminopyridin-3-yl) piperazine-1-carboxylate (63 mg 0.23 mmol). The mixture was stirred at rt for 2 h. The crude was purified by reverse phase column to give title product (20 mg, Y: 9.2%) as a gray solid. ESI-MS (M+H)+: 481.57.
A mixture of tert-butyl 4-(6-(7-ethoxy-2-methylimidazol[1,2-a]pyridine-6-carboxamido)pyridin-3-yl)piperazine-1-carboxylate (70 mg, 0.145 mmol) in 3M HCl/EA (2 mL) was stirred at it for 2 h. After concentration, the residue was dissolved in water (5 mL) and lyophilized to give title product (8 mg, Y: 47.1%) as a yellow solid. ESI-MS (M+H)+: 382. 1H NMR (400 MHz, MeOD-d4) δ 9.20 (s, 1H), 8.07-8.04 (m, 2H), 7.89-7.87 (m, 2H), 7.36 (s, 1H), 4.47 (q, J=6.8 Hz, 2H), 3.55-3.53 (m, 4H), 3.44-3.42 (m, 4H), 2.51 (s, 3H), 1.60 (t, J=6.8 Hz, 3H).
To a mixture of tert-butyl 4-(6-aminopyridin-3-yl) piperazine-1-carboxylate (100 mg, 0.36 mmol) in DMF (5 mL) were added 7-isopropoxy-2-methylimidazo [1,2-a]pyridine-6-carboxylic acid (100 mg, 0.43 mmol), DIEA (140 mg, 1.08 mmol) and HATU (202 mg, 0.54 mmol). The mixture was stirred at rt for 5 h and quenched with water (10 mL). The precipitate was filtered and purified by reverse phase column (0.1% FA in water/CH3CN) to give title product (30 mg, 34%) as a yellow solid. ESI-MS (M+H)+: 495.1
Tert-butyl 4-(6-(7-isopropoxy-2-methylimidazo [1,2-a]pyridine-6-carboxamido)pyridin-3-yl)piperazine-1-carboxylate (30 mg, 0.11 mmol) was dissolved in 3M HCl/EA (5 mL) at rt. The mixture was stirred for 1 h at rt. The precipitate was filtered and the solid was dried to give title product (3.0 mg, yield: 13%) as a yellow solid. ESI-MS (M+H)+: 395.0 1H NMR (400 MHz, MeOD-d4) δ 9.18 (s, 1H), 8.20-8.07 (m 2H), 7.87-7.75 (m, 2H), 7.38 (d, J=5.5 Hz, 1H), 5.09-5.03 (m, 1H), 3.66-3.57 (m, 4H), 3.47-3.40 (m, 4H), 2.52 (s, 3H), 1.53 (dd, J=5.9, 3.0 Hz, 6H).
To a mixture of tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate (100 mg, 0.358 mmol) in DMF (5 mL) were added 7-cyclobutoxy-2-methylimidazo [1,2-a]pyridine-6-carboxylic acid (133 mg, 0.54 mmol). DIEA (140 mg, 1.07 mmol) and HATU (205 mg, 0.54 mmol). The mixture was stirred at rt for 5 h and diluted with water (10 mL). The precipitate was filtered and purified by reverse phase column (0.1% FA in water/CH3CN) to give tide product (20 mg, 11%) as a yellow solid. ESI-MS (M+H)+: 507.2. 1H NMR (400 MHz, MeOD-d4) δ 9.06 (s, 1H), 8.25-8.14 (m, 2H), 8.06 (d, J=2.8 Hz, 1H), 7.60 (s, 1H), 7.48 (dd, J=9.1, 3.0 Hz, 1H), 6.83 (s, 1H), 5.03 (p, J=6.9 Hz, 1H), 3.60-3.58 (m, 4H), 3.18-3.15 (m, 4H), 2.66-2.64 (m, 2H), 2.41-2.39 (m, 5H), 2.01-1.83 (m, 2H), 1.48 (s, 9H).
To a solution of tert-butyl 4-(6-(7-cyclobutoxy-2-methylimidazo [1,2-a]pyridine-6-carboxamido)pyridin-3-yl)piperazine-1-carboxylate (20 mg, 0.04 mmol) in EA (I mL) was added 3M HCl/EA (5 mL) at rt. The mixture was stirred for 2 h at rt. The precipitate was filtered and the solid was dried under vacuum to give title product (4 mg, yield: 25%) as a yellow solid. ESI-MS (M+H)+: 407.0. 1H NMR (400 MHz, MeOD) δ 9.20 (s, 1H), 8.29-8.17 (m, 1H), 8.07 (d, J=2.4 Hz, 1H), 7.87 (s, 1H), 7.84-7.75 (m, 1H), 7.17 (d, J=3.6 Hz, 1H), 5.11 (p, J=7.1 Hz, 1H), 3.73-3.60 (m, 4H), 3.50-3.41 (m, 4H), 2.71-2.58 (m, 2H), 2.52 (d, J=0.9 Hz, 3H), 2.43-2.30 (m, 2H), 2.04-1.76 (m, 2H).
To a mixture of 6-methoxy-2H-indazole-5-carboxylic acid (72 mg, 0.35 mmol) and tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (100 mg, 0.35 mmol) in DMF (5 mL) were added HATU (267 mg, 0.7 mmol), DIEA (226 mg 1.75 mmol).
The mixture was stirred at rt for 2 h. After concentration, the crude was purified by prep-HPLC (0.05% FA in water/CH3CN) to give title product (50 mg, Y: 30.5%) as a yellow solid. ESI-MS (M+H)+: 482.1.
A mixture of tert-butyl (S)-4-(6-(6-methoxy-2-methyl-2H-indazole-5-carboxamido)pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (50 mg, 0.145 mmol) in 3M HCl/EA (4 mL) was stirred at rt for 2 h. The precipitate was filtered and the solid was dried in vacuo to give title product (19 mg, Y: 44.2%) as a yellow solid. ESI-MS (M+H)+:382.0. 1H NMR (400 MHz, MeOD-d4) δ 8.52 (s, 1H), 8.47 (s, 1H), 8.25-8.18 (m, 2H), 7.19 (s, 1H), 4.49 (d, J=14.0 Hz, 2H), 4.23 (s, 3H), 4.10 (s, 3H), 3.61-3.46 (m, 3H), 3.33-3.31 (m, 2H), 1.44 (d, J=7.2 Hz, 3H).
To a stirred solution of 2,8-dimethylimidazo[1,2-a]pyrazine-6-carboxylic acid (50 mg, 0.26 mmol), tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (84 mg, 0.28 mmol) and DIEA (100 mg, 0.78 mmol) in DMF (5 mL) was added HATU (128 mg, 0.33 mmol) at rt. The mixture was stirred at rt for 16 hrs. The reaction mixture was treated with EA/Water (50 mL, 1:1). The organic phase was separated and the aqueous phase was extracted with EA (50 mL×1). The combined organic layer was dried over sodium sulfate. After concentration, the crude was purified by pre-HPLC (0.05% FA in water/CH3CN) to provide title product (15 mg, Y: 12%) as an off-white solid. ESI-MS (M+H)+: 467.2. 1H NMR (400 MHz, CDCl3) δ 8.78 (s, 1H), 8.40 (d, J=9.8 Hz, 1H), 7.51 (s, 1H), 7.07 (d, J=9.8 Hz, 1H), 4.35-4.07 (m, 2H), 3.95-3.92 (m, 2H), 3.30-3.25 (m, 2H), 3.09-3.07 (m, 1H), 2.83 (s, 3H), 2.47 (s, 3H), 1.42 (s, 9H), 1.16 (d, J=6.8 Hz, 3H).
To a solution of tert-butyl (S)-4-(6-(2,8-dimethylimidazo[1,2-a]pyrazine-6-carboxamido)pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (15 mg, 0.03 nmol) in EA (1 mL) was added HCl-EA (3 M, 1.0 mL) at 0° C. and the mixture was allowed to warm to rt and stirred for 1 h. The solid was collected by filtration and washed with EA (2 mL×3). The solid was dried under vacuum at 55° C. to provide title product as light grey solid (5 mg, yield: 45%). ESI-MS (M+H)+: 367.0. 1H NMR (400 MHz, MeOD-d4) δ 9.48 (d, J=4.4 Hz, 1H), 8.56 (d, J=9.9 Hz, 1H), 8.29 (d, J=3.9 Hz, 1H), 8.14-8.07 (m, 1H), 4.50 (d, J=13.8 Hz, 2H), 3.57-3.51 (m, 3H), 3.37-3.35 (m, 2H), 3.03 (s, 3H), 2.68 (s, 3H), 1.45 (d, J=6.5 Hz, 3H).
To a stirred solution of 6-methoxy-2-methyl-2H-indazole-5-carboxylic acid (120 mg, 0.58 mmol), tert-butyl 4-(6-aminopyridazin-3-yl) piperazine-1-carboxylate (180 mg, 0.64 mmol) and DIEA (224 mg, 1.74 mmol) in DMF (5 mL) was added HATU (286 mg, 0.75 mmol) at rt. The mixture was stirred at it for 2 hours. The reaction mixture was treated with EA/Water (50 mL, 1:1). The organic phase was separated and the aqueous phase was extracted with EA (50 mL×1). The combined organic layer was dried over sodium sulfate. After concentration, the crude was purified by prep-HPLC (0.05% FA in water/CH3CN) to provide title product (60 mg, Y: 22%) as an off-white solid. ESI-MS (M+H)+: 468.2.
To a solution of tert-butyl 4-(6-(6-methoxy-2-methyl-2H-indazole-5-carboxamido)pyridazin-3-yl)piperazine-1-carboxylate (50 mg, 0.107 nmol) in EA (I mL) was added HCl/EA (3 M, 1.0 mL) at 0° C. and warmed to it for 1 h. The solid was collected by filtration and washed with EA (2 mL×3). The solid was concentrated in vacuo to provide title product as a yellow solid (15 mg, yield: 37%). ESI-MS (M+H)+: 368.1. 1H NMR (400 MHz, DMSO-d6) δ 10.98 (s, 1H), 9.53 (s, 2H), 8.47 (s, 1H), 8.38 (d, J=9.8 Hz, 1H), 8.30 (s, 1H), 7.76 (d, J=9.7 Hz, 1H), 7.15 (s, 1H), 4.15 (s, 3H), 3.98 (br s, 3H), 3.88 (br s, 4H), 3.24 (s, 4H).
To a mixture of 7-cyclobutoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (95 mg, 0.38 mmol) and tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (85 mg, 0.29 mmol) in DMF (3 mL) were added HATU (165 mg, 0.44 mmol) and DIPEA (112 mg, 0.87 mmol). The mixture was stirred at RT overnight. Water (10 mL) was added to the mixture and filtered, the crude product was purified by prep-HPLC (0.05% NH3H2O in water/CH3CN) to give title product (15 mg, Y: 9.9%) as a yellow solid. ESI-MS (M+H)+: 522.3. 1H NMR (400 MHz, CDCl3) δ 10.68 (s, 1H), 8.98 (s, 1H), 8.42 (d, J=9.8 Hz, 1H), 7.30 (s, 1H), 7.00 (d, J=9.9 Hz, 1H), 6.83 (s, 1H), 4.97-4.87 (m, 1H), 4.38 (s, 1H), 4.17 (d, J=12.2 Hz, 1H), 4.05-3.94 (m, 2H), 3.36-3.25 (m, 2H), 3.11-3.03 (m, 1H), 2.69-2.62 (m, 2H), 2.50-2.44 (m, 2H), 2.44 (s, 3H), 2.00-1.96 (m, 1H), 1.86-1.84 (m, 1H), 1.49 (s, 9H), 1.23 (d, J=6.7 Hz, 3H).
A mixture of tert-butyl (S)-4-(6-(7-cyclobutoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (15 mg, 0.029 mmol) in 3M EtOAc/HCl (2 mL) was stirred at rt for 2 h. After concentration, the residue was dissolved in water (5 mL) and lyophilized to give title product (11.2 mg, Y: 85.1%) as a yellow solid. ESI-MS (M+H)+: 422.2. 1H NMR (400 MHz, CD3OD) δ 9.19 (s, 1H), 8.51 (d, J=10.0 Hz, 1H), 8.01 (d, J=10.1 Hz, 1H), 7.85 (s, 1H), 7.15 (s, 1H), 5.15-5.09 (m, 1H), 4.51-4.39 (m, 2H), 3.64-3.52 (m, 3H), 3.41-3.33 (m, 2H), 2.70-2.59 (m, 2H), 2.51 (s, 3H), 2.43-2.31 (m, 2H), 2.02-1.95 (mz, 1H), 1.89-1.82 (m, 1H), 1.45 (d, J=6.6 Hz, 3H).
A mixture of tert-butyl (S)-4-(5-aminopyrazin-2-yl)-2-methylpiperazine-1-carboxylate (100 mg, 0.34 mmol), HATU (181 mg, 0.48 mmol). 7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (84 mg, 0.41 mmol) and DIEA (132 mg, 1.02 mmol) in DMF (5 mL) was stirred at r.t. for 16 h. The reaction mixture was diluted with water (20 mL), the precipitate was filtered and concentrated in vacuo. The residue was purification by reverse phase column (0.1% FA in water/CH3CN) to give title compound (40 mg, 24% yield) as a yellow solid. ESI-MS (M+H)+: 482.2. 1H NMR (400 MHz, CDCl3) δ 9.95 (s, 1H), 9.16 (s, 1H), 8.99 (s, 1H), 7.83 (s, 1H), 7.30 (s, 1H), 6.92 (s, 1H), 4.40-4.34 (m, 1H), 4.10 (s, 3H), 4.09-4.07 (m, 1H), 3.99-3.95 (m, 2H), 3.31-3.23 (m, 2H), 3.05-2.98 (m, 1H), 2.43 (s, 3H), 1.49 (s, 9H), 1.22 (d, J=6.8 Hz, 3H).
Tert-butyl (S)-4-(5-(7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyrazin-2-yl)-2-methylpiperazin-1-carboxylate (40 mg, 0.044 mmol) was dissolved in 3M HCl/EA (4 mL) and the mixture was stirred at r.t for 2 h. The solid was collected by filtration and washed EA three times (3 mL×3) to afford the title compound as hydrochloride salt (20 mg, 59% yield). ESI-MS (M+H)+: 382.0. 1H NMR (400 MHz, DMSO-d6) δ 10.82 (s, 1H), 9.59 (br s, 1H), 9.48 (br s, 1H), 9.19 (s, 1H), 8.93 (s, 1H), 8.27 (s, 1H), 7.95 (s, 1H), 7.33 (s, 1H), 4.35-4.31 (m, 2H), 4.07 (s, 3H), 3.38-3.25 (m, 3H), 3.09-3.03 (m, 2H), 2.46 (s, 3H), 1.33 (d, J=6.4 Hz, 3H).
To a stirred solution of 7-isopropoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (100 mg, 0.427 mmol), tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (125 mg, 0.427 mmol) and NMI (105 mg, 1.281 mmol) in MeCN (5 mL) was added TCFH (180 mg, 0.641 mmol) at rt. The mixture was stirred at it for 2 h. The reaction mixture was treated with EA/water (50 mL, 1:1). The organic phase was separated and the aqueous phase was extracted with EA (50 mL). The combined organic layer was dried over sodium sulfate. After concentration, the crude was purified by prep-HPLC (0.05% NH3·H2O in water/CH3CN) to provide title product (60 mg, Y: 27%) as off-white solid. ESI-MS (M+H)+: 510.3. 1H NMR (400 MHz, CDCl3) δ 10.71 (s, 1H), 9.01 (s, 1H), 8.41 (d, J=9.8 Hz, 1H), 7.30 (s, 1H), 7.13 (s, 1H), 7.02-6.98 (m, 1H), 4.94-4.86 (m, 1H), 4.17 (d, J=12.8 Hz, 1H), 4.04-3.95 (m, 2H), 3.36-3.27 (m, 2H), 3.11-3.03 (m, 1H), 2.48 (s, 3H), 2.01 (s, 1H), 1.62 (s, 3H), 1.61 (s, 3H), 1.49 (s, 9H), 1.22 (d, J=6.7 Hz, 3H).
To a solution of tert-butyl (S)-4-(6-(7-isopropoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido) pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (60 mg, 0.177 mmol) in EA (1 mL) was added 3M HCl/EA (1 mL), the mixture was stirred at r.t for 2 h. The solid was collected by filtration and washed EA three times (3 mL×3) to afford the title compound as hydrochloride salt (25 mg, yield: 52%). ESI-MS (M+H)+: 410.1. 1H NMR (400 MHz, DMSO-d6) δ 11.23 (s, 1H), 9.97-9.75 (m, 2H), 9.26 (s, 1H), 8.29 (d, J=9.3 Hz, 1H), 7.98 (s, 1H), 7.75 (d, J=10.0 Hz, 1H), 7.37 (s, 1H), 5.06-4.97 (m, 1H), 4.45-4.33 (m, 2H), 3.51-3.42 (m, 1H), 3.37-3.35 (m, 2H), 3.27-3.17 (m, 1H), 3.14-3.03 (m, 1H), 2.46 (s, 3H), 1.41 (d, J=6.0 Hz, 6H), 1.
To a mixture of 1 eq. of N-(4-bromo-2-fluorophenyl)-7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamide and 1.5 eq. Amine (B) in 1 ml Dioxane under an inert atmosphere were added 0.1 eq. RuPhosPdG4, 0.1 eq. RuPhos and 2 equiv Cs2CO3. The reaction mixture was stirred at 100° C. for 16 hours. After cooling to room temperature, the solvent was evaporated and 1 ml TFA was added and stirred at room temperature 4 h. The mixture was evaporated. The residue was dissolved in DMSO (appr. 1 ml), treated with scavenger SiliaMetS DMT and filtered. Resulting solution was purified by HPLC (Deionized Water (phase A) and HPLC-grade Acetonitrile (phase B) were used as an eluent to obtained final compound (C). In most cases, TFA was used as an additive.
To a mixture of 6-methoxy-2H-indazole-5-carboxylic acid (72 mg, 0.35 mmol) and tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (100 mg, 0.35 mmol) in DMF (5 mL) were added HATU (267 mg, 0.7 mmol), DIEA (226 mg 1.75 mmol). The mixture was stirred at it for 2 h. After concentration, the crude was purified by prep-HPLC (0.05% FA in water/CH3CN) to give title product (50 mg, Y: 30.5%) as a yellow solid. ESI-MS (M+H)+: 482.1.
A mixture of tert-butyl (S)-4-(6-(6-methoxy-2-methyl-2H-indazole-5-carboxamido)pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (50 mg, 0.145 mmol) in 3M HCl/EA (4 mL) was stirred at it for 2 h. The precipitate was filtered and the solid was dried in vacuo to give title product (19 mg, Y: 44.2%) as a yellow solid. ESI-MS (M+H)+:382.0. 1H NMR (400 MHz, MeOD-d4) δ 8.52 (s, 1H), 8.47 (s, 1H), 8.25-8.18 (m, 2H), 7.19 (s, 1H), 4.49 (d, J=14.0 Hz, 2H), 4.23 (s, 3H), 4.10 (s, 3H), 3.61-3.46 (m, 3H), 3.33-3.31 (m, 2H), 1.44 (d, J=7.2 Hz, 3H).
To a stirred solution of 2,8-dimethylimidazo[1,2-a]pyrazine-6-carboxylic acid (50 mg, 0.26 mmol), tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (84 mg, 0.28 mmol) and DIEA (100 mg, 0.78 mmol) in DMF (5 mL) was added HATU (128 mg, 0.33 mmol) at rt. The mixture was stirred at rt for 16 hrs. The reaction mixture was treated with EA/Water (50 mL, 1:1). The organic phase was separated and the aqueous phase was extracted with EA (50 mL×1). The combined organic layer was dried over sodium sulfate. After concentration, the crude was purified by pre-HPLC (0.05% FA in water/CH3CN) to provide title product (15 mg, Y: 12%) as an off-white solid. ESI-MS (M+H)+: 467.2. 1H NMR (400 MHz, CDCl3) δ 8.78 (s, 1H), 8.40 (d, J=9.8 Hz, 1H), 7.51 (s, 1H), 7.07 (d, J=9.8 Hz, 1H), 4.35-4.07 (m, 2H), 3.95-3.92 (m, 2H), 3.30-3.25 (m, 2H), 3.09-3.07 (m, 1H), 2.83 (s, 3H), 2.47 (s, 3H), 1.42 (s, 9H), 1.16 (d, J=6.8 Hz, 3H).
To a solution of tert-butyl (S)-4-(6-(2,8-dimethylimidazo[1,2-a]pyrazine-6-carboxamido)pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (15 mg, 0.03 nmol) in EA (1 mL) was added HCl-EA (3 M, 1.0 mL) at 0° C. and the mixture was allowed to warm to rt and stirred for 1 h. The solid was collected by filtration and washed with EA (2 mL×3). The solid was dried under vacuum at 55° C. to provide title product as light grey solid (5 mg . . . yield: 45%). ESI-MS (M+H)+: 367.0. 1H NMR (400 MHz, MeOD-d4) δ 9.48 (d, J=4.4 Hz, 1H), 8.56 (d, J=9.9 Hz, 1H), 8.29 (d, J=3.9 Hz, 1H), 8.14-8.07 (m, 1H), 4.50 (d, J=13.8 Hz, 2H), 3.57-3.51 (m, 3H), 3.37-3.35 (m, 2H), 3.03 (s, 3H), 2.68 (s, 3H), 1.45 (d, J=6.5 Hz, 3H).
To a stirred solution of 6-methoxy-2-methyl-2H-indazole-5-carboxylic acid (120 mg, 0.58 mmol), tert-butyl 4-(6-aminopyridazin-3-yl) piperazine-1-carboxylate (180 mg, 0.64 mmol) and DIEA (224 mg, 1.74 mmol) in DMF (5 mL) was added HATU (286 mg, 0.75 mmol) at rt. The mixture was stirred at rt for 2 hours. The reaction mixture was treated with EA/Water (50 mL, 1:1). The organic phase was separated and the aqueous phase was extracted with EA (50 mL×1). The combined organic layer was dried over sodium sulfate. After concentration, the crude was purified by prep-HPLC (0.05% FA in water/CH3CN) to provide title product (60 mg, Y: 22%) as an off-white solid. ESI-MS (M+H)+: 468.2.
To a solution of tert-butyl 4-(6-(6-methoxy-2-methyl-2H-indazole-5-carboxamido)pyridazin-3-yl)piperazine-1-carboxylate (50 mg, 0.107 nmol) in EA (1 mL) was added HCl/EA (3 M, 1.0 mL) at 0° C. and warmed to rt for 1 h. The solid was collected by filtration and washed with EA (2 mL×3). The solid was concentrated in vacuo to provide title product as a yellow solid (15 mg, yield: 37%). ESI-MS (M+H)+: 368.1. 1H NMR (400 MHz, DMSO-d6) δ 10.98 (s, 1H), 9.53 (s, 2H), 8.47 (s, 1H), 8.38 (d, J=9.8 Hz, 1H), 8.30 (s, 1H), 7.76 (d, J=9.7 Hz, 1H), 7.15 (s, 1H), 4.15 (s, 3H), 3.98 (br s, 3H), 3.88 (br s, 4H), 3.24 (s, 4H).
To a mixture of 7-cyclobutoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (95 mg, 0.38 mmol) and tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (85 mg, 0.29 mmol) in DMF (3 mL) were added HATU (165 mg, 0.44 mmol) and DIPEA (112 mg, 0.87 mmol). The mixture was stirred at RT overnight. Water (10 mL) was added to the mixture and filtered, the crude product was purified by prep-HPLC (0.05% NH3H2O in water/CH3CN) to give title product (15 mg, Y: 9.9%) as a yellow solid. ESI-MS (M+H)+: 522.3. 1H NMR (400 MHz, CDCl3) δ 10.68 (s, 1H), 8.98 (s, 1H), 8.42 (d, J=9.8 Hz, 1H), 7.30 (s, 1H), 7.00 (d, J=9.9 Hz, 1H), 6.83 (s, 1H), 4.97-4.87 (m, 1H), 4.38 (s, 1H), 4.17 (d, J=12.2 Hz, 1H), 4.05-3.94 (m, 2H), 3.36-3.25 (m, 2H), 3.11-3.03 (m, 1H), 2.69-2.62 (m, 2H), 2.50-2.44 (m, 2H), 2.44 (s, 3H), 2.00-1.96 (m, 1H), 1.86-1.84 (m, 1H), 1.49 (s, 9H), 1.23 (d, J=6.7 Hz, 3H).
A mixture of tert-butyl (S)-4-(6-(7-cyclobutoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (15 mg, 0.029 mmol) in 3M EtOAc/HCl (2 mL) was stirred at rt for 2 h. After concentration, the residue was dissolved in water (5 mL) and lyophilized to give title product (11.2 mg, Y: 85.1%) as a yellow solid. ESI-MS (M+H)+: 422.2. 1H NMR (400 MHz, CD3OD) δ 9.19 (s, 1H), 8.51 (d, J=10.0 Hz, 1H), 8.01 (d, J=10.1 Hz, 1H), 7.85 (s, 1H), 7.15 (s, 1H), 5.15-5.09 (m, 1H), 4.51-4.39 (m, 2H), 3.64-3.52 (m, 3H), 3.41-3.33 (m, 2H), 2.70-2.59 (m, 2H), 2.51 (s, 3H), 2.43-2.31 (m, 2H), 2.02-1.95 (mz, 1H), 1.89-1.82 (m, 1H), 1.45 (d, J=6.6 Hz, 3H).
To a stirred solution of 7-isopropoxy-2-methylimidazol[1,2-a]pyridine-6-carboxylic acid (100 mg, 0.427 mmol), tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (125 mg, 0.427 mmol) and NMI (105 mg, 1.281 mmol) in MeCN (5 mL) was added TCFH (180 mg, 0.641 mmol) at rt. The mixture was stirred at rt for 2 h. The reaction mixture was treated with EA/water (50 mL, 1:1). The organic phase was separated and the aqueous phase was extracted with EA (50 mL). The combined organic layer was dried over sodium sulfate. After concentration, the crude was purified by prep-HPLC (0.05% NH3·H2O in water/CH3CN) to provide title product (60 mg, Y: 27%) as off-white solid. ESI-MS (M+H)+: 510.3. 1H NMR (400 MHz, CDCl3) δ 10.71 (s, 1H), 9.01 (s, 1H), 8.41 (d, J=9.8 Hz, 1H), 7.30 (s, 1H), 7.13 (s, 1H), 7.02-6.98 (m, 1H), 4.94-4.86 (m, 1H), 4.17 (d, J=12.8 Hz, 1H), 4.04-3.95 (m, 2H), 3.36-3.27 (m, 2H), 3.11-3.03 (m, 1H), 2.48 (s, 3H), 2.01 (s, 1H), 1.62 (s, 3H), 1.61 (s, 3H), 1.49 (s, 9H), 1.22 (d, J=6.7 Hz, 3H).
To a solution of tert-butyl (S)-4-(6-(7-isopropoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido) pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (60 mg, 0.177 mmol) in EA (1 mL) was added 3M HCl/EA (I mL), the mixture was stirred at r.t for 2 h. The solid was collected by filtration and washed EA three times (3 mL×3) to afford the title compound as hydrochloride salt (25 mg, yield: 52%). ESI-MS (M+H)+: 410.1. 1H NMR (400 MHz, DMSO-d6) δ 11.23 (s, 1H), 9.97-9.75 (m, 2H), 9.26 (s, 1H), 8.29 (d, J=9.3 Hz, 1H), 7.98 (s, 1H), 7.75 (d, J=10.0 Hz, 1H), 7.37 (s, 1H), 5.06-4.97 (m, 1H), 4.45-4.33 (m, 2H), 3.51-3.42 (m, 1H), 3.36 (d, J=12.3 Hz, 2H), 3.27-3.17 (m, 1H), 3.14-3.03 (m, 1H), 2.46 (s, 3H), 1.41 (d, J=6.0 Hz, 6H), 1.35 (d, J=6.4 Hz, 3H).
To a mixture of tert-butyl 4-(6-aminopyridazin-3-yl)piperazine-1-carboxylate (100 mg, 0.358 mmol) in DMF (5 mL) were added 7-cyclobutoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (133 mg, 0.54 mmol), DIEA (140 mg, 1.07 mmol) and HATU (205 mg, 0.54 mmol). The mixture was stirred at rt for 5 h and quenched with water (10 mL). The precipitate was filtered and purified by C18 flash (0.1% FA in water/CH3CN) to give title product (11 mg, 6%) as a yellow solid. ESI-MS (M+H)+: 508.2.
To a solution of tert-butyl 4-(6-(7-cyclobutoxy-2-methylimidazol[1,2-a]pyridine-6-carboxamido)pyridazin-3-yl)piperazine-1-carboxylate (II mg, 0.02 mmol) was added 3M HCl/EA (5 mL) at rt. The mixture was stirred for 2 h at rt. The reaction mixture was concentrated in vacuo and the solid was washed with EA and dried to give title product (2 mg, yield: 22.7%) as a yellow solid. ESI-MS (M+H)+: 408.0. 1H NMR (400 MHz, MeOD-d4) δ 9.20 (s, 1H), 8.50 (d, J=9.9 Hz, 1H), 7.98-7.91 (m, 1H), 7.85 (s, 1H), 7.15 (s, 1H), 5.18-5.07 (m, 1H), 4.00 (br.s, 4H), 3.48-3.41 (m, 4H), 2.71-2.60 (m, 2H), 2.50 (s, 3H), 2.44-2.31 (m, 2H), 2.01-1.96 (m, 1H), 1.92-1.81 (m, 1H).
To a solution of tetrahydrofuran-3-ol (4.0 g, 45.4 mmol) in DCM (50 mL) was added TEA (13.6 g, 136 mmol) and MsCl (6.2 g, 54.5 mmol) at 0° C., the mixture was stirred for 2 h at RT. TLC (MeOH:DCM=1:4, Rf=0.3) showed the reaction was completed. The mixture was washed with water and brine, dried over Na2SO4, filtered and the filtrate was concentrated to afford crude and purified by silica gel column chromatography eluted with PE:EtOAc=1:2 to afford title compound (6.5 g, crude) as a colorless oil.
To a solution of 6-bromo-2-methylimidazo[1,2-a]pyridin-7-ol (650 mg, crude) in DMF (10 mL) was added K2CO3 (3.15 g, 23.2 mmol) and tetrahydrofuran-3-yl methanesulfonate (2.86 g, 17.2 mmol), the mixture was stirred for 16 h at 60° C. The mixture was diluted with water (50 mL) and extracted with EtOAc (50 mL×3), the combined organic layer was washed with brine, dried over Na2SO4, filtered and the filtrate was concentrated. The crude was purified by C18 column chromatography eluted with MeCN:H2O=20% to 60% to afford title compound (500 mg, 37.4% for two steps) as a grey solid. ESI-MS (M+H)+: 296.8. 1H NMR (400 MHz, CDCl3) δ 8.16 (s, 1H), 7.15 (s, 1H), 6.78 (s, 1H), 5.01-4.91 (m, 1H), 4.11-4.05 (m, 2H), 4.05-3.99 (m, 1H), 3.97-3.90 (m, 1H), 2.39 (s, 3H), 2.30-2.21 (m, 2H).
To a solution of 6-bromo-2-methyl-7-((tetrahydrofuran-3-yl)oxy)imidazo[1,2-a]pyridine (480 mg, 1.62 mmol) in MeOH (50 mL) were added Pd(dppf)Cl2 (237 mg, 0.32 mmol) and TEA (1.64 g, 16.2 mmol), the mixture was charged with CO for three times and stirred overnight at 80° C. under CO balloon. The mixture was filtered and the filtrate was concentrated to afford the title compound (crude 400 mg, 89.4% yield) as a grey solid. ESI-MS (M+H)+: 277.2.
To a solution of methyl 2-methyl-7-((tetrahydrofuran-3-yl)oxy)imidazo[1,2-a]pyridine-6-carboxylate (crude 400 mg, 1.45 mmol) in THF (8 mL) and H2O (4 mL) was added LiOH (174 mg, 7.2. mmol), the mixture was stirred for 1 h at RT. The mixture was diluted with water (10 mL) and extracted with EtOAc (20 mL×3). Aqueous phase was adjusted to pH=2 with 1M HCl and purified by C18 column chromatography eluted with MeCN:H2O (0.1% FA)=0% to 15% to afford title compound (270 mg, 70.8%) as a white solid, ESI-MS (M+H)+:263.0
To a solution of 2-methyl-7-((tetrahydrofuran-3-yl)oxy)imidazo[1,2-a]pyridine-6-carboxylic acid (100 mg, 0.38 mmol) and tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (111 mg, 0.38 mmol) in DMF (10 mL) were added DIPEA (147 mg, 1.14 mmol) and HATU (216 mg, 0.57 mmol), the mixture was stirred for 16 h at RT. The mixture was purified by pre-HPLC (0.05% FA in water/CH3CN) to afford title compound (70 mg, 34.1%) as a grey solid. ESI-MS (M+H)+:538.3. 1H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 9.06 (s, 1H), 8.21 (d, J=9.8 Hz, 1H), 7.67 (s, 1H), 7.42 (d, J=9.9 Hz, 1H), 7.02 (s, 1H), 5.34 (br s, 1H), 4.27-4.16 (m, 2H), 4.09-4.06 (m, 1H), 3.98-3.95 (m, 3H), 3.87-3.77 (m, 2H), 3.23-3.15 (m, 2H), 2.99-2.88 (m, 1H), 2.42-2.34 (m, 1H), 2.30 (s, 3H), 2.22-2.13 (m, 1H), 1.44 (s, 9H), 1.13 (d, J=6.7 Hz, 3H).
A solution of tert-butyl (2S)-2-methyl-4-(6-(2-methyl-7-((tetrahydrofuran-3-yl)oxy)imidazo[1,2-a]pyridine-6-carboxamido)pyridazin-3-yl)piperazine-1-carboxylate (70 mg, 0.13 mmol) in 3M HCl/EtOAc (3 mL) was stirred for 1 h at RT. The precipitate was filtered, washed with EtOAc (1 mL) and dried to afford title compound (50 mg, 82.2%) as a yellow solid. ESI-MS (M+H)+:438.1. 1H NMR (400 MHz, DMSO-d6) δ 11.18 (s, 1H), 9.77-9.68 (m, 2H), 9.25 (s, 1H), 8.26 (d, J=8.8 Hz, 1H), 7.98 (s, 1H), 7.68 (d, J=9.8 Hz, 1H), 7.34 (s, 1H), 4.38 (t, J=12.6 Hz, 2H), 3.96 (br s, 2H), 3.88-3.75 (m, 2H), 3.45-3.30 (m, 3H), 3.22-2.87 (m, 3H), 2.46 (s, 3H), 2.39-2.30 (m, 1H), 2.19-2.07 (m, 1H), 1.34 (d, J=6.4 Hz, 3H).
To a stirred solution of 6-cyclobutoxy-2-methyl-2H-indazole-5-carboxylic acid (40 mg, 0.14 mmol), tert-butyl 4-(6-aminopyridazin-3-yl)piperazine-1-carboxylate (40 mg, 0.14 mmol) and DIEA (55 mg, 0.429 mmol) in DMF (1 mL) was added HATU (70 mg, 0.18 mmol) at rt. The mixture was stirred for 16 h. The reaction mixture was diluted with water (10 mL), extracted with EA (25 mL). The combined organic layers was washed with brine and dried over sodium sulfate. After concentration under reduced pressure, the crude was purified by prep-HPLC (0.1% NH3·H2O in water/CH3CN) to give title product (34 mg. Y: 47.2%) as a yellow solid. ESI-MS (M+H)+: 508.3
To a solution of tert-butyl 4-(6-(6-cyclobutoxy-2-methyl-2H-indazole-5-carboxamido)pyridazin-3-yl)piperazine-1-carboxylate (20 mg, 0.04 mmol) in EA (2 mL) was added HCl/EA (3 M, 2 mL) at RT and the mixture was allowed to warm to rt and stirred for 2 h. After concentration, the solid was washed with EA three times (3 mL×3), and dried under vacuum to provide title product (9 mg, yield: 60%) as a light yellow solid. 1H NMR (400 MHz, MeOD-d4) δ 8.52 (d, J=8.8 Hz, 2H), 8.25-8.15 (m, 2H), 7.00 (s, 1H), 5.02-4.98 (m, 1H), 4.25 (s, 3H), 4.05-4.00 (m, 4H), 3.48-3.43 (m, 4H), 2.68-2.59 (m, 2H), 2.36-2.25 (m, 2H), 1.99-1.81 (m, 2H). ESI-MS (M+H)+: 408.1.
To a solution of 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (1 g, 4.545 mmol) and tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (1.3 g, 4.545 mmol) in MeCN (10 mL) was added NMI (1.1 g, 13.635 mmol) and TCFH (1.9 g, 6.815 mmol), the mixture was stirred for 2 h at RT. The precipitate was filtered to afford title compound (1 g, 45.4% yield) as a grey solid. 1H NMR (400 MHz, CDCl3) δ 10.94 (s, 1H), 8.72 (s, 1H), 8.53-8.50 (m, 1H), 8.00 (s, 1H), 7.09 (s, 1H), 7.03-6.98 (m, 1H), 4.49-4.22 (m, 2H), 4.20 (s, 3H), 4.03-3.94 (m, 2H), 3.48 (br.s, 2H), 3.32-3.25 (m, 2H), 3.09-3.01 (m, 1H), 1.70 (t, J=7.0 Hz, 3H), 1.49 (s, 9H), 1.23 (d, J=6.7 Hz, 3H). ESI-MS (M+H)+:496.0.
A solution of tert-butyl (S)-4-(6-(6-ethoxy-2-methyl-2H-indazole-5-carboxamido)pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (1 g, 2.02 mmol) in 3M HCl/EtOAc (10 mL) was stirred for 2 h at RT. The precipitate was filtered, washed with EtOAc (1 mL) to afford tide compound (740 mg, 85.4%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 9.79 (d, J=31.1 Hz, 2H), 8.53-8.41 (m, 2H), 8.36 (s, 1H), 7.88 (d, J=9.9 Hz, 1H), 7.14 (s, 1H), 4.39 (t, J=14.0 Hz, 2H), 4.30-4.23 (m, 2H), 4.15 (s, 3H), 3.53-3.10 (m, 5H), 1.48 (t, J=6.9 Hz, 3H), 1.34 (d, J=6.4 Hz, 3H). ESI-MS (M+H)+: 396.1.
To a solution of 6-cyclobutoxy-2-methyl-2H-indazole-5-carboxylic acid (100 mg, 0.406 mmol) and tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (120 mg, 0.406 mmol) in DMF (6 mL) were added DIEA (157 mg, 1.218 mmol) and HATU (200 mg, 0.528 mmol), the mixture was stirred for 16 h at RT. The mixture was purified by pre-HPLC (0.1% NH3·H2O in water/CH3CN) to afford title compound (110 mg, 52.3% yield) as a grey solid. ESI-MS (M+H)+:522.2
A solution of tert-butyl (S)-4-(6-(6-cyclobutoxy-2-methyl-2H-indazole-5-carboxamido)pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (100 mg, 0.19 mmol) in 3M HCl/EtOAc (4 mL) was stirred for 2 h at RT. The precipitate was filtered, washed with EtOAc (1 mL) and dried to afford title compound (66 mg, 82.5% yield) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.14 (s, 1H), 10.00-9.84 (m, 2H), 8.51-8.43 (m, 2H), 8.36 (s, 1H), 7.94 (d, J=10.0 Hz, 1H), 6.94 (s, 1H), 4.99-4.90 (m, 1H), 4.46-4.35 (m, 2H), 4.15 (s, 3H), 3.59-3.48 (m, 1H), 3.42-3.28 (m, 3H), 3.19-3.08 (m, 1H), 2.60-2.54 (m, 2H), 2.26-2.15 (m, 2H), 1.90-1.81 (m, 1H), 1.73 (d, J=9.6 Hz, 1H), 1.35 (d, J=6.2 Hz, 3H). ESI-MS (M+H)+: 422.1.
Preparation of Starting Material: 7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid
To a mixture of 2-amino-5-bromopyridin-4-ol (20.8 g, 110.05 mmol) and potassium carbonate (30.42 g, 220.09 mmol) in DMF (100 mL), iodoethane (20.6 g, 132.06 mmol) was added and the reaction mixture was stirred for two days at r.t. The resulting mixture was poured into water (200 mL) and extracted with EtOAc (2×100 mL). The organic layer was washed with H2O (100 mL), brine (100 mL), dried over Na2SO4 and evaporated to give crude 5-bromo-4-ethoxypyridin-2-amine (11.4 g, Y: 43%) which was used in the next step without purification. 1H NMR (500 MHz, DMSO-d6) δ 7.80 (s, 1H), 6.01 (s, 1H), 6.00 (br s, 2H) 4.02 (q, J=7.1 Hz, 2H), 1.33 (t, J=6.9 Hz, 3H).
5-Bromo-4-ethoxypyridin-2-amine (11.4 g, 52.52 mmol), 3-bromoprop-1-yne (7.5 g, 63.02 mmol) and 2-propanol (200 mL) were added to a round bottom flask with a rubber septum and stirred vigorously at 80° C. for 6 hours. After that, the mixture was allowed to cool to room temperature and the excess of solvent/propargyl bromide was removed under high vacuum. The resulting crude residue of 2-amino-5-bromo-4-ethoxy-1-(prop-2-yn-1-yl)pyridin-1-ium bromide (18.0 g, Y: 86.7%) was used in the next step without purification. 1H NMR (500 MHz, DMSO-d6) δ 8.53 (br s, 3H), 6.66 (s, 1H), 5.00 (d, J=2.6 Hz, 2H), 4.22 (q, J=7.1 Hz, 2H), 3.82-3.67 (m, 1H), 1.38 (t, J=6.9 Hz, 3H).
To a stirring solution of sodium hydroxide (2.57 g, 64.28 mmol) in deionized H2O (100 mL), 2-amino-5-bromo-4-ethoxy-1-(prop-2-yn-1-yl)pyridin-1-ium bromide (18.0 g, 53.57 mmol) was added over a period of 5 minutes and the reaction mixture was stirred for 30 min at r.t. Immediately after adding, the solution phase turned yellow and the yellow oil began to disperse as a separate distinct phase. The oil product was subsequently extracted with EtOAc (2×60 mL), dried over anhydrous MgSO4, filtered and concentrated under reduced pressure to afford 6-bromo-7-ethoxy-2-methylimidazo[1,2-a]pyridine (15.1 g, Y: 93.9%) as a pale yellow solid. 1H NMR (500 MHz, DMSO-d6) δ 8.73 (d, J=2.6 Hz, 1H), 7.40 (d, J=2.6 Hz, 1H), 6.88 (d, J=2.6 Hz, 1H), 4.09 (q, J=7.1 Hz, 2H), 2.23 (s, 3H), 1.36 (t, J=6.9 Hz, 3H).
6-Bromo-7-ethoxy-2-methylimidazo[1,2-a]pyridine (15.1 g, 59.19 mmol) was dissolved in MeOH (100 mL) and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane adduct (966.71 mg, 1.18 mmol) was added followed by triethylamine (11.98 g, 118.38 mmol). The reaction mixture was transferred into an autoclave and stirred at 130° C. under CO pressure (40 bar) overnight. Then the MeOH was evaporated, and the residue was partitioned between water (100 mL) and EtOAc (200 mL). The organic layer was separated, dried over Na2SO4 and evaporated under reduced pressure to give crude methyl 7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylate (12.5 g, Y: 72.1%) which was used in the next step without purification. 1H NMR (500 MHz, DMSO-d6) δ 8.92 (s, 1H), 7.55 (s, 1H), 6.85 (s, 1H), 4.07 (q, J=7.1 Hz, 2H), 3.78 (s, 3H), 2.24 (s, 3H), 1.33 (t, J=6.9 Hz, 3H).
A mixture of methyl 7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylate (12.5 g, 53.36 mmol) and potassium hydroxide (4.49 g, 80.05 mmol) was stirred in a mixture of methanol (80 mL) and H2O (60 mL) overnight. The resulting mixture was concentrated under reduced pressure to remove methanol, and the resulting aqueous solution was neutralized with 1 N HCl to pH=5 to precipitate the carboxylic acid. The solid 7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (6.0 g, Y: 48.5%) was isolated by filtration, dried, and used directly in the next step without further purification. 1H NMR (500 MHz, DMSO-d6) δ 8.85 (s, 1H), 7.52 (s, 1H), 6.81 (s, 1H), 4.06 (q, J=7.1 Hz, 2H), 2.24 (s, 3H), 1.33 (t, J=6.9 Hz, 3H).
3,6-Dichloropyridazine (842.11 mg, 5.65 mmol) in anhydrous toluene (10 mL) was treated with 4-Boc-4,7-diazaspiro[2.5]octane (1.2 g, 5.65 mmol) and triethylamine (1.72 g, 16.96 mmol). The reaction mixture was heated under reflux for 16 hours, concentrated under reduced pressure, and the residue was purified on SiO2 (5% MeOH/CH2Cl2/1% NH4OH) to provide the title tert-butyl 7-(6-chloropyridazin-3-yl)-4,7-diazaspiro[2.5]-octane-4-carboxylate (1.05 g. Y: 57.2%) as a white solid. ESI-MS (M+H)+: 325.2. 1H NMR (400 MHz, DMSO-d6) δ 7.48 (d, J=9.5, 1H), 7.33 (d, J=9.5, 1.6 Hz, 1H), 3.55 (m, 2H), 3.4 (s, 2H), 2.93 (m, 2H), 1.37 (s, 91H), 0.87 (m, 2H), 0.77 (m, 2H).
Tert-butyl 7-(6-chloropyridazin-3-yl)-4,7-diazaspiro[2.5]octane-4-carboxylate (949.7 mg, 2.92 mmol), diphenylmethanimine (582.9 mg, 3.22 mmol), Pd2(dba)3 (133.87 mg, 146.2 μmol), 1-[2-(diphenylphosphanyl)naphthalen-1-yl]naphthalen-2-yldiphenylphosphane (182.06 mg, 292.39 μmol) and cesium carbonate (1.91 g, 5.85 mmol) were suspended in dioxane (20.0 mL), and the suspension was stirred at 100° C. overnight. The resultant reaction mixture was cooled to room temperature and then filtered through Celite, and the precipitate was washed with ethyl acetate (30 mL). The filtrate was washed with saturated brine (10 mL), dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (Hex/EtOAc 2/1 as an eluent) to give pure tert-butyl 7-6-[(diphenylmethylidene)-amino]pyridazin-3-yl-4,7-diazaspiro[2.5]octane-4-carboxylate (1.0 g, Y: 72.8%). 1H NMR (500 MHz, DMSO-d6) δ 7.71-7.65 (m, 2H), 7.60-7.55 (m, 1H), 7.53-7.47 (m, 2H), 7.34 (s, 3H), 7.18-7.09 (m, 3H), 6.90-6.85 (m, 1H), 3.55-3.47 (m, 2H), 3.46-3.39 (m, 2H), 2.55-2.53 (m, 2H) 1.40 (s, 9H), 0.90-0.85 (m, 2H), 0.78-0.72 (m, 2H).
Tert-butyl 7-6-[(diphenylmethylidene)amino]pyridazin-3-yl-4,7-diazaspiro[2,5]octane-4-carboxylate (999.51 mg, 2.13 mmol) was dissolved in THF (10 mL). To the solution was added an aqueous solution 2-hydroxy-1,2,3-propanetricarboxylic acid monohydrate (2 M: 2.24 g, 10.64 mmol) and the mixture was stirred at room temperature overnight. The resultant reaction mixture was neutralized with a saturated aqueous sodium hydrogen carbonate solution (25 mL), and the mixture was extracted twice with ethyl acetate (2×15 mL). The organic phases were combined, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The crude residue was triturated with MTBE (10 mL) and the precipitate was filtered, washed with MTBE (5 mL) and dried in vacuo to yield tert-butyl 7-(6-aminopyridazin-3-yl)-4,7-diazaspiro[2.5]octane-4-carboxylate (260.0 mg, Y: 38% yield) as a white solid. ESI-MS (M+H)+: 306.2. 1H NMR (500 MHz, Chloroform-d) δ 6.90 (d, J=9.6 Hz, 1H), 6.81 (d, J=9.6 Hz, 1H), 4.69 (br s, 2H), 3.71-3.66 (m, 2H), 3.45-3.37 (m, 2H), 3.29 (s, 2H), 1.46 (s, 9H), 1.05-0.99 (m, 2H), 0.85-0.80 (m, 2H).
7-Ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (100.0 mg, 454.08 μmol) was suspended in DMF (4 mL) and ethylbis(propan-2-yl)amine (146.41 mg, 1.13 mmol) was added followed by [(dimethylamino)(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methylidene]dimethylazanium; hexafluoro-lambda5-phosphanide (HATU) (206.75 mg, 543.75 μmol). The resulting mixture was stirred for 30 min at r.t. After that, tert-butyl 7-(6-amino-pyridazin-3-yl)-4,7-diazaspiro[2.5]octane-4-carboxylate (138.37 mg, 453.13 μmol) was added in one portion and the reaction mixture was stirred overnight at r.t. The precipitate formed was filtered, washed with MeCN (2 mL), MTBE (2 mL), dried in vacuo to give pure tert-butyl 7-(6-7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-amidopyridazin-3-yl)-4,7-diazaspiro[2.5]octane-4-carboxylate (88.0 mg, 173.37 μmol, 38.3% yield). ESI-MS (M+H)+: 508.4.
To a solution of tert-butyl 7-(6-7-ethoxy-2-methylimidazol[1,2-a]pyridine-6-amidopyridazin-3-yl)-4,7-diazaspiro-[2.5]octane-4-carboxylate (87.99 mg, 173.35 μmol) in dichloromethane (20 mL) 2,2,2-trifluoroacetic acid (197.66 mg, 1.73 mmol) was added and the resulting mixture was stirred overnight at r.t. Then the solvents were evaporated to dryness under reduced pressure. The crude residue was crystallized from MTBE/MeCN (4/1, ˜5 mL) to obtain N-(6-4,7-diazaspiro[2.5]octan-7-ylpyridazin-3-yl)-7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamide as TFA salt (64.5 mg, Y: 67.8%). ESI-MS (M+H)+: 408.2. 1H NMR (500 MHz, DMSO-d6): δ 11.01 (s, 1H), 9.51 (br s, 1H), 9.2 (s, 1H), 8.22 (s, 1H), 7.92 (s, 1H), 7.53 (s, 1H), 7.34 (s, 1H), 4.35 (q, J=7.1 Hz, 2H), 3.87 (s, 2H), 3.55 (s, 2H), 2.43 (s, 3H), 1.45 (t, J=6.9 Hz, 3H), 1.15-1.05 (m, 2H), 0.98-90 (m, 2H).
To a solution of tetrahydro-2H-pyran-4-ol (5.0 g, 49 mmol) in DCM (50 mL) was added TEA (15 g, 147 mmol) and MsCl (6.8 g, 58.8 mmol) at 0°, the mixture was stirred for 2 h at RT. The mixture was washed with water and brine, dried over Na2SO4 and filtered. The crude was purified by silica gel column chromatography eluted with PE:EtOAc=1:2 to afford title compound (8.3 g, 86.1% yield) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 4.91-4.90 (m, 1H), 3.99-3.89 (m, 2H), 3.61-3.50 (m, 2H), 3.04 (s, 3H), 2.10-2.00 (m, 2H), 1.93-1.83 (m, 2H).
To a solution of 6-bromo-2-methylimidazol[1,2-a]pyridin-7-ol (500 mg, 2.19 mmol) in DMF (10 mL) were added K2CO3 (0.92 g, 6.6 mmol) and tetrahydro-2H-pyran-4-yl methanesulfonate (800 mg, 4.8 mmol), the mixture was stirred for 3 h at 75° C. The mixture was diluted with water (50 mL) and extracted with EtOAc (50 mL×3). The organics were washed and brine, dried over Na2SO4 and filtered. The filtrate was concentrated and the crude was purified by C18 column chromatography eluted with MeCN:H2O=20% to 60% to afford title compound (400 mg, 58.8% yield) as a grey solid. ESI-MS (M+H)+:310.8.
To a solution of 6-bromo-2-methyl-7-((tetrahydrofuran-3-yl)oxy)imidazo[1,2-a]pyridine (400 mg, 1.29 mmol) in MeOH (50 mL) were added Pd(dppf)Cl2 (141 mg, 0.2 mmol) and TEA (1.0 g, 10 mmol), the mixture was charged with CO for three times and stirred overnight at 80° C. under CO balloon. The mixture was filtered and the filtrate was concentrated to afford title compound (600 mg, crude) as a grey solid. ESI-MS (M+H)+: 290.9
To a solution of methyl 2-methyl-7-((tetrahydro-2H-pyran-4-yl)oxy)imidazo[1,2-a]pyridine-6-carboxylate (600 mg, crude) in THF (8 mL) and H2O (4 mL) was added LiOH (200 mg, 8 mmol), the mixture was stirred for 2 h at RT. The mixture was diluted with water (10 mL) extracted with EtOAc (20 mL×3). Aqueous phase was adjusted to pH=2 with 1M HCl and purified by C18 column chromatography eluted with MeCN:H2O (FA)=0% to 15% to afford title compound (70 mg, 19.7% yield for two steps) as a white solid. ESI-MS (M+H)+: 277.0.
To a solution of 2-methyl-7-((tetrahydro-2H-pyran-4-yl)oxy)imidazo[1,2-a]pyridine-6-carboxylic acid (60 mg, 0.22 mmol) and tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (60 mg, 0.20 mmol) in DMF (1.5 mL) were added DIPEA (100 mg, 0.775 mmol) and HATU (120 mg, 0.31 mmol), the mixture was stirred for 16 h at RT. The mixture was purified by pre-HPLC (0.05% FA in water/CH3CN) to afford title compound (10 mg, 8.8% yield) as a grey solid, ESI-MS (M+H)+:552.2.
A solution of tert-butyl (S)-2-methyl-4-(6-(2-methyl-7-((tetrahydro-2H-pyran-4-yl)oxy)imidazo[1,2-a]pyridine-6-carboxamido)pyridazin-3-yl)piperazine-1-carboxylate (10 mg, 0.022 mmol) in 3M HCl/EtOAc (2 mL) was stirred for 1 h at RT. The precipitate was filtered, washed with EtOAc (1 mL) and dried to afford title compound (5.5 mg, 63.0% yield) as a yellow solid, ESI-MS (M+H) 452.2. 1H NMR (400 MHz, MeOD-d4) δ 9.16 (s, 1H), 8.57 (d, J=9.2 Hz, 1H), 8.14 (d, J=9.4 Hz, 1H), 7.87 (s, 1H), 7.52 (s, 1H), 5.12 (br s, 1H), 4.51-4.42 (m, 2H), 3.94 (br s, 2H), 3.73-3.57 (m, 5H), 3.48-3.37 (m, 2H), 2.52 (s, 3H), 2.21 (br s, 2H), 1.92 (br s, 2H), 1.29 (br s, 3H).
2-methyl-N-(6-((S)-3-methylpiperazin-1-yl)pyridazin-3-yl)-7-((tetrahydrofuran-3-yl)oxy)imidazo[1,2-a]pyridine-6-carboxamide (20 mg) was obtained by SFC to afford title compound 212 (7 mg, 35%) as a white solid and compound 213 (7.64 mg, 38.2%) as a gray solid. ESI-MS (M+H)+:438.1.
1H NMR (400 MHz, MeOD-d4) δ 8.89 (s, 1H), 8.19 (d, J=9.8 Hz, 1H), 7.43 (s, 1H), 7.23 (d, J=9.9 Hz, 1H), 6.79 (s, 1H), 5.28-5.21 (m, 1H), 4.11-3.96 (m, 5H), 3.89-3.79 (m, 1H), 2.98 (d, J=10.0 Hz, 1H), 2.87-2.73 (m, 3H), 2.51-2.44 (m, 1H), 2.41-2.23 (m, 5H), 1.06 (d, J=6.3 Hz, 3H).
1H NMR (400 MHz, MeOD-d4) δ 8.98 (s, 1H), 8.29 (d, J=9.6 Hz, 1H), 7.53 (s, 1H), 7.32 (d, J=9.9 Hz, 1H), 6.88 (s, 1H), 5.38-5.26 (m, 1H), 4.19-4.02 (m, 5H), 3.99-3.86 (m, 1H), 3.09-3.02 (m, 1H), 2.97-2.81 (m, 3H), 2.55 (dd, J=24.4, 13.2 Hz, 1H), 2.48-2.29 (m, 5H), 1.16 (d, J=6.1 Hz, 3H).
3-Ethylpyrazin-2-amine (10.73 g, 87.13 mmol) and pyridine (7.58 g, 95.84 mmol, 7.75 mL) were mixed in CHCl3(200 mL), and bromine (14.62 g, 91.48 mmol, 4.69 mL) was added dropwise, the mixture was left to stir at r.t. overnight. After that the resulting mixture was washed with water and brine and evaporated under reduced pressure to give pure 5-bromo-3-ethylpyrazin-2-amine (15.8 g, Y: 80.8%). 1H NMR (500 MHz, Chloroform-d) δ 7.93 (s, 1H), 4.61 (br s, 2H), 2.62 (q, J=7.4 Hz, 2H), 1.29 (t, J=7.6 Hz, 3H).
To a mixture of 4-methylbenzene-1-sulfonic acid hydrate (1.49 g, 7.82 mmol) and pyridine (618.53 mg, 7.82 mmol, 630.0 μL) in i-PrOH (100 mL) was added 5-bromo-3-ethylpyrazin-2-amine (15.8 g, 78.2 mmol) and 1-bromo-2,2-dimethoxypropane (16.46 g, 89.93 mmol, 12.16 mL) and the reaction mixture was heated to 90° C. for 4 h. The resulting mixture was diluted with DCM, washed with a saturated sodium hydrogen carbonate solution, dried (Na2SO4) and concentrated under reduced pressure to give pure 6-bromo-8-ethyl-2-methylimidazo[1,2-a]pyrazine (16.0 g, Y: 85.2%). ESI-MS (M+H)+: 240.0. 1H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H), 8.13 (s, 1H), 3.11 (q, J=7.4 Hz, 2H), 2.50 (s, 3H), 1.28 (t, J=7.6 Hz, 3H).
6-Bromo-8-ethyl-2-methylimidazo[1,2-a]pyrazine (5.0 g, 20.82 mmol), triethylamine (2.53 g, 24.98 mmol) and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane (1.7 g, 2.08 mmol) were dissolved in dry MeOH (200 mL). The reaction mixture was heated at 125° C. in high pressure vessel under CO pressure (20 atm) for 48 h. The solvent was evaporated, and the mixture was poured into water (250 mL). The resulting mixture was extracted with EtOAc (2×100 mL) and the organics were dried over Na2SO4 and evaporated to dryness to give pure methyl 8-ethyl-2-methylimidazo[1,2-a]pyrazine-6-carboxylate (4.0 g, Y: 87.6%). ESI-MS (M+H)+: 220.2. 1H NMR (400 MHz, DMSO-d6) δ 9.21 (s, 1H), 7.97 (s, 1H), 3.88 (s, 3H), 3.12 (q, J=7.5 Hz, 2H), 2.42 (s, 3H), 1.33 (t, J=7.6 Hz, 3H).
Methyl 8-ethyl-2-methylimidazo[1,2-a]pyrazine-6-carboxylate (400.0 mg, 1.82 mmol) was dissolved in NH3/MeOH (10 mL) and the reaction mixture was sealed and heated to 90° C. overnight. After work-up with EtOAc pure 8-ethyl-2-methylimidazo[1,2-a]pyrazine-6-carboxamide (300.0 mg, Y: 80.4%) was obtained. 1H NMR (400 MHz, DMSO-d6) δ 8.97 (s, 1H), 7.93 (s, 1H), 7.89 (s, 1H), 7.65 (s, 1H), 3.09 (q, J=7.5 Hz, 2H), 2.37 (s, 3H), 1.33 (t, J=7.6 Hz, 3H).
To a solution of 3,6-dichloropyridazine (5.0 g, 33.56 mmol) in DMF (100 mL) were added ethylbis(propan-2-yl)-amine (4.54 g, 35.16 mmol, 6.12 mL) and tert-butyl (2S)-2-methylpiperazine-1-carboxylate (6.4 g, 31.96 mmol). The reaction mixture was heated to 90° C. for 17 h and after cooling to room temperature, partitioned between ethyl acetate (200 mL) and H2O (100 mL). The aqueous layer was extracted with ethyl acetate (2-80 mL) and the combined organic layers were washed with H2O (200 mL), brine (200 mL), dried (Na2SO4), filtered and the solvent was removed under reduced pressure to give the crude product. Purification by flash silica chromatography gave the desired tert-butyl (2S)-4-(6-chloropyridazin-3-yl)-2-methylpiperazine-1-carboxylate (5.88 g, Y: 56%). ESI-MS (M+H)+: 313.2
8-Ethyl-2-methylimidazo[1,2-a]pyrazine-6-carboxamide (118.57 mg, 580.57 μmol), tert-butyl (2S)-4-(6-chloro-pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (199.76 mg, 638.63 μmol), tris((1E,4E)-1,5-diphenylpenta-1,4-dien-3-one) dipalladium (53.16 mg, 58.06 μmol), [5-(diphenylphosphanyl)-9,9-dimethyl-9H-xanthen-4-yl]-diphenylphosphane (67.19 mg, 116.11 μmol), and cesium carbonate (567.48 mg, 1.74 mmol) were mixed in dioxane under an argon atmosphere and the reaction mixture was heated to 100° C. for 17 h. After evaporation and HPLC, tert-butyl (2S)-4-(6-8-ethyl-2-methylimidazo[1,2-a]pyrazine-6-amidopyridazin-3-yl)-2-methyl-piperazine-1-carboxylate (200.0 mg, Y: 61.6%) was obtained. ESI-MS (M+H)+: 481.4.
Tert-butyl (2S)-4-(6-8-ethyl-2-methylimidazo[1,2-a]pyrazine-6-amidopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (0.2 g, 0.42 mmol) was dissolved in a mixture of DCM (1 mL) and TFA (1 mL) and the reaction mixture was stirred at r.t. overnight. After full evaporation and HPLC, pure 8-ethyl-2-methyl-N-6-[(3S)-3-methylpiperazin-1-yl]pyridazin-3-ylimidazol[1,2-a]pyrazine-6-carboxamide as trifluoroacetic acid salt (117.9 mg, Y: 58% yield) was obtained. ESI-MS (M+H)+: 381.2. 1H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 9.25 (s, 1H), 9.05 (s, 1H), 8.75 (s, 1H), 8.32 (d, J=9.8 Hz, 1H), 8.07 (s, 1H), 7.58 (d, J=9.8 Hz, 1H), 4.36 (d, J=13.2 Hz, 2H), 3.46-3.35 (m, 2H), 3.29-3.07 (m, 4H), 3.00 (q, J=7.6 Hz, 1H), 2.45 (s, 3H), 1.43 (t, J=7.6 Hz, 3H), 1.28 (d, J=6.1 Hz, 3H).
To a mixture of 2-bromo-5-fluoroisonicotinic acid (3.0 g, 13.6 mmol) in EtOH (30 mL) was added EtONa (2.8 g, 54.6 mmol). The mixture was stirred at 65° C. for 16 h. After cooling to rt, to the reaction mixture was added SOCl2 (3.0 mL, 4.9 g, 41.3 mmol). The reaction mixture was stirred for 2 days. After concentration, the residue was diluted with EA (100 mL), washed with water and brine, dried with Na2SO4. After concentration, the crude product was purified by silica gel column (PE/EA=5:1) to afford title product (3.0 g, Y: 80.5%) as a white solid. ESI-MS (M+H)+: 273.9.
A mixture of ethyl 2-bromo-5-ethoxyisonicotinate (0.36 g, 1.3 mmol), trimethyl(prop-1-yn-1-yl)silane (0.16 g, 1.4 mmol), CuI (76 mg, 0.4 mmol), Pd(PPh3)4 (150 mg, 0.13 mmol), TEA (0.56 mL, 3.9 mmol) and TBAF (1.0 M, 1.3 mL, 1.3 mmol) in toluene (10 mL) was stirred at r.t. for 5 h. The reaction mixture was diluted with EA (200 mL), washed with brine and water, dried with Na2SO4. After concentration, the residue was purified by silica gel column (PE/EA=3:1) to afford title product (100 mg, Y: 33%) as yellow solid. ESI-MS (M+H)+: 234.1. 1H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 7.64 (s, 1H), 4.37 (q, J=7.1 Hz, 2H), 4.23 (q, J=7.0 Hz, 2H), 2.06 (s, 3H), 1.47 (t, J=7.1 Hz, 3H), 1.38 (t, J=7.1 Hz, 3H).
A mixture of ethyl 5-ethoxy-2-(prop-1-yn-1-yl)isonicotinate (160 mg, 0.67 mmol) and O-(mesitylsulfonyl)hydroxylamine (740 mg, 3.4 mmol) in trichloromethane (5 mL) was stirred at r.t. for 16 h. The reaction mixture was concentrated, the residue was diluted with DMF (5 mL), K2CO3 (180 mg, 1.3 mmol) was added and stirred at r.t. for 5 h. The reaction mixture was diluted with EA (120 mL), washed with brine and water, dried with Na2SO4. After concentration, the crude product was purified by silica gel column (PE/EA=3:1) to afford title product (100 mg, Y: 60%) as a colorless oil. ESI-MS (M+H)+: 249.0.
To a mixture of ethyl 6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxylate (100 mg, 0.4 mmol) in THF/water (10 mL, 2:1) was added LiOH·H2O (50 mg, 1.2 mmol). The mixture was stirred at rt for 2 h. After concentration, the residue was adjusted to pH=5 with 1M HCl. The crude was purified by pre-HPLC to give title product (80 mg, Y: 90%) as a colorless oil. ESI-MS (M+H)+: 221.0. 1H NMR (400 MHz, CDCl3) δ 8.38-8.24 (m, 2H), 6.48 (s, 1H), 4.26 (q, J=6.9 Hz, 2H), 2.49 (s, 3H), 1.59 (t, J=6.9 Hz, 3H).
To a mixture of 6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxylic acid (100 mg, 0.45 mmol) in DMF (5 mL) were added tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (146 mg, 0.5 mmol), DIEA (116 mg, 0.9 mmol) and HATU (190 mg, 0.5 mmol). The mixture was stirred at rt for 5 h and quenched with water (20 mL), extracted with EA (60 mL×2). Combined the organic layers, dried with Na2SO4. After concentration, the residue was purified by silica gel column (PE/EA=2:1) to afford title product (100 mg, 45%) as a yellow solid. ESI-MS (M+H)+: 496.1.
To a solution of tert-butyl (S)-4-(6-(6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxamido)pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (50 mg, 0.1 mmol) in EA (2 mL) was added HCl-EA (3 M, 2 mL) at 0° C. and the mixture was allowed to warm to rt and stirred for 2 h. After concentration, the residue purified by prep-HPLC (0.05% HCl in water/MeCN) to give title compound (20.0 mg, yield: 50%) as a yellow solid. ESI-MS (M+H)+: 396.1. 1H NMR (400 MHz, DMSO-d6) δ 10.98 (s, 1H), 9.50 (s, 1H), 9.36 (s, 1H), 8.51 (s, 1H), 8.32 (d, J=9.7 Hz, 1H), 8.10 (s, 1H), 7.64 (d, J=9.9 Hz, 1H), 6.56 (s, 1H), 4.36 (t, J=12.0 Hz, 2H), 4.25-4.19 (m, 2H), 3.39-3.33 (m, 3H), 3.15-3.08 (m, 2H), 2.39 (s, 3H), 1.45 (t, J=6.9 Hz, 3H), 1.32 (d, J=6.5 Hz, 3H).
To a mixture of 6-bromo-2-methylimidazol[1,2-a]pyridin-7-ol (1.5 g, 6.6 mmol) in DMF (20 mL) was added NaH (0.48 g, 13.2 mmol) at 0° C. and the mixture was stirred at 0° C. for 2 h. 2,2,2-trifluoroethyl trifluoromethanesulfonate (2.23 g, 9.9 mmol) was added to the mixture and stirred at it for another 16 h. The mixture was diluted with water (20 mL) and extracted with EA (3×30 mL). The organic layer washed with brine (3×40 mL) and dried over anhydrous Na2SO4. The filtrate was concentrated in vacuo. The residue was purified by column chromatography (MeOH/DCM=1:10) to give title product (1.1 g, 53.8%) as a white solid. ESI-MS (M+H)+: 310.9. 1H NMR (400 MHz, DMSO-d6) δ 8.85 (s, 1H), 7.50 (s, 1H), 7.19 (s, 1H), 4.94-4.90 (m, 2H), 2.28 (s, 3H).
To a mixture of 6-bromo-2-methyl-7-(2,2,2-trifluoroethoxy)imidazo[1,2-a]pyridine (300 mg, 1 mmol) in MeOH (30 mL) were added Pd(dppf)Cl2(143 mg, 0.2 mmol) and TEA (2.7 mL, 0.2 mmol). The mixture was stirred at 60° C. for 20 h under CO atmosphere. The mixture was filtered and the filtrate was concentrated in vacuo to give title product (crude 240 mg, 84.6%) as a grey solid. ESI-MS (M+H)+: 289.1.
To a solution of methyl 2-methyl-7-(2,2,2-trifluoroethoxy)imidazo[1,2-a]pyridine-6-carboxylate (300 mg, 1 mmol) in THF/H2O (8 mL/4 mL) was added LiOH (100 mg, 4.1 mmol). The mixture was stirred at rt for 2 h. The mixture was adjusted to pH=5 with 1M HCl. The aqueous layer was extracted with EA (3×50 mL). The organic layer was concentrated in vacuo, the residue was purified by C18 flash (0.1% FA in water/CH3CN) to give title product (174 mg, 57.8%) as a yellow solid. ESI-MS (M+H)+: 275.1.
To a solution of 2-methyl-7-(2,2,2-trifluoroethoxy)imidazo[1,2-a]pyridine-6-carboxylic acid (120 mg, 0.44 mmol) and tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (107 mg, 0.36 mmol) in DMF (10 mL) were added HATU (208 mg, 0.54 mmol) and DIEA (0.25 mL, 1.44 mmol). The mixture was stirred at rt for 1 h. The reaction mixture was diluted with water (20 mL) and extracted with EA (3×50 mL). The organic layer washed with brine (3×50 mL), dried over anhydrous Na2SO4, filtered and the filtrated was concentrated in vacuo. The residue was purified by prep-HPLC (0.1% FA in water/CH3CN) to give title product (90 mg, 37.3%) as a gray solid. ESI-MS (M+H)+: 550.3. 1H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.95 (s, 1H), 8.14 (s, 1H), 7.67 (s, 1H), 7.41 (d, J=9.9 Hz, 1H), 7.17 (s, 1H), 4.97 (dd, J=17.4, 8.7 Hz, 2H), 4.19 (d, J=12.4 Hz, 2H), 4.08 (d, J=13.1 Hz, 1H), 3.82 (d, J=13.3 Hz, 1H), 3.17 (dd, J=13.0, 3.8 Hz, 2H), 2.97-2.90 (m, 1H), 2.31 (s, 3H), 1.43 (s, 9H), 1.12 (d, J=6.7 Hz, 3H).
To a mixture of tert-butyl (S)-2-methyl-4-(6-(2-methyl-7-(2,2,2-trifluoroethoxy)imidazo[1,2-a]pyridine-6-carboxamido)pyridazin-3-yl)piperazine-1-carboxylate (70 mg, 0.13 mmol) in EA (5 mL) was added EA/HCl (5 mL) The mixture was stirred at rt for 2 h. The reaction mixture was concentrated in vacuo. The residue was purified by pre-HPLC (0.1% NH3·H2O in water/CH3CN) to give title product (20 mg, 34.9%) as a white solid. ESI-MS (M+H)+: 450.2. 1H NMR (400 MHz, MeOD-d4) δ 8.95 (s, 1H), 8.29 (d, J=9.9 Hz, 1H), 7.59 (s, 1H), 7.37 (d, J=9.9 Hz, 1H), 7.07 (s, 1H), 4.90 (d, J=8.2 Hz, 2H), 4.17 (dd, J=15.1, 6.6 Hz, 2H), 3.10 (d, J=11.2 Hz, 1H), 2.92 (dd, J=13.2, 2.9 Hz, 3H), 2.62 (dd, J=12.8, 10.6 Hz, 1H), 2.38 (s, 3H), 1.17 (d, J=6.4 Hz, 3H).
To a solution of 6-bromo-7-ethoxy-2-methylimidazo[1,2-a]pyridine (2 g, 7.87 mmoL) in toluene (40 mL) were added tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (2.31 g, 7.87 mmoL), Pd(OAc)2 (176 mg, 0.787 mmoL), xantphos (455 mg, 0.787 mmoL) and Na2CO3 (1.31 g, 15.78 mmoL). The resulting solution was stirred at 80° C. for 5 h under the atmosphere of CO. The resulting solution was concentrated under vacuum. The residue was purified by C18 column (0.1% NH3·H20 in water/CH3CN) to give title product (2.1 g, Y: 53.8%) as an off-white solid. ESI-MS (M+H)+: 496.2. 1H NMR (400 MHz, CDCl3) δ 10.48 (s, 1H), 9.11 (s, 1H), 8.36 (d, J=9.8 Hz, 1H), 7.66 (s, 1H), 7.38 (s, 1H), 7.01 (d, J=9.9 Hz, 1H), 4.50 (q, J=7.0 Hz, 2H), 4.38-4.37 (m, 1H), 4.17-4.16 (m, 1H), 4.07-3.89 (m, 2H), 3.44-3.21 (m, 2H), 3.09-3.08 (m, 1H), 2.59 (s, 3H), 1.74 (t, J=7.0 Hz, 3H), 1.49 (s, 9H), 1.22 (d, J=6.7 Hz, 3H).
To a solution of in tert-butyl (S)-4-(6-(7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (2.1 g, 4.24 mmoL) in EA (10 mL) was added 3M HCl/EA (10 mL), the resulting solution was stirred for 2 h at RT. The precipitate was filtered and dried to give title product (650 mg, Y: 35.7%) as a yellow solid. ESI-MS (M+H)+: 396.1. 1H NMR (400 MHz, MeOD-d4) δ 9.18 (s, 1H), 8.53 (d, J=10.1 Hz, 1H), 8.16 (d, J=10.2 Hz, 1H), 7.86 (s, 1H), 7.38 (s, 1H), 4.62-4.30 (m, 4H), 3.76-3.55 (m, 3H), 3.47-3.32 (m, 2H), 2.52 (s, 3H), 1.57 (t, J=7.0 Hz, 3H), 1.46 (d, J=6.5 Hz, 3H).
To a solution of tetrahydro-2H-pyran-3-ol (150 mg, 1.46 mmol) and TEA (445 mg, 4.4 mmol) in DCM (5 mL) was added MsCl (200 mg, 1.2 mmol), the reaction mixture was stirred for 2 h at 0° C. After cooling to rt and diluting with water, the mixture was extracted with EA. The combined organics were washed with brine and water, dried over Na2SO4 and concentrated to give desired product as colorless oil (250 mg, crude), which was used to next step without further purification. 1H NMR (400 MHz, CDCl3) δ 4.77-4.68 (m, 1H), 3.89-3.80 (m, 1H), 3.73-3.59 (m, 3H), 3.04 (s, 3H), 2.13-2.02 (m, 1H), 1.99-1.86 (m, 2H), 1.65-1.55 (m, 1H).
A solution of tetrahydro-2H-pyran-3-yl methanesulfonate (1.3 g, 7.2 mmol), 6-bromo-2-methylimidazo[1,2-a]pyridin-7-ol (1.1 g, 5.0 mmol) and K2CO3 (2.6 g, 20.0 mmol) in DMF (5 mL) was stirred for 1 h at 75° C. in a Sealed tube under microwave. After cooling to it and diluting with water (80 mL), the mixture was extracted with EA (100 mL×2). The combined organics were washed with brine and water, dried over Na2SO4, concentrated. The crude was purified by prep-HPLC (0.05% FA in water/CH3CN) to give title product (200 mg, yield: 15%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.16 (s, 1H), 7.14 (s, 1H), 6.85 (s, 1H), 4.44-4.32 (m, 1H), 4.00-3.92 (m, 1H), 3.79-3.61 (m, 3H), 2.38 (s, 3H), 2.19-2.07 (m, 1H), 2.01-1.85 (m, 2H), 1.73-1.56 (m, 1H).
To a mixture of 6-bromo-2-methyl-7-((tetrahydro-2H-pyran-3-yl)oxy)imidazo[1,2-a]pyridine (155 mg, 0.5 mmol) in MeOH (5 mL) were added TEA (510 mg, 5.0 mmol) and Pd(dppf)Cl2 (65 mg, 0.1 mmol). The resulting mixture was stirred overnight at 60° C. under CO (balloon). The mixture was allowed to cooling down to room temperature and concentrated. The residue was purified by silica gel column chromatography (PE:EA=3:1) to give title product (100 mg, 69%) as a brown solid. ESI-MS (M+H)+: 291.0.
To a mixture of methyl 2-methyl-7-((tetrahydro-2H-pyran-3-yl)oxy)imidazo[1,2-a]pyridine-6-carboxylate (150 mg, 0.5 mmol) in THF/water (5 mL, 2:1) was added LiOH (48 mg, 2.0 mmol). The mixture was stirred at rt for 2 h. After concentration, the residue was adjusted to pH=5 with 1M HCl. The cured was purified by pre-HPLC to give title product (32 mg, Y: 23.1%) as a colorless oil. ESI-MS (M+H)+: 277.0.
To a mixture of 2-methyl-7-((tetrahydro-2H-pyran-3-yl)oxy)imidazo[1,2-a]pyridine-6-carboxylic acid (30 mg, 0.11 mmol) in DMF (2 mL) were added tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (40 mg, 0.13 mmol), DIEA (30 mg, 0.29 mmol) and HATU (50 mg, 0.13 mmol). The mixture was stirred at rt for 1 h. After diluting with water, the mixture was filtered and the cake was purified by silica gel column chromatography (DCM:MeOH=20:1) to give title product (30 mg, 50%) as a white solid. ESI-MS (M+H)+: 552.4.
A solution of tert-butyl (2S)-2-methyl-4-(6-(2-methyl-7-((tetrahydro-2H-pyran-3-yl)oxy)imidazo[1,2-a]pyridine-6-carboxamido)pyridazin-3-yl)piperazine-1-carboxylate (30 mg, 0.05 mmol) in HCl/EA (1.0M, 3 mL) was stirred at rt for 2 h. The mixture was concentrated and the residue was purified by prep-HPLC (0.05% NH3·H2O in water/MeCN) to give title product (24 mg, Y: 98%) as a yellow solid. ESI-MS (M+H)+: 452.1, 1H NMR (400 MHz, MeOD-d4) δ 9.31 (s, 1H), 8.45 (br s, 1H), 8.11 (br s, 1H), 7.90 (s, 1H), 7.51 (s, 1H), 5.07-4.99 (m, 1H), 4.54-4.42 (m, 2H), 4.13-4.02 (m, 1H), 3.98-3.87 (m, 2H), 3.76-3.55 (m, 4H), 3.44-3.34 (m, 2H), 2.53 (s, 3H), 2.23-2.09 (m, 2H), 2.06-1.97 (m, 1H), 1.66-1.57 (m, 1H), 1.46 (s, 3H).
To a mixture of tert-butyl 4-(6-aminopyridazin-3-yl)-2,2-dimethylpiperazine-1-carboxylate (35 mg, 0.114 mmol) and 7-cyclobutoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (42 mg, 0.171 mmol) in DMF (3 mL) were added HATU (65 mg, 0.171 mmol) and DIEA (44 mg, 0.342 mmol). The mixture was stirred at rt for 1 hour. After diluting with water, the precipitate was filtered and dried to give title product (30 mg, 49.2%) as a white solid. ESI-MS (M+H)+: 536.2. 1H NMR (400 MHz, CDCl3) δ 10.67 (s, 1H), 8.97 (s, 1H), 8.43 (d, J=9.8 Hz, 1H), 7.29 (s, 1H), 6.85 (d, J=9.8 Hz, 1H), 6.76 (s, 1H), 4.93-4.86 (m, 1H), 3.94-3.86 (m, 4H), 3.61-3.54 (m, 2H), 2.69-2.59 (m, 2H), 2.46-2.41 (m, 4H), 2.06-1.78 (m, 3H), 1.50 (s, 9H), 1.43 (s, 6H).
To a mixture of tert-butyl 4-(6-(7-cyclobutoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyridazin-3-yl)-2,2-dimethylpiperazine-1-carboxylate (25 mg, 0.047 mmol) in EA (2 mL) was added EA/HCl (2 mL, 3 M). The mixture was stirred at rt for 1 h. After concentration, the residue was lyophilized to give title product (18.18 mg, 82.6%) as a white solid. ESI-MS (M+H)+: 436.1. 1H NMR (400 MHz, MeOD-d4) δ 9.21 (d, J=13.7 Hz, 1H), 8.47 (d, J=10.0 Hz, 1H), 8.01 (d, J=10.1 Hz, 1H), 7.85 (s, 1H), 7.16 (s, 1H), 5.20-5.06 (m, 1H), 4.05-3.97 (m, 2H), 3.87 (s, 21H), 3.53-3.45 (m, 2H), 2.64-2.61 (m, 2H), 2.51 (d, J=1.0 Hz, 3H), 2.43-2.31 (m, 2H), 2.03-1.84 (m, 2H), 1.51 (s, 6H).
To a mixture of tert-butyl 4-(6-aminopyridazin-3-yl)-2,2-dimethylpiperazine-1-carboxylate (42 mg, 0.136 mmol) and 6-ethoxy-2-methyl-2H-indazole-5-carboxylic (30 mg, 0.136 mmol) in MeCN (5 mL) were added NMI (33.7 mg, 0.411 mmol) and TCFH (59 mg, 0.20 mmol). The mixture was stirred at rt for 2 hours. The precipitate was filtered and dried to give title product (50 mg, 72%) as a white solid. ESI-MS (M+H)+: 510.3.
To a mixture of 4-(6-(6-ethoxy-2-methyl-2H-indazole-5-carboxamido) pyridazin-3-yl)-2,2-dimethylpiperazine-1-carboxylate (50 mg, 0.1 mmol) in EA (5 mL) was added EA/HCl (5 mL, 3 mol). The mixture was stirred at rt for 1 h. After concentration, the residue was lyophilized to give title product (24 mg, 58%) as a white solid. ESI-MS (M+H)+: 410.1. 1H NMR (400 MHz, MeOD-d4) δ 8.49 (s, 1H), 8.44 (s, 1H), 8.18-8.16 (m, 2H), 7.15 (s, 1H), 4.32 (q, J=7.0 Hz, 2H), 4.22 (s, 3H), 4.02-3.98 (m, 2H), 3.86 (s, 2H), 3.50-3.46 (m, 2H), 1.57 (t, J=7.0 Hz, 3H), 1.50 (s, 6H).
To a mixture of tert-butyl 4-(6-aminopyridazin-3-yl)-2,2-dimethylpiperazine-1-carboxylate (42 mg, 0.136 mmol) and 7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (30 mg, 0.136 mmol) in MeCN (5 mL) were added NMI (33.7 mg, 0.411 mmol) and TCFH (59 mg, 0.20 mmol). The mixture was stirred at rt for 2 hours. The precipitate was filtered to give title product (50 mg, 72%) as a white solid. ESI-MS (M+H)+: 510.3.
To a mixture of tert-butyl 4-(6-(7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyridazin-3-yl)-2,2-dimethylpiperazine-1-carboxylate (50 mg, 0.1 mmol) in EA (5 mL) was added EA/HCl (5 mL, 3 mol). The mixture was stirred at rt for 1 h. After concentration, the residue was purified by pre-HPLC to give title product (20 mg, 55%) as a white solid. ESI-MS (M+H)+: 410.1. 1H NMR (400 MHz, MeOD-d4) δ 9.19 (s, 1H), 8.48 (br s, 1H), 8.10 (br s, 1H), 7.87 (s, 1H), 7.38 (s, 1H), 4.46 (br s, 2H), 4.04 (br.s, 2H), 3.90 (br.s, 2H), 3.51 (br.s, 2H), 2.52 (s, 3H), 1.59 (br.s, 3H), 1.52 (s, 6H).
A solution of 6-chloro-5-methylpyridazin-3-amine (1.2 g, 8.4 mmol) in tert-butyl (S)-2-methylpiperazine-1-carboxylate (13.44 g, 67.2 mmol) was stirred at 170° C. for 24 h. After cooling to rt, the mixture was concentrated and purified by silica gel column chromatography eluted with (EA/PE=1:5) to give title compound (1 g, 38%) as a yellow oil. ESI-MS (M+H)+: 308.3.
To a solution of tert-butyl (S)-4-(6-amino-4-methylpyridazin-3-yl)-2-methylpiperazine-1-carboxylate (50 mg, 0.163 mmol), 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (36 mg, 0.163 mmol) in MeCN (3 mL) were added NMI (40.1 mg, 0.489 mmol) and TCFH (68.5 mg, 0.2445 mmol), the mixture was stirred at RT for 16 h. The precipitate was filtered to give crude title compound (20 mg, 24.3%) as a white solid. ESI-MS (M+H)+: 510.2.
To a solution of tert-butyl (S)-4-(6-(6-ethoxy-2-methyl-2H-indazole-5-carboxamido)-4-methylpyridazin-3-yl)-2-methylpiperazine-1-carboxylate (20 mg, 0.04 mmol) in EtOAc (1 mL) was added 3M HCl/EtOAc (1 mL) at room temperature. The reaction mixture was stirred for 2 h. The precipitate was filtered and dried to give to give title compound (5 mg, 31%) as a white solid. ESI-MS (M+H)+: 410.3. 1H NMR (400 MHz, MeOD-d4) δ 8.53 (s, 1H), 8.49 (s, 1H), 7.92 (s, 1H), 7.18 (s, 1H), 4.35 (q, J=6.9 Hz, 2H), 4.25 (s, 3H), 3.90-3.88 (m, 2H), 3.66-3.65 (m, 1H), 3.54-3.53 (m, 1H), 3.44-3.43 (m, 2H), 3.26-3.09 (m, 1H), 2.63 (s, 3H), 1.56 (t, J=6.9 Hz, 3H), 1.43 (d, J=6.3 Hz, 3H).
To a solution of 5-bromo-4-methoxypyrimidin-2-amine (4.5 g, 22.057 mmol) in EtOH (100 mL) was added 1-bromopropan-2-one (6 g, 44.113 mmol). The mixture was stirred at 90° C. for 16 h. After cooling to RT, the mixture was concentrated under reduced pressure, then the residue was washed by EA and filtered. The residue was dissolved in NaOH (2 mol/L, 100 mL) and stirred at RT for 2 b. Then the solid was collected by filtration to give the title compound (1.1 g, 20%) as a white solid. ESI-MS (M+H)+: 242.0/243.9. 1H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 7.04 (s, 1H), 4.10 (s, 3H), 2.39 (s, 3H).
To a solution of 6-bromo-7-methoxy-2-methylimidazo[1,2-a]pyrimidine (1 g, 4.15 mmol) in DCM (20 mL) was added BBr3 (1 mol/L in DCM, 50 mL) at ice bath temperature. The mixture was stirred at RT for 48 h. After quenching with MeOH (30 mL), the mixture was basified with saturated aqueous sodium bicarbonate solution and extracted with DCM (50 mL×3). The aqueous phase was concentrated in vacuo and the residue was purified by silica gel column chromatography (DCM:MeOH=10:1) to give title product (307 mg, 32%) as a brown solid. ESI-MS (M+H)+:227.9. 1H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H), 6.99 (d, J=1.2 Hz, 1H), 2.15 (d, J=11 Hz, 3H).
To a solution of 6-bromo-2-methylimidazo[1,2-a]pyrimidin-7-ol (150 mg, 0.658 mmol) in DMF (10 mL) were added iodoethane (205 mg, 1.316 mmol) and K2CO3(272 mg, 1.974 mmol). The mixture was stirred at rt for 2 h. After diluting with water (30 mL), the mixture was extracted with EA (50 mL×3). The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo to give the title product (145 mg, 86%) as a white solid. ESI-MS (M+H)+: 257.9. 1H NMR (400 MHz, DMSO-d6) δ 8.% (s, 1H), 7.18 (d, J=1.1 Hz, 1H), 4.21 (q, J=7.1 Hz, 2H), 2.24 (d, J=1.0 Hz, 3H), 1.29 (t, J=7.1 Hz, 3H).
To a solution of 6-bromo-8-ethyl-2-methylimidazo[1,2-a]pyrimidin-7(8H)-one (105 mg, 0.412 mmol), tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (121 mg, 0.412 mmol) and Na2CO3 (66 mg, 0.618 mmol) in toluene (15 mL) were added Xantphots (9 mg, 0.016 mmol) and Pd(OAc)2 (4 mg, 0.016 mmol). The mixture was charged with CO three times and stirred at 80° C. under CO for 16 h. The mixture was concentrated under reduced pressure to give title product (100 mg, crude) as a dark solid, which was used to next step without further purification. ESI-MS (M+H)+: 497.2.
To a solution of tert-butyl (S)-4-(6-(8-ethyl-2-methyl-7-oxo-7,8-dihydroimidazo[1,2-a]pyrimidine-6-carboxamido)pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (crude 100 mg, 0.202 mmol) in EA (4 mL) was added EA/HCl (4 mL, 3 mol/L). The mixture was stirred at rt for 1 h. The mixture was concentrated in vacuo and the residue was purified by prep-HPLC (0.05% HCl in water/CH3CN) to give title compound (33 mg, yield: 38% for two steps) as a yellow solid. ESI-MS (M+H)+:397.1. 1H NMR (400 MHz, MeOD-<d4) δ 9.29 (s, 1H), 8.51 (d, J=10.0 Hz, 1H), 7.89 (d, J=10.0 Hz, 1H), 7.36 (d, J=1.1 Hz, 1H), 4.45 (d, J=14.4 Hz, 2H), 4.39-4.34 (m, 2H), 3.60-3.53 (m, 2H), 3.52-3.40 (m, 2H), 3.25-3.21 (m, 1H), 2.34 (s, 3H), 1.44-1.40 (m, 6H).
To a solution of 4-chloropyrimidin-2-amine (15 g, 115.385 mmol) in methanol (210 mL) and acetonitrile (150 mL), N-bromosuccinimide (20.4 g, 115.385 mmol) was added. The mixture was stirred at rt for 2 h then diethyl ether (300 mL) was added and stirred for 1 h at 5° C. Then the solid was collected by filtration and dried to give the title compound (18.4 g, Y: 77%) as a white solid. ESI-MS (M+H)+: 209.9. 1H NMR (400 MHz, DMSO-d6) δ 8.41 (s, 1H), 7.34 (s, 2H).
A solution of 5-bromo-4-chloropyrimidin-2-amine (10 g, 48.309 mmol) and sodium ethoxide (20% in ethanol, 6.57 g, 96.61 mmol) in ethanol (200 mL) stirred at RT for 6 h. Then the mixture was concentrated in vacuo, the residue was diluted with water (100 mL) and extracted with DCM (200 mL×3). The organic layer was concentrated in vacuo to give title compound (11.4 g. yield: 95%). ESI-MS (M+H)+:218.0. 1H NMR (400 MHz, CDCl3) δ 8.10 (s, 1H), 4.98 (s, 2H), 4.40 (q, J=7.1 Hz, 2H), 1.41 (t, J=7.1 Hz, 3H).
To a solution of 5-bromo-4-ethoxypyrimidin-2-amine (2 g, 9.217 mmol) in EtOH (40 mL) was added 1-bromopropan-2-one (2.5 g, 18.433 mmol). The mixture was stirred at 90° C. for 16 h. After cooling to RT, the mixture was concentrated under reduced pressure, then the residue was washed by EA and filtered. The residue was dissolved in NaOH (2 mol/L, 20 mL) and stirred at RT for 2 h. Then the solution was extracted with EA (40 mL×3) and the organic layer was washed with saturated NaCl solution and concentrated in vacuo to get crude compound. The crude was purified by silica gel column (PE:EA=4:1˜1:1) to give the title compound (500 mg, Y: 17%) as a white solid. ESI-MS (M+H)+: 257.9. 1H NMR (400 MHz, CDCl3) δ 8.31 (s, 1H), 7.02 (d, J=0.9 Hz, 1H), 4.55 (q, J=7.1 Hz, 2H), 2.38 (d, J=0.8 Hz, 3H), 1.46 (t, J=7.1 Hz, 3H).
To a solution of 6-bromo-7-ethoxy-2-methylimidazo[1,2-a]pyrimidine (100 mg, 0.389 mmol), tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (114 mg, 0.389 mmol) and Na2CO3 (62 mg, 0.584 mmol) in toluene (6 mL) were added Xantphots (10 mg, 0.016 mmol) and Pd(OAc)2 (4 mg, 0.016 mmol). The mixture was charged CO three times and stirred at 80° C. under CO for 16 h. The mixture was concentrated under reduced pressure to give title product (300 mg, crude) as a dark solid, which was used to next step without further purification. ESI-MS (M+H)+: 497.2.
To a solution of tert-butyl (S)-4-(6-(7-ethoxy-2-methylimidazo[1,2-a]pyrimidine-6-carboxamido)pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (250 mg, crude) in EA (4 mL) was added EA/HCl (4 mL, 3 mol/L). The mixture was stirred at rt for 1 h. The mixture was concentrated in vacuo and the residue was purified by prep-HPLC (0.05% HCl in water/CH3CN) to give title compound (40 mg, yield: 26%) as a yellow solid. ESI-MS (M+H)+:397.1. 1H NMR (400 MHz, MeOD-d4) δ 9.49 (s, 1H), 8.59 (d, J=10.1 Hz, 1H), 8.20 (d, J=10.1 Hz, 1H), 7.79 (d, J=1.0 Hz, 1H), 4.77 (d, J=7.1 Hz, 2H), 4.51-4.45 (m, 2H), 3.70-3.66 (m, 1H), 3.64-3.60 (m, 2H), 3.47-3.40 (m, 2H), 2.51 (s, 3H), 1.55 (t, J=7.1 Hz, 3H), 1.47 (d, J=6.5 Hz, 3H).
To a mixture of 6-chloro-2-methyl-2H-pyrazolo [3,4-b]pyridine (10.3 g, 67.3 mmol) in THF (250 mL) was added NaH (5.38 g, 224 mmol) at 0° C., the mixture was stirred at this temperature for 30 min. CH3I (36.3 g, 256 mmoL) was added and the resultant was stirred at RT for 2 h. LCMS showed the starting material was consumed completely. The mixture was diluted with water (100 mL), extracted with EA (150 mL), washed with brine (100 mL*3). The combined organic layer was dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (EA) to give title product (9.5 g, Y: 85%) as a white solid. ESI-MS (M+H+): 168.0. 1H NMR (400 MHz, CDCl3) δ 7% (d, J=8.6 Hz, 1H), 7.94 (s, 1H), 7.03 (d, J=8.6 Hz, 1H), 4.23 (s, 3H).
To a mixture of 6-ethoxy-2-methyl-2H-pyrazolo [3,4-b] pyridine (1.1 g, 6.6 mmol) in EtOH (20 mL) was added NaOEt (4.47 g, 24 mmol). The mixture was stirred at 80° C. for 16 h. LCMS showed the starting material was consumed completely. The mixture was diluted water (40 mL), extracted with EA (50 mL), wash with brine (40 mL*3). The combined organic layer was dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (EA) to give title product (1 g, Y: 94%) as a white solid. ESI-MS (M+H+): 178.1. 1H NMR (400 MHz, CDCl3) δ 7.80 (d, J=8.9 Hz, 1H), 7.72 (s, 1H), 6.55 (d, J=8.9 Hz, 1H), 4.50 (q, J=7.2 Hz 2H), 4.12 (s, 3H), 1.42 (t, J=7.2 Hz, 3H).
To a mixture of 6-ethoxy-2-methyl-2H-pyrazolo [3,4-b]pyridine (800 mg, 4.52 mmol) in DMF (20 mL) was added NBS (950 g, 5.37 mmol). The mixture was stirred at rt for 2 h. The mixture was diluted with water (30 mL), extracted with EA (40 mL), wash with brine (30 mL*3). The combined organic layer was dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (EA) to give title product (800 mg. Y: 69%) as a white solid. ESI-MS (M+H+): 257.9. 1H NMR (400 MHz, CDCl3) β 8.10 (s, 1H), 7.71 (s, 1H), 4.56 (q, J 6.8 Hz, 2H), 4.13 (s, 3H), 1.47 (t, J=6.8 Hz, 3H).
To a mixture of 5-bromo-6-ethoxy-2-methyl-2H-pyrazolo [3,4-b]pyridine (100 mg, 0.4 mmol) and tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (115 mg, 0.4 mmol) in toluene (10 mL) were added Pd(OAc)2 (3.5 mg, 0.02 mmol), Xantphots (10 mg, 0.02 mmol) and Na2CO3 (62 mg, 0.6 mmoL). The mixture was charged with CO for three times and stirred at 120° C. for 16 h. After cooling to rt, the mixture was filtered and the filtrate was concentrated to give title product (100 mg, crude) as a black solid, which was used in next step directly. ESI-MS (M+H+): 497.4.
A mixture of tert-butyl (S)-4-(6-(6-ethoxy-2-methyl-2H-pyrazolo [3,4-b]pyridine-5-carboxamido) pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (crude, 100 mg, 0.2 mmol) in HCl/EA (8.00 mL) was stirred at rt for 2 h. The mixture was concentrated in vacuo and the residue was purified by prep-HPLC (0.05% HCl in water/CH3CN) to give title product (13.00 mg, Y: 16.25% for two steps) as a yellow solid. ESI-MS (M+H+): 397.3. 1H NMR (400 MHz, MeOD-d4) δ 8.87 (s, 1H), 8.43 (s, 1H), 8.36-8.29 (m, 1H), 8.27-8.18 (m, 1H), 4.69 (q, J=6.6 Hz, 21H), 4.53-4.44 (m, 2H), 4.20 (s, 3H), 3.64-3.55 (m, 3H), 3.32-3.30 (m, 2H), 1.56-1.52 (m, 3H), 1.49-1.45 (m, 3H).
7-Ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (6.1 g, 27.7 mmol) was suspended in DMF (50 mL) and 6-bromopyridazin-3-amine (4.82 g, 27.7 mmol) was added followed by N,N-dimethylpyridin-4-amine (4.06 g, 33.24 mmol). To the resulting mixture N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (6.37 g, 33.24 mmol) was added in one portion and the reaction mixture was stirred overnight at r.t. The precipitate formed was filtered, washed with MeCN (30 mL), MTBE (30 mL), dried in vacuo to give pure N-(6-chloropyridazin-3-yl)-7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamide (9.06 g. Y: 87%). ESI-MS (M+H)+: 377.0 1H NMR (400 MHz, DMSO-d6) δ 11.03 (s, 1H), 9.01 (s, 1H), 8.4 (d, J=9 Hz, 1H), 8.01 (d, J=9 Hz), 7.64 (s, 1H), 6.97 (s, 1H), 4.29 (q, J=7.1 Hz, 2H), 2.29 (s, 3H), 1.49 (t, J=6.9 Hz, 3H).
To a mixture of 1 eq. of ArBr (A) and 1.5 eq. amine (B) in 1 mL Dioxane under an inert atmosphere were added 0.1 eq. RuPhosPdG4, 0.1 eq. RuPhos and 3 eq. tBuONa*. The reaction mixture was stirred at 100° C. for 16 hours. After cooling to room temperature, the solvent was evaporated and 1 ml of cleavage cocktail** was added. The mixture was left to react for 4 h and evaporated to dryness. The residue was dissolved in DMSO and subjected to HPLC purification***. *In case of using Reagent 1 and/or Reagent 2 as salt, an additional amount t-BuONa (1.1 eq. for each eq. of acid) was added to the reaction mixture to convert the reagent into a free form.**The cleavage cocktail was prepared by mixing TFA (92.5% v/v), water (5% v/v) and TIPS (2.5% v/v).***Resulting solution was purified by HPLC (Deionized Water (phase A) and HPLC-grade Acetonitrile (phase B) were used as an eluent to obtained final compound (D). In most cases. TFA was used as an additive.
To a solution of (S)-6-ethoxy-2-methyl-N-(6-(3-methylpiperazin-1-yl)pyridazin-3-yl)-2H-indazole-5-carboxamide (50 mg, 0.13 mmol) in MeOH (5 mL) were added PFA (19 mg, 0.63 mmol) and acetic acid (50 mg, 0.83 mmol). The mixture was stirred at RT for 1 h, then sodium cyanoborohydride (24 mg, 0.38 mmol) was added under ice bath and the mixture was stirred at rt for 16 h, then water (10 mL) was added and the solution and concentrated in vacuo. The crude was purified by prep-HPLC (0.05% HCl in water/CH3CN) to give title compound (17 mg, yield: 32.8%) as a white solid. ESI-MS (M+H)+: 410.3. 1H NMR (400 MHz, MeOD-d4) δ 8.50 (s, 1H), 8.41 (s, 1H), 8.15 (s, 1H), 8.12 (s, 1H), 7.14 (s, 1H), 4.54 (d, J=14.7 Hz, 2H), 4.36-4.31 (m, 2H), 4.21 (s, 3H), 3.77-3.69 (m, 1H), 3.58-3.46 (m, 2H), 3.42-3.34 (m, 2H), 3.01 (s, 3H), 1.60-1.56 (m, 3H), 1.54-1.47 (m, 3H).
To a mixture of (S)-6-ethoxy-2-methyl-N-(6-(3-methylpiperazin-1-yl)pyridazin-3-yl)-2H-indazole-5-carboxamide (100 mg, 0.25 mmol) and CH3CHO (56.3 mg, 1.28 mmol) in MeOH (8 mL) and HOAc (45.6 mg, 0.76 mmol) was added NaBH3CN (50 mg, 0.8 mmol). The mixture was stirred at RT for 5 h. The mixture was diluted with H2O (3 mL) and concentrated in vacuo. The residue was purified by prep-HPLC (0.05% HC in water/ACN) to give title product (33 mg, Y: 28%) as a yellow solid. ESI-MS (M+H+): 424.2. 1H NMR (400 MHz, MeOD-d4) δ 8.72 (s, 1H), 8.49 (s, 1H), 8.27 (br.s, 2H), 7.20 (s, 1H), 4.56 (d, J=8.1 Hz, 2H), 4.39-4.33 (m, 2H), 4.31 (s, 3H), 3.77 (br s, 2H), 3.69-3.45 (m, 3H), 3.44-3.32 (m, 1H), 3.30-3.12 (m, 1H), 1.56 (br.s, 6H), 1.43 (br.s, 3H).
To a solution of (S)-6-ethoxy-2-methyl-N-(6-(3-methylpiperazin-1-yl)pyridazin-3-yl)-2H-indazole-5-carboxamide (100 mg, 0.25 mmol) in MeOH (5 mL) were added tetrahydro-4H-pyran-4-one (253 mg, 2.5 mmol) and acetic acid (100 mg, 1.66 mmol). The mixture was stirred at RT for 1 h, then sodium cyanoborohydride (47 mg, 0.76 mmol) was added under ice bath and the mixture was stirred at 50° C. for 40 h. After concentration, the crude was purified by prep-HPLC (0.05% NH3·H2O in water/CH3CN) to give title compound (7 mg, yield: 5.8%) as a white solid. ESI-MS (M+H)+: 480.4. 1H NMR (400 MHz, DMSO-d6) δ 10.86 (s, 1H), 8.46 (d, J=4.9 Hz, 2H), 8.25 (d, J=9.9 Hz, 1H), 7.41 (d, J=9.8 Hz, 1H), 7.15 (s, 1H), 4.31-4.26 (m, 2H), 4.14 (s, 3H), 3.92-3.82 (m, 4H), 3.31-3.24 (m, 4H), 3.21-3.15 (m, 1H), 2.97-2.90 (m, 2H), 2.87-2.80 (m, 2H), 1.67-1.59 (m, 2H), 1.54-1.51 (m, 3H), 1.43-1.34 (m, 1H), 1.10-1.05 (m, 3H).
To a mixture of tert-butyl (S)-4-(6-amino-4-methylpyridazin-3-yl)-2-methylpiperazine-1-carboxylate (100 mg, 0.417 mmol) in toluene (15 mL) were added 6-bromo-7-methoxy-2-methylimidazo [1,2-a]pyridine (86 mg, 0.417 mmol), Pd(OAc)2 (9 mg, 0.042 mmol). Xantphos (48 mg, 0083 mmol) and Na2CO3 (88.4 mg, 0.834 mmol). The resulting mixture was stirred overnight at 100° C. under CO. The mixture was allowed to cool down to room temperature and concentrated. Then diluted with water (15 mL) and extracted with EA (30 mL*2). The combined organic layer was concentrated to provide the crude product (150 mg, crude), which was used to next step without further purification. ESI-MS (M+H)+: 496.2
To a solution of tert-butyl (S)-4-(6-(7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)-4-methylpyridazin-3-yl)-2-methylpiperazine-1-carboxylate (150 mg, crude) in EA (5 mL) was added 3M HCl in EA (5 mL) at rt. The mixture was stirred for 2 h at rt. The reaction was concentrated, the residue was purified by prep-HPLC (0.05% HCl in water/CH3CN) to give title product (7 mg, yield: 5% two steps) as a yellow solid. ESI-MS (M+H)+: 396.1. 1H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H), 8.20 (s, 1H), 7.91 (s, 1H), 7.38 (s, 1H), 4.07 (s, 3H), 3.60 (d, J=11.3 Hz, 2H), 3.53-3.47 (m, 1H), 3.43-3.37 (m, 1H), 3.29-3.19 (m, 2H), 3.06-2.97 (m, 1H), 2.46 (s, 3H), 2.38 (s, 3H), 1.32 (d, J=6.5 Hz, 3H).
To a solution of 2-fluoro-4-hydroxybenzaldehyde (30.0 g, 214.11 mmol) in acetic acid (250 mL) was added a solution of bromine (35.93 g, 224.82 mmol, 11.52 mL) in acetic acid (20 mL), and the mixture was stirred at 45° C. for 26 h. The reaction mixture was concentrated under reduced pressure, brine was added to the residue, and the resulting mixture was extracted with ethyl acetate. The extract was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give 5-bromo-2-fluoro-4-hydroxybenzaldehyde (45.4 g, 85.0% purity, 176.2 mmol, Y: 82.3%) as colorless crystals. 1H NMR (500 MHz, DMSO-d6) δ 11.95 (s, 1H), 9.92 (s, 1H), 7.88 (d, J=8.1, 1H), 6.82 (d, J=12.2 Hz, 1H).
To a solution of 5-bromo-2-fluoro-4-hydroxybenzaldehyde (45.4 g, 207.3 mmol) in DMF (300 mL) was added potassium carbonate (57.3 g, 414.59 mmol) and the solution was stirred at 25° C. for 30 min. Iodoethane (38.8 g, 248.76 mmol) was added and the reaction mixture was stirred at 50° C. for 16 h. A mixture of water (300 mL) and ethyl acetate (300 mL) was added, and the organic layer was separated, washed with water (100 mL), brine (100 mL), dried over Na2SO4 and evaporated under vacuo to afford 5-bromo-4-ethoxy-2-fluorobenzaldehyde (41.3 g, 85.0% purity, 142.09 mmol, Y: 68.5%) as a white solid. 1H NMR (500 MHz, DMSO-d6) δ 9.99 (s, 1H), 7.93 (d, J=7.5 Hz, 1H), 7.19 (d, J=12.7 Hz, 1H), 4.21 (q, J=7.2 Hz, 2H), 1.37 (t, J=6.8 Hz, 3H).
To a solution of 5-bromo-4-ethoxy-2-fluorobenzaldehyde (41.3 g, 167.17 mmol) in DMSO (350 mL) was added potassium carbonate (27.72 g, 200.6 mmol) and hydrazine hydrate (20.92 g, 417.91 mmol) and the reaction mixture was heated at 100° C. for 16 h. The cooled resulting mixture was diluted with water (500 mL) and extracted with EtOAc (3×250 mL). The combined organic layers were washed with brine (300 mL), dried over Na2SO4, filtered, and concentrated in vacuo to give 5-bromo-6-ethoxy-2H-indazole (26.0 g, 85.0% purity, 91.67 mmol, Y: 54.8%) as a yellow solid. ESI-MS (M+H)+: 241.0. 1H NMR (500 MHz, CDCl3) δ 10.30 (s, 1H), 7.94 (s, 1H), 6.89 (s, 1H), 4.16 (q, J=7.2 Hz, 2H), 1.49 (t, J=6.9 Hz, 3H).
To a stirred solution of 5-bromo-6-ethoxy-2H-indazole (26.6 g, 110.33 mmol) in EtOAc (400 mL) was added trimethyloxonium tetrafluoroborate (48.96 g, 331.01 mmol) at rt. The solution was stirred at rt for 2 days. The resulting mixture was poured into sat. NaHCO3 and the organic layer was separated, dried over Na2SO4, and evaporated to dryness. The residue was purified by FCC to give 5-bromo-6-ethoxy-2-methyl-2H-indazole (9.6 g, 95.0% purity, 35.75 mmol, Y: 32.4%). 1H NMR (400 MHz, DMSO-d6) δ 8.20 (s, 1H), 7.96 (s, 1H), 7.04 (s, 1H), 4.09 (d, J=7.5 Hz, 2H), 3.32 (s, 3H), 1.38 (t, J=6.9 Hz, 3H).
5-Bromo-6-ethoxy-2-methyl-2H-indazole (9.6 g, 37.63 mmol) was dissolved in MeOH (150 mL) and Pd(dppf)Cl¬2 (614.61 mg, 752.61 μmol) was added followed by triethylamine (7.62 g, 75.26 mmol). The resulting mixture was transferred into an autoclave and stirred at 130° C. under 40 bar pressure of CO overnight. Then the MeOH was evaporated, and the residue was partitioned between water (100 mL) and EtOAc (150 mL). The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure to give crude methyl 6-ethoxy-2-methyl-2H-indazole-5-carboxylate (8.2 g, 90.0% purity, 31.5 mmol. Y: 83.7%) which was used in the next step without purification. ESI-MS (M+H)+: 235.2. 1H NMR (500 MHz, DMSO-d6) δ 8.35 (s, 1H), 8.04 (s, 1H), 6.98 (s, 1H), 4.09 (s, 3H), 4.04 (q, J=8.6, 2H), 3.30 (s, 3H), 1.32 (t, J=7.1, 3H).
A mixture of methyl 6-ethoxy-2-methyl-2H-indazole-5-carboxylate (8.2 g, 35.01 mmol) and potassium hydroxide (2.36 g, 42.01 mmol) was stirred in methanol (150 mL) and H2O (30 mL) overnight. The reaction mixture was then concentrated under reduced pressure to remove methanol, and the resulting aqueous solution was neutralized with 1 N HCl to pH=5 to precipitate the carboxylic acid. The solid 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (6.5 g, 95.0% purity, 28.04 mmol, Y: 80.1%) was isolated by filtration, dried, and used directly in the next step without further purification. ESI-MS (M+H)+: 221.0. 1H NMR (500 MHz, DMSO-d6) δ 8.31 (s, 1H), 8.01 (s, 1H), 6.95 (s, 1H), 4.15-3.90 (m, 5H), 1.32 (d, J=7.2 Hz, 3H).
A mixture of 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (3.2 g, 14.53 mmol), 5-bromopyrazin-2-amine (2.53 g, 14.53 mmol), and pyridine (3.45 g, 43.6 mmol) was dissolved in anhydrous acetonitrile (4 mL) and stirred at room temperature for 5 minutes, followed by addition of T3P (18.5 g, 58.13 mmol) (50 wt % in EtOAc) over 10 minutes. The reaction mixture was stirred for 12 hours, then filtered and dried under vacuto to afford N-(5-bromopyrazin-2-yl)-6-ethoxy-2-methyl-2H-indazole-5-carboxamide (4.68 g, 95.0% purity, 11.82 mmol, Y: 81.3%). ESI-MS (M+H)+: 376.1. 1H NMR (400 MHz, DMSO-d6) δ 10.80 (s, 1H), 9.30 (s, 1H), 8.58 (s, 1H), 8.45 (s, 1H), 8.37 (s, 1H), 7.14 (s, 1H), 4.30 (q, J=7.2 Hz, 2H), 4.13 (s, 3H), 1.50 (t, J=6.8 Hz, 3H).
To a mixture of 1 eq. of ArBr (A) and 1.5 eq. Amine (B) in 1 ml dry Dioxane under an inert atmosphere were added 0.05 eq. RuPhosPdG4, 0.05 eq. RuPhos and 2.5 eq. Cs2CO3. The reaction mixture was stirred at 100° C. for 16 hours. After cooling to room temperature, the solvent was evaporated and 1 ml TFA was added and stirred at room temperature for 4 h. The mixture was evaporated. The residue was dissolved in DMSO (appr. 1 ml), treated with scavenger SiliaMetS DMT and filtered. Resulting solution was purified by HPLC (Deionized Water (phase A) and HPLC-grade Acetonitrile (phase B) were used as an eluent to obtain final compound (C). In most cases, TFA was used as an additive.
6-Ethoxy-2-methyl-2H-indazole-5-carboxylic acid (3.38 g, 15.35 mmol) was suspended in DMF (30 mL) and DIPEA (3.97 g, 30.7 mmol) was added followed by DIPEA (7.0 g, 18.42 mmol). The resulting mixture was stirred for 30 min and 2-bromopyrimidin-5-amine (2.67 g, 15.35 mmol) was added in one portion and the reaction mixture was stirred overnight at rt. The precipitate formed was filtered, washed with MeCN (60 mL), MTBE (60 mL), dried in vacuo to give pure N-(2-bromopyrimidin-5-yl)-6-ethoxy-2-methyl-2H-indazole-5-carboxamide (1.57 g, 95.0% purity, 3.96 mmol, Y: 25.8%). ESI-MS (M+H)+: 376.2. 1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 9.02 (s, 2H), 8.36 (s, 1H), 8.16 (s, 1H), 7.08 (s, 1H), 4.22 (q, J=6.7 Hz, 2H), 4.13 (s, 3H), 1.43 (t, J=6.7 Hz, 3H).
To a mixture of 1 eq. of ArBr (A) and 1.5 eq. Amine (B) in 1 ml dry Dioxane under an inert atmosphere were added 0.05 eq. RuPhosPdG4, 0.05 eq. RuPhos and 2.5 eq. Cs2CO3. The reaction mixture was stirred at 100° C. for 16 hours. After cooling to room temperature, the solvent was evaporated and 1 ml TFA was added and stirred at room temperature for 4 h. The mixture was evaporated. The residue was dissolved in DMSO (appr. 1 ml), treated with scavenger SiliaMetS DMT and filtered.
Resulting solution was purified by HPLC (Deionized Water (phase A) and HPLC-grade Acetonitrile (phase B) were used as an eluent to obtain final compound (C). In most cases, TFA was used as an additive.
A solution of 2-bromo-5-fluoropyridine-4-carboxylic acid (30.0 g, 136.37 mmol) in EtOH (100 mL) was added to a freshly prepared EtONa from sodium (9.41 g, 409.1 mmol) and EtOH (300 mL). The reaction mixture was heated at 60° C. for 24 hours then cooled to room temperature and the solvent was removed in vacuo to give crude 2-bromo-5-ethoxypyridine-4-carboxylic acid (32.0 g, 95.0% purity, 123.55 mmol, Y: 90.6%) as sodium salt. 1H NMR (400 MHz, DMSO-d6) δ 8.00 (s, 1H), 7.24 (s, 1H), 4.08 (q, J=7.0 Hz, 2H), 1.27 (t, J=6.9 Hz, 3H).
To 2-bromo-5-ethoxypyridine-4-carboxylic acid (32.0 g, 130.05 mmol) in EtOH (400 mL) thionyl chloride (46.42 g, 390.15 mmol, 28.3 mL) was added dropwise and the reaction mixture was stirred overnight at 60° C. After that the solvent was evaporated and the residue was partitioned between DCM (300 mL) and water (200 mL), and the aqueous phase was extracted with DCM (200 mL). The combined organic extracts were washed with a saturated aqueous NaHCO3 solution (100 mL), brine (100 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give ethyl 2-bromo-5-ethoxypyridine-4-carboxylate (32.0 g, 90.0% purity, 105.07 mmol, Y: 80.8%). 1H NMR (500 MHz, DMSO-d6) δ 8.34 (s, 1H), 7.71 (s, 1H), 4.29 (q, J=7.0 Hz, 2H), 4.19 (q, J=7.0 Hz, 2H), 1.35-1.21 (m, 6H).
A 500 mL four-necked flask was charged with ethyl 2-bromo-5-ethoxypyridine-4-carboxylate (32.0 g, 116.74 mmol), 1-(trimethylsilyl)-1-propyne (15.72 g, 140.09 mmol), copper(I) iodide (2.22 g, 11.67 mmol), and tetrabutyl ammonium fluoride (61.05 g, 233.48 mmol) in THF (150 mL) and triethylamine (150 mL). After the solution was degassed and purged with argon. Pd(dppf)Cl2 (4.1 g, 5.84 mmol) was added to the reaction flask. The reaction mixture was stirred at 40° C. for 15 h under an argon atmosphere. After that the solvent was evaporated under reduced pressure and the residue was partitioned between ethyl acetate (300 mL) and H2O (150 mL). The organic phase was washed with water (50 mL), dried with anhydrous magnesium sulfate, filtered, concentrated, and purified by FCC to give crude ethyl 5-ethoxy-2-(prop-1-yn-1-yl)-pyridine-4-carboxylate (18.17 g, 95.0% purity, 74.0 mmol, Y: 63.4%). ESI-MS (M+H)+: 234.0. 1H NMR (400 MHz, DMSO-d6) δ 8.47 (s, 1H), 7.54 (s, 1H), 4.35-4.18 (m, 4H), 2.04 (s, 3H), 1.37-1.24 (m, 6H).
Amino 2,4,6-trimethylbenzene-1-sulfonate (13.37 g, 62.11 mmol) was dissolved in dry DCM (200 mL) and cooled under argon atmosphere to 0° C. then ethyl 5-ethoxy-2-(prop-1-yn-1-yl)pyridine-4-carboxylate (13.17 g, 56.46 mmol) was added dropwise over 1 min. The reaction mixture was slowly (without removal of the cooling bath) warmed to RT (about 3 h) and stirred at RT overnight (18 h) under an argon atmosphere. The solvent was evaporated to give crude 1-amino-5-ethoxy-4-(methoxycarbonyl)-2-(prop-1-yn-1-yl)pyridin-1-ium 2,4,6-trimethylbenzene-1-sulfonate (25 g, 55.74 mmol) which was used in the next step without purification. ESI-MS (M)+: 249.2.
1-Amino-5-ethoxy-4-(methoxycarbonyl)-2-(prop-1-yn-1-yl)pyridin-1-ium 2,4,6-trimethylbenzene-1-sulfonate (25.0 g, 55.74 mmol) and potassium carbonate (23.11 g, 167.21 mmol) were mixed in anhydrous DMF (250 mL). The reaction mixture was stirred at ambient temperature for 3 days. A mixture of water (300 mL) and ethyl acetate (300 mL) was added and the organic layer was separated, washed with water (100 mL), brine (100 mL), dried over Na2SO4 and evaporated in vacuo. The residue was purified by FCC to give ethyl 6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxylate (10.1 g, 95.0% purity, 38.65 mmol, Y: 69.3%). ESI-MS (M+H)+: 249.2. 1H NMR (500 MHz, DMSO-d6) δ 8.35 (s, 1H), 7.90 (s, 1H), 6.47 (s, 1H), 4.27 (q, J=7.1 Hz, 3H), 4.02 (q, J=6.9 Hz, 2H), 2.35 (s, 3H), 1.35-1.25 (m, 6H).
A mixture of ethyl 6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxylate (14.0 g, 56.39 mmol) and sodium hydroxide (2.48 g, 62.03 mmol) was stirred in methanol (150 mL) and H2O (50 mL) overnight. The reaction mixture was then concentrated under reduced pressure to remove methanol, and the resulting aqueous solution was neutralized with 1 N HCl to pH=5 to precipitate the carboxylic acid. Solid 6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxylic acid (7.0 g, 95.0% purity, 30.2 mmol, Y: 53.6%) was isolated by filtration, dried, and used directly in the next step without further purification. ESI-MS (M+H)+: 221.0. 1H NMR (400 MHz, DMSO-d6) δ 8.33 (s, 1H), 7.87 (s, 1H), 6.45 (s, 1H), 4.03 (q, J=6.9 Hz, 2H), 2.35 (s, 3H), 1.32 (t, J=6.9, 3H).
A mixture of 6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxylic acid (2.8 g, 12.71 mmol), 5-bromo-pyrazin-2-amine (2.21 g, 12.71 mmol), and pyridine (3.02 g, 38.14 mmol) was dissolved in anhydrous acetonitrile (40 mL). The mixture was stirred at room temperature for 5 minutes, followed by addition of T3P (16.18 g, 50.85 mmol, 50 wt % in EtOAc) over 10 minutes. The reaction mixture was stirred for 12 hours, then the resulting mixture was filtered and dried in vacuto to afford N-(5-bromopyrazin-2-yl)-6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxamide (3.83 g, 95.0% purity, 9.67 mmol, Y: 76.1%). ESI-MS (M+H)+: 376.2. 1H NMR (400 MHz, Chloroform-d) δ 10.56 (s, 1H), 9.48 (s, 1H), 8.41 (s, 1H), 8.35 (s, 1H) 8.10 (s, 1H), 6.46 (s, 1H), 4.23 (q, J=6.9 Hz, 2H), 2.47 (s, 3H), 1.65 (t, J=6.9 Hz, 3H).
To a mixture of 1 eq. of ArBr (A) and 1.5 eq. Amine (B) in 1 ml dry Dioxane under an inert atmosphere were added 0.05 eq. RuPhosPdG4, 0.05 eq. RuPhos and 2.5 eq. Cs2CO1. The reaction mixture was stirred at 100° C. for 16 hours. After cooling to room temperature, the solvent was evaporated and 1 ml TFA was added and stirred at room temperature for 4 h. The mixture was evaporated. The residue was dissolved in DMSO (appr. 1 ml), treated with scavenger SiliaMetS DMT and filtered.
Resulting solution was purified by HPLC (Deionized Water (phase A) and HPLC-grade Acetonitrile (phase B) were used as an eluent to obtain final compound (C). In most cases, TFA was used as an additive.
6-Ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxylic acid (3.5 g, 15.89 mmol) was suspended in DMF (30 mL) and DIPEA (4.11 g, 31.79 mmol) was added followed by HATU (7.25 g, 19.07 mmol). The resulting mixture was stirred for 30 min and 2-bromopyrimidin-5-amine (2.77 g, 15.89 mmol) was added in one portion and the reaction mixture was stirred overnight at rt. The precipitate formed was filtered, washed with MeCN (60 mL), MTBE (60 mL), dried in vacuo to give pure N-(2-bromopyrimidin-5-yl)-6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxamide (4.45 g, 95.0% purity, 11.24 mmol, Y: 70.7%). ESI-MS (M+H)+: 376.0. 1H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H), 9.02 (s, 2H), 8.42 (s, 1H), 7.95 (s, 1H), 6.51 (s, l-H), 4.18 (q, J=7.1, 2H), 2.40 (s, 3H), 1.45-1.39 (t, J=7.0, 3H).
To a mixture of 1 eq. of ArBr (A) and 1.5 eq. Amine (B) in 1 ml dry Dioxane under an inert atmosphere were added 0.05 eq. RuPhosPdG4, 0.05 eq. RuPhos and 2.5 eq. Cs2CO3. The reaction mixture was stirred at 100° C. for 16 hours. After cooling to room temperature, the solvent was evaporated and 1 ml TFA was added and stirred at room temperature for 4 h. The mixture was evaporated. The residue was dissolved in DMSO (appr. 1 ml), treated with scavenger SiliaMetS DMT and filtered.
Resulting solution was purified by HPLC (Deionized Water (phase A) and HPLC-grade Acetonitrile (phase B) were used as an eluent to obtain final compound (C). In most cases, TFA was used as an additive.
The 7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (4.0 g, 18.16 mmol) was suspended in DMF (2 mL) and DIPEA (4.69 g, 36.33 mmol) was added followed by HATU (8.29 g, 21.8 mmol). The resulting mixture was stirred for 30 min and 2-bromopyrimidin-5-amine (3.16 g, 18.16 mmol) was added in one portion and the reaction mixture was stirring overnight at rt. The precipitate formed was filtered, washed with MeCN (60 ml), MTBE (60 mL), dried under vacuo to give pure N-(2-bromopyrimidin-5-yl)-7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamide (4.9 g, Y: 68.1%). ESI-MS (M+H)+: 376.0. 1H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 9.01 (s, 2H), 8.89 (s, 1H), 7.60 (s, 1H), 6.95 (s, 1H), 4.23 (d, J=7.8 Hz, 2H), 2.29 (s, 3H), 1.44 (t, J=6.7 Hz, 3H).
To a mixture of 1 eq. of ArBr (A) and 1.5 eq. Amine (B) in 1 ml dry Dioxane under an inert atmosphere were added 0.05 eq. RuPhosPdG4, 0.05 eq. RuPhos and 2.5 eq. Cs2CO3. The reaction mixture was stirred at 100° C. for 16 hours. After cooling to room temperature, the solvent was evaporated and 1 ml TFA was added and stirred at room temperature for 4 h. The mixture was evaporated. The residue was dissolved in DMSO (appr. 1 ml), treated with scavenger SiliaMetS DMT and filtered.
Resulting solution was purified by HPLC (Deionized Water (phase A) and HPLC-grade Acetonitrile (phase B) were used as an eluent to obtain final compound (C). In most cases, TFA was used as an additive.
Mixture of 7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (3.6 g, 16.35 mmol), 5-bromopyrazin-2-amine (2.84 g, 16.35 mmol), and pyridine (3.88 g, 49.04 mmol) were dissolved in anhydrous acetonitrile (4 mL). The reaction was stirred at room temperature for 5 minutes, followed by addition of 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide (T3P) (20.81 g, 65.39 mmol) (50 wt % in EtOAc) over the course of 10 minutes. The reaction continued to stir for 12 hours, then the reaction mixture was filtered and solid was dried under vacuo to afford N-(5-bromopyrazin-2-yl)-7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamide (4.65 g, Y: 71.8%). ESI-MS (M+H)+: 376.0. 1H NMR (500 MHz, Chloroform-d) δ 10.53 (s, 1H), 9.45 (s, 1H), 8.96 (s, 1H), 8.37 (s, 1H), 7.29 (s, 1H), 6.92 (s, 1H), 4.31 (J=7.8 Hz, 2H), 2.41 (s, 3H), 1.66 (J=6.7 Hz, 3H).
To a mixture of 1 eq. of ArBr (A) and 1.5 eq. Amine (B) in 1 ml dry Dioxane under an inert atmosphere were added 0.05 eq. RuPhosPdG4, 0.05 eq. RuPhos and 2.5 eq. Cs2CO3. The reaction mixture was stirred at 100° C. for 16 hours. After cooling to room temperature, the solvent was evaporated and 1 ml TFA was added and stirred at room temperature for 4 h. The mixture was evaporated. The residue was dissolved in DMSO (appr. 1 ml), treated with scavenger SiliaMetS DMT and filtered.
Resulting solution was purified by HPLC (Deionized Water (phase A) and HPLC-grade Acetonitrile (phase B) were used as an eluent to obtained final compound (C). In most cases, TFA was used as an additive.
The 7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (100.0 mg, 454.08 μmol) was suspended in DMF (4 mL) and DIPEA (146.7 mg, 1.14 mmol) was added followed by HATU (207.16 mg, 544.82 μmol). The resulting mixture was stirred for 30 min at rt. After that tert-butyl (2S)-4-(5-aminopyrazin-2-yl)-2-methylpiperazine-1-carboxylate (133.19 mg, 454.02 μmol) was added in one portion and the reaction mixture was stirring overnight at rt. The precipitate formed was filtered, washed with MeCN (2 ml). MTBE (2 mL), dried under vacuo to give pure tert-butyl (2S)-4-(5-7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-amidopyrazin-2-yl)-2-methylpiperazine-1-carboxylate (40.0 mg, Y: 17.8%). ESI-MS (M+H)+: 496.4.
To a solution of tert-butyl (2S)-4-(5-7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-amidopyrazin-2-yl)-2-methylpiperazine-1-carboxylate (40.0 mg, 80.71 μmol) in dichloromethane (3 mL) TFA (91.98 mg, 806.7 μmol) was added and the mixture was stirred overnight. Then the mixture was evaporated to dryness under reduced pressure. The obtained crude residue was crystallized from MTBE/MeCN to give 7-ethoxy-2-methyl-N-5-[(3S)-3-methylpiperazin-1-yl]pyrazin-2-ylimidazo[1,2-a]pyridine-6-carboxamide as TFA salt (18.6 mg, Y: 45.3%). ESI-MS (M+H)+: 396.2. 1H NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H), 9.19 (br s, 2H), 8.96 (s, 1H), 8.90 (br s, 1H), 8.28 (s, 1H), 7.90 (s, 1H), 7.33 (s, 1H), 4.35 (q, J=7.1 Hz, 2H), 3.65-3.25 (m, 4H), 3.20-3.07 (m, 2H), 2.99-2.95 (m, 1H) 2.43 (s, 3H), 1.45 (t, J=6.9 Hz, 3H)), 1.28 (d, J=8.0 Hz, 3H).
The 6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxylic acid (130.0 mg, 591 μmol) was suspended in DMF (4 mL) and DIPEA (146.7 mg, 1.14 mmol) was added followed by HATU (270 mg, 710 μmol). The resulting mixture was stirred for 30 min at rt. After that tert-butyl (2S)-4-(5-aminopyrazin-2-yl)-2-methylpiperazine-1-carboxylate (173 mg, 590 μmol) was added in one portion and the reaction mixture was stirring overnight at rt. The precipitate formed was filtered, washed with MeCN (2 ml), MTBE (2 mL), dried under vacuo to give pure tert-butyl (2S)-4-(5-7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-amidopyrazin-2-yl)-2-methylpiperazine-1-carboxylate (40.0 mg, Y: 17.8%). ESI-MS (M+H)+: 496.2.
To a solution of tert-butyl (2S)-4-(5-6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-amidopyrazin-2-yl)-2-methylpiperazine-1-carboxylate (79.95 mg, 161.33 μmol) in dichloromethane (3 mL) TFA (91.98 mg, 806.66 μmol) was added and the mixture was stirred overnight. Then the mixture was evaporated to dryness under reduced pressure. The obtained crude residue was crystallized from MTBE/MeCN to give 6-ethoxy-2-methyl-N-5-[(3S)-3-methylpiperazin-1-yl]pyrazin-2-ylpyrazolo[1,5-a]pyridine-5-carboxamide as TFA salt (62.0 mg, Y: 87.2%). ESI-MS (M+H)+: 396.2. 1H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 9.09 (br m, 1H) 9.01 (s, 1H), 8.76 (br s, 1H), 8.49 (s, 1H), 8.27 (s, 1H), 8.09 (s, 1H), 6.53 (s, 1H), 4.39-4.29 (m, 2H), 4.21 (d, J=6.9 Hz, 2H), 3.21-3.06 (m, 3H), 2.96 (t, J=12.4 Hz, 1H), 2.38 (s, 3H), 1.43 (t, J=7.1 Hz, 3H), 1.27 (d, J=6.3 Hz, 3H).
The 7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (100.0 mg, 454.08 μmol) was suspended in DMF (40 mL) and DIPEA (146.53 mg, 1.13 mmol) was added followed by HATU (206.92 mg, 544.19 μmol). The resulting mixture was stirred for 30 min at rt. After that tert-butyl (2S)-4-(4-amino-3-fluorophenyl)-2-methylpiperazine-1-carboxylate (140.3 mg, 453.49 μmol) was added in one portion and the reaction mixture was stirring overnight at rt. The precipitate formed was filtered, washed with MeCN (20 ml), MTBE (20 mL), dried under vacuo to give pure tert-butyl (2S)-4-(4-7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-amido-3-fluorophenyl)-2-methylpiperazine-1-carboxylate (78.0 mg, Y: 33.6%) ESI-MS (M+H)+: 512.4.
The tert-butyl (2S)-4-(4-7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-amido-3-fluorophenyl)-2-methylpiperazine-1-carboxylate (78.0 mg, 152.47 μmol) was dissolved in dichloromethane (10 mL) and TFA (173.8 mg, 1.52 mmol) was added. The resulting mixture was stirred ar rt overnight and then evaporated to dryness to provide crude material. The residue was triturated with MeCN/MTBE (1/4) and the resulting precipitate was filtered and dried under vacuo to give pure 7-ethoxy-N-2-fluoro-4-[(3S)-3-methylpiperazin-1-yl]phenyl-2-methylimidazo[1,2-a]pyridine-6-carboxamide as TFA salt (67.0 mg, 127.5 μmol, 83.6% yield). ESI-MS (M+H)+: 412.0. 1H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 1H), 9.22 (s, 1H), 9.11 (br s, 1H), 8.73 (br s, 1H), 7.91 (s, 1H), 7.81 (t, J=9.0 Hz, 1H), 7.33 (s, 1H), 7.03 (dd, J=14.0, 2.5 Hz, 1H), 6.88 (dd, J=8.9, 2.3 Hz, 1H), 3.90-3.70 (m, 2H), 3.40-3.30 (m, 2H), 3.13-3.08 (m, 1H), 2.97-2.90 (m, 1H), 2.74-2.70 (m, 1H), 2.41 (s, 3H), 1.46 (t, J=7.1 Hz, 3H), 1.27 (d, J=6.3 Hz, 3H).
2,4-Difluoro-1-nitrobenzene (374.89 mg, 2.36 mmol), tert-butyl 4,7-diazaspiro[2.5]octane-4-carboxylate (500.25 mg, 2.36 mmol) and potassium carbonate (651.35 mg, 4.71 mmol) were dissolved in dimethylformamide (5 mL) and stirred at 60° C. overnight. Then the reaction mixture was poured into ice water and extracted with EtOAc (20 mL). The organic layer was separated, washed with H2O (10 mL), brine (10 mL) dried and evaporated to give crude, which was purified by flash chromatography (EA:PE=1:10, v/v) to give title product tert-butyl 7-(3-fluoro-4-nitrophenyl)-4,7-diazaspiro[2.5]octane-4-carboxylate (260.0 mg, Y: 31.4%). ESI-MS (M-56+H)+: 296.0.
tert-butyl 7-(3-fluoro-4-nitrophenyl)-4,7-diazaspiro[2.5]octane-4-carboxylate (260.0 mg, 739.96 μmol) was dissolved in methanol and treated with 10% Pd/C (0.17 g). The resulting mixture was stirred vigorously at ambient hydrogen pressure and room temperature until the reaction was complete. The catalyst was filtered off, the filtrate was evaporated and purified by column chromatography using hexane/EtOAc (2:1) as an eluent to afford tert-butyl 7-(4-amino-3-fluorophenyl)-4,7-diazaspiro[2.5]octane-4-carboxylate (200.0 mg, Y: 84%). ESI-MS (M+H)+: 322.0. 1H NMR (500 MHz, DMSO-d6) δ 6.69-6.56 (m, 2H), 6.48 (d, J=8.5 Hz, 1H), 4.52 (br s, 2H), 3.50 (t, J=4.9 Hz, 2H), 2.88 (t, J=4.9 Hz, 2H), 2.74 (s, 2H), 0.91-0.86 (m, 2H), 0.81-0.76 (m, 2H).
The 7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (100.0 mg, 454.08 μmol) was suspended in DMF (3 mL) and DIPEA (176.24 mg, 1.36 mmol) was added followed HATU (207.4 mg, 545.46 μmol). The resulting mixture was stirred for 30 min at rt. After that tert-butyl 7-(4-amino-3-fluorophenyl)-4,7-diazaspiro[2.5]octane-4-carboxylate (146.09 mg, 454.55 μmol) was added in one portion and the reaction mixture was stirring overnight at rt. The precipitate formed was filtered, washed with MeCN (5 ml). MTBE (2 mL), dried under vacuo to give pure tert-butyl 7-(4-7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-amido-3-fluorophenyl)-4,7-diazaspiro[2.5]octane-4-carboxylate (75.0 mg. Y: 31.5% yield). ESI-MS (M+H)+: 524.0.
The tert-butyl 7-(4-7-ethoxy-2-methylimidazol[1,2-a]pyridine-6-amido-3-fluorophenyl)-4,7-diazaspiro[2.5]octane-4-carboxylate (75.01 mg, 143.25 μmol) was dissolved in DCM (10 mL) and TFA (163.34 mg, 1.43 mmol) was added. The resulting mixture was stirred ar it overnight and then evaporated to dryness to provide crude material. The residue was triturated with MeCN/MTBE (1:4) and the resulting precipitate was filtered and dried under vacuo to give pure N-(4-4,7-diazaspiro[2.5]octan-7-yl-2-fluorophenyl)-7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamide as TFA salt (72.0 mg, Y: 93.5%). ESI-MS (M+H)+: 424.2. 1H NMR (400 MHz, DMSO-d6) δ 9.96 (s, 1H), 9.21 (br s, 3H), 7.89 (s, 1H), 7.83 (t, J=8.9 Hz, 1H), 7.30 (s, 1H), 6.98 (d, J=13.8 Hz, 1H), 6.82 (d, J=9.2 Hz, 1H), 4.33 (d J=7.3 Hz, 2H), 3.31-3.21 (m, 6H), 2.40 (s, 3H), 1.44 (t, J=7.1 Hz, 3H), 1.01 (s, 2H), 0.89 (s, 2H).
The 7-cyclobutoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (260.18 mg, 1.06 mmol) was suspended in DMF (4 mL) and DIPEA (341.36 mg, 2.64 mmol) was added followed by HATU (482.05 mg, 1.27 mmol). The resulting mixture was stirred for 30 min at rt. After that tert-butyl (2S)-4-(4-amino-3-fluorophenyl)-2-methylpiperazine-1-carboxylate (326.86 mg, 1.06 mmol) was added in one portion and the reaction mixture was stirring overnight at it. The precipitate formed was filtered, washed with MeCN (5 ml), MTBE (2 mL), dried under vacuo to give pure tert-butyl (2S)-4-(4-7-cyclobutoxy-2-methylimidazo[1,2-a]pyridine-6-amido-3-fluorophenyl)-2-methylpiperazine-1-carboxylate (130.0 mg, Y: 17.9%). ESI-MS (M+H)+: 538.4.
The tert-butyl (2S)-4-(4-7-cyclobutoxy-2-methylimidazo[1,2-a]pyridine-6-amido-3-fluorophenyl)-2-methylpiperazine-1-carboxylate (100.4 mg, 186.75 μmol) was dissolved in DCM (10 mL) and TFA (212.94 mg, 1.87 mmol) was added. The resulting mixture was stirred ar rt overnight and then evaporated to dryness to provide crude material. The residue was triturated with MeCN/MTBE (1/4) and the resulting precipitate was filtered and dried under vacuo to give pure 7-cyclobutoxy-N-2-fluoro-4-[(3S)-3-methylpiperazin-1-yl]phenyl-2-methylimidazo[1,2-a]pyridine-6-carboxamide as TFA salt (13.0 mg, Y:12.6%). ESI-MS (M+H)+: 438.0. 1H NMR (400 MHz, DMSO-d6) δ 9.96 (s, 1H), 9.20 (s, 1H), 9.09 (br s, 1H), 8.71 (br s, 1H), 7.90-7.83 (m, 2H), 7.11 (s, 1H), 7.01 (d, J=14.0 Hz, 1H), 6.85 (d, J=9.1 Hz, 1H), 5.13-4.96 (m, 1H), 3.78 (dd, J=22.4, 13.2 Hz, 2H), 3.16-3.02 (m, 3H), 2.93 (t, J=12.3 Hz, 1H), 2.71 (t, J=11.9 Hz, 1H), 2.38 (s, 3H), 2.28-2.16 (m, 2H), 1.94-1.84 (m, 1H), 1.77-1.68 (m, 1H), 1.23 (d, J=6.6 Hz, 3H).
A mixture of 3,6-dichloropyridazine (100 g, 0.67 mol). DIEA (174 g, 1.34 mol) and tert-butyl (2R,6S)-2,6-dimethylpiperazine-1-carboxylate (144 g, 0.67 mol) in 1,4-dioxane (1.5 L) was stirred at 100° C. for 16 h. The mixture was cooled to room temperature and concentrated in vacuum. The residue was recrystallized with MTBE to give tert-butyl(2R,6S)-4-(6-chloropyridazin-3-yl)-2,6-dimethylpiperazine-1-carboxylate (130 g, 59% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.21 (d, J=9.5 Hz, 1H), 6.89 (d, J=9.5 Hz, 1H), 4.34-4.27 (m, 2H), 4.15 (d, J=13.2 Hz, 2H), 3.21 (dd, J=13.1, 4.4 Hz, 2H), 1.48 (s, 9H), 1.25 (d, J=6.9 Hz, 6H). ESI-MS (M+H)+: 327.2.
To a solution of tert-butyl(2R,6S)-4-(6-chloropyridazin-3-yl)-2,6-dimethylpiperazine-1-carboxylate (100 g, 306 mmol), diphenylmethanimine (66.5 g, 367 mmol), BINAP (38.1 g, 61.2 mmol) and Cs2CO3(299 g, 918 mmol) in 1,4-dioxane (2.0 L) was added Pd(OAc)2 (6.87 g, 30.6 mmol) at rt and the mixture was stirred at 120° C. for 16 h. The reaction mixture was diluted with ethyl acetate (3 L) and washed with water (1.5 L) and brine (1.5 L). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (15% EA in PE) to give tert-butyl (2R,6S)-4-(6-((diphenylmethylene)amino) pyridazin-3-yl)-2,6-dimethylpiperazine-1-carboxylate (70 g, 49% yield) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.81 (d, J=7.0 Hz, 2H), 7.51-7.28 (m, 6H), 7.21-7.19 (m, 2H), 6.79 (d, J=9.5 Hz, 1H), 6.71 (d, J=9.5 Hz, 1H), 4.29-4.21 (m, 2H), 4.03 (d, J=13.0 Hz, 2H), 3.10 (dd, J=13.0, 4.5 Hz, 2H), 1.48 (s, 9H), 1.24 (d, J=6.8 Hz, 6H).
The mixture of tert-butyl (2R,6S)-4-(6-((diphenylmethylene)amino)pyridazin-3-yl)-2,6-dimethylpiperazine-1-carboxylate (70 g, 148 mmol), NaOAc (36.5 g, 445 mmol) and NH2OH·HCl (51.6 g, 742 mmol) in MeOH (500 mL) was stirred at 25° C. for 15 min. The mixture was adjusted to pH 4 with solid Citric Acid, then the mixture was diluted with brine (300 mL) and extracted with EtOAc (300 mL×2). Then the aqueous phase was adjusted to pH 10 with solid Na2CO3 and extracted with EtOAc (300 mL×2). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to give tert-butyl (2R,6S)-4-(6-aminopyridazin-3-yl)-2,6-dimethylpiperazine-1-carboxylate (25 g, 55% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.15 (d, J=9.6 Hz, 1H), 6.76 (d, J=9.5 Hz, 1H), 5.69 (s, 2H), 4.14-4.06 (m, 2H), 3.84 (d, J=12.6 Hz, 2H), 2.78 (dd, J=12.6, 4.3 Hz, 2H), 1.42 (s, 9H), 1.20 (d, J=6.8 Hz, 6H). ESI-MS (M+H)+: 308.1
A mixture of 6-chloropyridazin-3-amine (306 mg, 2.36 mmol) and tert-butyl 4,7-diazaspiro[2.5]octane-4-carboxylate (500 mg, 2.36 mmol) was stirred at 150° C. for 5 hours. The mixture was purified by silica gel column chromatography (EA) to provide the title compound (200 mg, yield: 27.8%). ESI-MS (M+H)+:306.1.
To a mixture of tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (1.0 g, 3.24 mmol) and 6-chloropyridazin-3-amine (626 mg, 4.85 mmol) in 1,4-dioxane (20 mL) and water (4 mL) were added K2CO3 (1.34 g, 9.72 mmol) and Pd(dppf)Cl2 (236 mg, 0.32 mmol). The mixture was stirred at 80° C. for 3 h. The reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (PE/EA=2:1) to give title product (700 mg, yield 79.3%) as a brown solid. ESI-MS (M+H)+: 277.3. 1H NMR (400 MHz, DMSO-d6) δ 7.54 (d, J=9.3 Hz, 1H), 6.75 (d, J=9.3 Hz, 1H), 6.36 (br s, 3H), 4.01 (br s, 2H), 3.52 (t, J=5.5 Hz, 2H), 2.65-2.55 (m, 2H), 1.43 (s, 9H).
To a solution of tert-butyl 4-(6-aminopyridazin-3-yl)-3,6-dihydropyridine-1(2H)-carboxylate (1 g, 3.62 mmol) in MeOH (10 mL) was added Pd/C (100 mg, 10% w.t.). The mixture was stirred at rt for 16 h under H2. The mixture was filtered and the filtrate was concentrated in vacuo to give title product (1 g, 99.28%) as a brown oil. ESI-MS (M+H)+:279.2. 1H NMR (400 MHz, DMSO-d6) δ 7.19 (d, J=9.1 Hz, 1H), 6.73 (d, J=9.1 Hz, 1H), 6.13 (s, 2H), 4.22-3.83 (m, 4H), 2.84-2.81 (m, 1H), 1.77 (d, J=13.8 Hz, 2H), 1.56-1.49 (m, 2H), 1.41 (s, 9H).
To a solution of 6-bromo-2-methylimidazo[1,2-a]pyridin-7-ol (500 mg, 2.25 mmol) in DMA (15 mL) were added bromocyclopropane (2.75 g, 22.50 mmol), K2CO3 (2.2 g, 6.75 mmol) and KI (187 mg 1.6 mmol). The mixture was stirred at 140° C. overnight. The reaction mixture was diluted with water (20 mL), extracted with EtOAc (40 mL×2). The organic layer was washed with brine (50 mL), dried over sodium sulfate, filtered and concentrated in vacuum. The residue was purified by C18 flash (0.1% NH3·H2O in water/CH3CN) to give title product (660 mg, Y: 62%) as a gray solid. ESI-MS (M+H)+: 267.0. 1H NMR (400 MHz, CDCl3) δ 8.06 (s, 1H), 7.16 (s, 1H), 7.08 (s, 1H), 3.78-3.71 (m, 1H), 2.33 (d, J=0.8 Hz, 3H), 0.81-0.78 (m, 4H).
To a solution of 6-bromo-7-cyclopropoxy-2-methylimidazo[1,2-a]pyridine (640 mg, 2.40 mmol) in MeOH (10 mL) were added TEA (3.6 g, 36.00 mmol) and Pd(dppf)Cl2 (176 mg, 0.24 mmol). The mixture was charged with CO for three times and stirred at 60° C. for 16 h. The reaction mixture was diluted with water (50 mL), extracted with ethyl acetate (100 mL). The organic layer was washed with brine (50 mL), dried over sodium sulfate, filtered and concentrated in vacuum to give title product (410 mg, crude) as a gray solid, which was used to next step without further purification. ESI-MS (M+H)+:246.9.
To a solution of methyl 7-cyclopropoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylate (400 mg, 1.63 mmol) in MeOH (8 mL) and H2O (4 mL) was added LiOH (77 mg, 3.26 mmol), the mixture was stirred for 1 h at it. After concentration, the residue was diluted with water (10 mL) and adjusted pH to 5-6 with 1M HCl. The precipitate was collected by filtration, washed with water and dried under vacuum to provide title compound (240 mg, 63.6%) as a white solid, ESI-MS (M+H)+:233.0. 1H NMR (400 MHz, DMSO-d6) δ 9.27 (s, 1H), 7.91 (s, 1H), 7.49 (s, 1H), 4.29-4.08 (m, 1H), 2.44 (d, J=1.0 Hz, 3H), 0.97-0.92 (m, 2H), 0.83-0.76 (m, 2H).
To a suspension of tert-butyl (S)-2-ethylpiperazine-1-carboxylate (4.8 g, 22.40 mmol) and 2,5-dibromopyrazine (5.34 g, 22.40 mmol) in NMP (50 mL) was added DIEA (8.67 g, 67.2 mmol), and the reaction mixture was stirred for 16 h at 110° C. The mixture was diluted with water (100 mL), extracted with EA (80 mL×3). The combined organic was washed with brine, dried over Na2SO4 and concentrated in vacuo. The crude was purified by silica gel column (PE/EA=3:1) to provide title product (6.2 g, Y: 74.5%) as a yellow solid. ESI-MS (M+H)+: 371.2. 1H NMR (400 MHz, CDCl3) δ 8.11 (d, J=4.0 Hz, 1H), 7.84 (d, J=4.0 Hz, 1H), 4.16-4.08 (m, 2H), 4.08-3.96 (m, 2H), 3.19-3.08 (m, 2H), 3.05-2.93 (m, 1H), 1.72-1.60 (m, 1H), 1.56-1.51 (m, 11), 1.48 (s, 9H), 0.90 (t, J=8 Hz, 3H).
To a solution of tert-butyl (S)-4-(5-((diphenylmethylene)amino)pyrazin-2-yl)-2-ethylpiperazine-1-carboxylate (5 g, 13.48 mmol) in 1,4-dioxane (70 mL) were added diphenylmethanimine (2.92 g, 16.17 mmol), Pd(OAc)2 (421.5 mg, 1.88 mmol), BINAP (2.35 g, 3.77 mmol) and Cs2CO3 (12.68 g, 37.74 mmoL). The mixture was stirred at 130° C. for 16 h. The mixture was concentrated in vacuo. The crude was purified by silica gel column (EtOAc/PE=3:1) to give title product (6 g, Y: 94.3%) as a grey solid. ESI-MS (M+H)+: 472.3.
To a solution of tert-butyl (S)-4-(5-((diphenylmethylene)amino)pyrazin-2-yl)-2-ethylpiperazine-1-carboxylate (5.8 g, 13.48 mmol) in MeOH (80 mL) were added NaOAc (3.02 g, 36.86 mmol) and NH2OH·HCl (4.27 g, 61.44 mmol). The mixture was stirred at r.t. for 1 h. The mixture was concentrated in vacuo. The crude was purified by silica gel column (EtOAc/PE=3:1) to give title product (3.47 g. Y: 91.8%) as a grey solid. ESI-MS (M+H)+: 308.2. 1H NMR (400 MHz, DMSO-d6) δ 7.66 (s, 1H), 7.57 (s, 1H), 5.55 (s, 2H), 4.00-3.96 (m, 1H), 3.87-3.79 (m, 3H), 3.07-2.97 (m, 1H), 2.72-2.66 (m, 1H), 2.59-2.54 (m, 1H), 1.69-1.56 (m, 2H), 1.41 (s, 9H), 0.83 (t, J=7.4 Hz, 3H).
To a solution of tert-butyl (S)-2-methyl-4-oxopiperidine-1-carboxylate (5.0 g, 23.47 mmol) in dry THF (50 mL) was added LiHMDS (28.16 mL, 28.16 mmol, 1.0 M) dropwise at −78° C. The mixture was stirred at this temperature for 30 min. 1,1,1-trifluoro-N-phenyl-N-((trifluoromethyl)sulfonyl)methanesulfonamide (10.89 g, 30.51 mmol) in dry THF (20 mL) was added to the solution. The mixture was allowed to warm to room temperature and stirred at rt for 24 h. The mixture was diluted with water (40 mL) and extracted with EtOAc (50 mL×3). The combined organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo. The crude was purified by silica gel column chromatography (EA/PE=1:20) to give title product (3 g, 37.05%) as a yellow oil. ESI-MS (M-t-Bu+H)+: 290.0.
To a solution of tert-butyl (S)-6-methyl-4-(((trifluoromethyl)sulfonyl)oxy)-3,6-dihydropyridine-1(2H)-carboxylate (3 g, 8.70 mmol) in 1,4-dioxane (30 mL) were added B2Pin2 (5.5 g, 21.75 mmol) and KOAc (3 g, 30.45 mmol). The mixture was charged with N2 for three times and stirred at 100° C. for 2 h. The reaction mixture was diluted with water (30 mL) and extracted with EtOAc (50 mL×3). The organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo. The crude was purified by silica gel column chromatography (EA/PE=1:10) to give title product (1.2 g, 42.86%) as a yellow oil. ESI-MS (M-56+H)+:268.2.
To a solution of tert-butyl (S)-6-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (1.2 g, 3.72 mmol) in 1,4-dioxane (14 mL) and H2O (2 mL) were added 6-chloropyridazin-3-amine (480 mg, 3.72 mmol), K2CO3 (1.5 g, 11.16 mmol) and Pd(dppf)Cl2 (302 mg, 0.37 mmol). The mixture was charged with N2 for three times and stirred at 110° C. for 16 h. The reaction mixture was diluted with water (20 mL) and extracted with EA (30 mL×3). The organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo. The crude was purified by silica gel column chromatography (EA/PE=1:3) to give title product (400 mg, 37.14%) as a yellow solid. ESI-MS (M+H)+: 291.2.
To a mixture of tert-butyl (S)-4-(6-aminopyridazin-3-yl)-6-methyl-3,6-dihydropyridine-1(2H)-carboxylate (1 g, 3.45 mmol) in MeOH (10 mL) was added Pd/C (200 mg, 10% w.t), the mixture was charged with H2 for three times and stirred at 40° C. for 16 h. The mixture was filtered and the filtrate was concentrated in vacuo to give title product (400 mg, crude) as a yellow solid. ESI-MS (M+H+): 293.1.
To mixture of 5-bromo-2-nitropyridine (2.50 g, 12.40 mmol) in NMP (15 mL) were added tert-butyl (2R,6S)-2,6-dimethylpiperazine-1-carboxylate (1.64 g, 7.66 mmol) and TEA (2.49 g, 24.8 mmol), and the mixture was stirred at 120° C. overnight. The mixture was diluted with water (50 mL), extracted with EA (50 mL×3). The combined organic layer was washed with brine (50 mL×2), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography (PE/EA=100/1 to 5/1) to afford tert-butyl (2R,6S)-2,6-dimethyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate (2.3 g, 55.3% yield) as a yellow solid. ESI-MS (M+H+):337.1.
To a mixture of tert-butyl (2R,6S)-2,6-dimethyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate (2.30 g, 6.84 mmol) in MeOH (20 mL) was added Pd/C (10%) (230 mg), the mixture was stirred under H2 atmosphere overnight. The mixture was filtered and the filtrate was concentrated under vacuum to afford tert-butyl (2R,6S)-4-(6-aminopyridin-3-yl)-2,6-dimethylpiperazine-1-carboxylate (1.1 g, 52.5% yield) as a yellow solid. ESI-MS (M+H+):307.2.
A solution of 6-chloro-4-methylpyridazin-3-amine (0.80 g, 5.59 mmol) in tert-butyl (S)-2-methylpiperazine-1-carboxylate (4.40 g, 22.37 mmol) was stirred at 150° C. for 3 h. The crude was purified by C18 flash (0.1% FA in water/CH3CN) to give title product (0.90 g, 53%) as a yellow solid. ESI-MS (M+H)+:308.1. 1H NMR (400 MHz, CDCl3) δ 6.72 (s, 1H), 4.31-4.29 (m, 3H), 3.96-3.94 (m, 2H), 3.80-3.79 (m, 1H), 3.27-3.17 (m, 1H), 3.15-3.02 (m, 1H), 2.95-2.84 (m, 1H), 2.15 (s, 3H), 1.48 (s, 9H), 1.23 (d, J=6.8 Hz, 3H).
To a solution of 1-(tert-butoxycarbonyl)-2-methylpiperidin-4-one (10.00 g, 47 mmol) in THF (100 mL) was added LiHMDS (56 mL, 56 mmol, 1.0 M in THF) at −78° C. The mixture was stirred for 10 min, then a solution of N-phenylbis(trifluoromethanesulfonimide) (19 g, 56 mmol) in THF (50 mL) was added dropwise to the mixture. The reaction mixture was gradually warmed to room temperature and stirred for 16 hours. The mixture was diluted with water (50 mL), extracted with EA (80 mL×3). The organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography (PE/EA=5:1) to give the title compound (16 g, crude) as a yellow oil.
A solution of tert-butyl 6-methyl-4-(((trifluoromethyl)sulfonyl)oxy)-3,6-dihydropyridine-1(2H)-carboxylate (5 g, 14.50 mmol), B2Pin2 (7.5 g, 29.00 mmol), KOAc (4.3 g, 43.50 mmol) and Pd(dppf)Cl2 (1 g, 1.45 mmol) in 1,4-dioxane (50 mL) was stirred at 120° C. for 16 h under N2. The mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (PE:EA=5:1) to give tittle product (4 g, crude) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 6.40 (d, J=31.5 Hz, 1H), 4.42-4.41 (m, 1H), 4.28-3.87 (m, 1H), 2.11-2.08 (m, 3H), 1.45 (s, 9H), 1.26-1.24 (m, 15H).
A mixture of 6-chloropyridazin-3-amine (670 mg, 5.20 mmol), tert-butyl 6-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (2 g, 6.24 mmol), Pd(dppf)Cl2 (377 mg, 0.52 mmol) and Na2CO3 (2.15 g, 15.60 mmol) in 1,4-dioxane (20 mL) and H2O (4 mL) was stirred at 120° C. for 16 h under N2 atmosphere. After cooling to rt, the mixture was diluted with water (20 mL), extracted with EA (20 mL×3). The organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (SiO2, Ethyl acetate/Petroleum ether=0% to 50%) to give title product (200 mg, yield: 13.21%) as a yellow solid. ESI-MS (M+H)+: 291.0 1H NMR (400 MHz, CDCl3) δ 7.49-7.33 (m, 1H), 6.74 (d, J=9.2 Hz, 1H), 6.34-6.16 (m, 1H), 4.89-4.56 (m, 3H), 3.10-2.68 (m, 2H), 1.49 (s, 9H), 1.27 (d, J=6.8 Hz, 3H).
To a mixture of 5-bromo-4-chloropyrimidin-2-amine (6.4 g, 30.92 mmol) in EtOH (130 mL) was added EtONa (3.154 g, 46.38 mmol) and the mixture was stirred at rt for 6 h. The mixture was concentrated in vacuo. The residue was diluted with water (100 mL), extracted with DCM (100 mL×3). The organic phase was washed with brine, dried over sodium sulfate and concentrated in vacuo to give title product (6.5 g, Y: 97%) as a white solid. ESI-MS (M+H+):219.9.
A mixture of 5-bromo-4-ethoxypyrimidin-2-amine (14.4 g, 66.36 mmol) and 1-bromopropan-2-one (22.73 g, 165.90 mmol) in EtOH (100 mL) was stirred at 80° C. for 16 h. The mixture concentrated in vacuo. The residue was basified with 2M NaOH and the solid was filtered and further purified by flash chromatography (silica gel, DCM:MeOH=10:1) to give title product (6 g, Y:28%) as a white solid. ESI-MS (M+H+):256.2. 1H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H), 7.35 (s, 1H), 4.42 (q, J=7.0 Hz, 2H), 2.26 (s, 3H), 1.38 (t, J=7.1 Hz, 3H).
To a mixture of 6-bromo-7-ethoxy-2-methylimidazo[1,2-a]pyrimidine (5.5 g, 21.48 mmol) in MeOH (400 mL) were added and Pd(OAc)2 (481 mg, 2.15 mmol). Xantphos (2.485 g, 4.30 mmol) and TEA (400 mL). The resulting mixture was stirred overnight at 70° C. under CO for 16 h. The mixture was allowed to cool down to room temperature and concentrated. The residue was diluted with water and extracted with DCM (100 mL×3). The organic layer was washed with brine, dried over sodium sulfate and concentrated to provide the crude product (6 g, crude). The crude was used to next step without further purification. ESI-MS (M+H)+: 236.0.
To a mixture of methyl 7-ethoxy-2-methylimidazo [1,2-a]pyrimidine-6-carboxylate (5 g, 21.22 mmol) in THF/H2O (200:50 mL), LiOH (2.128 g, 106.383 mmol) was added and the mixture was stirred at rt for 4 h. The mixture was concentrated in vacuo, the residue was purified with reversed phase column chromatography to give title product (800 mg, Y: 17%) as a white solid. ESI-MS (M+H+):222.0. 1H NMR (400 MHz, DMSO-d6) δ 9.29 (d, J=7.5 Hz, 1H), 7.47 (s, 1H), 4.42 (q, J=7.0 Hz, 2H), 2.26 (s, 3H), 1.36 (t, J=7.0 Hz, 3H).
To a mixture of 2-bromo-5-fluoroisonicotinic acid (6.0 g, 0.027 mol) in MeOH (30 mL) was added MeONa (4.4 g, 0.082 mol). The mixture was stirred at 65° C. for 16 h. The mixture was concentrated in vacuo. The residue was dissolved in MeOH (30 mL), SOCl2 (9.8 g, 0.082 mol) was added dropwise to the mixture and stirred at rt for 16 h. After concentration, the residue was diluted with EA (300 mL), washed with water and brine, dried with Na2SO4 and concentrated in vacuo. The crude product was purified by silica gel column (PE/EA=5:1) to afford title product (6 g, 94.8%) as a white solid. ESI-MS (M+H)+: 245.9.
To a mixture of methyl 2-bromo-5-methoxyisonicotinate (6.0 g, 24.39 mmol) in toluene (60 mL) were added trimethyl(prop-1-yn-1-yl)silane (2.7 g, 24.39 mmol), CuI (1.3 g, 7.31 mmol), Pd(PPh3)4 (2.8 g, 2.43 mmol), TEA (7.3 g, 73.17 mmol) and TBAF (6.3 g, 24.39 mmol). The mixture was stirred at 50° C. for 12 h. LCMS showed the reaction was completed. The mixture was concentrated in vacuo, the residue was diluted with water (50 mL), extracted with EA (60 mL×2). The organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column (PE:EA=4:1) to give the title product (1.7 g, Y: 33%) as a yellow solid. ESI-MS (M+H+):206.0. 1H NMR (400 MHz, CDCl3) δ 8.35 (s, 1H), 7.66 (s, 1H), 4.01 (s, 3H), 3.92 (s, 3H), 2.07 (s, 3H).
To a mixture of methyl 5-methoxy-2-(prop-1-yn-1-yl)isonicotinate (1.7 g, 8.29 mmol) in CHCl3 (50 mL) was added O-(mesitylsulfonyl)hydroxylamine (5.3 g, 24.87 mmol). The mixture was stirred at 25° C. for 16 h. The mixture was concentrated in vacuo. The residue was diluted with DMF (30 mL), K2CO3 (2.1 g, 14.92 mmol) was added and the mixture was stirred at 25° C. for 5 h. LCMS showed the reaction was completed. The mixture was diluted with water (50 mL), extracted with EA (50 mL×2). The organic layer was washed with brine, dried with Na2SO4 and concentration in vacuo. The residue was purified by silica gel column (PE:EA=3:1) to give the title product (500 mg, Y: 27%) as a yellow solid. ESI-MS (M+H+):221.0.
To a mixture of methyl 6-methoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxylate (500 mg, 2.27 mmol) in THF (5 mL) and H2O (5 mL) was added LiOH (104 mg, 4.54 mmol). The mixture was stirred at 50° C. for 3 h. LCMS showed the reaction was completed. The reaction mixture was concentrated in vacuo to remove most THF. The mixture was adjusted to pH=5 with 1M HCl. The precipitate was filtered and dried under vacuum to give title product (180 mg, Y: 38%) as a white solid. ESI-MS (M+H+):207.1. 1H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 8.21 (s, 1H), 6.48 (s, 1H), 4.05 (s, 3H), 2.49 (s, 3H).
To a solution of tert-butyl 2,2-dimethyl-4-oxopiperidine-1-carboxylate (1 g, 4.41 mmol) in dry THF (10 mL) was added LiHMDS (5.29 mL, 5.29 mmol, 1.0 M in THF) dropwise at −78° C. The mixture was stirred at this temperature for 30 min. 1,1,1-trifluoro-N-phenyl-N-((trifluoromethyl)sulfonyl)methanesulfonamide (2.05 g, 5.73 mmol) in dry THF (5 mL) was added. The mixture was allowed to warm to room temperature and stirred at rt for 24 h. The mixture was diluted with H2O (10 mL) and extracted with EtOAc (50 mL×3). The combined organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo. The crude was purified by silica gel column chromatography (EA/PE=1:20) to give title product (500 mg, 31.66%) as a yellow oil. ESI-MS (M-100+H)+: 260.0. 1H NMR (400 MHz, CDCl3) δ 5.80-5.73 (m, 1H), 4.09-4.04 (m, 2H), 2.41-2.36 (m, 2H), 1.49 (s, 6H), 1.46 (s, 9H).
To a solution of tert-butyl 6,6-dimethyl-4-(((trifluoromethyl)sulfonyl)oxy)-3,6-dihydropyridine-1(2H)-carboxylate (500 mg, 1.39 mmol) in 1,4-dioxane (10 mL) were added B2Pin2 (884 mg, 3.48 mmol), Pd(dppf)Cl2 (102 mg, 0.14 mmol) and KOAc (477 mg, 4.87 mmol). The mixture was charged with N2 for three times and stirred at 100° C. for 2 h. The reaction mixture was diluted with water (10 mL) and extracted with EtOAc (20 mL×3). The organic layer was washed with bine, dried over Na2SO4 and concentrated in vacuo. The crude was purified by silica gel column chromatography (EA/PE=1:10) to give title product (200 mg, 42.64%) as a yellow oil. ESI-MS (M+H)+:338.2.
To a solution of tert-butyl 6,6-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (200 mg, 0.59 mmol) in 1,4-dioxane (7 mL) and H2O (1 mL) were added 6-chloropyridazin-3-amine (76 mg, 0.59 mmol), K2CO3 (244 mg, 1.77 mmol) and Pd(dppf)Cl2 (49 mg, 0.06 mmol). The mixture was charged with N2 for three times and stirred at 110° C. for 16 h. The reaction mixture was diluted with water (10 mL) and extracted with EA (20 mL×3). The organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo. The crude was purified by silica gel column chromatography (EA/PE=1:3) to give title product (70 mg, 38.89%) as a yellow solid. ESI-MS (M+H)+: 305.2.
To a solution of tert-butyl (R)-2-methyl-4-oxopiperidine-1-carboxylate (3 g, 14.08 mmol) in dry THF (30 mL) was added LiHMDS (16.90 mL, 16.90 mmol, 1.0 M in THF) dropwise at −78° C. The mixture was stirred at this temperature for 30 min. 1,1,1-trifluoro-N-phenyl-N-((trifluoromethyl)sulfonyl)methanesulfonamide (6.5 g, 18.30 mmol) in dry THF (30 mL) was added. The mixture was allowed to warm to room temperature and stirred at rt for 24 h. The mixture was diluted with H2O (50 mL) and extracted with EtOAc (70 mL×3). The combined organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo. The crude was purified by silica gel column chromatography (EA/PE=1:20) to give title product (1.5 g, 30.86%) as a yellow oil. ESI-MS (M-t-Bu+ACN+H)+: 331.1.
To a solution of tert-butyl (R)-6-methyl-4-(((trifluoromethyl)sulfonyl)oxy)-3,6-dihydropyridine-1(2H)-carboxylate (1.5 g, 4.18 mmol) in 1,4-dioxane (20 mL) were added B2pin2 (2.7 g, 10.45 mmol) Pd(dppf)Cl2 (300 mg, 0.41 mmol) and KOAc (1.4 g, 14.63 mmol). The mixture was charged with N2 for three times and stirred at 100° C. for 2 h. The reaction mixture was diluted with water (20 mL) and extracted with EtOAc (30 mL×3). The organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo. The crude was purified by silica gel column chromatography (EA/PE=1:10) to give title product (800 mg, 59.26%) as a yellow oil. ESI-MS (M-Boc+H)+:224.0
To a solution of tert-butyl (R)-4-(6-aminopyridazin-3-yl)-6-methyl-3,6-dihydropyridine-1(2H)-carboxylate (800 mg, 2.48 mmol) in 1,4-dioxane (14 mL) and H2O (2 mL) were added 6-chloropyridazin-3-amine (320 mg, 2.48 mmol), K2CO3 (1 g, 7.44 mmol) and Pd(dppf)Cl2 (204 mg, 0.25 mmol). The mixture was charged with N2 for three times and stirred at 120° C. for 16 h. The reaction mixture was diluted with water (10 mL) and extracted with EA (20 mL×3). The organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo. The crude was purified by silica gel column chromatography (EA/PE=1:3) to give title product (400 mg, 55.71%) as a yellow solid. ESI-MS (M+H)+: 291.1.
A mixture of tert-butyl 4-(6-aminopyridazin-3-yl)-3,6-dihydropyridine-1(2H)-carboxylate (150 mg, 0.54 mmol) 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (132 mg, 0.60 mmol), TCFH (234 mg, 0.82 mmol) and NMI (134 mg, 1.64 mmol) in ACN (10 mL) 4 as stirred at rt for 16 h. The mixture was diluted with H2a (20 mL) and extracted with EA (20 ml 3). The organic phase was washed with brine (20 mL) dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=5/1 to 1/1) to afford title product (75 mg, 0.15 mmol, 29.18%) as a yellow % solid. ESI-MS (M+H+): 479.2.
To a solution of tert-butyl 4-(6-(6-ethoxy-2-methyl-2H-indazole-5-carboxamido)pyridazin-3-yl)-3,6-dihydropyridine-1(2H)-carboxylat (75 mg, 0.16 mmol) in EA (4 mL) was added 3M HCl/EA (4 mL). The mixture was stirred at rt for 2 h. The mixture was diluted with water (15 mL) and extracted with EA (20 mL×3). The aqueous phase was concentrated in vacuo. The residue was purified by prep-HPLC (0.05% FA in water/ACN) to give title product (37 mg, 54.4%) as a white solid. ESI-MS (M+H+): 379.2. 1H NMR (400 MHz, MeOD-d4) a 8.67 (d, J=9.5 Hz, 1H), 8.61 (s, 1H), 8.52 (s, 1H), 8.35 (s, 1H), 8.01 (d, J=9.4 Hz, 1H), 7.15 (s, 1H), 6.70 (s, 1H), 4.38 (q, J=7.0 Hz, 2H), 4.20 (s, 3H), 3.90 (s, 2H), 3.46 (t, J=6.1 Hz, 2H), 3.00-2.98 (m, 2H), 1.66 (t, J=7.0 Hz, 3H).
To a solution of tert-butyl 4-(6-aminopyridazin-3-yl)-3,6-dihydropyridine-1(2H)-carboxylate (200 mg, 0.72 mmol) and 7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (237.6 mg, 1.08 mmol) in ACN (5 mL) were added NMI (358.4 mg, 4.32 mmol) and TCFH (604.8 mg, 2.16 mmol). The resulting mixture was stirred at 50° C. for 16 h. The reaction mixture was diluted with water (20 mL) and extracted with EtOAc (20 mL×3). The organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo to give title product (150 mg, 47.9%) as a white solid. ESI-MS (M+H)+:479.4. 1H NMR (400 MHz, CDCl3) δ 10.95 (s, 1H), 8.99 (s, 1H), 8.58 (d, J=9.4 Hz, 1H), 7.66 (d, J=9.6 Hz, 1H), 7.31 (s, 1H), 6.98 (s, 1H), 6.57 (s, 1H), 4.39-4.34 (m, 2H), 4.18 (br s, 2H), 3.68 (br s, 2H), 2.81 (br s, 2H), 2.44 (s, 3H), 1.74 (d, J=7.0 Hz, 3H), 1.50 (s, 9H).
To a solution of tert-butyl 4-(6-(7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyridazin-3-yl)-3,6-dihydropyridine-1(2H)-carboxylate (100 mg, 0.20 mmol) in 3M HCl/EA (2 mL) was stirred at rt for 2 h. The precipitate was filtered, washed with EtOAc (3 mL) and dried in vacuo to afford title compound (51.18 mg, 64.5%) as a yellow solid. ESI-MS (M+H)+: 379.1. 1H NMR (400 MHz, DMSO-d6) δ 11.51 (s, 1H), 9.36 (s, 2H), 9.26 (s, 1H), 8.44 (d, J=9.2 Hz, 1H), 8.12 (d, J=9.6 Hz, 1H), 7.97 (s, 1H), 7.33 (s, 1H), 6.75 (s, 1H), 4.37 (q, J=6.8 Hz, 2H), 3.85 (br s, 2H), 3.35 (br s, 2H), 2.90 (br s, 2H), 2.46 (s, 3H), 1.44 (t, J=6.8 Hz, 3H).
To a mixture of tert-butyl (S)-4-(5-aminopyrazin-2-yl)-2-methylpiperazine-1-carboxylate (300 mg, 0.971 mmol) in DMF (6 mL) were added HATU (443 mg, 1.17 mmol), DIEA (501 mg, 3.88 mmol) and 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (320 mg, 1.46 mmol). The mixture was stirred at 45° C. for 3 h. LCMS showed the reaction was completed. The reaction was diluted with water (10 mL), extracted with EA (30 mL), and the organic phase was washed with brine, dried and concentrated to give title product (400 mg, 83%) as a brown solid. ESI-MS (M+H)+: 496.4.
To a mixture of tert-butyl (S)-4-(5-(6-ethoxy-2-methyl-2H-indazole-5-carboxamido) pyrazin-2-yl)-2-methylpiperazine-1-carboxylate (100 mg, 0.202 mmol) in EA (2 mL) was added 3M HCl/EA (2 mL). The mixture was stirred at RT for 2 h. The mixture was concentrated in vacuo, the residue was purified by prep-HPLC (0.05% FA in water/ACN) to give title product (51 mg, Y: 57.3%) as a white solid. ESI-MS (M+H)+:396.3. 1H NMR (400 MHz, DMSO-d6) δ 10.50 (s, 1H), 9.01 (s, 1H), 8.45 (d, J=6.9 Hz, 2H), 8.19 (s, 1H), 8.14 (s, 1H), 7.13 (s, 1H), 4.27 (q, J=6.8 Hz, 2H), 4.16-4.09 (m, 5H), 3.06-3.02 (m, 1H), 2.90-2.76 (m, 4H), 1.50 (t, J=6.9 Hz, 3H), 1.08 (d, J=6.3 Hz, 3H).
To a mixture of 7-ethoxy-2-methylimidazo[1,2-a]pyrimidine-6-carboxylic acid (50 mg, 0.22 mmol) in DMF (5 mL) were added HATU (128.9 mg, 0.34 mmol) and DIEA (145.9 mg, 1.13 mmol), the mixture was stirred at rt for 0.5 h. tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate (63 mg, 0.22 mmol) was added. The resulting mixture was stirred at rt for 2 h. The mixture was diluted with water (15 mL), the precipitate was filtered, washed with water and dried in vacuo to provide the crude title product (90 mg, crude) as a yellow solid. ESI-MS (M+H)+: 482.1.
To a solution of tert-butyl 4-(6-(7-ethoxy-2-methylimidazo[1,2-a]pyrimidine-6-carboxamido)pyridin-3-yl)piperazine-1-carboxylate (70 mg, crude) in EA (3 mL) was added 3M HCl in EA (3 mL) at rt. The mixture was stirred for 2 h at rt. The reaction was concentrated, the residue was purified by prep-HPLC (0.1% FA in water/CH3CN) to give title product (39 mg, yield: 70%) as a yellow solid. ESI-MS (M+H)+: 382.1. 1H NMR (400 MHz, DMSO-d6) δ 10.26 (s, 1H), 9.33 (s, 1H), 8.22 (s, 1H), 8.11-8.08 (m, 2H), 7.58-7.45 (m, 2H), 4.55 (q, J=7.0 Hz, 2H), 3.18-3.14 (m, 4H), 2.99-2.92 (m, 4H), 2.29 (s, 3H), 1.46 (t, J=7.0 Hz, 3H).
To a mixture of 7-cyclopropoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (60 mg, 0.26 mmol) in DMF (4 mL) were added HATU (198 mg, 0.52 mmol), DIEA (180 mg, 1.32 mmol) and tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate (72 mg, 0.26 mmol). The mixture was stirred at rt for 1 h. After concentration, the residue was purified by reverse phase column (0.1% NH3·H20 in water/CH3CN) to give title product (32 mg, Y: 45%) as a gray solid. ESI-MS (M+H)+: 493.1. 1H NMR (400 MHz, CDCl3) δ 9.95 (s, 1H), 8.89 (s, 1H), 8.16 (d, J=9.0 Hz, 1H), 7.95 (d, J=2.8 Hz, 1H), 7.30-7.22 (m, 3H), 3.93-3.92 (m, 1H), 3.56-3.51 (m, 4H), 3.06 (d, J=4.8 Hz, 4H), 2.37 (s, 3H), 1.42 (s, 9H), 0.97-0.93 (m, 4H).
A mixture of tert-butyl 4-(6-(7-cyclopropoxy-2-methylimidazol[1,2-a]pyridine-6-carboxamido)pyridin-3-yl)piperazine-1-carboxylate (32 mg, 0.065 mmol) in 3M HCl/EA (4 mL) was stirred at rt for 2 h. After concentration, the residue was dissolved in water (5 mL) and lyophilized to give tide product (15 mg, 65%) as a yellow solid. ESI-MS (M+H)+: 393.0. 1H NMR (400 MHz, MeOD-d4) δ 9.13 (s, 1H), 8.13 (s, 1H), 8.04 (d, J=9.1 Hz, 1H), 7.88-7.81 (m, 1H), 7.74-7.70 (m, 2H), 4.27-4.26 (m, 1H), 3.60-3.51 (m, 4H), 3.49-3.41 (m, 4H), 2.54 (s, 3H), 1.06 (d, J=16.7 Hz, 4H).
To a mixture of tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (244 mg, 0.83 mmol) and 6-bromo-7-methoxy-2-methylimidazo[1,2-a]pyridine (200 mg, 0.83 mmol) in toluene (15 mL) were added Pd(OAc)2 (20 mg, 0.08 mmol), Xantphos (96 mg, 0.17 mmol) and Na2CO3 (264 mg, 2.49 mmol), the mixture was charged with CO for three times and stirred at 100° C. for 16 h. The mixture was filtered and the filtrate was concentrated to give 431-2(200 mg, 64%) as a solid, which was used to next step without further purification. ESI-MS (M+H+): 482.1.
A mixture of tert-butyl (S)-4-(6-(7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (200 mg, 0.414 mmol) in EA/HCl (6 mL) was stirred at rt for 2 h. The mixture was concentrated in vacuo. The residue was purified by prep-HPLC (0.1% HCl in water/ACN) to give title product (66 mg, 38%) as a red solid. ESI-MS (M+H+): 382.1. 1H NMR (400 MHz, MeOD-d4) δ 9.17 (s, 1H), 8.41 (d, J=9.8 Hz, 1H), 7.80 (s, 1H), 7.52 (d, J=9.9 Hz, 1H), 7.31 (s, 1H), 4.50-4.38 (m, 2H), 4.22 (s, 3H), 3.56-3.43 (m, 2H), 3.40-3.32 (m, 1H), 3.29-3.23 (m, 1H), 3.13-3.05 (m, 1H), 2.49 (s, 3H), 1.42 (d, J=6.6 Hz, 3H).
To a stirred solution of 7-ethoxy-2-methylimidazo[1,2-a]pyrimidine-6-carboxylic acid (40 mg, 0.181 mmol) in DMF (1.5 mL) under argon with an ice bath was added HATU (104 mg, 0.274 mmol). The reaction was stirred under argon with an ice bath for 10 minutes. Tert-butyl (2R,6S)-4-(6-aminopyridazin-3-yl)-2,6-dimethylpiperazine-1-carboxylate (72 mg, 0.234 mmol) was added, followed by DIPEA (0.15 mL, 0.908 mmol). The reaction was then stirred under argon at room temperature for 1 hour and then at 35° C. for 1 hour. The reaction mixture was diluted with water (20 mL), stirred at room temperature for 30 minutes and filtered. The filter cake was washed with water, dissolved in EtOAc (50 mL), dried over sodium sulfate and concentrated to dryness to afford crude desired product (18 mg, yield: 15%) as a yellow solid. ESI-MS (M+H+): 511.4.
To a stirred solution of tert-butyl (2R,6S)-4-(6-(7-ethoxy-2-methylimidazo[1,2-a]pyrimidine-6-carboxamido)pyridazin-3-yl)-2,6-dimethylpiperazine-1-carboxylate (18 mg, 0.035 mmol) in DCM (2.0 mL) under argon with an ice bath was added HCl (4 M in 1,4-dioxane) (0.5 mL, 2.0 mmol). The reaction was stirred under argon at room temperature for 1 hour, solid precipitated. The reaction mixture was concentrated. The residue was suspended in DCM (2.0 mL), the precipitate was filtered to afford title product (11 mg, yield: 69%) as a pale yellow solid. ESI-MS (M+H+):411.3. 1H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 9.73-9.67 (m, 1H), 9.54 (s, 1H), 9.37-9.26 (m, 1H), 8.22 (d, J=9.6 Hz, 1H), 7.87 (s, 1H), 7.61 (d, J=9.6 Hz, 1H), 4.62 (q, J=6.9 Hz, 2H), 4.46 (d, J=12.4 Hz, 2H), 3.40-3.27 (m, 2H), 3.01 (t, J=12.6 Hz, 2H), 2.44 (s, 3H), 1.45 (t, J=7.0 Hz, 3H), 1.34 (d, J=6.4 Hz, 6H).
To a mixture of 6-ethoxy-2-methyl-2H-pyrazolo[3,4-b]pyridine-5-carboxylic acid (100 mg, 0.45 mmol) in DMF (3 mL) were added tert-butyl 4-(6-aminopyridazin-3-yl)piperazine-1-carboxylate (151 mg, 0.54 mmol), HATU (258 mg, 0.68 mmol) and DIEA (292 mg, 2.26 mmol). The mixture was stirred at rt for 3 h. LCMS showed the reaction was completed. The mixture was poured into H2O (5 mL) and the precipitate was filtered, washed with water and dried in vacuo to give title product (80 mg, Y: 37%) as a yellow solid. ESI-MS (M+H)+: 483.2
A mixture of tert-butyl 4-(6-(6-ethoxy-2-methyl-2H-pyrazolo[3,4-b]pyridine-5-carboxamido)pyridazin-3-yl)piperazine-1-carboxylate (80 mg, 0.14 mmol) in 3M HCl/EA (3 mL) was stirred at rt for 1 h. LCMS showed the reaction was completed. The mixture was concentrated in vacuo and the residue was purified by prep-HPLC (0.05% NH2H2O in water/ACN) to give title product (22.6 mg, Y: 36.9%) as a yellow solid. ESI-MS (M+H+): 383.1. 1H NMR (400 MHz, DMSO-d6) δ 10.76 (s, 1H), 8.75 (s, 1H), 8.46 (s, 1H), 8.22 (d, J=9.2 Hz, 1H), 7.38 (d, J=9.8 Hz, 1H), 4.54 (q, J=7.0 Hz, 2H), 4.13 (s, 3H), 3.49-3.42 (m, 4H), 2.85-2.76 (m, 4H), 1.47 (t, J=7.0 Hz, 3H).
To a stirred solution of tert-butyl (S)-4-(5-aminopyrazin-2-yl)-2-ethylpiperazine-1-carboxylate (200 mg, 0.65 mmol), 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (143.3 mg, 0.65 mmol) and NMI (160 mg, 1.95 mmol) in MeCN (3 mL) was added TCFH (273 mg, 0.98 mmol). The mixture was stirred at rt for 16 h. The precipitate was filtered and washed with ACN (2×15 mL), dried in vacuo to give title product (200 mg, Y: 60.24%) as an off-white solid. ESI-MS (M+H)+: 510.3.
A mixture of tert-butyl (S)-4-(5-(6-ethoxy-2-methyl-2H-indazole-5-carboxamido)pyrazin-2-yl)-2-ethylpiperazine-1-carboxylate (100 mg, 0.20 mmol) in 3M EtOAc/HCl (3 mL) was stirred at rt for 2 h. After concentration, the residue was dissolved in water (5 mL) and lyophilized to give title product (37.46 mg, Y: 46.6%) as a yellow solid. ESI-MS (M+H)+: 410.2. 1H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H), 9.30-9.10 (m, 2H), 9.07 (s, 1H), 8.47 (s, 1H), 8.44 (s, 1H), 8.29 (s, 1H), 7.14 (s, 1H), 4.38-4.24 (m, 4H), 4.14 (s, 3H), 3.36 (d, J=12 Hz, 1H), 3.28-3.14 (m, 2H), 3.12-2.97 (m, 2H), 1.74-1.62 (m, 2H), 1.50 (t, J=8 Hz, 3H), 1.02 (t, J=8 Hz, 3H).
To a mixture of 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (53 mg, 0.24 mmol) in DMF (2 mL) were added tert-butyl (2S)-4-(6-aminopyridazin-3-yl)-2-methylpiperidine-1-carboxylate (70 mg, 0.24 mmol), HATU (137 mg, 0.36 mmol) and DIEA (154 mg, 1.20 mmol). The mixture was stirred at rt for 16 h. The mixture was diluted with H2O (5 mL) and the precipitate was filtered, washed with water and dried in vacuo to give title product (50 mg, Y: 38.6%) as a yellow solid. ESI-MS (M+H+): 495.2.
A mixture of tert-butyl (2S)-4-(6-(6-ethoxy-2-methyl-2H-indazole-5-carboxamido) pyridazin-3-yl)-2-methylpiperidine-1-carboxylate (50 mg, 0.10 mmol) in 3 M HCl/EA (2 mL) was stirred at rt for 2 h. The mixture was diluted with H2O (5 mL), extracted with EA (10 mL×3). The aqueous layer was concentrated in vacuo and the residue was purified by prep-HPLC (0.1% NH3·H2O in water/ACN) to give title product (5.6 mg, 14.1%) as a yellow solid. ESI-MS (M+H+): 395.2. 1H NMR (400 MHz, MeOD-d4) δ 8.60 (d, J=7.2 Hz, 1H), 8.58 (s, 1H), 8.33 (s, 1H), 7.68 (d, J=7.3 Hz, 1H), 7.12 (s, 1H), 4.35 (q, J=7.0 Hz, 2H), 4.19 (s, 3H), 3.22-3.03 (m, 2H), 2.87-2.76 (m, 2H), 1.94 (t, J=14.1 Hz, 2H), 1.76-1.68 (m, 1H), 1.65 (t, J=7.0 Hz, 3H), 1.41 (dd, J=23.9, 12.4 Hz, 1H), 1.16 (d, J=6.3 Hz, 3H).
To a mixture of tert-butyl (S)-4-(6-aminopyridin-3-yl)-2-methylpiperazine-1-carboxylate (100 mg, 0.30 mmol) in DMF (5 mL) were added HATU (148 mg, 0.36 mmol), DIEA (168 mg, 1.20 mmol) and 7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (80 mg, 0.36 mmol). The mixture was stirred at 45° C. for 16 h. LCMS showed the starting material was consumed completely. The mixture was diluted with water (10 mL), extracted with EA (20 mL-3). The organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo to give title product (120 mg, 69.36%) as a yellow solid. ESI-MS (M+H)+: 481.3.
To a mixture of tert-butyl (S)-4-(6-(7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyridin-3-yl)-2-methylpiperazine-1-carboxylate (100 mg, 0.20 mmol) in EA (3 mL) was added 3 M HCl/EA (3 mL). The mixture was stirred at rt for 2 h. LCMS showed the starting material was consumed completely. The mixture was concentrated in vacuo. The crude was purified by prep-HPLC (0.05% FA in water/CH3CN) to give title product (38 mg, Y: 48.10%) as a yellow solid. ESI-MS (M+H)+: 381.2. 1H NMR (400 MHz, MeOD-d4) δ 9.06 (s, 1H), 8.43 (s, 1H), 8.26 (d, J=9.0 Hz, 1H), 8.15-8.07 (m, 1H), 7.62-7.53 (m, 2H), 7.01 (s, 1H), 4.16 (s, 3H), 3.82 (t, J=15.3 Hz, 2H), 3.57-3.48 (m, 2H), 3.32-3.27 (m, 1H), 3.08 (t, J=10.9 Hz, 1H), 2.89-2.79 (m, 1H), 2.40 (s, 3H), 1.42 (d, J=6.5 Hz, 3H).
To mixture of 7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (72 mg, 0.33 mmol), tert-butyl (2R,6S)-4-(5-aminopyrazin-2-yl)-2,6-dimethylpiperazine-1-carboxylate (100 mg, 0.33 mmol) and NMI (80 mg, 0.99 mmol) in ACN (5 mL) was added TCFH (137 mg, 0.49 mmol) and the mixture was stirred at rt for 16 h. The precipitate was collected by filtration, washed with water and dried to give title product (130 mg, Y: 78.3%) as a yellow solid. ESI-MS (M+H)+:510.3.
A mixture of tert-butyl (2S,6R)-4-(5-(7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyrazin-2-yl)-2,6-dimethylpiperazine-1-carboxylate (100 mg, 0.20 mmol) in 3M EA/HCl (4 mL) was stirred at rt for 1 h. The mixture was concentrated in vacuo, the residue was purified by prep-HPLC (0.1% FA in water/CH3CN) to give title compound (40 mg, yield: 50%) as a yellow solid. ESI-MS (M+H+): 410.2. 1H NMR (400 MHz, DMSO-d6) δ 10.41 (s, 1H), 9.05 (s, 1H), 9.00 (s, 1H), 8.27 (s, 1H), 7.65 (s, 1H), 7.00 (s, 1H), 4.39 (d, J=11.7 Hz, 2H), 4.27 (q, J=6.8 Hz, 2H), 3.27-3.20 (m, 2H), 2.80 (t, J=12.4 Hz, 2H), 2.29 (s, 3H), 1.48 (t, J=6.8 Hz, 3H), 1.26 (d, J=6.4 Hz, 6H).
To a mixture of tert-butyl (2R,6S)-4-(5-aminopyrazin-2-yl)-2,6-dimethylpiperazine-1-carboxylate (100 mg, 0.33 mmol) in CH3CN (2 mL) was added 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (71.7 mg, 0.33 mmol), TCFH (136.92 mg, 0.49 mmol) and NMI (80.2 mg, 0.98 mmol). The mixture was stirred at rt for 16 h. LCMS showed the starting material was consumed completely. The reaction mixture was filtered and the filter cake was washed with CH3CN (15 mL×3), dried in vacuum to give title product (85 mg, Y: 51.3%) as a yellow solid.
To a mixture of tert-butyl (2R,6S)-4-(5-(6-ethoxy-2-methyl-2H-indazole-5-carboxamido)pyrazin-2-yl)-2,6-dimethylpiperazine-1-carboxylate (85 mg, 0.157 mmol) in EtOAc (5 mL) was added 4 M EtOAC-HCl (5 mL). The mixture was stirred at R.T. for 1 h. LCMS showed the starting material was consumed completely. The solid was collected by filtration, washed with DCM and dried under vacuum to afford the title compound. ESI-MS (M+H+): 410.3. 1H NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 9.58 (s, 1H), 9.14 (s, 1H), 9.05 (s, 1H), 8.47 (s, 1H), 8.43 (s, 1H), 8.28 (s, 1H), 7.14 (s, 1H), 4.42 (d, J=12.3 Hz, 2H), 4.27 (q, J=6.8 Hz, 2H), 4.14 (s, 3H), 3.38-3.26 (m, 2H), 2.99-2.85 (m, 2H), 1.50 (t, J=6.9 Hz, 3H), 1.32 (d, J=6.5 Hz, 6H).
To a mixture of tert-butyl (2R,6S)-4-(5-aminopyrazin-2-yl)-2,6-dimethylpiperazine-1-carboxylate (556 mg, 1.81 mmol) in DMF (10 mL) were added 7-ethoxy-2-methylimidazo[1,2-a]pyrimidine-6-carboxylic acid (400 mg, 1.81 mmol), DIEA (1.17 g, 9.05 mmol) and HATU (2.1 g, 5.43 mmol). The mixture was stirred at rt for 16 h. After diluting with water, the mixture was filtered and the cake was washed with MeOH (6 mL) and dried to give title product (380 mg, 41.2%) as a white solid ESI-MS (M+H)+: 511.2.
To a solution of tert-butyl (2R,6S)-4-(5-(7-ethoxy-2-methylimidazo[1,2-a]pyrimidine-6-carboxamido)pyrazin-2-yl)-2,6-dimethylpiperazine-1-carboxylate (380 mg, 0.75 mmol) in EA (3 mL) was added 3M HCl/EA (3 mL) was stirred at it for 2 h. After concentration, the residue was filtered and the solid was dried under vacuum to give title product (200 mg, 60.2%) as a white solid. ESI-MS (M+H)+: 411.1. 1H NMR (400 MHz, DMSO-d6) δ 10.72 (s, 1H), 9.84-9.72 (m, 1H), 9.55 (s, 1H), 9.42 (d, J=9.9 Hz, 1H), 8.93 (s, 1H), 8.31 (s, 1H), 7.87 (d, J=1.1 Hz, 1H), 4.61 (q, J=7.0 Hz, 2H), 4.43 (d, J=12.6 Hz, 2H), 3.29 (s, 2H), 3.04-2.95 (m, 2H), 2.44 (d, J=0.9 Hz, 3H), 1.45 (t, J=7.0 Hz, 3H), 1.35 (d, J=6.4 Hz, 6H).
To a mixture of tert-butyl (S)-4-(5-aminopyrazin-2-yl)-2-methylpiperazine-1-carboxylate (100 mg, 0.34 mmol) in DMF (2 mL) were added HATU (155 mg, 0.41 mmol), DIEA (176 mg, 1.37 mmol) and 7-ethoxy-2-methylimidazo[1,2-a]pyrimidine-6-carboxylic acid (113 mg, 0.51 mmol). The mixture was stirred at 25° C. for 12 h. LCMS showed the reaction was completed. The reaction was diluted with water (10 mL), extracted with EA (30 mL), and the organic phase was washed with brine, dried and concentrated to give title product (150 mg, 89%) as a yellow solid. ESI-MS (M+H)+: 497.1.
A mixture of tert-butyl (S)-4-(5-(7-ethoxy-2-methylimidazo[1,2-a]pyrimidine-6-carboxamido)pyrazin-2-yl)-2-methylpiperazine-1-carboxylate (150 mg, 0.30 mmol) in EA/HCl (4 mL) was stirred at RT for 2 h. LCMS showed the reaction was completed, the mixture was concentrated and the residue was purified by prep-HPLC (0.05% FA in water/ACN) to give title product (24 mg, Y: 20%) as an off-white solid. ESI-MS (M+H+): 397.1. 1H NMR (400 MHz, DMSO-d6) δ 10.27 (s, 1H), 9.33 (s, 1H), 8.95 (s, 1H), 8.19 (s, 1H), 8.15 (s, 1H), 7.55 (s, 1H), 4.53 (q, J=7.0 Hz, 2H), 4.22-4.16 (m, 2H), 3.15-3.12 (m, 1H), 2.99-2.93 (m, 2H), 2.91-2.88 (m, 1H), 2.67-2.64 (m, 1H), 2.29 (s, 3H), 1.46 (d, J=6.8 Hz, 3H), 1.14 (d, J=6.3 Hz, 3H).
The N-(6-(3-(tert-butylamino)pyrrolidin-1-yl)pyridazin-3-yl)-7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamide (40 mg, 0.092 mmol) was separated by chiral SFC (EtOH/n-Hexane) to afford (R)—N-(6-(3-(tert-butylamino)pyrrolidin-1-yl)pyridazin-3-yl)-7-ethoxy-2-methylimidazol[1,2-a]pyridine-6-carboxamide (14.85 mg, Y: 37.1%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.62 (s, 1H), 9.06 (s, 1H), 8.18 (d, J=9.7 Hz, 1H), 7.66 (s, 1H), 7.00 (s, 2H), 4.28 (q, J=6.8 Hz, 2H), 3.75-3.68 (m, 1H), 3.57-3.48 (m, 2H), 3.38-3.36 (m, 1H), 3.26-2.86 (m, 1H), 2.29 (s, 3H), 2.20-2.17 (m, 1H), 1.77-1.75 (m, 1H), 1.50 (t, J=6.9 Hz, 3H), 1.11 (s, 9H). ESI-MS (M+H)+: 438.2.
To a mixture of tert-butyl (2R,6S)-4-(6-aminopyridin-3-yl)-2,6-dimethylpiperazine-1-carboxylate (48.0 mg, 0.16 mmol) in DMF (5 mL) was added DIEA (60.4 mg, 0.47 mmol), HATU (89.0 mg, 0.23 mmol) and 7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (32.1 mg, 0.17 mmol) and the mixture was stirred at RT for 1 h. The mixture was diluted with H2O (20 mL) and the resulting mixture was extracted with EA (50 mL×2). The organic layer was combined, washed with brine (20 mL×3), dried over anhydrous Na2SO4, filtered and concentrated in vacuum to afford tert-butyl (2R,6S)-4-(6-(7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyridin-3-yl)-2,6-dimethylpiperazine-1-carboxylate (60 mg, 69.7% yield) as a white solid. ESI-MS (M+H+):495.4.
A mixture of tert-butyl (2R,6S)-4-(6-(7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyridin-3-yl)-2,6-dimethylpiperazine-1-carboxylate (60.0 mg, 0.12 mmol) in 1N HCl-dioxane (5 mL) was stirred at rt for 1 h. The mixture was concentrated and purified by prep-HPLC (0.05% NH4HCO3 in water/CH3CN) to afford the title compound to afford title product (12.48 mg, 26.7% yield) as a white solid. ESI-MS (M+H+): 395.2. 1H NMR (400 MHz, DMSO-d6) δ 10.22 (s, 1H), 8.97 (s, 1H), 8.07 (d, J=8.9 Hz, 1H), 8.03 (d, J=3.0 Hz, 1H), 7.64 (s, 1H), 7.44 (dd, J=9.1, 3.1 Hz, 1H), 7.00 (s, 1H), 3.99 (s, 3H), 3.57-3.51 (m, 2H), 2.90-2.82 (m, 2H), 2.29 (d, J=1.0 Hz, 3H), 2.13 (t, J=10.8 Hz, 2H), 1.03 (d, J=6.3 Hz, 6H).
To a mixture of 7-ethoxy-2-methylimidazo[1,2-a]pyrimidine-6-carboxylic acid (54.34 mg, 0.25 mmol) in DMF (5 mL) were added HATU (93 mg, 0.25 mmol) and DIEA (105.7 mg, 0.82 mmol), the mixture was stirred at rt for 0.5 h. tert-butyl 7-(6-aminopyridazin-3-yl)-4,7-diazaspiro[2.5]octane-4-carboxylate (50 mg, 0.16 mmol) was added. The resulting mixture was stirred at rt for 2 h. The mixture was purified by reversed phase column chromatography to provide the title compound (50 mg, yield: 60%). ESI-MS (M+H)+:509.3.
To a solution of tert-butyl 7-(6-(7-ethoxy-2-methylimidazo[1,2-a]pyrimidine-6-carboxamido)pyridazin-3-yl)-4,7-diazaspiro[2.5]octane-4-carboxylate (50 mg, 0.098 mmol) in EA (3 mL) was added 3M HCl in EA (3 mL). The mixture was stirred for 2 h at rt. The reaction mixture was concentrated, the residue was purified by prep-HPLC (0.05% FA in water/CH3CN) to give title product (5.6 mg, yield: 14%) as a yellow solid. ESI-MS (M+H)+: 409.1. 1H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H), 9.33 (s, 1H), 8.20 (s, 1H), 8.15 (d, J=9.9 Hz, 1H), 7.56 (s, 1H), 7.37 (d, J=9.9 Hz, 1H), 4.54 (q, J=7.1 Hz, 2H), 3.52-3.49 (m, 2H), 3.40 (s, 2H), 2.91-2.84 (m, 2H), 2.29 (s, 3H), 1.46 (t, J=7.0 Hz, 3H), 0.56-0.44 (m, 4H).
To a mixture of 6-methoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxylic acid (160 mg, 0.77 mmol) in DMF (3 mL) were added tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (336 mg, 1.16 mmol), HATU (354 mg, 0.92 mmol) and DIEA (400 mg, 3.10 mmol). The mixture was diluted with H2O (10 mL), extracted with EA (20 mL×3), the combined organic layer was concentrated in vacuo to give the title product (250 mg, Y: 62%) as a yellow solid. ESI-MS (M+H+):482.2.
A mixture of tert-butyl (S)-4-(6-(6-methoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxamido)pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (250 mg, 0.519 mmol) in 3M HCl/EA (4 mL) was stirred at 25° C. for 2 h. LCMS showed the reaction was completed. The precipitate was filtered to give the title product (70 mg, Y: 35%) as a yellow solid. ESI-MS (M+H+):382.5. 1H NMR (400 MHz, MeOD-d4) δ 8.33-8.30 (m, 2H), 8.22-8.15 (m, 2H), 6.61 (s, 1H), 4.48 (d, J=13.1 Hz, 2H), 4.05 (s, 3H), 3.62-3.51 (m, 3H), 3.41-3.32 (m, 2H), 2.47 (s, 3H), 1.45 (d, J=6.6 Hz, 3H).
To a solution of tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methyl-3,6-dihydropyridine-1(2H)-carboxylate (400 mg, 1.38 mmol) in DMF (5 mL) were added 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (365 mg, 1.66 mmol), DIEA (712 mg, 5.52 mmol) and HATU (630 mg, 1.66 mmol). The mixture was stirred at 50° C. for 16 h. The mixture was diluted with water (10 mL), the precipitate was filtered, washed with water (20 mL) and concentrated in vacuo to give title product (300 mg, 44.12%) as a yellow solid. ESI-MS (M+H)+: 493.2.
To a solution of tert-butyl (S)-4-(6-(6-ethoxy-2-methyl-2H-indazole-5-carboxamido)pyridazin-3-yl)-6-methyl-3,6-dihydropyridine-1(2H)-carboxylate (300 mg, 0.61 mmol) in EA (5 mL) was added 3 M HCl/EA (6 mL). The mixture was stirred at rt for 2 h. The mixture was concentrated in vacuo. The crude was purified by prep-HPLC (0.05% FA in water/CH3CN) to give title product (48.75 mg, Y: 18.26%) as a yellow solid. ESI-MS (M+H+): 393.3. 1H NMR (400 MHz, MeOD-d4) δ 8.65 (d, J=9.5 Hz, 1H), 8.59 (s, 1H), 8.52 (s, 1H), 8.33 (s, 1H), 8.00 (t, J=9.1 Hz, 1H), 7.11 (s, 1H), 6.70-6.59 (m, 1H), 4.36 (q, J=6.9 Hz, 2H), 4.18 (s, 3H), 3.97 (br s, 1H), 3.67-3.56 (m, 1H), 3.42-3.35 (m, 1H), 3.28-3.05 (m, 1H), 3.04-2.96 (m, 1H), 1.65 (t, J=6.9 Hz, 3H), 1.52 (t, J=7.1 Hz, 3H).
To a mixture of tert-butyl 4-(6-aminopyridazin-3-yl)piperidine-1-carboxylate (240 mg, 0.86 mmol) and 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (241.2 mg, 1.1 mmol) in MeCN (10 mL) were added NMI (212.4 mg, 2.59 mmol) and TCFH (363.9 mg, 1.29 mmol). The mixture was stirred at 50° C. for 2 h. The precipitate was filtered and dried under vacuum to give title product (200 mg, 48.4%) as a white solid, which was used to next step without further purification. ESI-MS (M+H)+: 481.3. 1H NMR (400 MHz, DMSO-d6) δ 11.14 (s, 1H), 8.51-8.40 (m, 3H), 7.71 (d, J=9.2 Hz, 1H), 7.16 (s, 1H), 4.29 (q, J=6.9 Hz, 2H), 4.15 (s, 3H), 4.13-4.03 (m, 2H), 3.06 (t, J=12.0 Hz, 1H), 2.98-2.72 (m, 2H), 1.92-1.84 (m, 2H), 1.69-1.58 (m, 2H), 1.53 (t, J=6.9 Hz, 3H), 1.43 (s, 9H).
A mixture of tert-butyl 4-(6-(6-ethoxy-2-methyl-2H-indazole-5-carboxamido)pyridazin-3-yl)piperidine-1-carboxylate (200 mg, 0.42 mmol) in EA/HCl (5 mL) was stirred at rt for 2 h. The precipitate was filtered and lyophilized to give title product (132 mg, 75.6%) as a white solid. ESI-MS (M+H)+: 381.3. 1H NMR (400 MHz, DMSO-d6) δ 11.23 (s, 1H), 9.26 (s, 1H), 9.03 (s, 1H), 8.58-8.40 (m, 3H), 7.74 (d, J=9.3 Hz, 1H), 7.16 (s, 1H), 4.29 (q, J=6.9 Hz, 2H), 4.15 (s, 3H), 3.38 (d, J=12.6 Hz, 2H), 3.27-3.17 (m, 1H), 3.13-2.97 (m, 2H), 2.16-1.97 (m, 4H), 1.52 (t, J=6.9 Hz, 3H).
To a mixture of 7-ethoxy-2-methyl-N-(6-(piperazin-1-yl)pyridazin-3-yl)imidazol[1,2-a]pyridine-6-carboxamide (70 mg, 0.18 mmol) and (HCHO)n (27.6 mg, 0.92 mmol) in MeOH (5 mL) were added HOAc (33 mg, 0.8 mmol) and NaBH3CN (58 mg, 1.34 mmol). The mixture was stirred at 50° C. for 2 h. The mixture was poured into water (2 mL). Filtered and concentrated in vacuo. The residue was purified by prep-HPLC (0.05% NH3·H2O in water/ACN) to give title product (26.32 mg, Y: 32.8%) as a yellow solid. ESI-MS (M+H+): 396.2. 1H NMR (400 MHz, MeOD-d4) δ 9.03 (s, 1H), 8.37 (d, J=9.8 Hz, 1H), 7.55 (s, 1H), 7.38 (d, J=9.9 Hz, 1H), 6.95 (s, 1H), 4.37 (q, J=6.9 Hz, 2H), 3.66-3.59 (m, 4H), 2.62-2.56 (m, 4H), 2.38-2.34 (m, 6H), 1.64 (t, J=7.0 Hz, 3H).
To a mixture of 6-ethoxy-2-methyl-2H-pyrazolo[3,4-b]pyridine-5-carboxylic acid (150 mg, 0.68 mmol) in DMF (3 mL) were added tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate (226 mg, 0.81 mmol), HATU (773 mg, 2.04 mmol) and DIEA (438 mg, 3.4 mmol). The mixture was stirred at rt for 3 h. LCMS showed the reaction was completed. The mixture was diluted with water (5 mL) and the precipitate was filtered, washed with water and dried in vacuo to give crude product (150 mg, Y: 45.9%) as a yellow solid. ESI-MS (M+H)+: 482.3.
A mixture of tert-butyl 4-(6-(6-ethoxy-2-methyl-2H-pyrazolo[3,4-b]pyridine-5-carboxamido)pyridin-3-yl)piperazine-1-carboxylate (150 mg, 0.31 mmol) in 3M HC/EA (3 mL) was stirred at rt for 2 h. LCMS showed the reaction was completed. The mixture was added H2O (5 mL), extracted with EA (10 mL*3), the aqueous layer was concentrated in vacuo and the residue was purified by prep-HPLC (0.05% NH3·H2O in water/ACN) to give title product (72.6 mg, Y: 55.8%) as a yellow solid. ESI-MS (M+H+): 382.1. 1H NMR (400 MHz, DMSO-d6) δ 8.77 (s, 1H), 8.46 (s, 1H), 8.13 (d, J=8.9 Hz, 1H), 8.07-8.02 (m, 1H), 7.47-7.41 (m, 1H), 4.55 (q, J=7.0 Hz, 2H), 4.13 (s, 3H), 3.09-3.04 (m, 4H), 2.87-2.82 (m, 4H), 1.47 (t, J=7.0 Hz, 3H).
To a mixture of 7-ethoxy-2-methyl-N-(6-(piperazin-1-yl)pyridazin-3-yl)imidazo[1,2-a]pyrimidine-6-carboxamide (110 mg, 0.288 mmol), AcOH (52 mg, 0.864 mmol) in MeOH (10 mL) were added NaBH(OAc)3 (305 mg, 1.440 mmol) and (HCHO)n (43 mg, 1.44 mmoL). The mixture was stirred at 50° C. for 5 h. The mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by prep-HPLC (0.1% FA in water/ACN) to give title product (11.0 mg, Y: 10%) as a yellow solid. ESI-MS (M+H+): 397.1. 1H NMR (400 MHz, MeOD-d4) δ 9.34 (s, 1H), 8.50 (s, 1H), 8.40 (d, J=9.9 Hz, 1H), 7.50 (s, 1H), 7.42 (d, J=9.9 Hz, 1H), 4.76 (q, J=7.1 Hz, 2H), 3.72-3.66 (m, 4H), 2.73-2.68 (m, 4H), 2.45 (s, 3H), 2.39 (s, 3H), 1.62 (t, J=7.1 Hz, 3H).
To a mixture of 7-ethoxy-2-methylimidazo[1,2-a]pyrimidine-6-carboxylic acid (159 mg, 0.72 mmol) in DMF (10 mL) were added HATU (408 mg, 1.08 mmol) and DIEA (462 mg, 3.58 mmol) and the mixture was stirred at 40° C. for 0.5 h. Then tert-butyl 4-(6-aminopyridazin-3-yl)piperazine-1-carboxylate (200 mg, 0.72 mmol) was added and the mixture was stirred at 40° C. for 16 h. The mixture was diluted with H2O (15 mL) and stirred for 10 min. The precipitate was filtered, washed with water and concentrated in vacuo to give title product (250 mg, crude) as a yellow solid. ESI-MS (M+H)+:483.1.
A mixture of tert-butyl 4-(6-(7-ethoxy-2-methylimidazo [1,2-a]pyrimidine-6-carboxamido) pyridazin-3-yl)piperazine-1-carboxylate (90 mg, 0.187 mmol) in 3M HCl/EA (2 mL) was stirred at rt for 1 h. The mixture was concentrated in vacuo and the residue was purified by prep-HPLC (0.1 FA in water/CH3CN) to give title product (19.96 mg, Y: 28%) as a yellow solid. ESI-MS (M+H+):383.1. 1H NMR (400 MHz, DMSO-d6) δ 10.64 (s, 1H), 9.34 (s, 1H), 8.28-8.08 (m, 2H), 7.56 (d, J=0.9 Hz, 1H), 7.44 (d, J=9.9 Hz, 1H), 4.54 (q, J=7.0 Hz, 2H), 3.61-3.56 (m, 4H), 3.01-2.94 (m, 4H), 2.29 (s, 3H), 1.46 (t, J=7.0 Hz, 3H).
A dry vial was charged with 1-benzylpyrrolidin-3-one (20 g, 0.114 mol), 2-methylpropan-2-amine (19.2 g, 0.263 mol) and Titanium tetraisopropanolate (29.2 mL, 0.103 mol). The mixture was purged with N2 for 15 min and then allowed to stir at room temperature for 2 h. The resulting (E)-1-benzyl-N-(tert-butyl)pyrrolidin-3-imine was used to next step without further purification (30 g. crude). ESI-MS (M+H)+: 231.2.
To the mixture of (E)-1-benzyl-N-(tert-butyl)pyrrolidin-3-amine (26.3 g, 0.11 mol) was added dry methanol (260 mL) and the reaction mixture was cooled to 0° C. NaBH4 (8 g, 0.22 mol) was added slowly in portions (caution: very exothermic reaction). Once evolution of the gas subsided, the mixture was warmed to room temperature and stirred for 2 h at RT. Upon completion, 0.1 M NaOH solution (100 mL) was added to precipitate the titanium salts. The biphasic mixture was filtered through celite and washed with methanol. The solvent was removed under vacuum and the crude oil was purified by silica gel column chromatography (DCM/MeOH=10:1) to give title compound (10 g, 37.7% yield) as a black solid. ESI-MS (M+H)+: 233.1. 1H NMR (400 MHz, CDCl3) δ 7.33-7.30 (m, 4H), 7.26-7.22 (m, 1H), 3.62-3.53 (m, 2H), 3.47-3.39 (m, 1H), 2.96-2.91 (m, 1H), 2.71-2.63 (m, 1H), 2.51-2.43 (m, 1H), 2.24-2.14 (m, 1H), 2.11 (dd, J=8.9, 7.3 Hz, 1H), 1.51-1.42 (m, 1H), 1.07 (s, 9H).
To an oven-dry round bottom flask containing palladium hydroxide on activated carbon (300 mg, 10% wt) was added 1-benzyl-N-(tert-butyl)pyrrolidin-3-amine (3 g, 0.013 mol) and MeOH (20 mL). The mixture was charged with H2 for 5 minutes and a balloon of H2 was placed on top of the flask and the reaction was stirred for 16 h at 45° C. The reaction mixture was filtered through Celite, washed with MeOH and concentrated to afford N-(tert-butyl)pyrrolidin-3-amine (1.5 g, 81.7% yield) as a colorless oil. ESI-MS (M+H)+: 143.2. 1H NMR (400 MHz, CDCl3) δ 3.43-3.38 (m, 1H), 3.18-3.08 (m, 2H), 2.98-2.90 (m, 1H), 2.70-2.62 (m, 1H), 2.17-2.05 (m, 1H), 1.57-1.46 (m, 1H), 1.14-1.08 (m, 9H).
A mixture of N-(tert-butyl)pyrrolidin-3-amine (I g, 6.99 mmol) and 6-chloropyridazin-3-amine (757 mg, 6.99 mmol) was heated at 140° C. for 16 h. After cooling to rt, the reaction mixture was diluted with MeOH (10 mL), The precipitate was filtered, washed with MeOH (2 mL) and dried in vacuo to give 6-(3-(tert-butylamino)pyrrolidin-1-yl)pyridazin-3-amine (500 mg, 30.2% yield) as a brown solid. ESI-MS (M+H)+: 236.2. 1H NMR (400 MHz, DMSO-d6) δ 7.22 (d, J=9.6 Hz, 1H), 7.09 (d, J=9.7 Hz, 1H), 6.69 (s, 2H), 4.08-4.00 (m, 1H), 3.82-3.74 (m, 1H), 3.66-3.61 (m, 1H), 3.61-3.56 (m, 1H), 3.37-3.33 (m, 1H), 2.45-2.24 (m, 2H), 1.37 (s, 9H).
To a mixture of 6-(3-(tert-butylamino)pyrrolidin-1-yl)pyridazin-3-amine (200 mg, 0.85 mmol) and 7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (187 mg, 0.85 mmol) in ACN (10 mL) were added TCFH (359 mg, 1.28 mmol) and NMI (209 mg, 2.55 mmol). The mixture was stirred at RT overnight. The mixture was filtered, the crude solid was purified by prep-HPLC (0.05% NH3·H2O in water/CH3CN) to give title product (60 mg, Y: 16.1%) as a white solid. ESI-MS (M+H)+: 438.1. 1H NMR (400 MHz, MeOD-d4) δ 9.03 (s, 1H), 8.33 (d, J=9.7 Hz, 1H), 7.55 (s, 1H), 7.02 (d, J=9.8 Hz, 1H), 6.94 (s, 1H), 4.37 (q, J=6.9 Hz, 2H), 3.87-3.80 (m, 1H), 3.70-3.61 (m, 2H), 3.50-3.41 (m, 1H), 3.20-3.14 (m, 1H), 2.38 (s, 3H), 2.37-2.30 (m, 1H), 1.94-1.86 (m, 1H), 1.66 (t, J=7.0 Hz, 3H), 1.22 (s, 9H).
To a mixture of tert-butyl 4-(5-aminopyrazin-2-yl)-2,2-dimethylpiperazine-1-carboxylate (556 mg, 1.81 mmol) and 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (400 mg, 1.81 mmol) in MeCN (15 mL) were added DIEA (1.17 g, 9.050 mmol) and HATU (2.1 g, 5.43 mmol). The mixture was stirred at rt for 3 h. After diluting with water, the mixture was filtered and the cake was washed with MeOH (6 mL) and dried to give title product (300 mg, 32.4%) as a white solid. ESI-MS (M+H)+: 510.3. 1H NMR (400 MHz, CDCl3) δ 10.45 (s, 1H), 9.22 (d, J=1.4 Hz, 1H), 8.74 (s, 1H), 7.98 (s, 1H), 7.69 (d, J=1.4 Hz, 1H), 7.08 (s, 1H), 4.31 (q, J=7.0 Hz, 2H), 4.20 (s, 3H), 3.89 (t, J=5.7 Hz, 2H), 3.81 (s, 2H), 3.56 (t, J=5.7 Hz, 2H), 1.69-1.66 (m, 3H), 1.50 (s, 9H), 1.42 (s, 6H).
To a mixture of tert-butyl 4-(5-(6-ethoxy-2-methyl-2H-indazole-5-carboxamido)pyrazin-2-yl)-2,2-dimethylpiperazine-1-carboxylate (300 mg, 0.59 mmol) in EA (5 mL) was added EA/HCl (5 mL). The mixture was stirred at rt for 1 h. After concentration, the residue was purified by pre-HPLC to give title product (97 mg, 37%) as a white solid. ESI-MS (M+H)+: 410.1. 1H NMR (400 MHz, DMSO-d6) δ 10.56 (s, 1H), 9.51 (s, 2H), 9.02 (s, 1H), 8.47 (s, 1H), 8.43 (s, 1H), 8.25 (s, 1H), 7.14 (s, 1H), 4.27 (q, J=6.6 Hz, 2H), 4.14 (s, 3H), 3.79 (br s, 2H), 3.65 (s, 2H), 3.22 (br s, 2H), 1.50 (t, J=6.8 Hz, 3H), 1.37 (s, 6H).
A mixture of 7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (74 mg, 0.36 mmol), HATU (103 mg, 0.27 mmol), tert-butyl 4-(6-aminopyridin-3-yl)piperidine-1-carboxylate (50 mg, 0.18 mmol) and DIEA (70 mg, 0.54 mmol) in DMF (5 mL) was stirred at r.t. for 1 h. The reaction mixture was diluted with water (10 mL) and extracted with EtOAc (30 mL). The organic layer was washed with brine, dried with Na2SO4 and evaporated to give crude title compound. The crude was purified by reverse phase column (0.1% FA in water/CH3CN) to give title compound (40 mg, 48% yield). ESI-MS (M+H)+: 466.3.
tert-Butyl 4-(6-(7-methoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyridin-3-yl)piperidine-1-carboxylate (40 mg, 0.086 mmol) was dissolved in 3M HCl/EA solution (5 mL) and the mixture was stirred at r.t. for 2 h. The precipitate was collected by filtration, washed with EA (3 mL×3) and dried under vacuum to afford the title compound as hydrochloride salt (15 mg, 48% yield). ESI-MS (M+H)+: 366.2. 1H NMR (400 MHz, DMSO-d6) δ 10.94 (s, 1H), 9.20 (br s, 3H), 8.27 (s, 1H), 8.17 (d, J=8.1 Hz, 1H), 7.96 (s, 1H), 7.78 (dd, J=8.6, 2.1 Hz, 1H), 7.32 (s, 1H), 4.07 (s, 3H), 3.44-3.27 (m, 2H), 3.07-2.86 (m, 3H), 2.46 (s, 3H), 2.02-1.80 (m, 4H).
To a solution of 6-methoxy-2-methyl-2H-indazole-5-carboxylic acid (214 mg, 1.04 mmol) in DCM (10 mL) were added DIEA (0.48 mL, 3.12 mmol), HATU (488 mg, 1.56 mmol), and tert-butyl (S)-4-(5-aminopyrazin-2-yl)-2-methylpiperazine-1-carboxylate compound (300 mg, 1.04 mmol), and the mixture was stirred at room temperature overnight. The reaction mixture was diluted with DCM (50 mL) and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered, concentrated under vacuum. The residue was purified by silica gel column chromatography (EA/PE=100/1 to 40/1) to afford the title compound (460 mg, 0.96 mmol) as a yellow solid. ESI-MS (M+H+): 482.1.
A suspension of tert-butyl (S)-4-(5-(6-methoxy-2-methyl-2H-indazole-5-carboxamido)pyrazin-2-yl)-2-methylpiperazine-1-carboxylate (460 mg, 096 mmol) in 4M HCl 1,4-dioxance (20 mL) was stirred at room temperature overnight. The mixture was filtered and the solid was dried in vacuum to afford title product (350 mg, 87.5%) as a yellow solid. ESI-MS (M+H+): 382.3. 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.45 (s, 1H), 9.27 (s, 1H), 9.02 (s, 1H), 8.45 (s, 1H), 8.31 (s, 1H), 8.23 (s, 1H), 7.14 (s, 1H), 4.31 (t, J=11.0 Hz, 2H), 4.14 (s, 3H), 3.98 (s, 3H), 3.40-3.30 (m, 2H), 3.28-3.19 (m, 1H), 3.13-2.97 (m, 2H), 1.31 (d, J=6.5 Hz, 3H).
To a solution of 7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (188 mg, 0.85 mmol), tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (200 mg, 0.68 mmol) and DIEA (551 mg, 4.30 mmol) in DMF (5 mL) was added HATU (486 mg, 1.28 mmol) at rt. the mixture was stirred for 16 h. After diluting with water, the solid was collected by filtration, washed with H2O and dried under vacuum to give title product (200 mg, Y: 59.2%) as a yellow solid. ESI-MS (M+H)+: 496.2.
To a solution of the tert-butyl (S)-4-(6-(7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyridazin-3-yl)-2-methylpiperazine-1-carboxylatetert-butyl (200 mg, 0.40 mmol) in EtOAc (3 mL) was added HCl-EA (3M, 3 mL), the mixture was stirred at rt for 1 h. After concentration, title product (160 mg, yield: 94%) was obtained as a yellow solid. ESI-MS (M+H)+: 396.5.
To a solution of (S)-7-ethoxy-2-methyl-N-(6-(3-methylpiperazin-1-yl)pyridazin-3-yl)imidazo[1,2-a]pyridine-6-carboxamide (180 mg, 0.46 mmol) in MeOH (5 mL) were added (HCHO)n (68 mg, 2.28 mmol) and acetic acid (82 mg, 1.37 mmol). The mixture was stirred at 50° C. for 1 h, then sodium cyanoborohydride (24 mg, 0.38 mmol) was added and the mixture was stirred at 50° C. for 16 h. Water (5 mL) was added and the solution was concentrated in vacuo. The crude was purified by prep-HPLC (0.1% FA in water/CH3CN) to give title compound (42 mg, yield: 22.6%) as a yellow solid. ESI-MS (M+H)+: 410.2. 1H NMR (400 MHz, MeOD-d4) δ 9.04 (s, 1H), 8.41 (s, 1H), 8.39 (s, 1H), 7.56 (s, 1H), 7.43 (d, J=9.9 Hz, 1H), 6.97 (s, 1H), 4.37 (q, J=6.9 Hz, 2H), 4.24 (d, J=9.3 Hz, 2H), 3.28 (br s, 2H), 3.05-2.95 (m, 1H), 2.89-2.86 (m, 2H), 2.67 (s, 3H), 2.37 (s, 3H), 1.63 (t, J=6.9 Hz, 3H), 1.33 (d, J=6.3 Hz, 3H).
A mixture of 6-chloropyridazin-3-amine (150 mg, 1.16 mmol) and (S)-octahydropyrrolo[1,2-a]pyrazine (293 mg, 2.33 mmol) was stirred at 150° C. under Ar atmosphere for 18 h. The mixture was diluted with water (10 mL), extracted with EA (20 mL×3). The organic layer was washed with brine, dried with Na2SO4 and concentration in vacuo to give title product (260 mg, crude) as a black solid. ESI-MS (M+H)+: 220.2.
To a solution of (S)-6-(hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl)pyridazin-3-amine (200 mg, 0.91 mmol), 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (201 mg, 0.91 mmol) and HATU (530 mg, 1.39 mmol) in DMF (4 mL) was added DIEA (235 mg, 1.82 mmol). The mixture was stirred at rt under Ar atmosphere for 16 h. The mixture was diluted with water (20 mL), extracted with EA (40 mL×2). The organic layer was washed with brine (8 mL), dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by prep-HPLC (0.1% FA in H2O/MeCN) to give title product (30 mg, 7.83% yield) as a white solid. ESI-MS (M+H)+: 422.2. 1H NMR (400 MHz, CDCl3) δ 10.94 (s, 1H), 8.71 (s, 1H), 8.49 (d, J=9.6 Hz, 1H), 7.99 (s, 1H), 7.08 (s, 1H), 7.04 (d, J=9.6 Hz, 1H), 4.44-4.29 (m, 3H), 4.25-4.20 (m, 4H), 3.17-3.09 (m, 3H), 2.76 (dd, J=10.4 Hz, 1H), 2.37-2.31 (m, 1H), 2.20-2.08 (m, 2H), 1.92-1.77 (m, 3H), 1.70 (t, J=7.2 Hz, 3H), 1.53-1.49 (m, 1H).
A mixture of 6-chloropyridazin-3-amine (200 mg, 1.55 mmol) and (R)-octahydropyrrolo[1,2-a]pyrazine (390 mg, 3.10 mmol) was stirred at 150° C. under Ar atmosphere for 20 h. The mixture was diluted with water (10 mL), extracted with EA (20 mL×3). The organic layer was washed with brine, dried with Na2SO4 and concentrated in vacuo to give title product (350 mg, crude) as a black solid. ESI-MS (M+H+): 220.2.
To a mixture of (R)-6-(hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl)pyridazin-3-amine (200 mg, 0.91 mmol), 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (201 mg, 0.91 mmol) and HATU (530 mg, 1.39 mmol) in DMF (4 mL) was added DIEA (235 mg, 1.82 mmol). The mixture was stirred at rt under Ar atmosphere for 16 h. The mixture was diluted with water (20 mL), extracted with EA (40 mL×2). The organic layer was washed with brine (20 mL), dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by prep-HPLC (0.1% FA in H2O/MeCN) to give title product (32 mg, 8.35% yield) as a white solid. ESI-MS (M+H)+: 422.2. 1H NMR (400 MHz, CDCl3) δ 10.94 (s, 1H), 8.71 (s, 1H), 8.49 (d, J=10 Hz, 1H), 8.09-8.06 (m, 1H), 7.99 (s, 1H), 7.08 (s, 1H), 7.04 (d, J=9.6 Hz, 1H), 4.44-4.29 (m, 4H), 4.25-4.20 (m, 4H), 3.17-3.09 (m, 3H), 2.75 (t, J=12 Hz, 1H), 2.37-2.31 (m, 1H), 2.20-2.09 (m, 2H), 1.94-1.75 (m, 3H), 1.70 (t, J=7.2 Hz, 3H), 1.54-1.51 (m, 1H).
To a mixture of N-(6-((3S,5R)-3,5-dimethylpiperazin-1-yl)pyridazin-3-yl)-7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamide (70 mg, 0.17 mmol) and AcOH (26 mg, 0.43 mmol) in MeOH (7 mL) were added NaBH3CN (54 mg, 0.80 mmol) and (HCHO)n (25.3 mg, 0.80 mmol), the mixture was stirred at 50° C. for 2 hours. The mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by prep-HPLC (0.1% FA in water/ACN) to give title product (12 mg, 16.7%) as a white solid. ESI-MS (M+H+): 424.3. 1H NMR (400 MHz, CD3OD-d4) δ 9.03 (s, 1H), 8.36 (d, J=10.0 Hz, 1H), 7.54 (s, 1H), 7.39 (d, J=9.9 Hz, 1H), 6.95 (s, 1H), 4.36 (q, J=7.0 Hz, 2H), 4.16 (d, J=12.6 Hz, 2H), 2.85-2.67 (m, 2H), 2.46-2.19 (m, 8H), 1.63 (t, J=6.9 Hz, 3H), 1.22 (d, J=6.2 Hz, 6H).
To a solution of 6-bromo-7-ethoxy-2-methylimidazo[1,2-a]pyridine (200 mg, 0.79 mmol) in toluene (15 mL) were added Pd(OAc)2 (18 mg, 0.079 mmol), Xantphos (91 mg, 0.16 mmol), Na2CO3 (251 mg, 2.37 mmol) and tert-butyl (S)-4-(6-amino-4-methylpyridazin-3-yl)-2-methylpiperazine-1-carboxylate (244 mg, 1.18 mmol), the mixture was charged with CO for three times and stirred at 100° C. for 16 h under CO balloon. The mixture was filtered and the filtrate was concentrated to afford the title compound (250 mg, crude) as a grey solid. ESI-MS (M+H)+: 510.2.
A solution of tert-butyl (S)-4-(6-(7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)-4-methylpyridazin-3-yl)-2-methylpiperazine-1-carboxylate (250 mg, 0.49 mmol) in 3M HCl/EtOAc (5 mL) was stirred for 2 h at RT. The mixture was concentrated in vacuo. The residue was purified by prep-HPLC (0.05% FA in water/ACN) to give title product (6 mg, 1.7% with two steps) as a yellow solid. ESI-MS (M+H)+: 410.5. 1H NMR (400 MHz, DMSO-d6) δ 10.83 (s, 1H), 9.04 (s, 1H), 8.25 (s, 1H), 8.15 (s, 1H), 7.67 (s, 1H), 7.01 (s, 1H), 4.28 (q, J=6.8 Hz, 2H), 3.52 (d, J=11.4 Hz, 2H), 3.28-3.25 (m, 2H), 3.16-3.07 (m, 2H), 2.92-2.84 (m, 1H), 2.36 (s, 3H), 2.29 (s, 3H), 1.50 (t, J=6.9 Hz, 3H), 1.23 (d, J=6.4 Hz, 3H).
To a mixture of tert-butyl 4-(5-aminopyrazin-2-yl)piperazine-1-carboxylate (150 mg, 0.69 mmol) in DMF (5 mL) were added HATU (314 mg, 0.83 mmol), DIEA (356 mg, 2.76 mmol) and 6-ethoxy-2-methyl-2H-pyrazolo[3,4-b]pyridine-5-carboxylic acid (203 mg, 1.56 mmol). The mixture was stirred at 25° C. for 16 h. LCMS showed the starting material was consumed completely. The mixture was diluted with water (10 mL), extracted with EA (20 mL×3). The organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo to give title product (120 mg, 46.33%) as a yellow solid. ESI-MS (M+H)+: 483.3.
To a mixture of tert-butyl 4-(5-(6-ethoxy-2-methyl-2H-pyrazolo[3,4-b]pyridine-5-carboxamido)pyrazin-2-yl)piperazine-1-carboxylate (120 mg, 0.25 mmol) in EA (3 mL) was added 3 M HCl/EA (3 mL). The mixture was stirred at rt for 2 h. LCMS showed the starting material was consumed completely. The mixture was concentrated in vacuo. The crude was purified by prep-HPLC (0.05% HCl in water/CH3CN) to give title product (83 mg, Y: 87.28%) as a yellow solid. ESI-MS (M+H+): 383.1. 1H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H), 9.21 (brs, 2H), 9.04 (s, 1H), 8.73 (s, 1H), 8.47 (s, 1H), 8.25 (s, 1H), 4.54 (q, J=6.9 Hz, 2H), 4.13 (s, 3H), 3.77-3.74 (m, 4H), 3.21-3.19 (m, 4H), 1.45 (t, J=7.0 Hz, 3H).
To a mixture of (S)-6-ethoxy-2-methyl-N-(5-(3-methylpiperazin-1-yl)pyrazin-2-yl)-2H-indazole-5-carboxamide (100 mg, 0.25 mmol) in MeOH (10 mL) were added HOAc (76 mg, 1.27 mmol) and (HCHO)n (38 mg, 1.27 mmol). The mixture was stirred at 45° C. for 1 h. Then NaBH3CN (49 mg, 0.76 mmol) was added and the mixture was stirring at 45° C. for 2 h. The reaction mixture was diluted with H2O (3 mL) and concentrated, the residue was purified by prep-HPLC (0.05% FA in water/ACN) to give title product (44 mg, Y: 38.3%) as a white solid. ESI-MS (M+H)+:410.2. 1H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H), 9.01 (s, 1H), 8.45-8.44 (m, 2H), 8.15 (d, J=13.9 Hz, 1H), 7.13 (s, 1H), 6.51 (s, 1H), 4.27 (q, J=6.8 Hz, 2H), 4.14 (s, 3H), 4.12-4.03 (m, 2H), 3.13-2.86 (m, 3H), 2.76-2.58 (m, 2H), 2.30 (s, 3H), 1.50 (t, J=6.9 Hz, 3H), 1.10 (d, J=6.1 Hz, 3H).
To a solution of tert-butyl (2R,6S)-4-(5-aminopyrazin-2-yl)-2,6-dimethylpiperazine-1-carboxylate (100 mg, 0.32 mmol) in ACN (10 mL) were added 6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxylic acid (78.82 mg, 0.36 mmol) and TCFH (140 mg, 0.48 mmol) and NMI (80 mg, 0.98 mmol). The mixture was stirred at r.t. for 16 hours. LCMS showed the reaction was completed. The mixture was diluted with H2O (10 mL), extracted with EA (20 mL×3). The organic phase was washed with brine, dried over sodium sulfate and concentrated in vacuo to give title compound (100 mg, 61.7%) as a yellow oil. ESI-MS (M+H)+: 510.4. 1H NMR (400 MHz, CDCl3) δ 10.25 (s, 1H), 9.19 (s, 1H), 8.44 (s, 1H), 8.11 (s, 1H), 7.90 (s, 1H), 6.45 (s, 1H), 4.33-4.27 (m, 2H), 4.23 (q, J=7.0 Hz, 2H), 4.15-4.13 (m, 2H), 3.13-3.11 (m, 2H), 2.48 (s, 3H), 2.01 (s, 3H), 1.50 (s, 9H), 1.29 (d, J=6.8 Hz, 6H).
A solution of tert-butyl (2S,6R)-4-(5-(6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxamido)pyrazin-2-yl)-2,6-dimethylpiperazine-1-carboxylate (100 mg, 0.19 mmol) in 3M HCl/EA (2 mL) was stirred at rt for 1 h. The mixture was diluted with water (15 mL) and extracted with EA (20 mL×3), the aqueous phase was concentrated in vacuo and lyophilized to give title compound (85.07 mg, 93.4%) as a yellow solid. ESI-MS (M+H)+: 410.2. 1H NMR (400 MHz, DMSO-d6) δ 10.54 (s, 1H), 9.67 (s, 1H), 9.24 (s, 1H), 9.00 (s, 1H), 8.49 (s, 1H), 8.28 (s, 1H), 8.10 (s, 1H), 6.54 (s, 1H), 4.42 (d, J=12.7 Hz, 2H), 4.21 (d, J=6.5 Hz, 2H), 3.31-3.30 (m, 2H), 2.95 (t, J=12.4 Hz, 2H), 2.39 (s, 3H), 1.44 (s, 3H), 1.33 (d, J=6.0 Hz, 6H).
To a mixture of 7-ethoxy-N-(6-(hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyridazin-3-yl)-2-methylimidazo[1,2-a]pyridine-6-carboxamide formate (130 mg, 0.32 mmol), AcOH (57 mg, 0.96 mmol) in MeOH (10 mL) were added NaBH3CN (100 mg, 0.64 mmol) and (HCHO)n (135 mg, 1.60 mmol). The mixture was stirred at 50° C. for 5 h. The mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by prep-HPLC (0.1% FA in water/ACN) to give title product (52.0 mg, Y: 38.7%) as a yellow solid. ESI-MS (M+H+): 422.2. 1H NMR (400 MHz, MeOD-d4) δ 9.04 (s, 1H), 8.40 (s, 1H), 8.37 (d, J=9.8 Hz, 1H), 7.56 (s, 1H), 7.16 (d, J=9.8 Hz, 1H), 6.95 (s, 1H), 4.36 (q, J=6.9 Hz, 2H), 3.69 (dd, J=11.0, 1.7 Hz, 2H), 3.58 (dd, J=10.6, 7.0 Hz, 4H), 3.30-3.19 (m, 4H), 2.87 (s, 3H), 2.38 (s, 3H), 1.64 (t, J=6.9 Hz, 3H).
To a mixture of tert-butyl 4-(5-aminopyrazin-2-yl)piperazine-1-carboxylate (200 mg, 0.72 mmol) in DMF (6 mL) were added HATU (327 mg, 0.86 mmol), DIEA (370 mg, 2.87 mmol) and 6-methoxy-2-methyl-2H-indazole-5-carboxylic acid (222 mg, 1.08 mmol). The mixture was stirred at 45° C. for 3 h. LCMS showed the reaction was completed. The reaction mixture was diluted with H2O (10 mL) and extracted with EA (30 mL). The organic phase was washed with brine, dried over sodium sulfate and concentrated to give title product (200 mg, 60%) as a brown solid. ESI-MS (M+H)+: 468.2.
To a mixture of tert-butyl 4-(5-(6-methoxy-2-methyl-2H-indazole-5-carboxamido)pyrazin-2-yl)piperazine-1-carboxylate (100 mg, 0.21 mmol) in EA (2 mL) was added 3M HCl/EA (2 mL). The mixture was stirred at RT for 2 h. LCMS showed the reaction was completed. The mixture was concentrated in vacuo, the residue was purified by prep-HPLC (0.05% FA in water/ACN) to give title product (35 mg, Y: 40%) as a yellow solid. ESI-MS (M+H)+:368.2. 1H NMR (400 MHz, DMSO-dr) δ 10.32 (s, 1H), 8.98 (s, 1H), 8.44 (s, 1H), 8.32 (s, 1H), 8.20 (s, 1H), 8.11 (s, 1H), 7.14 (s, 1H), 4.14 (s, 3H), 3.98 (s, 3H), 3.49-3.47 (m, 4H), 2.90-2.87 (m, 4H).
To a solution of (S)-6-ethoxy-2-methyl-N-(6-(3-methylpiperazin-1-yl)pyridazin-3-yl)-2H-indazole-5-carboxamide hydrogen chloride (80 mg, 0.20 mmol) in DMF (5 mL) were added 1-(((4-nitrophenoxy)carbonyl)oxy)ethyl isobutyrate (90 mg, 0.30 mmol) and TEA (78 mg, 0.60 mmol). The mixture was stirred at rt overnight. Then mixture was concentrated in vacuo, and the residue was purified by prep-HPLC (0.05% NH3·H2O in H2O/CH3CN) to give title product (50 mg, Y: 44.6%) as a white solid. ESI-MS (M+H)+: 554.2. 1H NMR (400 MHz, DMSO-d6) δ 10.88 (s, 1H), 8.46 (d, J=6.3 Hz, 2H), 8.28 (d, J=9.9 Hz, 1H), 7.42 (d, J=9.9 Hz, 1H), 7.15 (s, 1H), 6.74-6.66 (m, 1H), 4.32-4.18 (m, 4H), 4.13-4.12 (m, 4H), 3.89-3.82 (m, 1H), 3.29-3.15 (m, 21H), 3.01-2.90 (m, 1H), 2.59-2.52 (m, 1H), 1.52 (t, J=6.9 Hz, 3H), 1.46 (d, J=5.4 Hz, 3H), 1.15 (t, J=6.9 Hz, 3H), 1.09-1.07 (m, 6H).
A solution of tert-butyl hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (5 g, 23.58 mmol) and 6-chloropyridazin-3-amine (1.533 g, 11.79 mmol) was stirred at 150° C. for 6 h. The mixture was purified by C18 flash (0.1% FA in water/CH3CN) to give title product (3.2 g, 89%) as a yellow solid. ESI-MS (M+H)+: 306.2.
To a solution of 7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (173 mg, 0.79 mmol), tert-butyl 5-(6-aminopyridazin-3-yl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (200 mg, 0.66 mmol) and DIEA (423 mg, 3.28 mmol) in DMF (5 mL) was added HATU (374 mg, 0.99 mmol) at rt, the mixture was stirred for 2 h. After diluting with water, the solid was collected by filtration, washed with H2O and dried to give title product (300 mg, Y: 90%) as a yellow solid. ESI-MS (M+H)+: 508.5.
To a solution of the tert-butyl 5-(6-(7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxamido)pyridazin-3-yl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (300 mg, 0.59 mmol) in EtOAc (3 mL) was added HCl-EA (3M, 3 mL), the mixture was stirred at rt for 1 h. After concentration, the crude was purified by prep-HPLC (0.1% FA in water/CH3CN) to give title product (71 mg, yield: 29.4%) as a yellow solid. ESI-MS (M+H)+: 408.2. 1H NMR (400 MHz, MeOD-d4) δ 9.02 (s, 1H), 8.40 (s, 1H), 8.35 (d, J=9.7 Hz, 1H), 7.56 (s, 1H), 7.09 (d J=9.8 Hz, 1H), 6.93 (s, 1H), 4.35 (q, J=6.9 Hz, 2H), 3.72-3.58 (m, 6H), 3.30-3.25 (m, 4H), 2.37 (s, 3H), 1.63 (t, J=6.9 Hz, 3H).
To a mixture of 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (200 mg, 0.90 mmol) in ACN (10 mL) were added tert-butyl 4-(6-aminopyridazin-3-yl)piperazine-1-carboxylate (228 mg, 0.81 mmol), NMI (298 mg, 3.63 mmol) and TCFH (510 mg, 1.81 mmol). The mixture was stirred at rt for 2 h. The precipitate was filtered and concentrated in vacuo to give title product (280 mg, Y: 75.2%) as a white solid. ESI-MS (M+H)+: 482.4. 1H NMR (400 MHz, CDCl3) δ 10.96 (s, 1H), 8.72 (s, 1H), 8.53 (d, J=9.8 Hz, 1H), 8.00 (s, 1H), 7.09 (s, 1H), 7.04 (d, J=9.8 Hz, 1H), 4.32 (t, J=7.0 Hz, 2H), 4.20 (s, 3H), 3.59 (br s, 8H), 1.70 (t, J=7.0 Hz, 3H), 1.49 (s, 9H).
To a solution of tert-butyl 4-(6-(6-ethoxy-2-methyl-2H-indazole-5-carboxamido)pyridazin-3-yl)piperazine-1-carboxylate (250 mg, 0.51 mmol) in EA (5 mL) was added 4M HCl/EA (10 mL). The mixture was stirred at rt for 2 h. The precipitate was filtered, washed with EA (10 mL) and lyophilized to give title product (261 mg, yield: 98.3%) as a yellow solid. ESI-MS (M+H)+: 382.2. 1H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H), 9.59 (s, 2H), 8.48 (s, 1H), 8.41 (d, J=9.9 Hz, 1H), 8.38 (s, 1H), 7.79-7.67 (m, 1H), 7.14 (s, 1H), 4.26 (q, J=6.8 Hz, 2H), 4.15 (s, 3H), 3.93-3.87 (m, 4H), 3.24-3.22 (m, 4H), 1.49 (t, J=6.9 Hz, 3H).
To a solution of 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (200 mg, 0.90 mmol), tert-butyl (R)-4-(6-aminopyridazin-3-yl)-2-methylpiperazine-1-carboxylate (266 mg, 0.90 mmol) and NMI (149 mg, 1.81 mmol) in ACN (5 mL) was added TCFH (380 mg 1.36 mmol). The mixture was stirred at it for 2 h. The precipitate was filtered and dried under vacuum to give title product (117 mg, 33%) as an off-white solid. ESI-MS (M+H)+: 496.3.
To a solution of tert-butyl (R)-4-(6-(6-ethoxy-2-methyl-2H-indazole-5-carboxamido) pyridazin-3-yl)-2-methylpiperazine-1-carboxylate (100 mg, 0.20 mmol) in EA (2 mL) was added 4M HCl in EA (2 mL). The mixture was stirred at rt. for 2 h. The precipitate was filtered, washed with EA (10 mL) and lyophilized to give title product (31.33 mg, 40%) as an off-white solid. ESI-MS (M+H)+: 396.6. 1H NMR (400 MHz, DMSO-d6) δ 11.09 (s, 1H), 9.85-9.77 (m, 2H), 8.51-8.34 (m, 3H), 7.83 (d, J=10.1 Hz, 1H), 7.14 (s, 1H), 4.38 (t, J=13.6 Hz, 2H), 4.26 (q, J=6.9 Hz, 2H), 4.15 (s, 3H), 3.47-3.46 (m, 1H), 3.38-3.37 (m, 2H), 3.26-3.25 (m, 1H), 3.12-3.11 (m, 1H), 1.48 (t, J=6.9 Hz, 3H), 1.34 (d, J=6.4 Hz, 3H).
To a mixture of 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (350 mg, 1.59 mmol), tert-butyl (2S,6R)-4-(6-aminopyridazin-3-yl)-2,6-dimethylpiperazine-1-carboxylate (420 mg, 1.36 mmol) and NMI (336 mg, 4.00 mmol) in ACN (15 mL) was added TCFH (573 mg, 2.05 mmol) at rt. The mixture was stirred at rt for 2 h. The precipitate was filtered, washed with MeCN and dried under vacuum to provide title product (450 mg, Y: 55.6%) as an off-white solid. ESI-MS (M+H)+: 510.4. 1H NMR (400 MHz, DMSO-d6) δ 10.86 (s, 1H), 8.46 (d, J=3.5 Hz, 2H), 8.27 (d, J=9.8 Hz, 1H), 7.47 (d, J=9.9 Hz, 1H), 7.15 (s, 1H), 4.26-4.24 (m, 6H), 4.14 (s, 3H), 3.05 (dd, J=13.0, 4.3 Hz, 2H), 1.53 (t, J=6.9 Hz, 3H), 1.44 (s, 9H), 1.19 (d, J=6.8 Hz, 6H).
To a mixture of tert-butyl (2S,6R)-4-(6-(6-ethoxy-2-methyl-2H-indazole-5-carboxamido)pyridazin-3-yl)-2,6-dimethylpiperazine-1-carboxylate (450 mg, 0.88 mmol) in EA (10 mL) was added 3 M HCl/EA (10 mL). The mixture was stirred at rt for 2 h. The precipitate was filtered, washed with EA (20 mL) and lyophilized to give title product (321 mg, Y: 88.9%) as a white solid. ESI-MS (M+H)+: 410.3. 1H NMR (400 MHz, MeOD-d4) a 8.48 (d, J=10.0 Hz, 2H), 8.20-8.19 (m, 2H), 7.16 (s, 1H), 4.56 (d, J=12.6 Hz, 2H), 4.34 (q, J=6.9 Hz, 2H), 4.23 (s, 3H), 3.56-3.55 (m, 2H), 3.19 (dd, J=14.3, 11.6 Hz, 2H), 1.57 (t, J=6.9 Hz, 3H), 1.46 (d, J=6.5 Hz, 6H).
To a solution of 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (185 mg, 0.84 mmol) in DMF (10 mL) were added DIEA (400 mg, 2.12 mmol), HATU (400 mg, 1.05 mmol) and 4-(6-aminopyridazin-3-yl)-6-methyl-3,6-dihydropyridine-1(2H)-carboxylate (200 mg, 0.70 mmol). The reaction mixture was stirred at it for 16 h. The mixture was diluted with water (30 mL). The precipitate was filtered, washed with water and dried in vacuo to give title product (45 mg, 10.87%) as a yellow solid. ESI-MS (M+H)+: 493.3.
A mixture of 6-ethoxy-2-methyl-N-(6-(6-methyl-1,2,3,6-tetrahydropyridin-4-yl)pyridazin-3-yl)-2H-indazole-5-carboxamide (45 mg, 0.091 mmol) in 3 M HCl/EA (3 mL) was stirred at it for 2 h. The mixture was concentrated in vacuo. The residue was purified by prep-HPLC (0.05% FA in water/CH3CN) to give title product (10 mg, Y: 28.03%) as a yellow solid. ESI-MS (M+H)+: 393.7. 1H NMR (400 MHz, MeOD-d4) δ 8.69-8.54 (m, 2H), 8.35 (s, 1H), 7.96 (dd, J=9.4, 5.5 Hz, 1H), 7.14 (s, 1H), 6.86-6.39 (m, 1H), 4.38 (q, J=7.0 Hz, 2H), 4.21 (s, 3H), 3.69 (d, J=18.9 Hz, 1H), 3.19-2.24 (m, 4H), 1.68 (t, J=6.9 Hz, 3H), 1.34 (t, J=6.7 Hz, 3H).
To a mixture of 6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxylic acid (4.2 g, 19.09 mmol) in ACN (80 mL) were added tert-butyl 4-(6-aminopyridazin-3-yl)piperazine-1-carboxylate (5.8 g, 20.90 mmol), NMI (4.7 g, 57.27 mmol), TCFH (8.1 g, 28.63 mmol). The mixture was stirred at rt for 2 h. LCMS showed the reaction was completed. The precipitate was filtered, washed with MeCN and concentrated in vacuo to give the title product (8.0 g, 87%) as a yellow solid. ESI-MS (M+H+):482.1. 1H NMR (400 MHz, CDCl3) δ 10.73 (s, 1H), 8.49 (d, J=9.8 Hz, 1H), 8.42 (s, 1H), 8.13 (s, 1H), 7.04 (d, J=9.8 Hz, 1H), 6.46 (s, 1H), 4.25 (q, J=6.9 Hz, 2H), 3.64-3.57 (m, 8H), 2.49 (s, 3H), 1.69 (t, J=7.0 Hz, 3H), 1.49 (s, 9H).
To a mixture of tert-butyl 4-(6-(6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxamido)pyridazin-3-yl)piperazine-1-carboxylate (4 g, 8.31 mmol) in EA (50 mL) was added 3M HCl/EA (50 mL). The mixture was stirred at rt for 2 h. LCMS showed the reaction was completed. The precipitate was filtered and dried in vacuo to give the title product (3.5 g, 86.4%) as a yellow solid. ESI-MS (M+H+):382.4. 1H NMR (400 MHz, DMSO-d6) δ 10.97 (s, 1H), 9.32 (s, 2H), 8.51 (s, 1H), 8.31 (d, J=9.7 Hz, 1H), 8.10 (s, 1H), 7.59 (s, 1H), 6.56 (s, 1H), 4.24-4.20 (m, 2H), 3.86-3.79 (m, 4H), 3.27-3.17 (m, 4H), 2.39 (s, 3H), 1.45 (t, J=6.9 Hz, 3H).
To a solution of 6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxylic acid (150 mg, 0.68 mmol) in CH3CN (20 mL) were added tert-butyl 4-(6-aminopyridazin-3-yl)-3,6-dihydropyridine-1(2H)-carboxylate (188.16 mg, 0.68 mmol), TCFH (285.60 mg, 1.03 mmol) and NMI (279.54 mg, 3.42 mmol). The mixture was stirred at room temperature for 16 hours. The precipitate was filtered, washed with CH3CN and dried to give title product (200 mg, yield: 61%) as a yellow solid. ESI-MS: [M+H]+:479.1.
To a solution of tert-butyl 4-(6-(6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxamido)pyridazin-3-yl)-3,6-dihydropyridine-1(2H)-carboxylate (200 mg, 0.42 mmol) in EA (5 mL) was added 3M HCl/EA (5 mL). The mixture was stirred at room temperature for 2 h. The precipitate was filtered, washed with EA (50 mL) and lyophilized to give title product (151.6 mg, yield: 95%) as a yellow solid. ESI-MS: [M+H]+:379.2. 1H NMR (400 MHz, DMSO-d6) δ 11.27 (s, 1H), 9.25-9.07 (m, 2H), 8.55-8.47 (m, 2H), 8.15-8.07 (m, 2H), 6.74 (s, 1H), 6.57 (s, 1H), 4.27-4.21 (m, 2H), 3.88-3.83 (m, 2H), 3.38-3.34 (m, 2H), 2.93-2.87 (m, 2H), 2.40 (s, 3H), 1.46 (t, J=6.9 Hz, 3H).
To a mixture of 6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxylic acid (150 mg, 0.68 mmol), tert-butyl 4-(6-aminopyridazin-3-yl)-2,2-dimethylpiperazine-1-carboxylate (210 mg, 0.68 mmol) and NMI (117 mg, 1.42 mmol) in ACN (3 mL) was added TCFH (381 mg, 1.36 mol). The mixture was stirred at rt. for 0.5 h. The precipitate was filtered, washed with CH3CN and dried to give title product (300 mg, 86.4% yield) as a white solid. ESI-MS (M+H)+: 510.3. 1H NMR (400 MHz, DMSO-d6) δ 10.77 (s, 1H), 8.48 (s, 1H), 8.30-8.02 (m, 2H), 7.22 (d, J=9.8 Hz, 1H), 6.54 (s, 1H), 4.22 (dd, J=13.6. 6.7 Hz, 2H), 3.80-3.76 (m, 4H), 3.65 (s, 2H), 3.51 (d, J=5.9 Hz, 3H), 2.37 (s, 3H), 1.45 (t, J=7.0 Hz, 3H), 1.42 (s, 9H), 1.34 (s, 6H).
To a mixture of tert-butyl 4-(6-(6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxamido)pyridazin-3-yl)-2,2-dimethylpiperazine-1-carboxylate (280 mg, 0.55 mmol) in EA (2 mL) was added HCl (2 mL, 4M in EA). The mixture was stirred at rt for 2 h. The precipitate was filtered, washed with EA (20 mL) and lyophilized to give title product (251.78 mg, 95.91% yield) as a white solid. ESI-MS (M+H)+: 410.3. 1H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 9.90 (s, 2H), 8.50 (s, 1H), 8.36 (d, J=9.8 Hz, 1H), 8.06 (s, 1H), 7.83 (d, J=9.9 Hz, 1H), 6.55 (s, 1H), 4.20 (d, J=6.9 Hz, 2H), 3.94 (br s, 2H), 3.81 (br s, 2H), 3.26 (br s, 2H), 2.39 (s, 3H), 1.46-1.38 (m, 9H).
To a solution of tert-butyl (S)-4-(6-aminopyridazin-3-yl)-2-methyl-3,6-dihydropyridine-1(2H)-carboxylate (400 mg, 1.38 mmol) in DMF (5 mL) were added 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (365 mg, 1.66 mmol), DIEA (712 mg, 5.52 mmol) and HATU (630 mg, 1.66 mmol). The mixture was stirred at 50° C. for 16 h. The mixture was diluted with H2O (10 mL) and the precipitate was filtered, washed with water (20 mL) and dried in vacuo to give title product (300 mg, 44.12%) as a yellow solid. ESI-MS (M+H)+: 507.4. 1H NMR (400 MHz, CDCl3) δ 11.24 (s, 1H), 8.74 (s, 1H), 8.67 (d, J=9.3 Hz, 1H), 8.01 (s, 1H), 7.72 (d, J=9.7 Hz, 1H), 7.11 (s, 1H), 6.65-6.59 (m, 1H), 4.39-4.32 (m, 2H), 4.21 (s, 3H), 4.18-4.14 (m, 2H), 2.89 (s, 2H), 1.52 (s, 9H).
To a solution of tert-butyl 4-(6-(6-ethoxy-2-methyl-2H-indazole-5-carboxamido)pyridazin-3-yl)-6,6-dimethyl-3,6-dihydropyridine-(2H)-carboxylate (70 mg, 0.14 mmol) in EA (2 mL) was added 3 M HCl/EA (1 mL). The mixture was stirred at rt for 2 h. The mixture was concentrated in vacuo. The crude was purified by prep-HPLC (0.05% FA in water/CH3CN) to give title product (6.78 mg, Y: 10.85%) as a yellow solid. ESI-MS (M+H+): 407.3. 1H NMR (400 MHz, MeOD-d4) δ 8.69 (d, J=9.4 Hz, 1H), 8.63 (s, 1H), 8.55 (s, 1H), 8.37 (s, 1H), 8.03 (d, J=9.5 Hz, 1H), 7.17 (s, 1H), 6.71 (s, 1H), 4.40 (q, J=7.0 Hz, 2H), 4.22 (s, 3H), 4.03-3.85 (m, 2H), 2.98-2.86 (m, 2H), 1.68 (t, J=6.9 Hz, 3H), 1.60-1.47 (m, 6H).
To a mixture of tert-butyl 4-(6-aminopyridazin-3-yl)piperidine-1-carboxylate (150 mg, 0.54 mmol), 6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxylic acid (130.58 mg, 0.54 mmol) in ACN (10 mL) were added NMI (132.84 mg, 1.62 mmol) and TCFH (227.61 mg, 0.81 mmol). The mixture was stirred at rt for 2 h. The precipitate was filtered and concentrated in vacuo to give title product (80 mg, 30.86%) as a white solid. ESI-MS (M+H+): 481.3. 1H NMR (400 MHz, DMSO-d6) δ 11.14 (s, 1H), 8.52 (s, 1H), 8.40 (d, J=9.2 Hz, 1H), 8.11 (s, 1H), 7.73 (d, J=9.2 Hz, 1H), 6.56 (s, 1H), 4.23 (q, J=6.9 Hz, 2H), 4.09 (d, J=12.3 Hz, 2H), 3.09-3.08 (m, 1H), 2.89-2.88 (m, 2H), 2.39 (s, 3H), 1.89-1.87 (m, 2H), 1.68-1.57 (m, 2H), 1.47-1.39 (m, 12H).
A mixture of tert-butyl 4-(6-(6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxamido)pyridazin-3-yl)piperidine-1-carboxylate (80 mg, 0.17 mmol) in EA/HCl (5 mL) was stirred at rt for 1 h. The precipitate was filtered and dried under vacuum to give title product (31.83 mg, 49%) as a yellow solid. ESI-MS (M+H+): 381.2. 1H NMR (400 MHz, DMSO-d6) δ 11.20 (s, 1H), 9.14 (s, 1H), 8.91 (s, 1H), 8.52 (s, 1H), 8.45 (d, J=9.2 Hz, 1H), 8.11 (s, 1H), 7.72 (d, J=9.2 Hz, 1H), 6.56 (s, 1H), 4.22 (t, J=6.9 Hz, 2H), 3.39 (d, J=12.6 Hz, 2H), 3.25-3.19 (m, 1H), 3.04 (d, J=11.1 Hz, 2H), 2.40 (s, 3H), 2.12-1.99 (m, 4H), 1.45 (t, J=6.9 Hz, 3H).
To a mixture of 2-chloro-6-methylpyrazine (4.85 g, 42.92 mmol) and Tributyl(vinyl)tin (13.2 g, 42.92 mmol) in DMF (100 mL) were added LiCl (1.75 g, 42.92 mmol), DIEA (5.37 g, 42.92 mmol) and Pd(PPh3)4(2.19 g, 1.90 mmol), the mixture was charged with N2 for three times and stirred at 120° C. for 16 h. The reaction mixture was diluted with water (200 mL), extracted with DCM (250 mL×3). The combined organics were washed with brine (200 mL×3), dried over Na2SO4 and evaporated in vacuo. The crude was purified by silica gel column chromatography (PE:EA=4:1) to give tittle product (900 mg, Y: 87.7%) as a yellow solid. ESI-MS (M+H)+: 121.2. 1H NMR (400 MHz, DMSO-d6) δ 8.55 (s, 1H), 8.42 (s, 1H), 6.85 (dd, J=17.5, 10.9 Hz, 1H), 6.35 (dd, J=17.5, 1.4 Hz, 11), 5.59 (d, J=10.9 Hz, 1H), 2.53-2.49 (m, 3H).
To a mixture of 2-methyl-6-vinylpyrazine (4 g, 33.30 mmol) in AcOH (50 mL) was added PtO2 (1 g, 4.40 mmol), the mixture was charged with H2 for three times and stirred at 50° C. for 24 h. The reaction mixture was filtered and the filtrate was evaporated in vacuo. The crude was purified by silica gel column chromatography eluted with (DCM/MeOH=10:1) to give title compound (2 g, 47%) as a yellow solid. ESI-MS (M+H)+: 129.2. 1H NMR (400 MHz, DMSO-d6) δ 2.88-2.65 (m, 3H), 2.19-2.04 (m, 2H), 1.84-1.82 (m, 3H), 1.33-1.17 (m, 2H), 0.96-0.89 (m, 3H), 0.88-0.82 (m, 3H).
To a mixture of 2-ethyl-6-methylpiperazine (2 g, 15.50 mmol) in 1,4-dioxane (50 mL) were added 3,6-dichloropyridazine (3.47 g, 23.26 mmol) and TEA (4.73 g, 46.50 mmol), the mixture was stirred at 120° C. for 16 h. The reaction mixture was evaporated in vacuo. The crude was purified by silica gel column chromatography eluted with (DCM/MeOH=10:1) to give title compound (1.5 g, 40%) as a yellow solid. ESI-MS (M+H)+: 241.0.
To a mixture of 3-chloro-6-(3-ethyl-5-methylpiperazin-1-yl)pyridazine (1 g, 4.17 mmol) in DCM (10 mL) were added Di-tert-butyl dicarbonate (7 g, 41.70 mmol) and DMAP (1.52 g, 12.5 mmol). The mixture was stirred at 50° C. for 16 h. The reaction was diluted with water (15 mL), extracted with DCM (20 mL×3). The combined organic was washed with brine (20 mL), dried over Na2SO4 and evaporated in vacuo. The crude was purified by silica gel column chromatography (PE:EA=4:1) to give tittle product (250 mg, Y: 17.5%) as a yellow solid. ESI-MS (M+H+): 341.3. 1H NMR (400 MHz, CDCl3) δ 7.21 (d, J=9.5 Hz, 1H), 6.87 (d, J=9.5 Hz, 1H), 4.37-4.27 (m, 2H), 4.13-4.03 (m, 2H), 3.25 (dd, J=13.2, 4.6 Hz, 1H), 3.11 (dd, J=13.3, 4.5 Hz, 1H), 1.64-1.62 (m, 2H), 1.49 (s, 9H), 1.27-1.20 (m, 3H), 0.97 (t, J=7.4 Hz, 3H).
To a mixture of tert-butyl 4-(6-chloropyridazin-3-yl)-2-ethyl-6-methylpiperazine-1-carboxylate (140 mg, 0.41 mmol) and Cs2CO3 (401 mg, 1.23 mmol) in 1,4 dioxane (5 mL) were added BINAP (51 mg, 0.24 mmol), Pd(dba)3 (38 mg, 0.04 mmol) and diphenylmethanimine (82 mg, 0.45 mmol). The mixture was stirred at 110° C. for 16 h. The reaction mixture was diluted with water (15 mL), extracted with DCM (20 mL×3). The combined organics were washed with brine (20 mL), dried over Na2SO4 and evaporated in vacuo to give crude title product (100 mg, Y: 51.5%) as a yellow oil. ESI-MS (M+H+):486.3.
To a mixture of tert-butyl 4-(6-((diphenylmethylene)amino)pyridazin-3-yl)-2-ethyl-6-methylpiperazine-1-carboxylate (100 mg, 0.21 mmol) in EtOAc (2 mL) was added 3M HCl/EtOAc (2 mL), the mixture was stirred for 1 h at rt. The precipitate was filtered to afford title compound (30 mg, 58.3%) as a yellow solid. ESI-MS (M+H)+: 222.2. 1H NMR (400 MHz, DMSO-d6) δ 7.11 (d, J=9.6 Hz, 1H), 6.72 (d, J=9.5 Hz, 1H), 5.59 (s, 2H), 3.90-3.77 (m, 2H), 2.79-2.73 (m, 1H), 2.61-2.55 (m, 1H), 2.15 (t, J=10.6 Hz, 2H), 1.42-1.30 (m, 2H), 1.01 (d, J=6.3 Hz, 3H), 0.92 (t, J=7.5 Hz, 3H).
To a solution of 6-(3-ethyl-5-methylpiperazin-1-yl)pyridazin-3-amine hydrochloride (30 mg, 0.11 mmol) and 7-ethoxy-2-methylimidazo[1,2-a]pyridine-6-carboxylic acid (35 mg, 0.16 mmol) in MeCN (3 mL) were added NMI (34 mg, 0.44 mmol) and TCFH (58 mg, 0.22 mmol), the mixture was stirred for 1 h at 50° C. for 2 h. The precipitate was filtered and purified by prep-HPLC (0.05% NH3·H2O in water/CH3CN) to give title compound (1.93 mg, 4.1% yield) as a yellow solid. 1H NMR (400 MHz, MeOD-d4) δ 9.03 (d, J=4.1 Hz, 1H), 8.40-8.33 (m, 1H), 7.55 (s, 1H), 7.44-7.36 (m, 1H), 6.95 (d, J=4.5 Hz, 1H), 4.61 (br s, 2H), 4.40-4.33 (m, 2H), 4.19 (d, J=12.8 Hz, 1H), 3.02-2.91 (m, 1H), 2.81-2.70 (m, 1H), 2.56-2.50 (m, 1H), 2.38 (s, 3H), 1.66 (t, J=6.9 Hz, 3H), 1.61-1.50 (m, 2H), 1.21 (d, J=6.4 Hz, 3H), 1.07 (t, J=7.5 Hz, 3H). ESI-MS (M+H)+:424.3
To a solution of 7-ethoxy-2-methyl-N-(6-(1,2,3,6-tetrahydropyridin-4-yl)pyridazin-3-yl)imidazo[1,2-a]pyridine-6-carboxamide (40 mg, 0.10 mmol), AcOH (32 mg, 0.52 mmol) and (HCHO)n (16 mg, 0.52 mmol) in MeOH (4 mL) was added NaBH3CN (20 mg, 0.31 mmol). The mixture was stirred at 40° C. for 4 h. The mixture was filtered and the filtrate was concentrated in vacuo, the residue was purified by prep-HPLC (0.05% NH3·H2O in water/CH3CN) to give title product (20.41 mg, 100%) as a yellow solid. ESI-MS (M+H)+: 393.2. 1H NMR (400 MHz, MeOD-d4) δ 9.16 (s, 1H), 8.65 (d, J=9.4 Hz, 1H), 8.06 (d, J=9.5 Hz, 1H), 7.70 (s, 1H), 7.14 (s, 1H), 6.71 (s, 1H), 4.44 (q, J=6.9 Hz, 2H), 4.06 (br s, 2H), 3.61 (t, J=5.8 Hz, 2H), 3.15 (br s, 2H), 3.06 (s, 3H), 2.45 (s, 3H), 1.67 (t, J=6.9 Hz, 3H).
To a solution of tert-butyl (R)-4-(6-aminopyridazin-3-yl)-6-methyl-3,6-dihydropyridine-1(2H)-carboxylate (400 mg, 1.38 mmol) in DMF (5 mL) were added 6-ethoxy-2-methyl-2H-indazole-5-carboxylic acid (365 mg, 1.66 mmol), DIEA (712 mg, 5.52 mmol) and HATU (630 mg, 1.66 mmol). The mixture was stirred at 70° C. for 16 h. The mixture was diluted with H2O (10 mL) and the precipitate was filtered, washed with H2O (20 mL) and dried in vacuo to give title product (250 mg, 36.76%) as a yellow solid. ESI-MS (M+H)+: 493.3. 1H NMR (400 MHz, CDCl3) δ 11.24 (s, 1H), 8.74 (s, 1H), 8.66 (d, J=9.1 Hz, 1H), 8.02 (s, 1H), 7.72-7.60 (m, 1H), 7.11 (s, 1H), 6.52 (d, J=11.2 Hz, 1H), 4.82-4.65 (m, 1H), 4.35 (q, J=13.9, 6.9 Hz, 2H), 4.21 (s, 3H), 3.02-2.79 (m, 2H), 1.74-1.71 (m, 2H), 1.50 (s, 9H), 1.31 (d, J=6.6 Hz, 3H), 1.17 (d, J=6.7 Hz, 3H).
To a solution of tert-butyl (R)-4-(6-(6-ethoxy-2-methyl-2H-indazole-5-carboxamido)pyridazin-3-yl)-6-methyl-3,6-dihydropyridine-1(2H)-carboxylate (250 mg, 0.51 mmol) in EA (5 mL) was added 3 M HCl/EA (5 mL). The mixture was stirred at it for 2 h. The mixture was concentrated in vacuo. The crude was purified by prep-HPLC (0.05% FA in water/CH3CN) to give title product (95.07 mg, Y: 42.79%) as a yellow solid. ESI-MS (M+H+): 393.2. 1H NMR (400 MHz, DMSO-d6) δ 11.23 (s, 1H), 8.52-8.44 (m, 3H), 8.27 (s, 1H), 8.02 (d, J=9.5 Hz, 1H), 7.17 (s, 1H), 6.68 (d, J=35.3 Hz, 1H), 4.30 (q, J=6.8 Hz, 2H), 4.15 (s, 3H), 3.12-2.98 (m, 2H), 2.94-2.85 (m, 1H), 2.75-2.61 (m, 2H), 1.54 (t, J=6.9 Hz, 3H), 1.27 (t, J=6.5 Hz, 3H).
To a mixture of 6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxylic acid (500 mg, 2.27 mmol), tert-butyl (2S,6R)-4-(6-aminopyridazin-3-yl)-2,6-dimethylpiperazine-1-carboxylate (696.89 mg, 2.27 mmol) in DMF (10 mL) were added HATU (1293.9 mg, 3.40 mmol) and DIEA (1171.32 mg, 9.08 mmol). The mixture was stirred at it for 2 h. The reaction mixture was diluted with H2O (30 mL), the precipitate was filtered, washed with water and dried in vacuo to give title product (520 mg, yield: 44.93%) as a brown solid. ESI-MS (M+H+): 510.2. 1H NMR (40) MHz, DMSO-d6) δ 10.81 (s, 1H), 8.50 (s, 1H), 8.29-8.05 (m, 2H), 7.48 (d, J=9.9 Hz, 1H), 6.55 (s, 1H), 4.29-4.22 (m, 4H), 4.20-4.14 (m, 2H), 3.06 (dd, J=13.0, 4.3 Hz, 2H), 2.39 (s, 3H), 1.50-1.42 (m, 12H), 1.18 (d, J=6.8 Hz, 6H).
A mixture of tert-butyl (2R,6S)-4-(6-(6-ethoxy-2-methylpyrazolo[1,5-a]pyridine-5-carboxamido)pyridazin-3-yl)-2,6-dimethylpiperazine-1-carboxylate (520 mg, 1.02 mmol) in EA/HCl (5 mL) was stirred at rt for 2 h. The precipitate was filtered, washed with EA and dried in vacuo to give title product (350 mg, 83.69%) as a yellow solid. ESI-MS (M+H+): 410.4. 1H NMR (400 MHz, DMSO-d6) δ 11.03 (s, 1H), 9.99 (s, 1H), 9.64 (s, 1H), 8.50 (s, 1H), 8.34 (d, J=9.9 Hz, 1H), 8.08 (s, 1H), 7.75 (d, J=10.0 Hz, 1H), 6.55 (s, 1H), 4.47 (d, J=13.0 Hz, 2H), 4.21 (q, J=6.9 Hz, 2H), 3.39-3.29 (m, 2H), 3.22-3.11 (m, 2H), 2.39 (s, 3H), 1.44 (t, J=6.9 Hz, 3H), 1.36 (d, J=6.4 Hz, 6H).
The biological activity of the compounds of the present disclosure was determined utilizing the assay described herein.
Mini-gene reporter constructs for each target site can be constructed by first PCR amplifying the region-of-interest including the sequence of the alternatively skipped, sequences of the immediate upstream and downstream introns, and sequences of the immediate upstream and downstream exons. Then the 3′ end of the amplified sequence can be ligated to a firefly luciferase reporter gene and cloned into the pcDNA3.1 vector backbone. The final reporter construct can be transiently transfected into HEK293 cells using Lipofectamine 3000 transfection reagents. Compounds that can induce the inclusion of the skipped exon in the reporter construct would increase the reporter firefly luciferase activity.
Test compounds may be diluted in duplicates and dispensed into an assay plate. Transient mini-gene reporter cell line stock is resuspended and dispensed into the assay plate. Assay plates are then centrifuged and incubated. A luciferase reagent (e.g. ONE-Glo firefly luciferase reagent from Promega®) may be added to the assay plate and the luminescence (RLU) signals recorded. EC50 values may be determined by curve fitting in Levenberg-Marquardt algorithm.
In Table D below, A indicates a IC50 (μM)<1 μM, B indicates a IC50 (μM) 1 μM to <10 μM, and C indicates a IC50 (μM) 10 μM to <50 μM. In Table D below, A indicates a >75% Effect at 5 μM μM. B indicates a 50 to <75% Effect at 5 μM μM, and C indicates a <50% Effect at 5 μM.
The details of one or more embodiments of the disclosure are set forth in the accompanying description above. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications cited in this specification are incorporated by reference.
The foregoing description has been presented only for the purposes of illustration and is not intended to limit the disclosure to the precise form disclosed, but by the claims appended hereto.
This application claims priority to and benefit of U.S. Provisional Application No. 63/280,939, filed Nov. 18, 2021, the contents of which are hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/080182 | 11/18/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63280939 | Nov 2021 | US |
