Patent Application
20250002460
The present invention generally relates to compounds suitable as sortilin inhibitors, and to uses thereof.
Sortilin is a type I transmembrane protein that acts both as a receptor of several ligands, and in the sorting of cargo from the trans-Golgi network (TGN) to late endosomes and lysosomes for degradation. Sortilin binds the secreted protein progranulin (PGRN) and targets it for lysosomal degradation, thus, negatively regulating extracellular levels of PGRN. PGRN is a secreted, growth factor-like, trophic, and anti-inflammatory protein, which also plays a role as an adipokine involved in diet-induced obesity and insulin resistance.
PGRN deficiency accounts for roughly 25% of all heritable forms of frontotemporal dementia (FTD), an early-onset neurodegenerative disease. Patients with heterozygous loss-of-function mutations in PGRN have about 50% reduced extracellular levels of the protein and they will invariably develop FTD, making PGRN a causal gene for the disease. In addition, PGRN mutant alleles have been identified in Alzheimer's disease patients. Importantly, PGRN acts protective in several disease models with increased PGRN levels accelerating behavioral recovery from ischemia, suppressing locomotor deficits in a Parkinson's disease model, attenuating pathology in a model of amyotrophic lateral sclerosis (ALS) and arthritis and preventing memory deficits in an Alzheimer's disease model.
Sortilin also binds pro-neurotrophins, such as pro-nerve growth factor (pro-NGF), pro-brain-derived neurotrophic factor (pro-BDNF), and pro-neurotrophin-3, which harbor a pro-domain and are typically pro-apoptotic. Such pro-neurotrophin precursors are released during stress, and sortilin is involved in regulating their release as well as stimulation of apoptosis in conjunction with p75NTR.
Sortilin also binds to p75NTR directly. Sortilin further binds neurotensin in a region that partially overlaps with PGRN binding and is therefore sometimes also referred to as NTR3 receptor. Sortilin also interacts with the Trk receptors NTRK1, NTRK2, and NTRK3 and can regulate their anterograde axonal transport and signaling. Sortilin additionally interacts with and regulates the processing and trafficking of amyloid precursor protein (APP) and the resulting production of pathological beta amyloid peptides.
Sortilin has been shown to bind to apolipoproteins and lipoprotein lipase and, thus, deficiency leads to reduced very low density lipoprotein (VLDL) release from liver and reduced cholesterol. Sortilin has also been implicated in binding to APP directly and also to the APP processing enzyme beta-secretase 1 (BACE1). Sortilin also binds to apolipoprotein E (APOE), and to the amyloid beta peptide. Sortilin has also been shown to bind to and regulate extracellular levels of proprotein convertase subtilisin/kexin type 9 (PCSK9), which directs low density lipoprotein receptor for degradation in lysosomes, resulting in increased levels of low density lipoprotein (LDL) cholesterol.
When present at intracellular vesicles, such as endosomes, the amino terminal extracellular domain of sortilin is directed towards the lumen, where cargo of the vesicle is present. The carboxy terminal intracellular/cytoplasmic domain of sortilin, however, binds to a series of adaptor proteins, which regulate its trafficking from the surface and within intracellular compartments. These include clathrin adaptor protein 2 (AP2) and the retromer complex/AP1, which modulate movement from early endosomes to Golgi for recycling, and interaction with GGA (Golgi-localizing, gamma-ear containing, ADP-ribosylation factor binding) family proteins for movement from Golgi directly to early endosomes, usually for subsequent degradation through lysosomes. Thus, sortilin can bind ligands at its luminal domain, while engaging the cytoplasmic adaptors that determine its destination to determine intracellular fates, such as degradation for PGRN and other factors.
Through its various interactions with proteins, such as PGRN, sortilin and its multiple ligands have been shown to be involved in various diseases, disorders, and conditions, such as FTD, ALS, ALS-FTD phenotypes, Alzheimer's disease, Parkinson's disease, depression, neuropsychiatric disorders, vascular dementia, seizures, retinal dystrophy, age related macular degeneration, glaucoma, traumatic brain injury, aging, seizures, wound healing, stroke, dermatology related diseases, autoimmune diseases, arthritis, atherosclerotic vascular diseases and cancers (WO 2009/140972, WO 2014/114779, WO 2016/164637, Breast Cancer Research 2018 20: 137, Bioorganic & Medicinal Chemistry Letters 2020 30: 127403).
Accordingly, there is a need for therapeutic compounds that bind to sortilin and block the binding of sortilin to its ligands, such as PGRN, or otherwise modulate the effective concentration of the ligands, in order to treat one or more diseases, disorders, and conditions associated with sortilin activity.
It is a general objective to provide therapeutic compounds that bind to sortilin and block the binding of sortilin to its ligands, such as PGRN.
It is a particular objective to provide such therapeutic compounds useful in treatment of diseases, disorders, and conditions associated with sortilin activity.
These and other objectives are met by embodiments as disclosed herein.
The invention is defined in the independent claims. Further embodiments of the invention are defined in dependent claims.
An aspect of the invention relates to a compound of formula I:
wherein
Another aspect of the invention relates to a pharmaceutical composition comprising a compound according to above and at least one pharmaceutically acceptable excipient, carrier or diluent.
Further aspects of the invention relate to a compound according to above for use as a medicament, for use in prevention or treatment of cancer and for use in prevention or treatment of a neurodegenerative disease, a psychiatric disease, a motor neuron disease, peripheral neuropathies, pain, neuroinflammation, atherosclerosis, hyperlipidemia, cardiovascular diseases, dermatology related disease, autoimmune diseases, preferably Alzheimer's disease and Parkinson's disease.
The present invention also relates to an intermediate for the production of a compound according to above. The intermediate is selected from the group consisting of:
The compounds of the invention are efficient sortilin inhibitors and bind to sortilin with a high affinity. Binding of the sortilin inhibitors to sortilin blocks or at least significantly inhibits the binding of other ligands, such as progranulin (PGRN), to sortilin. The sortilin inhibitors can thereby be used in the treatment of diseases, disorders, and conditions associated with such ligand-to-sortilin binding.
The embodiments, together with further objects and advantages thereof, may best be understood by making reference to the following description taken together with the accompanying drawings, in which:
The present invention generally relates to sortilin inhibitors and uses thereof.
The sortilin inhibitors of the present invention are capable of binding to sortilin and block or at least significantly inhibit the binding of other ligands, such as progranulin (PGRN), to sortilin. The sortilin inhibitors can thereby be used in the treatment of diseases, disorders, and conditions associated with sortilin activity.
Sortilin inhibitors are known in the art. WO 2014/114779 and Bioorganic & Medicinal Chemistry Letters 2014, 24: 177-180 disclose structurally unrelated N-substituted-5-substituted phthalic acids including N-(6-methyl-pyridin-2-yl)-5-trifluoromethyl-phthalamic acid also known as AF38469. AF38469 has in the literature a reported IC50 value of 330 nM for binding to sortilin. Bioorganic & Medicinal Chemistry Letters 2020, 30: 127403 discloses two series of inhibitors that disrupt PGRN-binding to sortilin. The best, optimized compounds have reported IC50 values of 20-90 nM.
The sortilin inhibitors of the present invention differ from the optimized compounds disclosed in Bioorganic & Medicinal Chemistry Letters 2020, 30: 127403 by, among others, having an unsaturated aliphatic moiety instead of such a saturated aliphatic moiety. Experimental data as presented herein shows that the binding affinity of the sortilin inhibitors can be unexpectedly increased (lower IC50 values) when comparing two sortilin inhibitors going from a saturated aliphatic moiety to an unsaturated aliphatic moiety.
Furthermore, AF38469 (referred to as RC3 herein) possessed sortilin agonistic properties when used alone, i.e., not in combination with PGRN. These agonistic properties resulted in increased cancer stem cell (sphere) formation in vitro and induced lung metastasis in vivo. The sortilin inhibitors of the present invention do not have these agonistic properties of AF38469 but rather have sortilin antagonistic properties when used alone resulting in no increase in sphere formation but rather a reduction in sphere formation.
The following abbreviations are used herein.
As used herein, the term “C1-C4 alkyl” includes methyl (—CH3), ethyl (—CH2CH3), propyl (—CH2CH2CH3), isopropyl (—CH(CH3)2), butyl (—CH2CH2CH2CH3), sec-butyl (—CH(CH3)(CH2CH3)), isobutyl (—CH2CH(CH3)2) and tert-butyl (—C(CH3)3).
As used herein, the term “C1-C4 alkoxy” includes methoxy (—OCH3), ethoxy (—OCH2CH3), propoxy (—OCH2CH2CH3), isopropoxy (—OCH(CH3)2), butoxy (—OCH2CH2CH2CH3), sec-butoxy (—OCH(CH3)(CH2CH3)), isobutoxy (—OCH2CH(CH3)2) and tert-butoxy (—OC(CH3)3).
As used herein, the term “halogen” includes fluorine (F), chlorine (Cl), bromine (Br) and iodine (I). In a particular embodiment, halogen as used herein includes F, Cl and Br.
As used herein, the term “heteroatom” relates to a non-carbon atom replacing a carbon atom in a ring structure. According to the invention, heteroatom is selected among nitrogen (N), oxygen (O) and sulfur (S). In a particular embodiment, heteroatom is selected among N and O. In a preferred embodiment, heteroatom is N.
As used herein, the term “sortilin” may refer to full length sortilin (also referred to as immature sortilin), comprising a signal peptide, a propeptide, a Vps10p domain, a 10 CC domain, a transmembrane domain and a large cytoplasmic tail, having an amino acid sequence according to SEQ ID NO: 1, or it may refer to mature sortilin comprising a Vps10p domain, a 10 CC domain, a transmembrane domain and a large cytoplasmic tail, having an amino acid sequence according to SEQ ID NO: 2, or a naturally occurring fragment, homologue or variant thereof. Sortilin as used herein also encompasses secreted or soluble sortilin (sSORT1), which lacks the transmembrane domain and the large cytoplasmic tail. It is understood that sortilin is capable of interacting with progranulin (PGRN) to form a sortilin/PGRN complex.
As used herein, the term “progranulin” or “PGRN” is a 593 amino acid long (SEQ ID NO: 3) and 68.5 kDa protein. Progranulin is precursor protein for granulin. Cleavage of progranulin produces a variety of smaller active cleavage products named granulin A, granulin B, granulin C, etc.
As used herein, the terms “sortilin antagonist” or “sortilin inhibitor” refer to a compound that interferes with, blocks, or otherwise attenuates the effect of, PGRN-binding to a sortilin molecule and preventing or at least inhibiting the formation of the complex between sortilin and PGRN. Compounds of formula I as disclosed herein act as sortilin antagonists or inhibitors by binding to sortilin.
As used herein, the term “enantiomer” is one of two stereoisomers that are mirror images of each other and that are non-superposable (not identical). A single chiral atom or similar structural feature in a compound causes that compound to have two possible structures which are non-superposable, each a mirror image of the other. A sample of a compound is considered enantiopure or enantiomerically pure when it has, within the limits of detection, molecules of only one chirality.
As used herein, the terms “racemate” or “racemic mixture” is a mixture having equal amounts of both enantiomers of a chiral molecule.
As used herein, the term “diastereomers” refer to stereoisomers that are not related as mirror images and are not enantiomers. Unlike enantiomers, which are mirror images of each other and non-superimposable, diastereomers are not mirror images. Diastereomers have two or more stereocenters and can have different physical properties and reactivity.
As used herein, the term “pharmaceutically acceptable salt” comprises therapeutically active non-toxic acid and base addition salt forms that the compounds of the invention are able to form. Compounds that have basic properties can be converted to their pharmaceutically acceptable acid addition salts by treating the base form with an appropriate acid. Exemplary acids include inorganic acids, such as hydrogen chloride, hydrogen bromide, hydrogen iodide, sulphuric acid, phosphoric acid; and organic acids, such as formic acid, acetic acid, propanoic acid, hydroxyacetic acid, lactic acid, pyruvic acid, glycolic acid, maleic acid, malonic acid, oxalic acid, benzenesulphonic acid, toluenesulphonic acid, methanesulphonic acid, trifluoroacetic acid, fumaric acid, succinic acid, malic acid, tartaric acid, citric acid, salicylic acid, p-aminosalicylic acid, pamoic acid, benzoic acid, ascorbic acid and the like. Compounds that have acidic properties can be converted to their pharmaceutically acceptable base addition salts by treating the acid form with an appropriate base. Suitable base addition salt forms are the sodium, potassium, calcium salts, and salts with pharmaceutically acceptable amines such as, for example, ammonia, alkylamines, benzathine, and amino acids, such as, e.g., arginine and lysine. The term “addition salt”, as used herein, also comprises solvates, which the compounds and salts thereof are able to form, such as, for example, hydrates, alcoholates and the like.
As used herein, the term “prodrug” refers to a biologically inactive derivative of a parent drug molecule, which is activated by a chemical or enzymatic transformation within the body, which results in the release of the active drug. In a particular embodiment, a prodrug of a compound or sortilin inhibitor of the invention is a short chain ester-containing prodrug, preferably a C1-C4 ester prodrug of the compound or sortilin inhibitor.
An aspect of the invention relates to a compound of formula I below
A particular embodiment relates to a compound of formula I according to above or an enantiomer thereof, a diastereomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof.
The compounds of formula I are capable of binding to sortilin with a high affinity (IC50 value in nM range). The compounds of formula I are thereby suitable as sortilin inhibitors. These sortilin inhibitors of the invention are capable of blocking or at least significantly inhibiting the binding of other ligands, such as PGRN, to sortilin. The sortilin inhibitors can thereby be used in the treatment of diseases, disorders, and conditions associated with the PGRN-sortilin axis or the interaction between sortilin and its other ligands. In fact, the sortilin inhibitors of the invention having an unsaturated aliphatic moiety, see hatched ring in formula II below, have higher binding affinity to sortilin as compared to optimized reference compounds having a saturated aliphatic moiety (Bioorganic & Medicinal Chemistry Letters 2020 30: 127403).
The sortilin inhibitors of the invention furthermore have biological effect as seen in reducing formation of cancer stem cells (spheres). Such cancer stem cells are a population of generally treatment resistant and aggressive cancer cells that may survive or circumvent traditional chemotherapy. Such cancer stem cells could then seed a new tumor that is resistant to chemotherapy. Hence, presence of cancer stem cells generally lead to cancer recurrences and metastasis, and thereby treatment failures. This means that there is a general need to target such aggressive cancer cells. PGRN has previously been identified as the most effective among over 500 cytokines in inducing cancer stem cells and lung metastasis in breast cancer (Breast Cancer Research 2018 20: 137). Furthermore, a highly malignant subgroup of cancer cells co-express PRGN and sortilin (BMC Cancer 2021 21: 185). Sortilin inhibitors of the invention are capable of inhibiting these effects.
Y is, as mentioned above, absent, —O—, —OCH2—, —CH2—, —NR3—, or —CH(NH2)—. As used herein —OCH2-represent —OCH2— and —CH2O—. Thus, according to the invention, Y is absent, —O—, —OCH2—, —CH2O—, —CH2—, —NR3—, or —CH(NH2)—.
In an embodiment, A is a 5 or 6 membered aromatic, heteroaromatic or heterocyclic ring with 0 to 2 heteroatom(s) selected among N and O or a 8 to 10 membered bicyclic heterocyclic or heteroaromatic ring with 1 or 2 heteroatom(s) selected among N and O. Hence, currently preferred heteroatoms for the heteroaromatic, heterocyclic or bicyclic ring A are nitrogen and/or oxygen atoms. In a particular embodiment, A is a 5 or 6 membered aromatic, heteroaromatic or heterocyclic ring with 0 to 2 nitrogen atom(s) or a 8 to 10 membered bicyclic heterocyclic or heteroaromatic ring with 1 or 2 nitrogen atoms. In this particular embodiment, any heteroatoms for the heteroaromatic, heterocyclic or bicyclic ring A are nitrogen atoms.
In an embodiment, A is a 5 or 6 membered aromatic, heteroaromatic or heterocyclic ring with 0 to 2 heteroatom(s) selected among N, O and S, preferably selected among N and O, and more preferably N.
In an embodiment, A is a 5 membered heteroaromatic or heterocyclic ring with 1 to 2 heteroatom(s) selected among N, O and S, preferably selected among N and O, and more preferably N. In a particular embodiment, A is 5 membered heteroaromatic ring with 1 to 2 heteroatom(s) selected among N, O and S, preferably selected among N and O, and more preferably N. In an another particular embodiment, A is a 5 membered heterocyclic ring with 1 to 2 heteroatom(s) selected among N, O and S, preferably selected among N and O, and more preferably N.
In another embodiment, A is a 6 membered aromatic, heteroaromatic or heterocyclic ring with 0 to 2 heteroatom(s) selected among N, O and S, preferably selected among N and O, and more preferably N. In a particular embodiment, A is 6 membered aromatic ring. In another particular embodiment, A is 6 membered heteroaromatic ring with 1 to 2 N as heteroatom(s). In a further particular embodiment, A is a 6 membered heterocyclic ring with 1 to 2 heteroatom(s) selected among N, O and S, preferably selected among N and O, and more preferably N.
In an embodiment, A is a 8 to 10 membered bicyclic heterocyclic or heteroaromatic ring with 1 or 2 heteroatom(s) selected among N, O and S, preferably selected among N and O, and more preferably N. In an embodiment, A is a 9 membered bicyclic heterocyclic or heteroaromatic ring with 1 or 2 heteroatom(s) selected among N, O and S, preferably selected among N and O, and more preferably N. In a particular embodiment, A is a 9 membered bicyclic heterocyclic ring with 1 or 2 heteroatom(s) selected among N, O and S, preferably selected among N and O, and more preferably N. In another particular embodiment, A is a 9 membered heteroaromatic ring with 1 or 2 heteroatom(s) selected among N, O and S, preferably selected among N and O, and more preferably N.
In the above described embodiments, the ring A is optionally substituted with one or more substituents independently selected from the group consisting of C1-C4 alkyl, —NR1R2, —C(O)CZ3, —OR4, halogen, and ═O.
In an embodiment, A is a 5 or 6 membered aromatic, heteroaromatic or heterocyclic ring with 0 to 2 heteroatom(s) selected among N and O, preferably N, or a 9 membered bicyclic heterocyclic ring with 1 or 2 heteroatom(s) selected among N and O, preferably N. The aromatic, heteroaromatic, heterocyclic or bicyclic ring A is, in this embodiment, optionally substituted with one or more substituents independently selected from the group consisting of C1-C4 alkyl, —NR1R2, —C(O)CZ3, —OR4, halogen, and ═O.
In an embodiment, any C1-C4 alkyl substituent of the aromatic, heteroaromatic, heterocyclic or bicyclic ring A is preferably independently selected from the group consisting of methyl, isobutyl and tert-butyl.
In an embodiment, R1 and R2 are independently selected from the group consisting of hydrogen, methyl and ethyl. In a preferred embodiment, R1 and R2 are independently selected from the group consisting of hydrogen and methyl. In a particular preferred embodiment, R1 and R2 are both methyl.
In a preferred embodiment, the aromatic, heteroaromatic, heterocyclic or bicyclic ring A is optionally substituted with one or more substituents independently selected from the group consisting of —CH3, —CH2CH(CH3)2, —C(CH3)3, —N(CH3)2, —C(O)CZ3, halogen, and ═O.
In an embodiment, Z is selected from the group consisting of F, Cl and Br. In a particular embodiment Z is F or Cl. In a particular preferred embodiment, Z is F. In such a particular embodiment, the aromatic, heteroaromatic, heterocyclic or bicyclic ring A is optionally substituted with one or more substituents independently selected from the group consisting of —CH3, —CH2CH(CH3)2, —C(CH3)3, —N(CH3)2, —C(O)CF3 and halogen.
In an embodiment, a halogen substituent of the ring A is preferably selected from the group consisting of F, Cl and Br. In a particular embodiment, the halogen substituent of the ring A is Cl or Br. In such a particular embodiment, the aromatic, heteroaromatic, heterocyclic or bicyclic ring A is optionally substituted with one or more substituents independently selected from the group consisting of —CH3, —CH2CH(CH3)2, —C(CH3)3, —N(CH3)2, —C(O)CZ3, —Cl and —Br.
In a currently preferred embodiment, the aromatic, heteroaromatic, heterocyclic or bicyclic ring A is optionally substituted with one or more substituents independently selected from the group consisting of —CH3, —CH2CH(CH3)2, —C(CH3)3, —N(CH3)2, —C(O)CF3, —Cl, and —Br.
As mentioned in the foregoing, Y is absent, —O—, —OCH2—, —CH2—, —NR3—, or —CH(NH2)—. In the case of absent, Y represents a direct link or bond between the aromatic, heteroaromatic, heterocyclic or bicyclic ring A and the aromatic or heteroaromatic ring B, if present.
In an embodiment, R3 is selected from the group consisting of hydrogen, methyl and ethyl. In a particular embodiment, R3 is selected from the group consisting of hydrogen and methyl. In a preferred particular embodiment, R3 is hydrogen.
In a particular embodiment, Y is absent, —O—, —OCH2—, —CH2—, —NH— or —CH(NH2)—, or preferably Y is absent, —O—, —OCH2—, —NH— or —CH(NH2)—. In another particular embodiment, Y is absent or O. In a preferred embodiment, Y is absent. Hence, in such an embodiment, the compound is represented by formula III below
In an embodiment, B is absent or a 5 or 6 membered aromatic or heteroaromatic ring with 0 to 4 heteroatom(s) selected among N and O. Hence, currently preferred heteroatoms for the heteroaromatic ring B are nitrogen and/or oxygen atoms. In a particular embodiment, B is absent or a 5 or 6 membered aromatic or heteroaromatic ring with 0 to 4 nitrogen atom(s). In this particular embodiment, any heteroatoms for the heteroaromatic ring B are nitrogen atoms. In these embodiment, the aromatic or heteroaromatic ring B is optionally substituted with one or more substituents independently selected from the group consisting of C1-C4 alkyl, —NR1R2, —C(O)CZ3, —OR4, halogen, and ═O.
In an embodiment, B is absent, a 5 membered heteroaromatic ring with 1 to 4 heteroatom(s) selected among N, O and S, preferably selected among N and O, and more preferably N, a 6 membered aromatic ring or a 6 membered heteroaromatic ring with 1 to 4 N as heteroatom(s). In this embodiment, the aromatic or heteroaromatic ring B is optionally substituted with one or more substituents independently selected from the group consisting of C1-C4 alkyl, —NR1R2, —C(O)CZ3, —OR4, halogen, and ═O.
In an embodiment, B is absent.
In another embodiment, B is a 6 membered aromatic ring. The aromatic ring B is optionally substituted with one or more substituents independently selected from the group consisting of C1-C4 alkyl, —NR1R2, —C(O)CZ3, —OR4, halogen, and ═O.
In a further embodiment, B is a 5 or 6 membered heteroaromatic ring with 1 to 4 heteroatom(s) selected among N, O and S, preferably selected among N and O, and more preferably N. The heteroaromatic ring B is optionally substituted with one or more substituents independently selected from the group consisting of C1-C4 alkyl, —NR1R2, —C(O)CZ3, —OR4, halogen, and ═O.
In an embodiment, B is absent or a 5 or 6 membered aromatic or heteroaromatic ring with 0 to 4 heteroatom(s) selected among N, O and S. The aromatic or heteroaromatic ring B is optionally substituted with one or more substituents independently selected from the group consisting of —OR4 and ═O. In a particular embodiment, B is absent or a 5 or 6 membered aromatic or heteroaromatic ring with 0 to 4 heteroatom(s) selected among N and O, preferably N. The aromatic or heteroaromatic ring B is optionally substituted with one or more substituents independently selected from the group consisting of —OR4 and ═O.
In an embodiment, R4 is selected from the group consisting of hydrogen, methyl and ethyl. Hence, in such an embodiment the substituent —OR4 is selected from the group consisting of hydroxyl (—OH), methoxy (—OCH3) and ethoxy (—OCH2CH3). In a particular embodiment, R4 is selected from the group consisting of hydrogen and methyl.
In a particular embodiment, the aromatic or heteroaromatic ring B is optionally substituted with one or more substituents independently selected from the group consisting of hydroxyl, methoxy or ═O.
As mentioned above, R5 is hydroxyl or C1-C4 alkoxy. In a particular embodiment, R5 is hydroxyl or C1-C2 alkoxy, i.e., R5 is selected from the group consisting of hydroxyl, methoxy and ethoxy. In a preferred particular embodiment, R5 is hydroxyl.
In an embodiment, A is a 5 or 6 membered aromatic, heteroaromatic or heterocyclic ring with 0 to 2 heteroatom(s) selected among N, O and S, preferably selected among N and O, and more preferably N. The aromatic, heteroaromatic or heterocyclic ring A is optionally substituted with one or more substituents independently selected from the group consisting of C1-C4 alkyl, —NR1R2, —C(O)CZ3, —OR4, halogen and ═O, preferably independently selected from the group consisting of methyl, isopropyl, isobutyl, —N(CH3)2, —N(CH2CH3)2, —C(O)CF3, C1 and Br. In this embodiment, Y is absent, —O—, —OCH2—, —CH2—, —NH— or —CH(NH2)—. In this embodiment, B is absent, a 6 membered aromatic ring or a 5 or 6 membered heteroaromatic ring with 0 to 4 heteroatom(s) selected among N, O and S, preferably selected among N and O, and more preferably N. The aromatic or heteroaromatic ring B is optionally substituted with one or more substituents independently selected from the group consisting of C1-C4 alkyl, —NR1R2, —C(O)CZ3, —OR4, halogen and ═O, preferably independently selected from the group consisting of hydroxyl, methoxy and ═O. In this embodiment, R5 is hydroxyl or C1-C4 alkoxy, preferably hydroxyl or C1-C2 alkoxy and more preferably hydroxyl.
Currently preferred compounds of formula I include the following compounds:
In a particular embodiment, preferred compounds of formula I include the following compounds:
In another preferred particular embodiment, preferred compounds of formula I include the following compounds:
In a more preferred particular embodiment, preferred compounds of formula I include the following compounds:
A currently preferred compound of the invention is (E)-2-[2-(dimethylamino)-4-methyl-5-pyrimidinylcarbonylamino]-5,5-dimethyl-3-hexenoic acid (SI1) or an enantiomer thereof, a diastereomer thereof, a racemate thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof, such as enantiomer thereof, a diastereomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof.
Another currently preferred compound of the invention is (E)-5,5-dimethyl-2-(6-phenoxynicotinoylamino)-3-hexenoic acid (SI5) or an enantiomer thereof, a diastereomer thereof, a racemate thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof, such as enantiomer thereof, a diastereomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof.
A further currently preferred compound of the invention is (E)-5,5-dimethyl-2[m-(1-imidazolyl)benzoylamino]-3-hexenoic acid (SI25), or an enantiomer thereof, a diastereomer thereof, a racemate thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof, such as enantiomer thereof, a diastereomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof.
Another preferred compound of the invention is (E)-2-(4-chloro-1-methyl-2-pyrrolylcarbonylamino)-5,5-dimethyl-3-hexenoic acid (SI31), or an enantiomer thereof, a diastereomer thereof, a racemate thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof, such as enantiomer thereof, a diastereomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof.
A further preferred compound of the invention is (E)-2-(4-chloro-2-thienylcarbonylamino)-5,5-dimethyl-3-hexenoic acid (SI32), or an enantiomer thereof, a diastereomer thereof, a racemate thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof, such as enantiomer thereof, a diastereomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof.
Yet another preferred compound of the invention is (E)-2-[6-(benzyloxy)nicotinoylamino]-5,5-dimethyl-3-hexenoic acid (SI39), or an enantiomer thereof, a diastereomer thereof, a racemate thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof, such as enantiomer thereof, a diastereomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof.
Another preferred compound of the invention is (E)-5,5-dimethyl-2-[p-(2-thienyl)benzoylamino]-3-hexenoic acid (SI51), or an enantiomer thereof, a diastereomer thereof, a racemate thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof, such as enantiomer thereof, a diastereomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof.
A further preferred compound of the invention is (E)-5,5-dimethyl-2-[m-(2-thienyl)benzoylamino]-3-hexenoic acid (SI52), or an enantiomer thereof, a diastereomer thereof, a racemate thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof, such as enantiomer thereof, a diastereomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof.
Yet another preferred compound of the invention is (E)-2-(4,5-dichloro-2-thienylamino)-5,5-dimethyl-3-hexenoic acid (SI62), or an enantiomer thereof, a diastereomer thereof, a racemate thereof, a prodrug thereof, or a pharmaceutically acceptable salt thereof, such as enantiomer thereof, a diastereomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof.
Compounds of the present invention can be manufactured, as is further described in Examples 1 to 37 below, from an intermediate as synthesized as disclosed in steps 1-3 of Example 1. The present invention also encompasses such intermediates, i.e., compounds D, E and F in Example 1, and the synthesis thereof. Hence, the invention also relates to an intermediate for the production of a compound of formula I. The intermediate is selected from the group consisting of:
In an embodiment, the intermediate is (E)-2-{[(p-methoxyphenyl)methyl]amino}-5,5-dimethyl-3-hexenoic acid (IC1). IC1 can be produced by adding 2-tert-butyl-E-vinylboronic acid to 4-methoxybenzylamine in dry dichloromethane followed by glyoxylic acid monohydrate to obtain IC1 (step 1 of Example 1).
In another embodiment, the intermediate is ethyl (E)-2-{[(p-methoxyphenyl)methyl]amino}-5,5-dimethyl-3-hexenoate (IC2). IC2 can be produced by adding sulphuric acid to a suspension of IC1 in ethanol to obtain IC2 (step 2 of Example 1).
In a preferred embodiment, the intermediate is ethyl (E)-2-amino-5,5-dimethyl-3-hexenoate (IC3). IC3 can be produced by adding a pre-dissolved solution of ceric ammonium nitrate in water to a solution of IC2 in acetonitrile to obtain IC3 (step 3 of Example 1).
The sortilin inhibitors of the present invention are capable of binding to sortilin with a high affinity (
The sortilin inhibitors of the invention can thereby be used as a medicament.
Through its various interactions with proteins, such as PGRN, sortilin and its multiple ligands have been shown to be involved in various diseases, disorders, and conditions, such as frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), ALS-FTD phenotypes, Alzheimer's disease, Parkinson's disease, depression, neuropsyciatric disorders, vascular dementia, seizures, retinal dystrophy, age related macular degeneration, glaucoma, traumatic brain injury, aging, seizures, wound healing, stroke, arthritis, atherosclerotic vascular diseases, dermatology related diseases and autoimmune diseases (WO 2009/140972, WO 2014/114779, WO 2016/164637, Bioorganic & Medicinal Chemistry Letters 2020 30: 127403).
The sortilin inhibitors of the invention can thereby be used in prevention or treatment of a neurodegenerative disease, a psychiatric disease, a motor neuron disease, peripheral neuropathies, pain, neuroinflammation, atherosclerosis, hyperlipidemia, cardiovascular diseases, dermatology related diseases or autoimmune diseases.
Illustrative, but non-limiting, examples of neurodegenerative diseases include FTD, ALS, ALS-FTD phenotypes, stroke, traumatic brain injury, retinal degeneration, light-induced photoreceptor degeneration, Alzheimer's disease and Parkinson's disease.
Illustrative, but non-limiting, examples of psychiatric disease include depression, neuropsychiatric disorders, epilepsy and bipolar disorder.
Illustrative, but non-limiting, examples of motor neuron diseases include ALS, progressive bulbar palsy (PBP), pseudobulbar palsy, progressive muscular atrophy (PMA), primary lateral sclerosis (PLS), spinal muscular atrophy (SMA) and monomelic amyotrophy (MMA).
Illustrative, but non-limiting, examples of peripheral neuropathies include diabetic neuropathy.
Illustrative, but non-limiting, examples of pain include acute pain, chronic pain, neuropathic pain, lower back pain, post operative pain and inflammatory pain.
Illustrative, but non-limiting, examples of neuroinflammatory diseases include rheumatoid arthritis, Crohns disease, ulcerative colitis and multiple sclerosis.
Illustrative, but non-limiting, examples of hyperlipidemias include hyperlipoproteinemia type I, such as Buerger-Gruetz syndrome, primary hyperlipoproteinemia or familial hyperchylomicronemia; hyperlipoproteinemia type Na, such as polygenic hypercholesterolemia or familial hypercholesterolemia; hyperlipoproteinemia type Nb, such as combined hyperlipidemia; hyperlipoproteinemia type III, such as familial dysbetalipoproteinemia; hyperlipoproteinemia type IV, such as endogenous hyperlipemia; hyperlipoproteinemia type V, such as familial hypertriglyceridemia. Other examples of diseases or disorder caused by hyperlipidemia or having a hyperlipidemia component include aneurysm, angina pectoris, atherosclerosis, cerebrovascular accident or disease, congenital heart disease, congestive heart failure, coronary artery disease, dilated cardiomyopathy, diastolic dysfunction, endocarditis, hypercholesterolemia, hypertension, hypertrophic cardiomyopathy, mitral valve prolapse, myocardial infarction and venous thromboembolism.
Illustrative, but non-limiting, examples of dermatology related disease include psoriasis, dermal fibrosis and dermal keratosis.
Illustrative, but non-limiting, examples of autoimmune disease include psoriasis, rheumatoid arthritis, diabetes mellitus and inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease.
The PGRN-sortilin axis is also of importance for cancers. In more detail, highly malignant cancer cells co-express PGRN and sortilin resulting in sphere (cancer stem cell) formation and metastasis. Blocking or at least inhibiting the interaction between PGRN and sortilin thereby prevents or at least significantly inhibits sphere formation (Example 42) and may also reduce metastasis (Breast Cancer Research 2018 20: 137). High PGRN levels have been found in various cancer types including, but non-limited, to breast cancer, bladder cancer, lymphomas, biliary cancer and pancreatic cancer.
The sortilin inhibitors of the invention can thereby be used in prevention or treatment of cancer.
In an embodiment, the cancer is selected from the group consisting of breast cancer, bladder cancer, lymphomas, biliary cancer, colon cancer, melanoma and pancreatic cancer. In a preferred embodiment, the cancer is selected from the group consisting of breast cancer, colon cancer and melanoma, more preferably breast cancer.
In a particular embodiment, the sortilin inhibitors of the invention are used in prevention or treatment of metastasis. Further uses of the sortilin inhibitors of the invention include in prevention or treatment of sphere formation and in inhibition of cancer stem cell formation and/or migration.
The present invention also relates to a pharmaceutical composition comprising a sortilin inhibitor according to the invention and at least one pharmaceutically acceptable excipient, carrier or diluent.
As used herein, pharmaceutically acceptable excipient, carrier or diluent encompass various pharmaceutically acceptable additives including, but not limited to, excipients, carriers, diluents, adjuvants, colorings, aromas, preservatives etc. that the person skilled in the art would consider using when formulating a sortilin inhibitor of the invention to make a pharmaceutical composition.
Pharmaceutically acceptable excipient, carrier or diluent include, but are not limited to, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine; lubricants, such as silica, talc, stearic acid including salts thereof, and polyethylene glycol; binders, such as magnesium and aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethyl cellulose, and polyvinylpyrrolidone; disintegrants, such as starch, agar, alginic acid, and sodium alginate; absorbents; dyes; flavoring agents; sweeteners; polypropylene glycol; liquid vehicles, such as water, physiological saline solution, aqueous dextrose, glycerol, ethanol, and oil.
The at least one excipient, carrier and/or diluent is pharmaceutically acceptable in terms of being compatible with the sortilin inhibitor of the invention (compound of formula I) and any other ingredients of the pharmaceutical composition, and not deleterious to a subject when the pharmaceutical composition is administered thereto. For instance, it is preferred that the pharmaceutical composition does not contain any material that may cause an adverse reaction, such as an allergic reaction, when administered to a subject.
In an embodiment, the pharmaceutical composition comprises from 1 to 99% by weight of the at least one pharmaceutically acceptable excipient, carrier and/or diluent and from 1 to 99% by weight of at least one sortilin inhibitor of the invention.
The pharmaceutical composition could comprise a single sortilin inhibitor according to invention or multiple sortilin inhibitors according to the invention, i.e., a mixture of two or more different sortilin inhibitors.
In an embodiment, the pharmaceutical composition may comprise at least one second active agent in addition to the one or more sortilin inhibitors of the invention.
The at least one second active agent may be an active agent traditionally used in treatment of a disease, disorder or condition associated with sortilin activity and ligand-to-sortilin binding, such as PGRN-to-sortilin binding. Hence, the at least one active agent could be an active agent used in prevention or treatment of a neurodegenerative disease, a psychiatric disease, a motor neuron disease, peripheral neuropathies, pain, neuroinflammation, atherosclerosis, hyperlipidemia, cardiovascular diseases, dermatology related diseases or autoimmune diseases.
In another embodiment, the at least one second active agent is a cytostatic agent or other anti-cancer agent. Non-limiting examples of such cytostatic agents that could be used in the pharmaceutical composition include alkylating agents, such as mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide, busulfan, N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, streptozotocin, dacarbazine, mitozolomide, temozolomide, thiotepa, mytomycin, diaziquone (AZQ), cisplatin, carboplatin and oxaliplatin; antimetabolites, such as methotrexate, pemetrexed, fluorouracil, capecitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, pentostatin, thioguanine and mercaptopurine; anti-microtubule agents, such as Vinca alkaloids derived from Catharanthus roseus, vincristine, vinblastine, vinorelbine, vindesine, vinflunine, paclitaxel, docetaxel, podophyllotoxin, etoposide and teniposide; topoisomerase inhibitors, such as irinotecan, topotecan, camptothecin, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin; and cytotoxic antibiotics, such as doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, mitoxantrone, actinomycin, bleomycin, mitomycin C and actinomycin.
In a particular embodiment, the at least one second active agent is progranulin.
The pharmaceutical composition comprising multiple active agents may be formulated in a unit dosage form comprising the multiple active agents or may be formulated as separated dosage forms with a respective active agent. In the latter case, the multiple separate dosage forms may be administered substantially simultaneously or separately, such as sequentially.
The pharmaceutical composition could be a solid pharmaceutical composition, such as a tablet, pill, powder, or granules, a semi solid pharmaceutical composition, such as a suppositories; or a liquid pharmaceutical composition, such as soft capsule or injection solution.
The sortilin inhibitor of the invention and in particular the pharmaceutical composition comprising at least one sortilin inhibitor of the invention may be administered to a subject using various administration routes. Illustrative, but non-limiting, examples of such administration routes include oral, intravenous, topical, intraperitoneal, nasal, buccal, sublingual, or subcutaneous administration, or administration via the respiratory tract. The pharmaceutical composition of the invention may be formulated based on the particular administration route. Illustrative, but non-limiting, examples of formulations of the pharmaceutical composition include tablets, capsules, powders, nanoparticles, crystals, amorphous substances, solutions, transdermal patches, (transdermal) creams or suppositories.
The present invention is also a method for treating a disease, disorder or condition selected from the group consisting of a neurodegenerative disease, a psychiatric disease, a motor neuron disease, peripheral neuropathies, pain, neuroinflammation, atherosclerosis, hyperlipidemia, cardiovascular diseases, dermatology related diseases, autoimmune diseases and cancer. The method comprising administering at therapeutically effective amount of a sortilin inhibitor of the invention or a pharmaceutical composition of the invention to a subject need thereof.
“Treatment” or “treating” as used herein means the management and care of a subject for the purpose of combating a disease, a disorder or a medical condition. The term is intended to include the full spectrum of treatments for a given disease, disorder or condition, from which the subject is suffering, such as administration of the sortilin inhibitor or the pharmaceutical composition of the invention to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and/or to cure or eliminate the disease, disorder or condition as well as to prevent the disease, disorder or condition. In such a case, prevention is to be understood as the management and care of a subject for the purpose of combating the disease, disorder or condition and includes the administration of the sortilin inhibitor or the pharmaceutical composition of the invention to prevent the onset of the symptoms or complications. The treatment may either be performed in an acute or in a chronic way.
“A therapeutically effective amount” of the sortilin inhibitor or the pharmaceutical composition of the invention as used herein means an amount sufficient to cure, inhibit, alleviate or partially arrest the clinical manifestations of a given disease, disorder or condition and its complications. An amount adequate to accomplish this is defined as therapeutically effective amount. Effective amounts for each purpose will depend on the severity of the disease, disorder or condition as well as parameters of the subject, such as weight, sex, age, general health status of the subject. It will be understood that determining an appropriate dosage may be achieved using routine experimentation, by constructing a matrix of values and testing different points in the matrix, which is all within the ordinary skills of a trained physician or veterinary.
A related aspect of the invention defines use of a sortilin inhibitor of the invention for the manufacture of a medicament for treatment of a disease, disorder or condition selected from the group consisting of a neurodegenerative disease, a psychiatric disease, a motor neuron disease, peripheral neuropathies, pain, neuroinflammation, atherosclerosis, hyperlipidemia, cardiovascular disease, dermatology related diseases, autoimmune diseases and cancer.
The subject treated with a sortilin inhibitor or pharmaceutical composition of the invention is preferably a human subject. The present invention can, however, be used for veterinary purposes by administering a sortilin inhibitor or pharmaceutical composition of the invention to a non-human mammal. Illustrative, but non-limiting, examples of such non-human mammals include dogs, cats, cows, horses, sheep, goat, pigs, rats, mice, rabbits and guinea pigs.
In the syntheses of the compounds of the present invention, as described below, the following analytical and chromatographic equipment were used.
2-tert-Butyl-E-vinylboronic acid (A) (1.86 g, 0.014 mol) was added to a stirred solution of 4-methoxybenzylamine (C) (2 g, 0.014 mol) in dry dichloromethane (DCM, 22 mol, 0.33 M with respect to amine) followed by glyoxylic acid monohydrate (B) (1.34 g, 0.014 mol) at 25-30° C. The resulting reaction mixture was stirred over a period of 24 h at 25-30° C. under argon atmosphere. Progress of the reaction was monitored by thin layer chromatography (TLC).
After completion of the reaction, the reaction mixture was filtered off, the solid obtained was washed with DCM and dried at 40° C. to obtain the expected product (D) as a white solid.
Yield: 1.78 g, (44.5%)
1H-NMR-CD3OD (400 MHz): δ: 1.04 (9H, s), 3.78 (3H, s), 3.86-3.89 (1H, m), 4.0-4.1 (2H, m), 5.38-5.45 (1H, m), 5.98 (1H, d, J=16.0 Hz), 6.95 (2H, dd, J1=2.4 Hz, J2=6.8 Hz), 7.35 (2H, dd, J1=2.0 Hz, J2=6.4 Hz);
LCMS: 278.2 [M+H]+
Sulphuric acid (3.4 ml) was added dropwise to a stirred suspension of compound D (1.7 g, 0.006 mol) in ethanol (34 ml) at 0-5° C. The resulting reaction mixture was stirred over a period of 24 h at 90° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was evaporated at 40° C. to remove ethanol. The remaining mixture was basified to pH ˜7-8 using saturated NaHCO3 solution, and the product was extracted into ethyl acetate (3×20 ml). The combined organic layer was dried over anhydrous sodium sulphate and concentrated at 40° C. under reduced pressure to obtain the crude product (E) as a pale brown oil. The crude product was used as such for the next reaction without any purification.
Yield: 1.3 g, (crude product)
1H-NMR-CD3OD (400 MHz): δ: 1.04 (9H, s), 1.22 (3H, t, J=7.2 Hz), 3.60-3.63 (2H, m), 3.69-3.71 (1H, m), 3.75 (3H, s), 4.15 (2H, q, J=7.2 Hz), 5.27-5.33 (1H, m), 5.71-5.77 (1H, m), 6.83-6.87 (2H, m), 7.17-7.21 (2H, m);
LCMS: 306.3 [M+H]+
A pre-dissolved solution of ceric ammonium nitrate (14.0 g, 0.0255 mol) in water (15.6 ml) was added dropwise over a period of 15 min to a stirred solution of compound E (1.3 g, 0.0042 mol) in acetonitrile (15.6 ml) at 0-5° C. The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was evaporated at 25° C. to remove acetonitrile, basified to pH ˜7-8 using saturated NaHCO3 solution. The thick suspension was filtered through a celite bed, and the celite bed was washed with ethyl acetate and the resulting layers were separated out. The combined organic layer was dried over anhydrous sodium sulphate and concentrated at 35° C. under reduced pressure to obtain the crude product. The crude product was purified by CombiFlash® using methanol/DCM as eluent to obtain the expected product (F) as a pale brown oil.
Yield: 255 mg, (32.3%)
1H-NMR-CD3OD (400 MHz): δ: 1.04 (9H, s), 1.25 (3H, t, J=7.2 Hz), 3.93-3.95 (1H, m), 4.18 (2H, q, J=7.2 Hz), 5.40-5.46 (1H, m), 5.77-5.82 (1H, m);
LCMS: 186.1 [M+H]+
2-(Dimethylamino)-4-methylpyrimidine-5-carboxylic acid (G) (244.4 mg, 1.3493 mmol) was added to a stirred solution compound F (250 mg, 1.3493 mmol) in dimethylformamide (DMF, 6.25 ml) followed by N,N-diisopropylethylamine (DIPEA, 0.94 mL, 5.3972 mmol) and propyl phosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (2.57 mL, 4.0479 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution until pH ˜7-8 and the product was then extracted into ethyl acetate (2×50 mL). The combined organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulphate and concentrated in vacuum at 35° C. to obtain the crude product. The crude product was purified by CombiFlash® using ethyl acetate/hexane as eluent to obtain the pure product (H) as a pale brown thick oil.
Yield: 62 mg, (13.2%)
1H-NMR-CD3OD (400 MHz): δ: 1.02 (9H, s), 1.25 (3H, t, J=7.2 Hz), 2.44 (3H, s), 3.18 (6H, s), 4.19 (2H, t, J=7.2 Hz), 4.94-4.92 (1H, m), 5.47-5.54 (1H, m), 5.87-5.91 (1H, m), 8.33 (1H, s);
LCMS: 349.3 [M+H]+
Lithium hydroxide monohydrate (33.11 mg, 0.7892 mmol) was added to a stirred solution of compound H (55 mg, 0.1578 mmol) in tetrahydrofuran (THF)/methanol/water (1.65 ml, 1:1:1, 0.55 ml each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the remaining residue was dissolved in water and washed with ethyl acetate, and the aqueous layer was acidified to pH ˜2-3 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was washed with saturated sodium chloride solution, dried over sodium sulphate and concentrated at 35° C. to obtain the crude product. The crude solid was triturated with hexanes and dried at 40° C. to afford pure final product (I) as off white solid.
Yield: 37 mg, (73.2%)
1H-NMR-DMSO-d6 (400 MHz): δ: 0.98 (9H, n), 2.40 (3H, n), 3.13 (6H, 4.78-4.82 (1H, n), 5.45-5.50 (1H, in), 5.80-5.84 (1H, in), 8.37 (1H, s), 8.57 (1H, d, J=7.2 Hz), 12.59 (1H, bs);
LCMS: 321.3 [M+H]+;
HPLC purity: 98.6%
(E)-2-[2-(dimethylamino)-4-methyl-5-pyrimidinylcarbonylamino]-5,5-dimethyl-3-hexenoic acid as synthesized in Example 1 (12 mg, 34 mmol) was dissolved in methanol (7 mL) and purified by preparative supercritical fluid chromatography (SFC) using a CHIRALPAK-ID column (250 mm×10 mm, 5 μM) eluting with methanol, 15% in CO2, flow 15 mL/min. Baseline separation was obtained. Enantiomer 1 eluted at 29.7-30.1 min and enantiomer 2 eluted at 30.2-30.9 min. The fractions for each enantiomer were combined and evaporated to give enantiomer 1 (3.2 mg) and enantiomer 2 (3.2 mg).
4-(Trifluoroacetyl)benzoic acid (B) (82.41 mg, 0.38 mmol) was added to a stirred solution of ethyl (E)-2-amino-5,5-dimethyl-3-hexenoate (A) (70 mg, 0.38 mmol) in DMF (1.75 mL) followed by DIPEA (0.263 mL, 1.52 mmol) and propyl phosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (0.72 mL, 1.14 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 48 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and product was then extracted into ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to obtain the crude product. The crude product was purified by CombiFlash® using ethyl acetate/hexane as eluent to afford the pure product (C) as a pale brown thick oil.
Yield: 68 mg, (46.7%)
1H-NMR-CD3OD (400 MHz): δ: 1.03 (9H, s), 1.27 (3H, t, J=7.2 Hz), 4.21 (2H, t, J=7.2 Hz), 4.85-4.89 (1H, m), 5.57-5.59 (1H, m), 5.90-5.94 (1H, m), 7.71-7.80 (2H, m), 7.78-7.93 (2H, m);
LCMS: 404.3 [M+H+H2O]+
Lithium hydroxide monohydrate (35.38 mg, 0.85 mmol) was added to a stirred solution of compound C (65 mg, 0.17 mmol) in THF/methanol/water (1.95 mL, 1:1:1, 0.65 mL each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the residue was dissolved in water and washed with ethyl acetate (10 mL), the aqueous layer was acidified to pH ˜3.4 using 1.5 N HCl, and the product was then extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to obtain a crude solid. The crude solid was washed with hexane (5 mL) and DCM (5 mL) to afford the final product D as an off white solid.
Yield: 18 mg, (29.9%)
1H-NMR-DMSO-d6 (400 MHz): δ: 1.0 (9H, s), 4.91 (1H, d, J=9.6 Hz), 5.52-5.56 (1H, m), 5.81-5.88 (1H, m), 7.68-7.81 (1H, m), 8.09-8.21 (3H, m), 9.13 (1H, m), 12.67 (1H, bs);
LCMS: 376.2 [M+H+H2O]+
HPLC purity: 86.6%
2,2′-Bis(diphenylphosphino)-1,1′-binaphthyl (BINAP, 0.084 g, 0.00013 mol), Pd(OAc)2 (0.03 g, 0.00013 mol) and potassium t-butoxide (0.30 g, 0.0027 mol) were added to a degassed solution of 2-bromopyridine (A) (0.21 g, 0.0013 mol) and N-Boc-piperazine-2-carboxylic acid methyl ester (B) (1.0 g, 0.0040 mol) in toluene (10 mL) at 25-30° C. Degassing was continued for another 5 mins. The resulting reaction mixture was heated at 85° C. over a period of 18 h. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was diluted with water (10 mL), ethyl acetate (10 mL) and the product was extracted into ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to obtain the crude product. The crude product was purified by CombiFlash® using ethyl acetate/hexane as eluent to afford the product C as pale yellow thick oil.
Yield: 0.18 g, (Impure)
LCMS: 322.2 [M+H]+
Lithium hydroxide monohydrate (0.11 g, 0.0028 mol) was added to a stirred solution of compound C (0.18 g, 0.00056 mol) in THF/methanol/water (5.4 mL, 1:1:1, 1.8 mL each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the residue was dissolved in water and washed with methyl tert-butyl ether (MTBE, 20 mL), aqueous layer was neutralized to pH ˜6-7 using 1.5 N HCl and concentrated to obtain the crude product. The crude product was purified by reverse-phase HPLC (RP-HPLC) to obtain pure D as a white solid.
Yield: 60 mg, (35.3%)
1H-NMR-DMSO-d6 (400 MHz): δ: 1.39 (9H, d, J=19.2 Hz), 2.78-2.80 (1H, m), 2.99-3.07 (2H, m), 3.77-3.79 (1H, m), 4.11-4.15 (1H, m), 4.59-4.69 (2H, m), 6.64-6.68 (1H, m), 6.78-6.81 (1H, m), 7.51-7.55 (1H, m), 8.08-8.10 (1H, m), 12.9 (1H, bs);
LCMS: 308.2 [M+H]+;
Ethyl (E)-2-amino-5,5-dimethyl-3-hexenoate (E) (39.18 mg, 0.22 mmol) was added to a stirred solution of compound D (65 mg, 0.2114 mmol) in DMF (1.62 mL) followed by DIPEA (0.14 mL, 0.85 mmol) and propyl phosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (0.40 mL, 0.64 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 48 h at 40° C. Progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution until pH ˜7-8 and product was extracted into ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to obtain the crude product. The crude product was purified by CombiFlash® using ethyl acetate/hexane as eluent to obtain the product F as a pale-yellow, thick oil.
Yield: 50 mg, (Impure)
1H-NMR-CD3OD (400 MHz): δ: 0.87-0.89 (9H, m), 1.24 (3H, t, J=3.6 Hz), 1.46 (9H, s), 3.46-3.51 (2H, m), 3.93-4.2 (5H, m), 4.67-4.79 (2H, m), 5.42-5.49 (2H, m), 5.69-5.82 (1H, m), 6.65-6.67 (1H, m), 6.80-6.86 (1H, m), 7.51-7.55 (1H, m), 8.08-8.10 (1H, m);
LCMS: 475.4 [M+H]+
Lithium hydroxide monohydrate (22.1 mg, 0.53 mmol) was added to a stirred solution of compound F (50 mg, 0.11 mmol) in THF/methanol/water (1.5 mL, 1:1:1, 0.5 mL each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the residue was dissolved in water and washed with ethyl acetate (10 mL), the aqueous layer was acidified to pH ˜3.4 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to obtain a crude solid product. The crude solid product was washed with hexane (5 mL) and DCM (5 mL) to obtain G as a pale brown solid.
Yield: 35 mg, (57.8%)
1H-NMR-CD3OD (400 MHz): δ: 0.98 (9H, s), 1.46 (9H, s), 3.59-3.65 (2H, m), 3.91-3.99 (1H, m), 4.01-4.06 (1H, m), 4.69-4.72 (2H, m), 5.44-5.51 (1H, m), 5.67-5.79 (1H, m), 6.66-6.69 (1H, m), 6.81-6.85 (1H, m), 7.55-7.59 (1H, m), 8.02-8.06 (1H, m);
LCMS: 447.4 [M+H]+
4 M HCl in a 1,4-dioxane solution was added to a stirred solution of compound G (35 mg, 0.0783 mmol) in 1,4-dioxane (0.2 mL) at 25-30° C. The resulting reaction mixture was stirred over a period of 5 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 5 h, the reaction mixture was concentrated; the solid obtained was washed with hexane (5 mL) and diethyl ether (5 mL) to obtain a crude mixture of two diastereomers of the product H. The crude mixture was purified by RP-HPLC to afford two pure diastereomers of H as white solids.
Yield: 4.2 mg (diastereomer-1) and 5.3 mg (diastereomer-2)
LCMS: 347.3 [M+H]+;
Step 1 Synthesis of tert-butyl 2-methoxycarbonyl-4-phenyl-1-piperazinecarboxylate (C)
BINAP (0.015 g, 0.00002 mol), Pd2(dba)3 (0.011 g, 0.00001 mol) and cesium carbonate (0.32 g, 0.0010 mol) were added to a solution of 4-bromopyridine (A) (0.1 g, 0.0006 mol) and tert-butyl 2-methoxycarbonyl-1-piperazinecarboxylate (B) (0.15 g, 0.0006 mol) in toluene (4 mL) at 25-30° C. The resulting reaction mixture was heated at 80° C. over a period of 40 h. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was diluted with ethyl acetate (10 mL) and filtered through a plug of celite. The filtrate was concentrated under reduced pressure at 35° C. to afford the crude product. This was purified by CombiFlash® using DCM/methanol as eluent to afford compound C as a yellow thick oil.
Yield: 0.19 g, (96.4%)
1H-NMR-DMSO-d6 (400 MHz): δ: 1.22-1.42 (9H, m), 2.87-3.08 (1H, m), 3.13-3.18 (2H, m), 3.55-3.59 (3H, m), 3.75-3.80 (2H, m), 4.23-4.33 (1H, m), 4.66-4.74 (1H, m), 6.81 (2H, d, J=6.4 Hz), 8.17 (2H, d, J=6.4 Hz);
LCMS: 322.2 [M+H]+
Lithium hydroxide monohydrate (0.12 g, 0.0029 mol) was added to a stirred solution of compound C (0.19 g, 0.0005 mol) in THF/methanol/water (5.7 mL, 1:1:1, 1.9 mL each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the residue was dissolved in water and washed with MTBE (20 mL). The aqueous layer was neutralized to pH ˜6-7 using 1.5 N HCl and concentrated to afford crude product. This was purified by RP-HPLC to afford pure compound D as white solid.
Yield: 0.15 g, (87.0%)
1H-NMR-DMSO-d6 (400 MHz): δ: 1.37-1.42 (9H, m), 3.25-3.27 (4H, m), 3.13-3.18 (11H, m), 3.55-3.61 (2H, m), 4.62-4.72 (2H, m), 7.16-7.32 (2H, m), 8.26-8.37 (2H, m) 13.45 (2H, bs);
LCMS: 308.0 [M+H]+
Ethyl (E)-2-amino-5,5-dimethyl-3-hexenoate (E) (90.42 mg, 0.4880 mmol) followed by DIPEA (0.33 mL, 1.9521 mmol) and propyl phosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (0.93 mL, 1.4640 mmol) were added to a stirred solution of compound D (150 mg, 0.4880 mmol) in DMF (3.75 mL) at 25-30° C. The resulting reaction mixture was stirred over a period of 48 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution until pH ˜7-8 and the product was extracted out using ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulphate and concentrated under reduced pressure at 35° C. to afford crude material. This was purified by CombiFlash® using DCM/methanol as eluent to afford compound F as a pale-yellow, thick oil.
Yield: 105 mg, (45.3%)
1H-NMR-DMSO-d6 (400 MHz): δ: 0.87-0.92 (9H, m), 1.18-1.21 (m, 3H), 1.34-1.41 (9H, m), 3.50-3.12 (1H, m), 3.47-3.52 (2H, m), 3.74-3.79 (2H, m), 4.04-4.23 (3H, m), 4.19-4.23 (0.5H, m), 4.49-4.51 (0.5H, m), 4.68-4.70 (1H, m), 5.36-5.41 (1H, m), 5.74-5.76 (1H, m), 6.76 (2H, bs), 8.13 (2H, bs), 8.67-8.68 (1H, m);
LCMS: 475.4 [M+H]+
Lithium hydroxide monohydrate (44.20 mg, 1.05 mmol) was added to a stirred solution of compound F (100 mg, 0.21 mmol) in THF/methanol/water (3 mL, 1:1:1, 1.0 mL each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the residue was dissolved in water and washed with ethyl acetate (10 mL). The aqueous layer was acidified to pH ˜3-4 using 1.5 N HCl and the product was extracted out with ethyl acetate (3×5 mL). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure at 40° C. to afford crude solid material. This was triturated with hexane (5 mL) and DCM (5 mL) respectively to afford compound G as an off-white solid.
Yield: 27 mg
1H-NMR-DMSO-d6 (400 MHz): δ: 0.88-0.98 (9H, m), 1.37-1.42 (9H, m), 3.59-3.65 (2H, m), 3.91-3.99 (1H, m), 4.01-4.06 (1H, m), 4.63-4.71 (1H, m), 5.36-5.44 (1H, m), 6.88-6.91 (2H, m), 8.14-8.17 (2H, m);
LCMS: 447.4 [M+H]+
4 M HCl in 1,4-dioxane solution (0.5 mL) was added to a stirred solution of compound G (25 mg, 0.0559 mmol) in 1,4-dioxane (0.3 mL) at 25-30° C. The resulting reaction mixture was stirred over a period of 5 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 5 h, the reaction mixture was concentrated, the solid material obtained was triturated with hexane (5 mL) and diethyl ether (5 mL) to afford a crude mixture. This was purified by RP-HPLC to afford two diastereomers as white solids.
Yield: 2 mg (isomer-1) and 1.9 mg (isomer-2)
LCMS: 347.3 [M+H]+
6-Phenoxynicotinic acid (A) (100 mg, 0.23 mmol) followed by DIPEA (0.32 mL, 0.92 mmol) and propyl phosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (0.88 mL, 0.69 mmol) were added to a stirred solution of ethyl (E)-2-amino-5,5-dimethyl-3-hexenoate (B) (86.08 mg, 0.23 mmol) in DMF (2.5 mL) at 25-30° C. The resulting reaction mixture was stirred over a period of 48 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted to ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulphate and concentrated in vacuum at 35° C. to afford the crude product. The crude product was purified by CombiFlash® using ethyl acetate/hexanes as eluent to afford compound C as yellow thick oil.
Yield: 71.3 mg, (40.2%)
1H-NMR-DMSO-d6 (400 MHz): δ: 0.99 (9H, s), 1.16 (3H, t, J=7.2 Hz), 4.10 (2H, m), 4.89-4.91 (1H, m), 5.52-5.54 (1H, m), 5.85 (1H, d, J=14.4 Hz), 7.08-7.10 (1H, m), 7.17-7.23 (2H, m), 7.24-7.27 (1H, m), 7.41-7.45 (2H, m), 8.28 (1H, d, J1=2.8 Hz, J2=8.8 Hz), 8.62-8.63 (1H, m), 8.96 (1H, s)
LCMS: 383.3 [M+H]+
Lithium hydroxide monohydrate (21.94 mg, 0.52 mmol) was added to a stirred solution of compound C (100 mg, 0.26 mmol) in THF/methanol/water (3 mL, 1:1:1, 1.0 mL each). The resulting reaction mixture was stirred over a period of 5 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, reaction mixture was concentrated, the residue was dissolved in water and washed with diethyl ether (10 mL), the aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid material. This was washed with hexane (5 mL) to afford the crude product (mixture of two isomers) as an off white solid. The crude material was purified by RP-HPLC to afford compound D as a white solid.
Yield: 19 mg
1H-NMR-DMSO-d6 (400 MHz): δ: 0.96 (9H, s), 4.68 (1H, t, J=6.0 Hz), 5.51-5.64 (2H, m), 7.06 (1H, d, J=8.8 Hz), 7.15-7.17 (2H, m), 7.22-7.25 (1H, m), 7.41-7.45 (2H, m), 8.26-8.28 (2H, m), 8.41 (1H, d, J=6.4 Hz), 8.61 (1H, d, J=2.4 Hz);
LCMS: 355.3 [M+H]+; HPLC purity −99.5%.
This separation was carried out in a similar way as described above in Example 1 starting with 250 mg of crude racemic (E)-2-[2-(dimethylamino)-4-methyl-5-pyrimidinylcarbonylamino]-5,5-dimethyl-3-hexenoic acid. The preparative column was CHIRALPAK-IG (250×30 mm) and the elution was carried out with hexanes/EtOAc/MeOH/TFA in a ratio of 70/15/15/0.1. The flow rate was 40 mL/min, detection UV 245 mm. The feed concentration was 10 mg/mL and the injection volume was 5 mL (on column 50 mg). The retention time for enantiomer 1 was 4.2 min and for enantiomer 2 4.6 min. CHIRALPAK-IG (250×4.6 mm) was used as analytical column.
The separation resulted in 60 mg of enantiomer 1 with HPLC purity of 99.9% and 58 mg of enantiomer 2 with HPLC purity of 99.4%.
m-(4H-1,2,4-Triazol-4-yl)benzoic acid (A) (120 mg, 0.64 mmol) followed by DIPEA (0.45 mL, 2.59 mmol) and propyl phosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.23 mL, 1.94 mmol) were added to a stirred solution of ethyl (E)-2-amino-5,5-dimethyl-3-hexenoate (B) (122.5 mg, 0.64 mmol) in DMF (3 mL) at 25-30° C. The resulting reaction mixture was stirred over a period of 48 h at 40° C. The progress of the reaction was monitored by TLC.
After the completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted to ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford the crude product. This was purified by RP-HPLC to afford compound C as clear thick oil.
Yield: 70 mg, (31.0%)
1H-NMR-DMSO-d6 (400 MHz): δ: 1.06 (9H, s), 1.17 (3H, t, J=7.2 Hz), 4.14 (2H, t, J=7.2 Hz), 4.92-4.94 (1H, m), 5.54-5.57 (1H, m), 5.90 (1H, d, J=14.4 Hz), 7.66-7.70 (1H, m), 7.87-7.93 (2H, m), 8.17 (1H, t, J=1.6 Hz), 9.02 (1H, m), 9.17 (2H, s);
LCMS: 357.3 [M+H]+
Lithium hydroxide monohydrate (16.57 mg, 0.39 mmol) was added to a stirred solution of compound C (70 mg, 0.19 mmol) in THF/methanol/water (2.1 mL, 1:1:1, 0.7 mL each). The resulting reaction mixture was stirred over a period of 5 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the residue was dissolved in water and washed with diethyl ether (10 mL), and the aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl. The precipitate formed was collected by vacuum filtration, washed with water (5 mL) and hexane (5 mL) to give compound D as a white solid.
Yield: 28 mg (43.2%)
1H-NMR-DMSO-d6 (400 MHz): δ: 1.00 (9H, s), 4.91-4.95 (1H, m), 5.52-5.58 (1H, m), 5.83-5.87 (1H, m), 7.65-7.69 (1H, m), 7.87-7.96 (2H, m), 8.16 (1H, t, J=1.6 Hz), 9.02 (1H, m), 9.17 (2H, s); 12.72 (1H, bs);
LCMS: 329.3 [M+H]+ HPLC purity −90.7%
Sodium methoxide solution (25 wt. % in methanol) (62.49 mL, 0.2898 mol) was added dropwise to a stirred solution of 2-chloro-6-methylnicotinaldehyde (A) (5.0 g, 0.032 mol) and ethyl azido acetate (B) (9.26 mL, 0.080 mol) in methanol (50 mL) at −20° C. to −30° C. over a period of 20 min. The resulting reaction mixture was stirred at 0-5° C. over a period of 5 h. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched with ice-cold NH4Cl solution and stirred at 5-10° C. over a period of 1 h to give a solid precipitation. The solid was collected by vacuum filtration, washed with water and dried to afford compound C as a pale yellow solid.
Yield: 1.88 g, (23.2%)
1H-NMR-DMSO-d6 (400 MHz): δ: 2.46 (3H, s), 3.87 (3H, s), 6.99 (1H, s), 7.36 (1H, d, J=10.8 Hz), 8.46 (1H, d, J=10.8 Hz);
LCMS: 352.8 [M+H]+
A solution of compound C (1.8 g, 0.0071 mol) in o-xylene (45 mL) was heated at 120° C. over a period of 4 h. After 4 h, the heating was switched off and the reaction mixture was left to stand at 25-30° C. for 16 h (solid precipitation was formed).
The solid was collected by vacuum filtration, washed with hexane (50 mL) and dried under reduced pressure to afford compound D as a pale yellow solid.
Yield: 0.85 g, (53.5%)
1H-NMR-DMSO-d6 (400 MHz): δ: 3.88 (3H, s), 7.12 (1H, s), 7.22 (1H, s), 12.59 (1H, s);
LCMS: 224.6 [M+H]+
10% Pd/C (0.17 g, 0.2% w/w) was added to a degassed solution of compound D (0.85 g, 0.0037 mol) in MeOH (30.6 mL) and THF (10.2 mL) at 25-30° C. The resulting reaction mixture was stirred under H2 atmosphere over a period of 4 h. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was filtered through a plug of celite and the celite plug was then washed with MeOH (50 mL). The combined filtrate was evaporated to dryness to afford the crude product (HCl salt) (0.8 g). The crude solid product was stirred with saturated NaHCO3 solution and DCM over a period of 1 h at 25-30° C. After 1 h, the layers were separated, the organic layer was dried over sodium sulphate and concentrated at 40° C. to afford compound E as a pale yellow solid.
Yield: 0.69 mg, (95.9%)
1H-NMR-DMSO-d6 (400 MHz): δ: 3.88 (3H, s), 7.12 (1H, s), 7.22 (1H, s), 12.59 (1H, s);
LCMS: 191.1 [M+H]+
Sodium hydride (60% dispersion in mineral oil) (0.16 g, 0.0039 mol) was added in portions (4 portions) over a period of 15 min to a stirred solution of compound E (0.69 g, 0.0036 mol) in THF (13.8 mL) and DMF (6.9 mL) at 0-5° C. After 10 min of stirring, SEM-CI (0.70 mL, 0.039 mol) was added dropwise at 0-5° C. The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was quenched by the addition of ice-cold water and the product was extracted to ethyl acetate (3×50 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford crude solid. The crude solid was purified by CombiFlash® using ethyl acetate/hexane as eluent to afford pure product F as a brown thick oil.
Yield: 0.55 g (47.4%)
1H-NMR-DMSO-d6 (300 MHz): δ: −0.13 (9H, s), 0.75 (2H, t, J=10.4 Hz), 2.55 (3H, s), 3.41 (2H, t, J=2.0 Hz), 3.85 (3H, s), 5.90 (2H, s), 7.43 (1H, s), 7.54 (1H, s), 8.86 (1H, s);
LCMS: 321.3 [M+H]+
Lithium hydroxide monohydrate (0.19 g, 0.0046 mol) was added to a stirred solution of compound F (0.3 g, 0.0009 mol) in THF/methanol/water (9 mL, 1:1:1, 3 mL each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, and the remaining residue was dissolved in water and washed with MTBE (20 mL), the aqueous layer was neutralized to pH ˜5-6 using 1.5 N HCl to afford a solid precipitate. The solid was collected by vacuum filtration, washed with water and dried at 40° C. to afford compound G as a pale brown solid.
Yield: 0.24 g (83.9%)
1H-NMR-DMSO-d6 (400 MHz): δ: −0.13 (9H, s), 0.80 (2H, t, J=8.0 Hz), 2.70 (3H, s), 3.47 (2H, t, J=7.6 Hz), 6.01 (2H, s), 7.59 (1H, s), 8.02 (1H, s), 9.20 (1H, s), 14.56 (1H, bs)
LCMS: 307.2 [M+H]+
Ethyl (E)-2-amino-5,5-dimethyl-3-hexenoate (H) (96.73 mg, 0.5221 mmol), followed by HATU (297.8 mg, 0.7832 mmol), HOBt (105.8 mg, 0.7832 mmol) and DIPEA (0.18 mL, 1.0442 mmol) were added to a stirred solution of compound G (160 mg, 0.5221 mmol) in DMF (3.2 mL) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution until pH ˜7-8 and then the product was extracted to ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford crude product. The crude product was purified by CombiFlash® using ethyl acetate/hexane as eluent to afford compound I as yellow thick oil.
Yield: 83 mg (29.4%)
1H-NMR-DMSO-d6 (400 MHz): δ: −0.14 (9H, S), 0.73 (2H, t, J=10.4 Hz), 0.99 (9H, S), 1.18 (3H, t, J=7.22 Hz), 2.54 (3H, s), 3.37 (2H, m), 4.04-4.14 (2H, m), 4.87 (1H, t, J=7.2 Hz), 5.55-5.72 (1H, m), 5.87-5.90 (3H, m), 7.33 (1H, s), 7.45 (1H, s), 8.83 (1H, s), 9.10 (1H, d, J=7.2 Hz);
LCMS: 474.4 [M+H]+
Lithium hydroxide monohydrate (35.4 mg, 0.8444 mmol) was added to a stirred solution of compound I (80 mg, 0.1688 mmol) in THF/methanol/water (2.4 mL, 1:1:1, 0.8 mL each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the residue was dissolved in water and washed with ethyl acetate (10 mL) and the aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl at 0-5° C. (solid precipitated). Solid was collected by vacuum filtration, washed with water and dried at 40° C. to afford pure compound J as off white solid.
Yield: 44 mg (58.5%)
1H-NMR-DMSO-d6 (400 MHz): δ: −0.13 (9H, S), 0.75 (2H, t, J=1.6 Hz), 1.0 (9H, S), 2.69 (3H, s), 3.44 (2H, t, J=6.8 Hz), 4.91 (1H, t, J=6.4 Hz), 5.51-5.55 (1H, m), 5.82-5.85 (1H, m), 6.0-6.02 (2H, m), 7.58 (1H, s), 7.97 (1H, s), 9.24-9.32 (1H, m), 12.87 (1H, bs), 15.34 (1H, bs);
LCMS: 446.2 [M+H]+
4 M HCl in 1,4-dioxane solution (0.8 mL) was added to a stirred solution of compound J (40 mg, 0.0897 mmol) in 1,4-dioxane (0.4 mL), at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated; the solid obtained was washed with hexane (5 mL) and diethyl ether (5 mL) to afford the crude product. This was purified by RP-HPLC to afford the pure compound K as a white solid.
Yield: 8 mg (28.3%)
1H-NMR-DMSO-d6 (400 MHz): δ: 1.0 (9H, S), 2.49 (3H, s), 4.90 (1H, t, J=6.0 Hz), 5.53-5.55 (1H, m), 5.79 (1H, t, J=15.6 Hz), 7.17 (1H, s), 7.35 (1H, s), 8.79-8.84 (3H, m), 11.83 (1H, bs);
LCMS: 316.3 [M+H]+; HPLC purity 93.5%.
K2CO3 (0.99 g, 0.0072 mol) and 2-(trimethylsilyl)ethoxymethyl chloride (SEM-CI) (0.43 mL, 0.0024 mol) were added to a stirred solution of 4-chloro-1H-pyrrole-2-carboxylic acid (A) (0.3 g, 0.0020 mol) in DMF (3 mL) at 25-30° C. The resulting reaction mixture was stirred at 25-30° C. over a period of 18 h. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was filtered through a plug of celite. The filtrate was concentrated under reduced pressure at 40° C. Upon concentration, a thick oily mass was obtained, and it was dissolved in water and the pH of the solution was adjusted to 3-4 using 1.5 N HCl solution. The product was extracted using ethyl acetate (3×10 mL). The organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure at 40° C. to afford the crude product. This was further purified by using CombiFlash® using ethyl acetate/hexane as eluent to afford the product B as a clear thick oil.
Yield: 0.22 g (38.7%)
1H-NMR-DMSO-d6 (400 MHz): δ: 0.11 (9H, s), 0.89 (2H, t, J=9.4 Hz), 3.72 (2H, t, J=8.8 Hz), 5.38 (2H, s), 6.79-6.80 (1H, m), 7.17-7.18 (1H, m), 12.3 (1H, bs);
LCMS: No ionization was observed
Oxalyl chloride (0.1 mL, 0.0011 mol) was added dropwise along with a catalytic amount of DMF (0.12 mL) to a stirred solution of compound B (0.3 g, 0.0009 mol) in DCM (6 mL) at 0-5° C. The resulting reaction mixture was stirred at 25-30° C. over a period of 2 h. The progress of the reaction was monitored by TLC.
After 2 h, the reaction mixture was concentrated to dryness at 35° C. and any remaining water was stripped off with toluene (3×5 mL). This resulted in a yellow thick oil. This was further dissolved in DCM (3 mL) and added to a stirred solution of ethyl (E)-2-amino-5,5-dimethyl-3-hexenoate (C) (0.16 g, 0.0009 mol) and DIPEA (0.62 mL, 0.0036 mol) in DCM (3 mL) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was quenched by the addition of saturated NaHCO3 solution (15 mL) and the product was extracted with ethyl acetate (3×10 mL). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure at 35° C. to afford the crude product as a pale brown thick oil. The crude product was purified by CombiFlash® using ethyl acetate/hexane as an eluent to afford the pure product D as a yellow thick oil.
Yield: 40 mg (14.3% over two steps)
1H-NMR-DMSO-d6 (400 MHz): δ: 0.99 (9H, s), 1.15 (3H, t, J=7.2 Hz), 4.05-4.14 (2H, m), 4.84-4.88 (1H, m), 5.45-5.50 (1H, m), 5.74-5.83 (1H, m), 6.94-6.98 (2H, m), 8.47 (1H, t, J=6.8 Hz); 11.89 (1H, bs).
Lithium hydroxide monohydrate (10.73 mg, 0.255 mmol) was added to a stirred solution of compound D (40 mg, 0.1278 mmol) in THF/methanol/water (1.2 mL, 1:1:1, 0.4 mL each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the remaining residue was dissolved in water and washed with ethyl acetate (10 mL), and the aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl at 0-5° C. The product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The crude mixture was purified by RP-HPLC to afford product E as a white solid.
Yield: 3.8 mg
LCMS: 285.2 [M+H]+
3-Isobutyl-1-methyl-5-pyrazolecarboxylic acid (A) (100 mg, 0.27 mmol) followed by DIPEA (0.38 mL, 1.09 mmol) and propyl phosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.04 mL, 0.82 mmol) were added to a stirred solution of ethyl (E)-2-amino-5,5-dimethyl-3-hexenoate (B) (101.6 mg, 0.27 mmol) in DMF (2.5 mL) at 25-30° C. The reaction mixture was stirred over a period of 48 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted with ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulphate and concentrated under reduced pressure at 35° C. to afford the crude product. This was purified by CombiFlash® using ethyl acetate/hexane as eluent to afford a mixture of compound C and an isomer as a yellow thick oil.
Yield: 66.6 mg (34.8%)
1H-NMR-DMSO-d6 (400 MHz): δ: (mixture of isomers) 0.88-0.90 (9H, s), 0.97 (9H, s), 1.20-1.23 (6H, m), 1.82-1.86 (1H, m), 2.37-2.40 (3H, m), 3.93 (3H, s), 4.07-4.14 (2H, m), 4.81-4.83 (1H, m), 5.49-5.51 (1H, m), 5.81-5.86 (1H, m), 6.58 (0.29H, m), 6.77 (1.31H, m), 8.79 (1H, m), 9.59 (0.29H, m);
LCMS: 350.3 [M+H]+
Lithium hydroxide monohydrate (24.01 mg, 0.57 mmol) was added to a stirred solution of compound C (100 mg, 0.28 mmol) in THF/methanol/water (3 mL, 1:1:1, 1.0 mL each). The resulting reaction mixture was stirred over a period of 5 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. This was washed with hexane (5 mL) to afford a crude mixture of two isomers as an off white solid. The mixture was purified by RP-HPLC to afford the pure compound D as a white solid.
Yield: 23.4 mg
1H-NMR-DMSO-d6 (400 MHz): δ: 0.83 (6H, d, J=6.4 Hz), 0.91 (9H, s), 1.78-1.82 (1H, m), 2.34 (2H, d, J=7.2 Hz), 3.93 (3H, s), 4.50-4.53 (1H, m), 5.46-5.58 (2H, m), 6.64 (1H, s), 8.04 (1H, d, J=5.2 Hz);
LCMS: 322.3 [M+H]+
HPLC Purity −99.76%
A pre-dissolved solution of 2-pyridinecarboxaldehyde (A) (2.0 g, 0.018 mol) in THF (6.0 mL) was added dropwise over a period of 15-20 min at −10° C. to −15° C. to a stirred solution of ethynyl magnesium bromide solution (0.5 M in THF) (44.7 mL, 0.0224 mol). The resulting reaction mixture was stirred over a period of 1 h at −5° C. to −10° C. and at 25-30° C. for 1 h. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched with saturated NH4Cl solution (100 mL) and the product was extracted with ethyl acetate (3×50 mL). The combined organic layer was dried over anhydrous sodium sulphate and concentrated in vacuo at 40° C. to afford crude product. The crude solid was purified by CombiFlash® using ethyl acetate/hexane as eluent to afford the product B as a brown oil.
Yield: 1.56 g (63.2%)
1H-NMR-CDCl3 (300 MHz): δ: 2.59 (1H, d, J=2.8 Hz), 5.53 (1H, d, J=2.8 Hz), 7.31-7.37 (1H, m), 7.56 (1H, d, J=10.4 Hz), 7.77-7.82 (1H, m), 8.58 (1H, t, J=5.2 Hz);
LCMS: 134.23 [M+H]+
Silver carbonate (0.414 g, 0.0015 mol) was added to stirred solution of compound B (1.5 g, 0.011 mol) and ethyl isocyanoacetate (0.849 ml, 0.011 mol) in 1,4-dioxane at 80° C. The resulting reaction mixture was stirred over a period of 16 h at 80° C. The progress of the reaction was monitored by TLC.
After completion, the reaction mixture was evaporated to dryness at 40° C. The obtained residue was dissolved in DCM (50 mL), and the solution was filtered through a celite plug. The DCM layer was washed with saturated sodium chloride solution (2×50 mL). The organic layer was dried over anhydrous sodium sulphate and concentrated in vacuo at 40° C. to afford a crude product. The crude product was purified by CombiFlash® using ethyl acetate/hexane as eluent to afford the product (C) as a brown thick oil.
Yield: 0.16 g (6%)
1H-NMR-CDCl3 (400 MHz): δ: 1.33 (3H, t, J=6.8 Hz), 4.35 (2H, q, J=5.6 Hz), 7.27-7.49 (1H, m), 7.71 (1H, d, J=1.2 Hz), 7.86-7.90 (1H, m), 8.13-8.15 (1H, m), 8.32 (1H, d, J=3.2 Hz), 8.71-8.74 (1H, m), 9.55 (1H, bs);
LCMS: 245.28 [M+H]+
K2CO3 (226.3 mg, 1.6377 mmol) followed by 2-(trimethylsilyl)ethoxymethyl chloride (SEM-CI) (0.13 ml, 0.786 mmol) were added to a stirred solution of compound C (160 mg, 0.655 mmol) in DMF (3.2 mL) at 25-30° C. The resulting reaction mixture was stirred at 25-30° C. over a period of 18 h. The progress of the reaction was monitored by TLC.
After completion of the reaction, the mixture was diluted with water (10 mL) and the product was extracted with ethyl acetate (3×10 mL). The organic layer was dried over anhydrous sodium sulphate and concentrated in vacuo at 40° C. to afford a crude product. The crude product was purified by CombiFlash® using ethyl acetate/hexane as eluent to afford the product D as a clear thick oil.
Yield: 50 mg, (20.4%)
1H-NMR-CDCl3 (400 MHz): δ: −0.08 (9H, s), 0.94 (2H, t, J=8.4 Hz), 1.38 (3H, t, J=6.8 Hz), 3.60 (2H, t, J=8.0 Hz), 4.34 (2H, q, J=7.2 Hz), 5.77 (2H, s), 7.46-7.49 (1H, m), 7.86 (1H, d, J=1.6 Hz), 7.88-7.90 (1H, m), 8.13-8.15 (1H, m), 8.72 (1H, d, J=1.6 Hz), 8.73 (1H, d, J=2.0 Hz);
LCMS: 375.3 [M+H]+
tert-Butanesulfinamide (126.2 mg, 1.041 mmol) and Ti(OEt)4 (0.36 mL, 1.735 mmol) were added to a stirred solution of compound D (130 mg, 0.347 mmol) in dry THF (1.95 mL) at 25-30° C. The resulting reaction mixture was heated at 80° C. over a period of 18 h. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched with ice-cold water (5 mL) and the product was extracted to ethyl acetate (3×5 mL). The organic layer was dried over anhydrous sodium sulphate and concentrated in vacuo at 40° C. to afford a crude product. The crude product was purified by CombiFlash® using ethyl acetate/hexane as eluent to afford compound E as a yellow oil.
Yield: 110 mg, (66.3%)
1H-NMR-DMSO-d6 (400 MHz): δ: −0.09 (9H, s), 0.92 (2H, t, J=8.2 Hz), 1.15 (9H, s), 1.25 (3H, t, J=6.8 Hz), 3.44 (2H, t, J=7.6 Hz), 4.23 (2H, q, J=7.2 Hz), 5.62 (2H, s), 7.06 (1H, bs), 7.51-7.56 (3H, m), 7.93 (1H, t, J=7.6 Hz), 8.66 (1H, d, J=4.4 Hz);
LCMS: 478.2 [M+H]+
NaBH4 (435 mg, 1.151 mmol) was added to a stirred solution of compound E (110 mg, 0.230 mmol) in dry THF (2.2 mL) at −78° C. The resulting reaction mixture was stirred at −40° C. to −60° C. over a period of 5 h and at 25-30° C. for 6 h. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched with ice-cold water (5 mL) and the product was extracted to ethyl acetate (3×5 mL). The organic layer was dried over anhydrous sodium sulphate and concentrated in vacuo at 40° C. to afford the crude compound F as pale yellow oil. The crude material was used as such for the next reaction.
Yield: 100 mg, (crude)
1H-NMR-DMSO-d6 (300 MHz): δ: −0.10 (9H, s), 0.75 (2H, t, J=10.4 Hz), 1.06-1.26 (12H, m), 3.37 (2H, t, J=2.8 Hz), 4.16 (2H, q, J=3.6 Hz), 5.38-5.42 (1H, s), 5.54 (2H, s), 5.89-5.92 (1H, s), 6.76-6.78 (1H, m), 7.15 (1H, d, J=2.8 Hz), 7.26-7.31 (1H, m), 7.49-7.55 (1H, m), 7.77-7.71 (1H, m), 8.48-8.51 (1H, m);
LCMS: 480.3 [M+H]+
Lithium hydroxide monohydrate (41.54 mg, 0.990 mmol) was added to a stirred solution of compound F (95 mg, 0.198 mmol) in a mixture of THF/methanol/water (2.85 mL, 0.95 ml each) at room temperature (20-25° C.). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was evaporated to dryness. The obtained residue was dissolved in purified water (5 mL) and washed with MTBE (2×10 mL). The aqueous layer was acidified to pH ˜5-6 using 0.5 N HCl solution and the product was extracted with ethyl acetate (3×15 mL). The combined organic layer was dried over anhydrous sodium sulphate and concentrated at 40° C. to afford a crude solid. The crude solid was washed with hexane (5 mL) and diethyl ether (10 mL) to afford crude compound G as an off white solid. The crude compound was used as such for next reaction.
Yield: 54 mg, (crude)
1H-NMR-DMSO-d6 (400 MHz): δ: −0.10 (9H, s), 0.75 (2H, t, J=7.6 Hz), 1.06-1.13 (9H, m), 3.39 (2H, t, J=5.6 Hz), 5.42-5.44 (1H, s), 5.54 (2H, s), 5.89-5.91 (1H, s), 6.72-6.79 (1H, m), 7.04-7.08 (1H, m), 7.25-7.28 (1H, m), 7.45-7.49 (1H, m), 7.52-7.61 (1H, m), 7.76-7.79 (1H, m), 8.46-8.49 (1H, m);
LCMS: 452.3 [M+H]+
The final step in the synthesis of compound I was carried in the same way as described above for Example 7, steps 7 and 8, starting with the coupling of ethyl (E)-2-amino-5,5-dimethyl-3-hexenoate (H) with the prepared compound G, followed by subsequent hydrolysis of the resulting ethyl ester and removal of the two remaining protecting group. The final product was isolated as a mixture of diastereomers.
4-Chloro-1H-pyridin-2-one (A) (200 mg, 1.54 mmol), ethyl 1H-pyrazole-4-carboxylate (B) (325 mg, 2.32 mmol) and cesium carbonate (760 mg, 2.33 mmol) were mixed in dry N-methyl-2-pyrrolidone (0.5 ml). The mixture was heated at 120° C. for 16 h.
After the reaction was complete, ethyl acetate (3 mL) was added. The mixture was shaken and then centrifuged. The supernatant was removed and more ethyl acetate (3 mL) was added and the mixture was again shaken, centrifuged and the supernatant was removed. Water and NaOH (0.5 mL) were added and the mixture was heated at 50° C. overnight. The mixture was then centrifuged and the supernatant was removed. Water was added, and the mixture was again shaken and centrifuged. The supernatant was removed. The solid material obtained was dried under vacuum at 45° C. to give a beige solid, 439 mg. Two compounds were detected in a 1:1 ratio according to LC-MS and NMR and assigned to a mixture of compound C and its corresponding ethyl ester.
The crude mixture (50 mg) was dissolved in methanol (0.5 mL) and aqueous (aq) NaOH (5 M, 0.5 mL) was added and the mixture was heated at 60° C. for 2 h. The resulting mixtures was acidified to pH 4-5 by addition of aq HCl (5 M). The mixture was centrifuged and the supernatant was removed. Water and acetonitrile were added and the solvents were evaporated. The crude solid compound C formed was used directly in the next step.
The final step in the synthesis of compound E was carried in the same way as described above for Example 5, starting with the coupling of ethyl (E)-2-amino-5,5-dimethyl-3-hexenoate (D) with the prepared compound C, followed by subsequent hydrolysis of the resulting ethyl ester giving the expected product.
2-(Dimethylamino)-5-pyrimidinecarboxylic acid (B) (90.10 mg, 0.53 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hexenoate (A) (100 mg, 0.53 mmol) in DMF (2.5 mL) followed by DIPEA (0.376 mL, 2.15 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.028 mL, 1.61 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted to ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution (15 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford crude product. The crude material was purified by RP-HPLC to afford pure product C as an off white solid.
Yield: 25 mg, 13.8%
LCMS: 335.26 [M+H]+
Lithium hydroxide monohydrate (15.69 mg, 0.37 mmol) was added to a stirred solution of compound C (25 mg, 0.07 mmol) in THF/methanol/water (0.75 mL, 1:1:1, 0.25 mL each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The crude material was washed with hexane (5 mL) to afford pure compound D as white solid.
Yield: 16.4 mg
1H-NMR-DMSO-d6 (400 MHz): δ: 0.98 (9H, s), 3.17 (6H, s), 4.78-4.81 (1H, m), 5.49-5.54 (1H, m), 5.76 (1H, d, J=16.4 Hz), 8.50 (1H, bs) 8.78 (2H, s);
LCMS: 307.3 [M+H]+; HPLC purity −94.39%
2-Isopropyl-4-methyl-5-pyrimidinecarboxylic acid (B) (144.16 mg, 0.80 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (A) (150 mg, 0.80 mmol) in DMF (3.75 mL) followed by DIPEA (0.55 mL, 3.2 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.52 mL, 2.4 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted to ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford crude product. The crude material was purified by CombiFlash® using ethyl acetate:hexanes as eluent to afford pure product C as a yellow, thick oil.
Yield: 66 mg (35.9%)
1H-NMR-CD3OD (400 MHz): δ: 1.12 (9H, s), 1.39-1.46 (9H, m), 2.73 (3H, s), 3.28-3.32 (1H, m), 4.35-4.38 (2H, m), 5.14 (1H, d, J=7.5 Hz), 5.62-5.70 (1H, m), 6.07 (1H, d, J=15.3 Hz), 6.94-7.26 (1H, m), 8.76 (1H, s);
LCMS: 348.3 [M+H]+
Lithium hydroxide monohydrate (39.9 mg, 1.04 mmol) was added to a stirred solution of compound C (66 mg, 0.19 mmol) in THF/methanol/water (2.4 mL, 1:1:1, 0.8 mL each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the residue was dissolved in water and washed with diethyl ether (10 mL), the aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The crude material was washed with hexane (5 mL) to afford a crude mixture of two isomers as an off white solid. The crude mixture was purified by RP-HPLC to afford pure product D as a white solid.
Yield: 12 mg
1H-NMR-DMSO-d6 (400 MHz): δ: 0.98 (9H, s), 1.24 (6H, d), 2.48 (3H, s), 3.05-3.12 (1H, m), 4.81-4.84 (1H, m), 5.42-5.48 (1H, m), 5.81-5.85 (1H, m), 8.59 (1H, s), 8.94 (1H, d, J=7.2 Hz), 12.74 (1H, bs);
LCMS: 320.3 [M+H]; HPLC purity: 94.9%.
p-(4H-1,2,4-Triazol-4-yl)benzoic acid (B) (50.88 mg, 0.26 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (A) (50 mg, 0.26 mmol) in DMF (1.25 mL) followed by DIPEA (0.18 mL, 1.07 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (0.51 mL, 0.807 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted to ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford crude product. The crude was purified by CombiFlash® using ethyl acetate:hexanes as eluent to afford pure compound C as thick yellow liquid.
Yield: 50 mg (52.0%)
1H-NMR-CD3OD (300 MHz): δ: 0.98 (9H, s), 1.30-1.34 (3H, m), 4.19-4.25 (2H, m), 5.07-4.89 (1H, m), 5.58-5.65 (1H, m), 5.92-5.97 (1H, m), 7.80 (2H, d, J=8.7 Hz), 8.09 (2H, d, J=9 Hz), 9.13 (2H, s);
LCMS: 357.4 [M+H]+
Lithium hydroxide monohydrate (29.46 mg, 0.70 mmol) was added to a stirred solution of compound C (50 mg, 0.14 mmol) in THF/methanol/water (1.5 mL, 1:1:1, 0.5 mL each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The crude material was washed with hexane (5 mL) to afford a mixture of two isomers as an off white solid. The crude material was purified by RP-HPLC to afford the pure compound D as a white solid.
Yield: 4.4 mg;
1H-NMR-DMSO-d6 (400 MHz): δ: 0.9 (9H, s), 4.89-4.90 (1H, t, J=6.4 Hz), 5.57-5.59 (1H, m), 5.80-5.84 (1H, m), 7.85-7.86 (2H, dd, J1=2 Hz, J2=7.2 Hz), 8.07-8.08 (1H, dd, J1=1.6 Hz, J2=6.8 Hz), 8.89-8.91 (1H, d), 9.23 (2H, s), 12.63 (1H, bs),
LCMS: 329.4 [M+H]+; HPLC purity 99.4%
m-(1H-1,2,4-Triazol-1-yl)benzoic acid (B) (101.96 mg, 0.53 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (A) (100 mg, 0.53 mmol) in DMF (2.5 mL) followed by DIPEA (0.37 mL, 2.15 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.02 mL, 1.61 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted to ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution (15 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford crude product. The crude material was purified by CombiFlash® using ethyl acetate:hexanes as eluent to afford compound C as a thick yellow liquid.
Yield: 64 mg, (33.3%)
1H-NMR-CD3OD (400 MHz): δ: 1.07 (9H, s), 1.24-1.30 (3H, m), 4.21-4.24 (2H, m), 5.05-5.07 (1H, d, J=7.6 Hz), 5.60-5.64 (1H, m), 5.93-5.97 (1H, m), 7.66-7.70 (1H, m), 7.94 (1H, d, J=7.6 Hz), 8.03-8.05 (1H, m), 8.21 (1H, s), 8.34 (1H, s), 9.17 (1H, s);
LCMS: 357.6 [M+H]+
Lithium hydroxide monohydrate (37.69 mg, 0.8975 mmol) was added to a stirred solution of compound C) (64 mg, 0.1795 mmol) in THF/methanol/water (1.92 mL, 1:1:1, 0.64 mL each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The crude material was washed with hexane (5 mL) to afford a mixture of two isomer as an off white solid. The mixture was purified by RP-HPLC to afford compound D as a white solid.
Yield: 29 mg
1H-NMR-DMSO-d6 (400 MHz): δ: 1.0 (9H, s), 4.90-4.94 (1H, m), 5.54-5.60 (1H, m), 5.83-5.87 (1H, m), 7.67 (1H, t, J=8 Hz), 7.94 (1H, d, J=8 Hz), 8.02-8.05 (1H, m), 8.28 (1H, s), 8.34 (1H, t, J=1.6 Hz), 9.00 (1H, d, J=7.2 Hz), 9.36 (1H, s), 12.60 (1H, bs);
LCMS: 329.3 [M+H]+; HPLC purity −99.6%
m-(1-Imidazolyl)benzoic acid (B) (101.5 mg, 0.53 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (A) (100 mg, 0.53 mmol) in DMF (2.5 mL) followed by DIPEA (0.37 mL, 2.15 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.03 mL, 1.61 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted with ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford a crude product. The crude material was purified by CombiFlash® using ethyl acetate:hexanes as eluent to afford pure compound C as a brown, thick oil.
Yield: 65 mg, (33.9%)
1H-NMR-CD3OD (400 MHz): δ: 1.04 (9H, s), 1.24 (3H, t, J=7.2 Hz), 4.18-4.21 (2H, m), 5.02-5.04 (1H, m), 5.69-5.61 (1H, m), 5.90-5.94 (1H, m), 7.16 (1H, s), 7.62-7.64 (2H, m), 7.77-7.79 (1H, m), 7.87-7.89 (1H, m), 8.05 (1H, s), 8.21 (1H, s);
LCMS: 356.3 [M+H]+
Lithium hydroxide monohydrate (32.5 mg, 0.77 mmol) was added to a stirred solution of compound C (61 mg, 0.17 mmol) in THF/methanol/water (1.83 mL, 1:1:1, 0.61 mL each) was added. The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the residue was dissolved in water and washed with diethyl ether (10 mL), The aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The obtained crude solid was washed with hexane (5 mL) to afford a crude mixture of two isomers as a white solid. The crude mixture was purified by RP-HPLC to afford the pure product D as a white solid.
Yield: 20 mg
1H-NMR-DMSO-d6 (400 MHz): δ: 1.22 (9H, s), 4.93-4.97 (1H, m), 5.52-5.60 (1H, m), 5.82-5.89 (1H, m), 7.23 (1H, s), 7.61-7.67 (1H, m), 7.83-7.91 (3H, m), 8.13 (1H, s), 8.47 (1H, s), 8.94 (1H, d, J=9.2 Hz), 12.63 (1H, bs);
LCMS: 328.3 [M+H]+; HPLC purity −99.6%
p-(2-Thienyl)benzoic acid (B) (108.1 mg, 0.53 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (A) (100 mg, 0.53 mmol) in DMF (2.5 mL) followed by DIPEA (0.37 mL, 2.15 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.03 mL, 1.61 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted with ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford crude product. The crude material was purified by CombiFlash® using ethyl acetate:hexanes as eluent to afford compound C as a pale yellow thick oil.
Yield: 117 mg (59.4%)
1H-NMR-CD3OD (400 MHz): δ: 1.04 (9H, s), 1.24 (3H, t, J=7.2 Hz), 4.19 (2H, d, J=5.6 Hz), 5.01-5.03 (1H, m), 5.48-5.53 (1H, m), 5.88-5.92 (1H, m), 7.10-7.14 (1H, m), 7.42-7.50 (2H, m), 7.71-7.73 (2H, m), 7.85-7.88 (2H, m);
LCMS: 372.10 [M+H]+
Lithium hydroxide monohydrate (31.6 mg, 0.75 mmol) was added to a stirred solution of compound C (112 mg, 0.30 mmol) in MeOH: THF:water (3.3 mL, 1:1:1, 1.1 mL each). The resulting reaction mixture was stirred over a period of 7 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜4-5 using saturated solution of potassium hydrogen sulphate and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The obtained solid was washed with hexane (5 mL) to afford a mixture of two isomers as a white solid. The crude product was purified by RP-HPLC to afford pure compound D as a white solid.
Yield: 48 mg;
1H-NMR-DMSO-d6 (400 MHz): δ: 1.00 (9H, s), 4.87-4.91 (1H, m), 5.53-5.61 (1H, m), 5.84 (1H, d, J=20.8 Hz), 7.16-7.19 (1H, m), 7.62-7.65 (2H, m), 7.74-7.77 (2H, m), 7.93-7.96 (2H, m), 8.81 (1H, d, J=9.6 Hz), 12.62 (1H, bs);
LCMS: 344.4 [M+H]+; HPLC purity −99.1%.
m-(2-Thienyl)benzoic acid (B) (109.96 mg, 0.53 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (A) (100 mg, 0.53 mmol) in DMF (2.5 mL) followed by DIPEA (0.37 mL, 2.15 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.02 mL, 1.61 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted to ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution (15 mL), dried over anhydrous sodium sulphate and concentrated in vacuum at 35° C. to afford the crude product, The crude material was purified by CombiFlash® using ethyl acetate:hexanes as eluent to afford compound C as a thick yellow liquid.
Yield: 100 mg, (49.8%)
1H-NMR-CD3OD (400 MHz): δ: 1.04 (9H, s), 1.25 (3H, t, J=6.8 Hz), 4.18-4.21 (2H, m), 5.03 (1H, m), 5.57-5.62 (1H, m), 5.89-5.94 (1H, m), 7.09-7.11 (1H, m), 7.39-7.41 (1H, m), 7.45-7.49 (2H, m), 7.73-7.79 (2H, m), 8.11-8.12 (1H, m);
LCMS: 372.08 [M+H]+
Lithium hydroxide monohydrate (56.51 mg, 1.34 mmol) was added to a stirred solution of compound C (100 mg, 0.26 mmol) in THF/methanol/water (3.0 mL, 1:1:1, 1.0 mL each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford crude solid. The obtained solid was washed with hexane (5 mL) to afford a crude mixture of two isomers as an off white solid. The crude mixture was purified by RP-HPLC to afford pure compound D
Yield: 52.8 mg;
1H-NMR-DMSO-d6 (400 MHz): δ: 1.01 (9H, s), 4.89-4.94 (1H, m), 5.54-5.62 (1H, m), 5.81-5.87 (1H, d, J=15.6 Hz), 7.16-7.19 (1H, m), 7.49-7.54 (1H, m), 7.60 (2H, d, J=5.7 Hz), 7.83 (2H, d, J=7.5 Hz), 8.14 (1H, s), 8.93 (1H, d, J=6.9 Hz), 12.72 (1H, bs);
LCMS: 344.2 [M+H]+; HPLC purity −99.8%.
SEM-CI (0.279 mL, 1.57 mmol) was added to a stirred solution of 4-bromo-2-pyrrolecarboxylic acid (A) (250 mg, 1.31 mmol) in DMF (2.5 mL) followed by K2CO3 (653.6 mg, 4.71 mmol). The resulting reaction mixture was stirred over a period of 2 h at 25-30° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of water and adjusted to pH ˜5-6 with 1.5 N HCl and the product was extracted to ethyl acetate (2×40 mL). The combined organic layer was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford crude product. The crude material was purified by CombiFlash® using ethyl acetate:hexanes as eluent to afford compound B as a thick, colorless liquid.
Yield: 239 mg (56.7%)
1H-NMR-DMSO-d6 (300 MHz): δ: −0.01 (9H, s), 0.89 (2H, t, J=7.8 Hz), 3.73 (2H, t, J=7.8 Hz), 5.37 (2H, s), 6.86 (1H, s), 7.21 (1H, s), 12.37 (1H, bs) Step 2: Synthesis of ethyl (E)-2-(4-bromo-1-{[2-(trimethylsilyl)ethoxy]methyl}-2-pyrrolylcarbonylamino)-5,5-dimethyl-3-hexenoate (D)
Oxalyl chloride (0.092 mL, 1.12 mmol) was added dropwise along with catalytic amount of DMF (0.12 mL) to a stirred solution of compound B (300 mg, 0.94 mmol) in DCM (6.0 mL) at 0-5° C. The resulting reaction mixture was stirred at 25-30° C. over a period of 2 h. The progress of the reaction was monitored by TLC (aliquot was diluted with MeOH and formation of methyl ester was confirmed).
After 2 h, the reaction mixture was concentrated to dryness at 35° C. and stripped off with toluene (5 mL×3). After complete concentration, a yellow, thick oil was obtained. This was further dissolved in DCM (3 mL) and was then added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (C) (0.16 g, 0.0009 mol) and DIPEA (0.62 mL, 0.0036 mol) in DCM (3 mL) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted to ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford crude material. The crude material was purified by CombiFlash© using ethyl acetate:hexanes as eluent to afford compound D as a thick yellow liquid.
Yield: 109 mg (21.9%);
1H-NMR-DMSO-d6 (400 MHz): δ: 1.02 (9H, s), 1.14-1.18 (3H, m), 4.10-4.13 (2H, m), 4.83-4.88 (1H, m), 5.44-5.52 (1H, m), 5.75-5.85 (1H, m), 7.02 (2H, s), 8.49 (1H, d, J=6.9 Hz), 11.88 (1H, bs);
LCMS: 381.1 [M+Na]+;
Lithium hydroxide monohydrate (58.8 mg, 1.39 mmol) was added to a stirred solution of compound D (100 mg, 0.27 mmol) in THF/methanol/water (3.0 mL, 1:1:1, 1.0 mL each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, and the residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The obtained solid was washed with hexane (5 mL) to afford a crude mixture of two isomers as an off white solid. The crude material was purified by RP-HPLC to afford pure compound E as a pale, yellow solid.
Yield: 5.2 mg;
1H-NMR-DMSO-d6 (300 MHz): δ: 0.99 (9H, s), 4.79 (1H, m), 5.48-5.55 (1H, m), 5.71-5.76 (1H, m), 6.99 (2H, s), 8.26 (1H, bs), 11.87 (1H, bs);
LCMS: 353.20 [M+Na]+; HPLC purity −98.5%
A pre-dissolved solution of t-butyl hypochlorite solution in CCl4 (40.1 mL) was added dropwise to a stirred solution of methyl 2-pyrrolecarboxylate (A) (1.18 g, 9.4 mmol) in CCl4 (200.6 mL) at 25-30° C. The resulting mixture was stirred over a period of 24 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated at 40° C. to afford a crude residue. The residue was purified by CombiFlash© using ethyl acetate:hexanes as eluent to afford compound B as a white solid.
Yield: 0.62 g (41.3%);
1H-NMR-DMSO-d6 (400 MHz): δ: 3.74 (3H, s), 6.13-6.15 (1H, m), 6.76-6.78 (1H, m), 12.70 (1H, bs);
LCMS: 159.85 [M+H]+
K2CO3 (0.46 g, 3.3 mmol) was added to a stirred solution of compound B (0.3 g, 1.8 mmol) in DMF (3 mL) at 25-30° C. After 5 min, SEM-chloride (0.39 mL, 2.2 mmol) was added dropwise at 25-30° C. The resulting mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted with ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford a crude residue. The residue was purified by CombiFlash© using ethyl acetate:hexanes as eluent to afford compound C as a colorless oil.
Yield: 0.47 g (86.4%)
1H-NMR-DMSO-d6 (400 MHz): δ: −0.09 (9H, s), 0.79 (2H, t, J=7.6 Hz), 3.49 (2H, t, J=7.6 Hz), 3.74 (3H, s), 5.69 (2H, s), 6.33 (1H, d, J=4.0 Hz), 6.98 (1H, d, J=4.0 Hz);
LCMS: 312.2 [M+Na]+
Lithium hydroxide monohydrate (0.16 g, 4.01 mmol) was added to a stirred solution compound C (0.46 g, 1.6 mmol) in MeOH/THF/water (13.8 mL, 1:1:1, 4.6 mL each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the resulting residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜4-5 using saturated solution of potassium hydrogen sulphate and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The solid was washed with hexane (5 mL) to afford compound D as a white solid.
Yield: 0.42 g (35.5%);
1H-NMR-DMSO-d6 (400 MHz): δ: −0.08 (9H, s), 0.79 (2H, t, J=7.6 Hz), 3.49 (2H, t, J=7.6 Hz), 5.72 (2H, s), 6.27 (1H, d, J=4.0 Hz), 6.90 (1H, d, J=4.0 Hz), 12.64 (1H, bs);
LCMS: 298.2 [M+Na]+
Oxalyl chloride at 0-5° C. was added to a stirred solution of compound D (0.2 g, 0.72 mmol) in DMF (4.0 mL) followed by DMF (0.08 mL). The resulting mixture was stirred over a period of 2 h at 25-30° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was concentrated and dried at 40° C. The pre-dissolved solution of the resulting residue in DCM (4.0 mL) was added to (E)-2-amino-5,5-dimethyl-3-hecenoate (E) (0.13 g, 0.72 mmol) followed by DIPEA (0.5 mL, 2.8 mmol) at 25-30° C. The resulting mixture was stirred over a period of 18 h at 25-30° C.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution (20 mL) to pH ˜7-8 and the product was extracted with DCM (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford a crude material. The crude material was purified by CombiFlash© using ethyl acetate:hexanes as eluent to afford compound F as a pale brown oil.
Yield: 0.228 g (70.9%);
1H-NMR-DMSO-d6 (400 MHz): δ: −0.09 (9H, s), 0.76 (2H, t, J=9.6 Hz), 0.98 (9H, s), 1.15 (3H, t, J=9.6 Hz), 3.44 (2H, t, J=10.4 Hz), 4.10 (2H, q, J=9.6 Hz), 4.82 (2H, t, J=9.6 Hz), 5.45-5.53 (1H, m), 5.74-5.83 (3H, m), 6.26 (1H, d, J=5.2 Hz), 7.01 (1H, d, J=5.6 Hz), 8.61 (1H, d, J=9.2 Hz); LCMS: 462.17 [M+Na]+
Lithium hydroxide monohydrate (0.052 g, 1.2 mmol) was added to a stirred solution of compound F (0.22 g, 0.49 mmol) in MeOH/THF/water (6.6 mL, 1:1:1, 2.2 mL each) was added. The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was concentrated, the resulting residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜4-5 using saturated solution of potassium hydrogen sulphate and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The resulting solid was washed with hexane (5 mL) to compound G as a white, gummy solid.
Yield: 0.19 g (95.9%);
1H-NMR-DMSO-d6 (400 MHz): δ: −0.09 (9H, s), 0.78 (2H, t, J=9.6 Hz), 0.98 (9H, s), 3.44 (2H, t, J=7.6 Hz), 4.78 (1H, t, J=6.4 Hz), 5.48-5.53 (1H, m), 5.69-5.79 (3H, m), 6.23 (1H, d, J=4.0 Hz), 6.99 (1H, d, J=4.0 Hz), 8.40 (1H, d, J=6.8 Hz), 12.71 (1H, s);
LCMS: 437.10 [M+Na]+
4 M HCl in 1,4-dioxane (3.8 mL) was added to a stirred solution of compound G (0.19 g, 0.45 mmol) in 1,4-dioxane (0.95 mL) at 25-30° C. The resulting mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated at 40° C. to afford a crude solid. The crude solid was washed with hexane (5 mL) a white solid (0.13 g).
Ammonium hydroxide solution (0.9 mL) was added to a crude solution of the solid material (90 mg, 0.28 mmol) in ACN (1.8 mL) at 25-30° C. The resulting mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated at 40° C. to afford a crude residue. The residue was washed with hexane (5 mL) to afford the pure product H, as a white solid.
Yield: 19.7 mg;
1H-NMR-DMSO-d6 (400 MHz): δ: 0.97 (9H, s), 4.81-4.85 (1H, m), 5.45-5.51 (1H, m), 5.74-5.78 (1H, m), 6.04 (1H, d, J=4.0 Hz), 6.88 (1H, d, J=4.0 Hz), 8.26 (1H, d, J=7.8 Hz); 12.2 4 (1H, bs), 12.60 (1H, bs);
LCMS: 285.3 [M+H]+; HPLC purity −98.4%
4,5-Dichloro-2-thenoic acid (B) (103.8 mg, 0.53 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (A) (100 mg, 0.53 mmol) in DMF (2.5 mL) followed by DIPEA (0.37 mL, 2.15 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.03 mL, 1.61 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted with ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford a crude product. The crude material was purified by CombiFlash© using ethyl acetate:hexanes as eluent to afford compound C as a pale yellow, thick oil.
Yield: 148 mg (76.6%);
1H-NMR-CD3OD (400 MHz): δ: 1.02 (9H, s), 1.22-1.26 (3H, m), 4.16-4.17 (2H, m), 4.93-4.96 (1H, m), 5.50-5.53 (1H, m), 5.86-5.90 (1H, m), 7.68-7.69 (1H, m);
LCMS: 363.97 [M+H]+
Lithium hydroxide monohydrate (40.9 mg, 0.97 mmol) was added to a stirred solution of compound C (142 mg, 0.38 mmol) in MeOH/THF/water (4.26 mL, 1:1:1, 1.42 mL each). The resulting reaction mixture was stirred over a period of 2.5 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, and the resulting residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜4-5 using a saturated solution of potassium hydrogen sulphate and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The obtained solid was washed with hexane (5 mL) to afford a mixture of two as a white solid. The crude mixture was purified by RP-HPLC to afford the pure product D as a white solid.
Yield: 65 mg;
1H-NMR-DMSO-d6 (400 MHz): δ: 0.99 (9H, s), 4.81-4.86 (1H, m), 5.44-5.52 (1H, m), 5.84 (1H, d, J=21.2 Hz), 8.01 (1H, s), 9.02 (1H, d, J=9.6 Hz), 12.81 (1H, bs);
LCMS: 336.3 [M+H]+; HPLC purity −98.4%.
4-Chloro-1-methyl-2-pyrrolecarboxylic acid (B) (86 mg, 0.53 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (A) (100 mg, 0.53 mmol) in DMF (2.5 mL) followed by DIPEA (0.35 mL, 2.12 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.03 mL, 1.61 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted to ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford crude product. The crude material was purified by RP-HPLC to afford pure compound C as a colorless, thick oil.
Yield: 11 mg, (6.5%)
LCMS: 327.5 [M+H]+
Lithium hydroxide monohydrate (7 mg, 0.15 mmol) was added to a stirred solution of compound C (11 mg, 0.03 mmol) in MeOH/THF/water (0.33 mL, 1:1:1). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The solid material was washed with hexane (5 mL) to afford a crude mixture of two isomers as a white solid. The crude material was purified by RP-HPLC to afford the product D as a white solid.
Yield: 0.4 mg
LCMS: 299.2 [M+H]+
4-Chloro-2-thenoic acid (B) (97.26 mg, 0.53 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (A) (100 mg, 0.53 mmol) in DMF (2.5 mL) followed by DIPEA (0.35 mL, 2.12 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.03 mL, 1.61 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted to ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution (15 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford a crude material. The crude material was purified by CombiFlash® using ethyl acetate:hexanes as eluent to afford compound C as a brown, thick oil.
Yield: 47 mg (27.0%)
1H-NMR-CD3OD (400 MHz): δ: 0.96 (9H, s), 1.26 (3H, t, J=7.2 Hz), 4.16-4.24 (2H, m), 4.96-4.98 (1H, m), 5.53-5.59 (1H, m), 5.93 (1H, d, J=1.2 Hz), 7.56 (1H, s), 7.73 (1H, s);
LCMS: 330.3 [M+H]+
Lithium hydroxide monohydrate (39.9 mg, 1.04 mmol) was added to a stirred solution compound C (47 mg, 0.14 mmol) in THF/methanol/water (1.41 mL, 1:1:1, 0.47 mL each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the resulting residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The obtained solid was washed with hexane (5 mL) to afford a crude mixture of two isomers as an off white solid. The crude mixture was purified by RP-HPLC to afford pure compound D as a white solid.
Yield: 22 mg;
1H-NMR-DMSO-d6 (400 MHz): δ: 0.97 (9H, s), 4.76 (1H, t, J=6.8 Hz), 5.46-5.51 (1H, m), 5.74 (1H, d, J=16.0 Hz), 7.80 (1H, s), 7.92 (1H, s), 8.76 (1H, bs);
LCMS: 302.6 [M+H]+; HPLC purity −99.2%.
p-(3-Pyridyloxy)benzoic acid (A) (116 mg, 0.53 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (B) (100 mg, 0.53 mmol) in DMF (2.5 mL) followed by DIPEA (0.35 mL, 2.12 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.03 mL, 1.61 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted to ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution (15 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford crude material. The crude material was purified by CombiFlash© using ethyl acetate:hexanes as eluent to afford compound C as a yellow, thick oil.
Yield: 50.0 mg, (24.2%);
1H-NMR-CD3OD (400 MHz): δ: 1.02 (9H, s), 1.24-1.31 (3H, m), 4.19-4.22 (2H, m), 5.04 (1H, d, J=1.2 Hz), 5.61-5.62 (1H, m), 5.94 (1H, d, J=1.2 Hz), 7.11 (2H, d, J=8.8 Hz), 7.46-7.66 (2H, m), 7.91-7.94 (2H, m), 8.37-8.38 (2H, m);
LCMS: 383.4 [M+H]+
Lithium hydroxide monohydrate (43.9 mg, 1.04 mmol) was added to a stirred solution of compound C (80 mg, 0.20 mmol) in THF/methanol/water (2.4 mL, 1:1:1, 0.8 mL each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the resulting residue was dissolved in water and washed with diethyl ether (10 mL): The aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The solid was washed with hexane (5 mL) to afford a crude mixture of two isomers as an off white solid. The crude product was purified by RP-HPLC to afford pure compound D as a white solid.
Yield: 3.0 mg;
1H-NMR-DMSO-d6 (300 MHz): δ: 1.1 (9H, s), 4.99 (1H, t, J=7.2 Hz), 5.62-5.70 (1H, m), 5.93 (1H, d, J=15.6 Hz), 7.21 (2H, d, J=8.7 Hz), 7.55-7.66 (2H, m), 8.06 (2H, d, J=8.7 Hz), 8.53 (2H, d, J=4.2 Hz), 8.88 (1H, d, J=7.2 Hz), 12.67 (1H, bs);
LCMS: 355.3 [M+H]+; HPLC purity −98.9%.
2-Phenoxyisonicotinic acid (A) (116.15 mg, 0.53 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (B) (100 mg, 0.53 mmol) in DMF (2.5 mL) followed by DIPEA (0.35 mL, 2.12 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.03 mL, 1.61 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted to ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution (15 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford crude product. The crude product was purified by CombiFlash® using ethyl acetate:hexanes as eluent to afford compound C as a brown, thick oil.
Yield: 103 mg, (49.9%);
1H-NMR-CD3OD (400 MHz): δ: 1.02 (9H, s), 1.23 (3H, t, J=6.8 Hz), 4.16-4.19 (2H, m), 5.00 (1H, d, J=1.2 Hz), 5.53 (1H, m), 5.89 (1H, d, J=1.2 Hz), 7.10 (2H, t, J=0.8 Hz), 7.12 (1H, d, J=0.8 Hz), 7.21 (1H, s), 7.29-7.46 (3H, m), 8.22 (1H, d, J=0.8 Hz);
LCMS: 383.3 [M+H]+
Lithium hydroxide monohydrate (54 mg, 1.3 mmol) was added to a stirred solution of compound C (100 mg, 0.26 mmol) in THF/methanol/water (3.0 mL, 1:1:1, 1.0 mL each) was added. The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, and the resulting residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The crude solid was washed with hexane (5 mL) to afford a crude mixture of two isomers as an off white solid. The crude mixture was purified by RP-HPLC to afford pure compound D as a white solid.
Yield: 45 mg;
1H-NMR-DMSO-d6 (400 MHz): δ: 0.97 (9H, s), 4.84 (1H, d, J=1.2 Hz), 5.54 (1H, d, J=1.2 Hz), 7.14 (2H, d, J=0.8 Hz), 7.20-7.24 (1H, m), 7.40-7.45 (3H, m), 7.52-7.54 (1H, d, J=1.2 Hz), 8.27 (1H, d, J=5.2 Hz), 9.11 (1H, bs), 12.68 (1H, bs);
LCMS: 355.3 [M+H]+; HPLC purity −98.4%.
6-(Benzyloxy)nicotinic acid (A) (123.7 mg, 0.53 mmol) was added to a stirred solution (E)-2-amino-5,5-dimethyl-3-hecenoate (B) (100 mg, 0.53 mmol) in DMF (2.5 mL) followed by DIPEA (0.35 mL, 2.12 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.03 mL, 1.61 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 48 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted to ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution (15 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford crude material. The crude product was purified by CombiFlash© using ethyl acetate:hexanes as eluent to afford compound C as a brown, thick oil.
Yield: 65 mg (30.4%);
1H-NMR-CD3OD (400 MHz): δ: 1.03 (9H, s), 1.22-1.26 (3H, m), 4.16-4.20 (2H, m), 5.01 (1H, m), 5.40 (2H, s), 5.58 (2H, d, J=7.2 Hz), 5.89 (2H, d, J=1.2 Hz), 6.88 (1H, m), 7.31-7.35 (3H, m), 7.41-7.43 (2H, m), 8.12 (1H, d, J=2.8 Hz), 8.66 (1H, s);
LCMS: 397.3 [M+H]+
Lithium hydroxide monohydrate (32.8 mg, 0.78 mmol) was added to a stirred solution of compound C (62 mg, 0.15 mmol) in THF/methanol/water (1.24 mL, 1:1:1, 0.62 mL each) The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford crude solid. The resulting solid was washed with hexane (5 mL) to afford a crude mixture of two isomers as an off white solid. The crude material was purified by RP-HPLC to afford pure compound D as a white solid.
Yield: 16 mg;
1H-NMR-DMSO-d6 (400 MHz): δ: 0.99 (9H, s), 4.87 (1H, d, J=7.2 Hz), 5.40 (2H, s), 5.53 (1H, d, J=7.2 Hz), 5.82 (1H, d, J=1.2 Hz), 6.95 (1H, d, J=8.8 Hz), 7.3 (3H, t, J=4.8 Hz), 7.35-7.46 (2H, m), 8.18 (1H, d, J=2.4 Hz), 8.70 (1H, s), 8.81 (1H, bs), 12.66 (1H, bs);
LCMS: 369.3 [M+H]+; HPLC purity 98.6%
p-(2-Pyrimidinyloxy)benzoic acid (B) (116.6 mg, 0.53 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (A) (100 mg, 0.53 mmol) in DMF (2.5 mL) followed by DIPEA (0.37 mL, 2.15 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.03 mL, 1.61 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After the completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted with ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford crude material. The crude material was purified by CombiFlash© using ethyl acetate:hexanes as eluent to afford compound C as a brown, thick oil.
Yield: 65 mg, (31.6%);
1H-NMR-DMSO-d6 (400 MHz): δ: 1.02 (9H, s), 1.16 (3H, t, J=7.2 Hz), 4.10 (2H, q, J=3.6 Hz), 4.88-4.92 (1H, m), 5.52-5.57 (1H, m), 5.83-5.87 (1H, m), 7.28-7.31 (3H, m), 7.95 (2H, d, J=8.4 Hz), 8.65-8.68 (2H, m), 8.91 (1H, d, J=6.8 Hz); LCMS: 384.3 [M+H]+
Lithium hydroxide monohydrate (16.7 mg, 0.39 mmol) was added to a stirred solution of compound C (61 mg, 0.15 mmol) in THF:water (1.83 mL, 2:1). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the resulting residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The obtained solid was washed with hexane (5 mL) to afford a crude mixture of two isomers. as a white solid. The crude solid was purified by RP-HPLC to afford pure product D as a white solid.
Yield: 20.0 mg;
1H-NMR-DMSO-d6 (400 MHz): δ: 1.02 (9H, s), 4.87-4.91 (1H, m), 5.53-5.59 (1H, m), 5.80-5.85 (1H, m), 7.28-7.30 (3H, m), 7.95 (2H, d, J=8.4 Hz), 8.65-8.68 (2H, m), 8.81 (1H, d, J=7.2 Hz), 12.64 (1H, bs);
LCMS: 356.03 [M+H]+; HPLC purity −99.8%.
K2CO3 (1.1 g, 8.02 mmol) at 25-30° C. was added to a stirred solution of ethyl 2-chloro-5-pyrimidinecarboxylate (A) (1 g, 5.35 mmol) and phenol (0.55 g, 5.89 mmol) in DMF (20 mL). The resulting reaction mixture was stirred over a period of 2 h at 60° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of ice-cold water and the product was extracted with ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulphate and concentrated in vacuo at 40° C. to afford a crude residue. The crude residue was purified by CombiFlash© using ethyl acetate:hexanes as eluent to afford compound B as a colorless, thick oil.
Yield: 1.1 g (84.6%);
1H-NMR-DMSO-d6 (400 MHz): δ: 1.30 (3H, t, J=7.2 Hz), 4.30 (2H, q, J=7.2 Hz), 7.23-7.30 (3H, m), 7.44-7.48 (2H, m), 9.06 (2H, m);
LCMS: 245.1 [M+H]+
1 M aqueous NaOH (0.98 mL, 0.9 mmol) was added to a stirred solution of compound B (0.2 g, 0.81 mmol) in THF (2 mL) at 0-5° C. The resulting reaction mixture was stirred over a period of 4 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 4 h, the reaction mixture was concentrated, the resulting residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜4-5 using saturated solution of potassium hydrogen sulphate and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The crude solid was washed with hexane (5 mL) to afford compound C as a white solid.
Yield: 0.14 g (83.4%);
1H-NMR-DMSO-d6 (400 MHz): δ: 7.23-7.31 (3H, m), 7.43-7.48 (2H, m), 9.03 (2H, m), 13.76 (1H, bs);
LCMS: 216.88 [M+H]+
Compound C (116.6 mg, 0.53 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (D) (100 mg, 0.53 mmol) in DMF (2.5 mL) followed by DIPEA (0.37 mL, 2.15 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.03 mL, 1.61 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted with ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford a crude residue. The crude residue was purified by CombiFlash© using ethyl acetate:hexanes as eluent to afford compound E as a pale green, thick oil.
Yield: 75 mg (43.6%);
1H-NMR-DMSO-d6 (400 MHz): δ: 1.02 (9H, s), 1.32 (3H, t, J=7.6 Hz), 4.13 (2H, q, J=7.2 Hz), 4.93-4.96 (1H, m), 5.47-5.55 (1H, m), 5.86-5.91 (1H, m), 7.23-7.41 (3H, m), 7.44-7.49 (2H, m), 9.04 (2H, s), 9.15 (1H, d, J=8.8 Hz);
LCMS: 384.3 [M+H]+
1 M aqueous NaOH (0.21 mL, 0.21 mmol) was added to a stirred solution of compound E (70 mg, 0.18 mmol) in THF (0.7 mL) at 0-5° C. The resulting reaction mixture was stirred over a period of 2 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 2 h, the reaction mixture was concentrated, the resulting residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜4-5 using saturated solution of potassium hydrogen sulphate and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The solid was washed with hexane (5 mL) to afford a white solid. The crude solid was purified by RP-HPLC to afford the pure product F as a white solid.
Yield: 19 mg;
1H-NMR-DMSO-d6 (400 MHz): δ: 1.02 (9H, s), 4.82-4.93 (1H, m), 5.49-5.55 (1H, m), 5.81-5.85 (1H, m), 7.22-7.29 (3H, m), 7.43-7.47 (2H, m), 9.03-9.05 (3H, m), 12.39 (1H, bs);
LCMS: 356.07 [M+H]+; HPLC purity −98.9%.
p-(p-Chlorophenoxy)benzoic acid (B) (131.7 mg, 0.53 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (A) (100 mg, 0.53 mmol) in DMF (2.5 mL) followed by DIPEA (0.37 mL, 2.15 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.03 mL, 1.61 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted with ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford a crude product. The crude material was purified by CombiFlash© using ethyl acetate:hexanes as eluent to afford compound C as a pale yellow, thick oil.
Yield: 35 mg (15.8%);
1H-NMR-CD3OD (400 MHz): δ: 1.02 (9H, s), 1.24 (3H, t, J=7.2 Hz), 4.17 (2H, d, J=5.2 Hz), 5.02-5.04 (1H, m), 5.58-5.60 (1H, m), 5.87-5.91 (1H, m), 7.00-7.04 (4H, m), 7.36-7.39 (2H, m), 7.85-7.87 (2H, m);
LCMS: 416.13 [M+H]+
Lithium hydroxide monohydrate (7.56 mg, 0.18 mmol) was added to a stirred solution of compound C (30 mg, 0.07 mmol) in MeOH/THF/water (0.9 mL, 1:1:1, 0.3 mL each) was added. The resulting reaction mixture was stirred over a period of 7 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the resulting residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜4-5 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The obtained solid was washed with hexane (5 mL) to afford the crude product as a white solid. The crude material was purified by RP-HPLC to afford the product D as a white solid.
Yield: 10 mg;
1H-NMR-DMSO-d6 (400 MHz): δ: 1.00 (9H, s), 4.84-4.88 (1H, m), 5.52-5.59 (1H, m), 5.81 (1H, d, J=20.8 Hz), 7.06-7.13 (4H, m), 7.48 (2H, d, J=12.0 Hz), 7.94 (2H, d, J=11.6 Hz), 8.70 (1H, d, J=9.2 Hz), 12.67 (1H, bs);
LCMS: 388.10 [M+H]+; HPLC purity −96.5%.
p-(m-Chlorophenoxy)benzoic acid (B) (100 mg, 0.40 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (A) (74.50 mg, 0.40 mmol) in DMF (2.5 mL) followed by DIPEA (0.28 mL, 1.60 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (0.76 mL, 1.2 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted with ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford crude material. The crude material was purified by CombiFlash© using ethyl acetate:hexanes as eluent to afford compound C as a pale yellow, thick oil.
Yield: 29 mg (17.3%);
1H-NMR-CD3OD (400 MHz): δ: 0.96 (9H, s), 1.22-1.28 (3H, m), 4.17-4.20 (2H, m), 4.99-5.01 (1H, m), 5.57 (1H, t, J=7.6 Hz), 5.90 (1H, d, J=1.2 Hz), 7.03-7.06 (5H, m), 7.35 (1H, d, J=8.0 Hz), 7.87 (2H, t, J=1.2 Hz);
LCMS: 416.4 [M+H]+
Lithium hydroxide monohydrate (6.30 mg, 0.15 mmol) was added to a stirred solution of compound C in MeOH/THF/water (0.75 mL, 1:1:1, 0.25 mL each) was added. The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the resulting residue was dissolved in water and acidified to pH ˜4-5 using 1.5 N HCl at 0-5° C. and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The obtained solid was washed with hexane (5 mL) to afford a crude mixture of two isomers as a white solid. The crude product was purified by RP-HPLC to afford the pure product D as a white solid.
Yield: 8.4 mg;
1H-NMR-DMSO-d6 (400 MHz): δ: 0.97 (9H, s), 5.50-5.56 (1H, m), 5.77-5.81 (1H, m), 7.01-7.09 (1H, m), 7.10-7.14 (3H, m), 7.22-7.25 (1H, m), 7.42 (1H, t, J=8.0 Hz), 7.92-7.94 (2H, m), 8.8 (1H, bs);
LCMS: 388.5 [M+H]+; HPLC purity: 97.9%.
p-(Phenoxymethyl)benzoic acid (B) (120 mg, 0.64 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (A) (147.83 mg, 0.64 mmol) in DMF (3.0 mL) was added followed by DIPEA (0.45 mL, 2.59 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.23 mL, 1.94 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted with ethyl acetate (2×20 mL). the combined organic layer was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford a crude material. The crude material was purified by CombiFlash© using ethyl acetate:hexanes as eluent to afford pure compound C as a brown, thick oil.
Yield: 34 mg (13.3%) Step 2: Synthesis of (E)-5,5-dimethyl-2-[p-(phenoxymethyl)benzoylamino]-3-hexenoic acid (D)
Lithium hydroxide monohydrate (8.73 mg, 0.20 mmol) was added to a stirred solution of compound C (32 mg, 0.08 mmol) in MeOH/THF/water (0.96 mL, 1:1:1, 0.32 mL each) was added. The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the resulting residue was dissolved in water and acidified to pH ˜4-5 using 1.5 N HCl at 0-5° C. and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a solid material. The solid material was washed with hexane (5 mL) to afford a crude mixture of two isomers as a white solid. The crude mixture was purified by RP-HPLC to afford pure product D as a pure white solid.
Yield: 12.5 mg;
1H-NMR-CDCl3 (400 MHz): δ: 1.00 (9H, s), 4.86-4.90 (1H, m), 5.18 (2H, s), 5.53-5.59 (1H, m), 5.81 (1H, d, J=1.2 Hz), 6.92-7.02 (3H, m), 7.27-7.31 (2H, m), 7.53 (2H, d, J=8.4 Hz), 7.91 (2H, d, J=8.4 Hz), 8.79 (1H, s);
LCMS: 368.3 [M+H]+; HPLC purity 99.0%,
1-Ethyl-3-isobutyl-5-pyrazolecarboxylic acid (B) (105.7 mg, 0.53 mmol) was added to a stirred solution (E)-2-amino-5,5-dimethyl-3-hecenoate (A) (100 mg, 0.53 mmol) in DMF (2.5 mL) followed by DIPEA (0.376 mL, 2.15 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.028 mL, 1.61 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted to ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution (15 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford crude material. The crude material was purified by CombiFlash© using ethyl acetate:hexanes as eluent to afford compound C as a thick, yellow liquid.
Yield: 47 mg, (24.0%);
1H-NMR-DMSO-d6 (400 MHz): δ: 0.94-0.97 (6H, m), 1.05 (9H, s), 1.23-1.36 (6H, m), 1.90-1.95 (1H, m), 2.48 (2H, d, J=7.2 Hz), 4.16-4.24 (2H, m), 4.43-4.48 (2H, m), 4.96 (1H, d, J=7.2 Hz), 5.54-5.59 (1H, m), 5.88-5.92 (1H, m), 6.66 (1H, s);
LCMS −363.49 [M+H]+
Lithium hydroxide monohydrate (27.15 mg, 0.64 mmol) was added to a stirred solution of compound C (47 mg, 0.12 mmol) in THF/methanol/water (1.41 mL, 1:1:1, 0.47 mL each) was added. The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the resulting residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The obtained solid was washed with hexane (5 mL) to afford a crude mixture of two isomers as an off white solid. The mixture was purified by RP-HPLC to afford pure product D as a white solid.
Yield: 28.9 mg;
1H-NMR-DMSO-d6 (400 MHz): δ: 0.90-0.88 (6H, m), 0.99 (9H, s), 1.23 (3H, t, J=6.8 Hz), 1.83-1.86 (1H, m), 2.49 (2H, d, J=2.0 Hz), 4.34-4.41 (2H, m), 4.80-4.82 (1H, m), 5.33-5.45 (1H, m), 5.79-5.83 (1H, m), 6.74 (1H, s) 8.67 (1H, d, J=7.2 Hz), 12.6 (1H, bs);
LCMS: 336.4 [M+H]+; HPLC purity −99.2%.
3-Chloro-1-methyl-5-pyrazolecarboxylic acid (B) (86.53 mg, 0.53 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (A) (100 mg, 0.53 mmol) in DMF (2.5 mL) followed by DIPEA (0.376 mL, 2.15 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.02 mL, 1.61 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted to ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution (15 mL), dried over anhydrous sodium sulphate and concentrated in vacuum at 35° C. to afford a crude residue. The crude residue was purified by CombiFlash© using ethyl acetate:hexanes as eluent to afford compound C as a thick, yellow liquid.
Yield: 52 mg, (29.4%);
1H-NMR-CD3OD (300 MHz): δ: 1.06 (9H, s), 1.25-1.35 (3H, m), 4.04 (3H, s), 4.15-4.25 (2H, m), 4.97 (1H, d, J=7.5 Hz), 5.50-5.58 (1H, m), 5.88-5.94 (1H, m), 6.83 (1H, s);
LCMS: 328.3 [M+H]+
Lithium hydroxide monohydrate (33.3 mg, 0.79 mmol) was added to a stirred solution of compound C (52 mg, 0.15 mmol) in THF/methanol/water (1.56 mL, 1:1:1, 0.52 mL each) was added. The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the resulting residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The crude solid was washed with hexane (5 mL) to afford a crude mixture of two isomers as an off white solid. The mixture was purified by RP-HPLC to afford pure product D as a white solid.
Yield: 28.5 mg;
1H-NMR-DMSO-d6 (400 MHz): δ: 0.97 (9H, s), 3.96 (3H, s), 4.80-4.84 (1H, m), 5.45-5.50 (1H, m), 5.77-5.81 (1H, m), 7.02 (1H, s), 8.84 (1H, d, J=7.6 Hz);
LCMS: 300.3 [M+H]+; HPLC purity −98.4%
3-Isopropyl-1-methyl-5-pyrazolecarboxylic acid (B) (90.7 mg, 0.53 mmol) followed by DIPEA (0.37 mL, 2.15 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (1.03 mL, 1.61 mmol) were added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (A) (100 mg, 0.53 mmol) in DMF (2.5 mL) was added at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted to ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford a crude residue. The crude residue was purified by CombiFlash© using ethyl acetate:hexanes as eluent to afford compound C as a brown, thick oil.
Yield: 56 mg, (31.0%);
1H-NMR-CD3OD (400 MHz): δ: 1.05 (9H, s), 1.25-1.28 (9H, m), 2.90-2.97 (1H, m), 4.02 (3H, s), 4.23 (2H, q, J=3.6 Hz), 4.95-4.98 (1H, m), 5.53-5.58 (1H, m), 5.92 (1H, d, J=14.4 Hz), 6.73 (1H, s);
LCMS: 366.5 [M+H]+
Lithium hydroxide monohydrate (32.5 mg, 0.77 mmol) was added to a stirred solution of compound C (52 mg, 0.15 mmol) in THF/methanol/water (1.56 mL, 1:1:1, 0.52 mL each) was added. The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The crude solid was washed with hexane (5 mL) to afford a crude mixture of two isomers as a white solid. The mixture was purified by RP-HPLC to afford pure product D as a white solid.
Yield: 23 mg;
1H-NMR-DMSO-d6 (400 MHz): δ: 1.0 (9H, s), 1.19 (6H, d, J=9.2 Hz), 2.84-2.89 (1H, m), 3.95 (3H, s), 4.81 (1H, t, J=9.6 Hz), 5.47-5.54 (1H, m), 5.82 (1H, d, J=16.4 Hz), 6.84 (1H, s), 8.68 (1H, d, J=9.6 Hz), 12.66 (1H, bs);
LCMS: 308.4 [M+H]+; HPLC purity −99.4%.
Diethyl oxalate (B) (6.7 g, 0.049 mmol) was washed with dry THF (4.5 mL) and added to a stirred solution of NaH (60% w/w, 2.95 g, 0.074 mmol) in dry THF (51 mL). The resulting mixture was heated to 75° C., then a solution of 1-(3-pyridyl)-1-ethanone (A) (3 g, 0.024 mmol) in dry THF (4.5 mL) was added slowly with stirring at reflux. The resulting reaction mixture was stirred over a period of 15 min at 25-30° C. The progress of the reaction was monitored by TLC.
The resulting reaction mixture was added slowly to ice cold 1.5 N HCl solution and then solid NaHCO3 was added until slightly basic. The resulting mixture was extracted with ethyl acetate (2×75 mL). The combined organic layer was washed with saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford crude material The obtained solid was dissolved in warm ethanol (100 mL) and 1 g of charcoal was added. After 5 min, the solution was filtered hot through a celite bed. The filtrate was concentrated under vacuo to afford compound C as a reddish brown solid.
Yield: 890 mg, Yield 16.3%
Methyl hydrazine (0.118 mL) was added to a stirred solution of compound C (450 mg, 2.034 mmol) in ethanol (5.4 mL) followed by p-toluensulfonic acid (PTSA) (699.69 mg, 4.06 mmol). The resulting reaction mixture was stirred over a period of 18 h at 80° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution, and the product was extracted to ethyl acetate (2×30 mL). The combined organic layer was dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford crude material. The crude material was purified by CombiFlash® to afford compound D as an off white solid.
Yield: 43 mg (9.2%);
1H-NMR-CDCl3 (400 MHz): δ: 1.42 (3H, t, J=7.2 Hz), 4.25 (3H, s), 4.39 (2H, q, J=7.2 Hz), 7.17 (1H, s), 7.34-7.37 (1H, m), 8.11-8.14 (1H, m), 8.57 (1H, d, J=4 Hz), 9.03 (1H, s);
LCMS: 232.1 [M+H]+
A stirred solution of compound D (34 mg, 0.14 mmol) in 1,4-dioxane (0.24 mL) was cooled to 0° C. NaOH (14.7 mg, 0.36 mmol) was dissolved in water (0.04 mL) and added to the above solution dropwise. The resulting reaction mixture was stirred over a period of 2 h at 25-30° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of 1.5 N HCl solution to pH ˜2-3. The reaction mixture was concentrated in vacuum at 45° C. and stripped with toluene to afford crude compound E as a white solid.
Yield: 50 mg (crude):
1H-NMR-DMSO-d6 (300 MHz): δ: 4.16 (3H, s), 7.55 (1H, s), 7.73-7.77 (1H, m), 8.56 (1H, d, J=8.4 Hz), 8.67 (1H, d, J=5.1 Hz), 9.18 (1H, d, J=2.0 Hz), 13.65 (1H, bs);
LCMS: 204.0 [M+H]+
Compound E (54.65 mg, 0.26 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (F) (50 mg, 0.26 mmol) in DMF (2.5 mL) followed by DIPEA (0.18 mL, 1.07 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (0.51 mL, 0.80 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted to ethyl acetate (2×20 mL). the combined organic layer was washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford crude material. The crude material was purified by CombiFlash© using ethyl acetate:hexanes as eluent to afford compound G as a white solid.
Yield: 15 mg, (7.5%);
H-NMR-DMSO-d6 (400 MHz): δ: 1.01 (9H, s), 1.15-1.17 (3H, m), 4.08 (3H, s), 4.11-4.16 (2H, m), 4.86-4.90 (1H, m), 5.47-5.51 (1H, m), 5.86-5.90 (1H, m), 7.40-7.45 (2H, m), 8.12 (1H, d, J=8.0 Hz), 8.53 (1H, d, J=2 Hz), 8.96 (1H, s), 9.00-9.02 (1H, m);
LCMS: 383.3 [M+H]+
Lithium hydroxide monohydrate (8.58 mg, 0.20 mmol) was added to a stirred solution of compound G (15 mg, 0.040 mmol) in THF/methanol/water (0.45 mL, 1:1:1, 0.15 mL each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the resulting residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜5-6 using 1.5 N HCl and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The obtained solid was washed with hexane (5 mL) to afford a crude mixture of two isomers as an off white solid. The crude mixture was purified by RP-HPLC to afford pure product H, as a white solid.
Yield: 7.1 mg;
1H-NMR-DMSO-d6 (400 MHz): δ: 0.99 (9H, s), 4.08 (3H, s), 4.77-4.81 (1H, m), 5.50-5.55 (1H, m), 5.79 (1H, d, J=16 Hz), 8.11-8.13 (1H, m), 8.14 (1H, s), 8.51-8.53 (1H, m), 8.71 (1H, bs), 8.97-8.99 (1H, m), 12.87 (1H, bs);
LCMS: 343.2 [M+H]+; HPLC purity −99.4%.
Methyl hydrazine (50.0 mg, 1.01 mmol) was added to a stirred solution of methyl 4-(3,4-dichlorophenyl)-2,4-dioxobutyrate (A) (300 mg, 1.01 mmol) in MeOH (3.0 mL) at 25-30° C. The resulting reaction mixture was heated at 70° C. for 2 h and allowed to stir at 25-30° C. over a period 18 h. The progress of the reaction was monitored by TLC
After completion of the reaction, the reaction mixture was evaporated to afford crude material containing two isomeric products. The crude material was purified by CombiFlash© using ethyl acetate:hexanes as eluent to afford compound B as an off white solid.
Yield: 0.11 g;
Int-2.2: 1H-NMR-DMSO-d6 (400 MHz): δ: 3.78 (3H, s), 3.92 (3H, s), 7.00 (1H, s), 7.58 (1H, d, J=2.0 Hz), 7.76 (1H, d, J=8.4 Hz), 7.89 (1H, s);
LCMS: 284.93 [M+H]+
Sodium hydroxide (61.72 mg, 1.54 mmol) was added to a stirred solution of compound B (2.2) (110 mg, 0.38 mmol) in MeOH: water (8.8 mL, 1:1, 4.4 mL each). The resulting reaction mixture was stirred over a period of 4 h at 75° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, most of the methanol was evaporated. The aqueous mixture was acidified to pH ˜3-4 using 1.5 N HCl and the product was extracted with ethyl acetate (3×10 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The solid was washed with hexane (15 mL) to afford crude compound C as a white solid. The crude material was used as such for next reaction.
Yield: 99 mg (crude);
1H-NMR-DMSO-d6 (400 MHz): δ: 3.90 (3H, s), 6.92 (1H, s), 7.56 (1H, d, J=2.0 Hz), 7.75 (1H, d, J=8.4 Hz), 7.88 (1H, s), 12.74 (1H. bs);
LCMS: 270.91 [M+H]+
Compound C (95 mg, 0.35 mmol) was added to a stirred solution of (E)-2-amino-5,5-dimethyl-3-hecenoate (D) (64.9 mg, 0.40 mmol) in DMF (2.37 mL) followed by DIPEA (0.24 mL, 1.40 mmol) and propylphosphonic anhydride (T3P, ˜50% solution in ethyl acetate) (0.66 mL, 1.05 mmol) at 25-30° C. The resulting reaction mixture was stirred over a period of 18 h at 40° C. The progress of the reaction was monitored by TLC.
After completion of the reaction, the reaction mixture was quenched by addition of saturated NaHCO3 solution to pH ˜7-8 and the product was extracted with ethyl acetate (2×20 mL). The combined organic layer was washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulphate and concentrated in vacuo at 35° C. to afford crude material. The crude material was purified by CombiFlash© using ethyl acetate:hexanes as eluent to afford compound E as a brown thick oil.
Yield: 60 mg 39.0%;
1H-NMR-CD3OD (300 MHz): δ: 0.99 (9H, s), 1.28 (3H, t, J=6.9 Hz), 3.96 (3H, s), 4.20-4.27 (2H, m), 5.07 (1H, d, J=6.0 Hz), 5.56-5.63 (1H, m), 5.91 (1H, d, J=15.6 Hz), 6.87 (1H, s), 7.48 (1H, d, J=1.2 Hz), 7.68 (1H, d, J=8.4 Hz), 7.75 (1H, s);
LCMS: 438.5 [M+H]+
Lithium hydroxide monohydrate (13.16 mg, 0.31 mmol) was added to a stirred solution of compound E (4.2) (55 mg, 0.12 mmol) in MeOH/THF/water (1.65 mL, 1:1:1, 0.55 mL each). The resulting reaction mixture was stirred over a period of 18 h at 25-30° C. The progress of the reaction was monitored by TLC.
After 18 h, the reaction mixture was concentrated, the resulting residue was dissolved in water and washed with diethyl ether (10 mL). The aqueous layer was acidified to pH ˜4-5 using 1.5 N HCl at 0-5° C. and the product was extracted with ethyl acetate (3×5 mL). The combined organic layer was dried over sodium sulphate and concentrated at 40° C. to afford a crude solid. The crude solid was purified by RP-HPLC to afford pure product F. as a white solid.
Yield: 42 mg (81.6%);
1H-NMR-DMSO-d6 (400 MHz): δ: 0.99 (9H, s), 3.94 (3H, s), 4.87 (1H, t, J=6.9 Hz), 5.58 (1H, d, J=6.9 Hz, 5.77 (1H, d, J=15.6 Hz), 6.92 (1H, s), 7.60 (1H, d, J=8.1 Hz), 7.79 (1H, d, J=8.4 Hz), 7.91 (1H, s), 8.17 (1H, d, J=7.5 Hz), 12.78 (1H, bs);
LCMS: 410.3 [M+H]+; HPLC purity −98.6%.
These compounds were prepared in the same way as described above for Example 5 starting with the coupling of ethyl (E)-2-amino-5,5-dimethyl-3-hexenoate with a suitable, commercially available aromatic, heteroaromatic or heterocyclic carboxylic acid, followed by subsequent hydrolysis of the resulting ethyl esters and giving the expected products.
2-Hydroxypyridine-1-oxide (26 mg, 0.276 mmol) and N,N′-diisopropylcarbodiimide (35 mg, 0.276 mmol) were added to a solution of 2-(dimethylamino)-4-methyl-5-pyrimidinecarboxylic acid (B) (50 mg, 0.276 mmol) in DMSO (1 mL) and the reaction mixture was stirred for 10 min at 0° C. A preformed solution of (S)-2-amino-5,5-dimethylhexanoic acid (A) (44 mg, 0.276 mmol) in acetonitrile:water (1:1, 0.5 mL) was added and the mixture was stirred at room temperature for 4 h. The crude material was purified by reverse phase HPLC (C18 column, gradient of 5-100% MeCN/H2O containing 0.05% HCOOH). The solvents were evaporated by freeze drying to provide pure compound C.
Yield: 6.0 mg (6.7%)
1H NMR (400 MHz, DMSO-d6) δ: 8.35 (s, 1H), 8.30 (d, J3=7.7 Hz, 1H), 4.25-4.13 (m, 1H), 3.14 (s, 6H), 2.41 (s, 3H), 1.82-1.57 (m, 2H), 1.34-1.16 (m, 2H), 0.86 (s, 9H).
This compound was prepared as described in the Supplementary data for the paper of Shawn J Stachel et al., Bioorganic & Medicinal Chemistry Letters (2020), 30(17): 127403.
This compound was prepared as described in WO 2014/114779
Synthesis of Secreted Sortilin (sSORT) and Neurotensin
The extracellular part of human sortilin (NCBI reference sequence: NM_002959.7), amino acids 1-756 in SEQ ID NO: 1 plus a C-terminal His6-tag, was produced in CHO—S cells by transient transfection as a secreted protein. Supernatant from two different transfection reagents were pooled; 150 ml FectoPro and 150 ml NovaCHOice. Purification was performed by using Immobilized Metal Ion Affinity Chromatography (IMAC) in buffer (50 mM HEPES pH 7.4, 100 mM NaCl and 2 mM CaCl2). Proteins were eluted using an imidazole-gradient (125-500 mM), fractions containing sSORT were pooled and protein size was confirmed by Western blot. Buffer was exchanged to 50 mM HEPES, pH 7.4; 100 mM NaCl; 2.0 mM CaCl2 prior to storage in −80° C. Neurotensin, amino acid sequence LYENKPRRPYIL, SEQ ID NO: 4, (Genescript), and Neurotensin-Ahx-FITC containing the same sequence with an additional N-terminal modification FITC-Ahx (Genescript) were used as competitive ligand for sSORT.
Fresh 0.1% bovine serum albumin (BSA) was added to the assay buffer to obtain the final concentration: 50 mM HEPES, pH 7.4; 100 mM NaCl; 2.0 mM CaCl2; 0.1% BSA; 0.1% TWEEN® 20. The sortilin inhibitors of the invention and reference compounds (see Table 1) analyzed in the screen and neurotensin (used as an assay control) were serially diluted in ten different concentrations in assay buffer. In each well of a Nunc® MaxiSorp™ 384-well plate (Sigma Aldrich), 100 nM sSORT was mixed with pre-diluted sortilin inhibitors and 10 nM Neurotensin-Ahx-FITC in assay buffer adjusted to contain 1% DMSO in a final volume of 20 μl. The plate was then briefly centrifuged prior to 1 h incubation at room temperature in the dark. The mPolarization values were obtained from a CLARIOstar plate reader (excitation at 482 nm and emission at 530-540 nm) with each well flashed 200 times. The Z′ value was calculated from a total of 16 positive controls and 16 negative controls.
To demonstrate binding of sortilin inhibitors to sortilin, a competitive binding assay was performed for sortilin using a fluorescence polarization-based (FPA) method with neurotensin as competitor ligand (
As can be seen by comparing sortilin inhibitor SI5 of the invention with reference compound RC2 and sortilin inhibitor SI1 of the invention with reference compound RC1, the sortilin inhibitors of the invention having an unsaturated aliphatic moiety instead of a saturated aliphatic moiety of the reference compounds had lower mean IC50 values and thereby a higher binding affinity to sortilin. This is even more accentuated by comparing the pure enantiomer SI5 with the pure enantiomer RC2.
C-terminal Progranulin (progranulin) (Casio, peptide sequence: EAPRWDAPLRDPALRQL, SEQ ID NO: 5) were reconstituted in sterile PBS upon delivery, aliquoted and stored at −20° C. Working stock concentrations were achieved by dilution in culture media. The MDA-MB-231, HT-29 or SK-MEL-30 cell lines were treated with or without 500 nM progranulin with and without indicated concentrations of sortilin inhibitors SI1, SI5, SI8, SI25, SI32, SI39, SI51 and SI62 of the invention or RC3 (AF38469, Bioorganic & Medicinal Chemistry Letters, 2014, 24(1): 177-180) for 48 hours at 37° C. 5% CO2 and 21% O2 before performing the primary followed by secondary sphere formation assay.
The sphere formation assay was performed as described previously (Mammary Gland Biol Neoplasia, 2012, 17(2): 111-117). Briefly, single cell suspensions were obtained following treatment with respective sortilin inhibitors SI1, SI5, SI8, SI25, SI32, SI39, SI51 and SI62 of the invention and seeded in phenol red-free DMEM/F-12 (Gibco®, Life Technologies), supplemented with 1% B27 supplement (Fisher Scientific, Invitrogen), 1% P/S and 20 ng/ml EGF (BD Biosciences) onto non-adherent polyhema-coated plates. After cultivation for five days, single cell suspensions were obtained from the primary spheres and seeded again in phenol red-free DMEM/F-12 supplemented with 1% B27 supplement, 1% P/S and 20 ng/ml EGF onto non-adherent polyhema-coated plates for 5-7 days. Subsequently, spheres greater than 50 μm in diameter were counted manually in the microscope.
To determine whether the increase in sphere formation triggered by progranulin could be blocked using sortilin inhibitors SI1, SI5 and SI8 of the invention, the triple negative breast cancer cell line MDA-MB-231 was treated with progranulin, alone or in combination with the sortilin inhibitors SI1, SI5 and SI8 of the invention, or the published sortilin binding small molecule RC3 (Breast Cancer Research 2018 20: 137). Results show that as expected, progranulin increased secondary sphere formation (
In addition to breast cancer, the sphere formation capacity using the colon cancer cell line HT-29 and melanoma cell line SK-MEL-30 were investigated. As is shown in
Luciferase tagged MDA-MB-231 cells were injected at a concentration of 0.2×106 cells subcutaneously into two sites of the flank of NOD SCID gamma mice (Taconic, Denmark). Cells were prepared in a 60% mixture of matrigel (growth factor reduced, BD Bioscience) and 40% complete media. The size of the tumors were determined by calliper measurement of the subcutaneous tumor mass two times per week and tumor volume was calculated according to the formula volume=(length×(width)2/2). Assessments of metastasis were performed using the IVIS® whole body imager (PerkinElmer) based on luciferase expression from stably transfected cell lines. For sortilin inhibition studies, mice were given vehicle (DMSO) or 5-50 μg RC3 (MedChem Express)/day/mouse in drinking water with a weekly refill for 21 days before assessments of metastasis were performed.
The agonistic property of RC3, observed in the sphere assay (
The embodiments described above are to be understood as a few illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations and changes may be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in the different embodiments can be combined in other configurations, where technically possible. The scope of the present invention is, however, defined by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2151469-0 | Dec 2021 | SE | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SE2022/051131 | 12/1/2022 | WO |
