Pyridine-3-carboxylate compounds as CaV1.2 activators

Abstract

The present disclosure relates to a method for the treatment of schizophrenia, bipolar disorder, major depressive disorder, substance use disorder, ADHD, Phelan-McDermid syndrome, autism spectrum disorder, multiple sclerosis, frontotemporal dementia, Alzheimer's disease, Brugada syndrome, short QT syndrome, or early repolarization syndrome. v 1.2 Provide a compound of formula (I) or a pharma- ceutically acceptable salt thereof as an active agent.

Description

[Technical field]

[0001] The present disclosure relates to pyridine-3-carboxylate compounds, pharmaceutical compositions containing them, and compositions for the treatment of calcium signaling deficiencies and / or synaptic dysfunction in neuropsychiatric disorders, including schizophrenia, bipolar disorder, major depressive disorder, and substance use disorders; neurodevelopmental disorders, such as attention deficit hyperactivity disorder, Phelan-McDermid syndrome, and other autism spectrum disorders; neurodegenerative disorders, such as multiple sclerosis, frontotemporal dementia, and Alzheimer's disease; and cardiac conditions, such as Brugada syndrome, short QT syndrome, and early repolarization syndrome. V 1.2 Concerning the use of such compounds as active agents. [Background technology]

[0002] Advances in human genomics are shedding light on the genetic basis of neuropsychiatric disorders. Genome-wide association studies (GWAS) of schizophrenia have revealed that Ca2+, which is involved in neuronal calcium signaling, is involved in the pathogenesis of schizophrenia. V Over 100 disease-associated loci have been identified, including 1.2 and other genes. Cross-disorder GWAS analyses have identified Ca as strongly associated with schizophrenia, bipolar disorder, major depressive disorder, ADHD, and autism spectrum disorder. V In addition to evidence from GWAS, exome sequencing in patients with schizophrenia has identified Ca V We showed an enrichment of disruptive mutations in 1.2 and other gene members of the neuronal calcium signaling pathway, including the CACNA2D, CACNB, and CAMK2 genes. V 1.2 has been shown to be important for neuronal differentiation and migration, neurite outgrowth, synaptic signaling, gene expression and brain plasticity. It has been shown to play a role in emotion, learning and memory, executive function, and the brain's reward response.

[0003] Ca V1.2 is widely expressed throughout the body and plays a major role in multiple organ systems, including the cardiovascular system, but Ca V The physiological function of Ca 1.2 is different from its function in the brain. V Studies have shown that p.G406R Ca2+ is a key contributor to action potential generation in the heart, while p.1.2 is a key driver of intracellular signaling and gene expression in neurons with a minimal role in action potential generation. V 1.2 mutations result in distinct cell phenotypes between cardiomyocytes and neurons. Ca2+-dependent deficiencies that cause cardiovascular-specific disorders (Brugada syndrome and long QT syndrome type 8) V 1.2 mutation reduces Ca in the heart and brain V Further evidence of 1.2's diverse capabilities.

[0004] Although calcium channel activators have been reported previously, further research into their use for neuropsychiatric disorders has been limited due to their effects on the cardiovascular system. Indeed, many of these molecules were initially studied and developed for their potential therapeutic use in heart failure. V 1.2 The majority of SNPs reside in the introns of genes, and these risk SNPs have been shown to be associated with reduced mRNA expression that often leads to an overall decrease in calcium current amplitude. Therefore, small molecules that can increase the overall current amplitude may be of most benefit to patients. Summary of the Invention

[0005] The present disclosure relates to a compound of formula (I), or a pharma- ceutically acceptable salt thereof: [ka] During the ceremony, R 1 But, C 1~6 Alkyl and C1~6 Haloalkyl, e.g., C 1~6 fluoroalkyl; R 2 is selected from H and halo, e.g., F; R 3 But, C 1~6 Alkyl, e.g., C 1~4 Alkyl, and C 3~8 cycloalkyl, each of which is optionally substituted with one to three halo, e.g., F, or a pharma- ceutically acceptable salt thereof.

[0006] In a second aspect, the disclosure provides a pharmaceutical composition comprising a compound of formula (I), or a pharma- ceutically acceptable salt thereof.

[0007] In a third aspect, the disclosure provides a compound of formula (I), or a pharma- ceutically acceptable salt thereof, for use in the treatment of neuropsychiatric disorders, including schizophrenia, bipolar disorder, major depressive disorder, or substance use disorder; neurodevelopmental disorders, such as attention deficit hyperactivity disorder, Phelan-McDermid syndrome, or other autism spectrum disorders; neurodegenerative disorders, such as multiple sclerosis, frontotemporal dementia or Alzheimer's disease; and cardiac conditions, such as Brugada syndrome, short QT syndrome, or early repolarization syndrome, in therapy.

[0008] In a fourth aspect, the disclosure provides a method of treating a neuropsychiatric disorder, such as schizophrenia, bipolar disorder, major depressive disorder, or substance use disorder; a neurodevelopmental disorder, such as ADHD, Phelan-McDermid syndrome, or autism spectrum disorder; a neurodegenerative disorder, such as multiple sclerosis, frontotemporal dementia, or Alzheimer's disease; or a cardiac condition, such as Brugada syndrome, short QT syndrome, or early repolarization syndrome, in a subject in need thereof, the method comprising administering a therapeutically effective amount of a compound of the disclosure, or a pharma- ceutically acceptable salt thereof.

[0009] In a fifth aspect, the disclosure provides a method of treating a neuropsychiatric disorder, such as schizophrenia, bipolar disorder, major depressive disorder, or substance use disorder; a neurodevelopmental disorder, such as ADHD, Phelan-McDermid syndrome, or autism spectrum disorder; a neurodegenerative disorder, such as multiple sclerosis, frontotemporal dementia, or Alzheimer's disease; or a cardiac condition, such as Brugada syndrome, short QT syndrome, or early repolarization syndrome, in a subject in need thereof, the method comprising administering a compound of the disclosure, or a pharma- ceutically acceptable salt thereof.

[0010] In a sixth aspect, the disclosure provides the use of a compound of the disclosure, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a neuropsychiatric disorder, such as schizophrenia, bipolar disorder, major depressive disorder, or substance use disorder; a neurodevelopmental disorder, such as ADHD, Phelan-McDermid syndrome, or autism spectrum disorder; a neurodegenerative disorder, such as multiple sclerosis, frontotemporal dementia, or Alzheimer's disease; or a cardiac condition, such as Brugada syndrome, short QT syndrome, or early repolarization syndrome. [Brief description of the drawings]

[0011] [Figure 1] FIG. 13 is a graph of simulated cardiac action potentials from an epicardial environment showing the effect of shifting the voltage of CaV1.2 activation to more negative membrane potentials in voltage (mV) versus time (ms). [Diagram 2] 1 is an image showing the X-ray crystal structure of Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] 5.1.Definition Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, and pharmacology described herein are those well known and commonly used in the art. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0013] Unless otherwise specified, the terms "compounds of the present disclosure", "compounds of the disclosure", or "compound of the disclosure" refer to compounds of formula (I), (II), (Ia), or (Ib), exemplified compounds, salts thereof, particularly their pharma- ceutically acceptable salts, hydrates, solvates, and all stereoisomers (including diastereoisomers and enantiomers), rotamers, tautomers, and isotopically labeled compounds (including deuterium substitutions), and moieties formed inherently.

[0014] The term "and / or" means either "and" or "or" unless otherwise indicated.

[0015] The term "substituted" means that the specified group or moiety has one or more suitable substituents that may be attached to the specified group or moiety at one or more positions. For example, a cyclopropyl substituted with a fluoro group (F) indicates that the fluoro is attached to an atom of the cyclopropyl by a bond.

[0016] As used herein, "C 1~6 The term "alkyl" refers to a straight or branched hydrocarbon chain radical containing no unsaturation, having from 1 to 6 carbon atoms, and connected to the remainder of the molecule by a single bond, consisting solely of carbon and hydrogen atoms. 1~6Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, 1-methylpropyl (sec-butyl), 2-methylpropyl (iso-butyl), 1,1-dimethylethyl (t-butyl), n-pentyl, and n-hexyl. 1~4 The term alkyl should be interpreted accordingly.

[0017] As used herein, "C 1~6 The term "haloalkyl" refers to a C alkyl group, as defined above, substituted by one or more halo groups, as defined herein. 1~6 Refers to an alkyl group. 1~6 Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 1,1-difluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 2-fluoropropyl, 3,3-difluoropropyl, and 1-fluoromethyl-2-fluoroethyl, 1,3-dibromopropan-2-yl, 3-bromo-2-fluoropropyl, and 1,4,4-trifluorobutan-2-yl.

[0018] As used herein, the term "halo" means fluorine, chlorine, bromine, or iodine.

[0019] As used herein, the term "cycloalkyl" refers to a monocyclic or polycyclic saturated or partially unsaturated carbocyclic ring containing 3 to 18 carbon atoms and no shared delocalized pi electrons (aromaticity) between ring carbons. 3~8 The term "cycloalkyl" should be interpreted accordingly. The term polycyclic includes bridged (e.g., norbornene), fused (e.g., decalin), and spirocyclic cycloalkyls. Preferably, cycloalkyls, e.g., "C 3~8 "Cycloalkyl" is a monocyclic hydrocarbon group of 3 to 8 carbon atoms.

[0020] C 3~8Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, spiro[3.3]heptanyl, and cyclooctyl.

[0021] As used herein, "administering" refers to the method by which a compound described herein is presented to a subject.

[0022] As used herein, "optionally substituted" means that the referenced group can be substituted at one or more positions with any one or any combination of the groups listed thereafter.

[0023] As used herein, "subject" or "patient" refers to a living organism suffering from one or more of the diseases or disorders described herein (e.g., neuropsychiatric disorders including schizophrenia, bipolar disorder, major depressive disorder, and substance use disorder; neurodevelopmental disorders such as attention deficit hyperactivity disorder, Phelan-McDermid syndrome, and other autism spectrum disorders; neurodegenerative disorders such as multiple sclerosis, frontotemporal dementia, and Alzheimer's disease; and cardiac conditions such as Brugada syndrome, short QT syndrome, and early repolarization syndrome) that can be treated by administration of the pharmaceutical compositions described herein. Examples of subjects include mammals (e.g., humans and animals such as dogs, cows, horses, monkeys, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals). In certain embodiments, the subject is a human, e.g., a human who has, is at risk of having, or may have a disease or disorder described herein (e.g., neuropsychiatric disorders including schizophrenia, bipolar disorder, major depressive disorder, and substance use disorders; neurodevelopmental disorders, e.g., attention deficit hyperactivity disorder, Phelan-McDermid syndrome, and other autism spectrum disorders; neurodegenerative disorders, e.g., multiple sclerosis, frontotemporal dementia, and Alzheimer's disease; and cardiac conditions, e.g., Brugada syndrome, short QT syndrome, and early repolarization syndrome).

[0024] Unless the context otherwise requires, throughout this specification and in the claims which follow, the word "comprise" as well as variations such as "comprises" or "comprising" are to be understood as implying the inclusion of stated elements or steps, or group of elements or steps, but not the exclusion of any other elements or steps, or group of elements or steps.

[0025] In the event of a discrepancy between a depicted structure and a chemical name given to that structure, the depicted structure is given more weight. In addition, if the stereochemistry of a structure or portion of a structure is not shown, for example, in bold or dashed lines, the structure or portion of a structure is to be interpreted as encompassing all stereoisomers of the structure of the portion of the structure.

[0026] 5.2. Compounds A compound of formula (I) or a pharma- ceutically acceptable salt thereof, [ka] During the ceremony, R 1 But, C 1~6 Alkyl and C 1~6 Haloalkyl, e.g., C 1~6 fluoroalkyl; R 2 is selected from H and halo, e.g., F; R 3 But, C 1~6 Alkyl, e.g., C 1~4 Alkyl, and C 3~8 cycloalkyl, each of which is optionally substituted with 1 to 3 halo, e.g., F, or a pharma- ceutically acceptable salt thereof.

[0027] One embodiment is a compound of formula (I), or a pharma- ceutically acceptable salt thereof: [ka] During the ceremony, R 1 is selected from CH3, CF3, CHF2 and CH2F; R 2 is selected from H and F; R 3 But, C 1~4 and selected from alkyl, cyclopropyl, and cyclobutyl, each of which is optionally substituted with 1 to 3 F.

[0028] One embodiment is a compound of formula (Ia): [ka] or a pharma- ceutically acceptable salt thereof.

[0029] Another embodiment is a compound of formula (Ib): [ka] or a pharma- ceutically acceptable salt thereof.

[0030] The following embodiments relate to compounds of formula (I), including formulae (Ia) and (Ib). In one embodiment, R 1 is CH3.

[0031] In another embodiment, R 1 is CF3.

[0032] In another embodiment, R 1 is CHF2.

[0033] In another embodiment, R 1 is CH2F.

[0034] In another embodiment, R 2 is H.

[0035] In another embodiment, R 2 is F.

[0036] In another embodiment, R 3 is C optionally substituted with 1 to 3 F 1~4 It is an alkyl.

[0037] In another embodiment, R 3 is cyclopropyl optionally substituted with 1 to 3 F.

[0038] In another embodiment, R 3 is cyclobutyl optionally substituted with 1 to 3 F.

[0039] Specific compounds of formula (I) include the following: 4-(5-fluoro-4-(1-fluoroethyl)pyridin-3-yl)-2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl (S)-4-(5-fluoro-4-((R)-1-fluoroethyl)pyridin-3-yl)-2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl (S)-4-(5-fluoro-4-((S)-1-fluoroethyl)pyridin-3-yl)-2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl (R)-4-(5-fluoro-4-((R)-1-fluoroethyl)pyridin-3-yl)-2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl, (R)-4-(5-fluoro-4-((S)-1-fluoroethyl)pyridin-3-yl)-2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl, 4-(5-fluoro-4-(1-fluoroethyl)pyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl (R)-4-(5-fluoro-4-((R)-1-fluoroethyl)pyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl, (R)-4-(5-fluoro-4-((S)-1-fluoroethyl)pyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl, (S)-4-(5-fluoro-4-((R)-1-fluoroethyl)pyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl, (S)-4-(5-fluoro-4-((S)-1-fluoroethyl)pyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl, 4-(4-ethyl-5-fluoropyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl (R)-4-(4-ethyl-5-fluoropyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl, (S)-4-(4-ethyl-5-fluoropyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl 4-(4-ethyl-5-fluoropyridin-3-yl)-2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl (S)-4-(4-ethyl-5-fluoropyridin-3-yl)-2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl (R)-4-(4-ethyl-5-fluoropyridin-3-yl)-2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl, 4-(4-ethylpyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl (R)-4-(4-ethylpyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl, (S)-4-(4-ethylpyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl 2-(difluoromethyl)-4-(5-fluoro-4-(1-fluoroethyl)pyridin-3-yl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl (R)-2-(difluoromethyl)-4-(5-fluoro-4-((R)-1-fluoroethyl)pyridin-3-yl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl, (R)-2-(difluoromethyl)-4-(5-fluoro-4-((S)-1-fluoroethyl)pyridin-3-yl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl, (S)-2-(difluoromethyl)-4-(5-fluoro-4-((R)-1-fluoroethyl)pyridin-3-yl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl, (S)-2-(difluoromethyl)-4-(5-fluoro-4-((S)-1-fluoroethyl)pyridin-3-yl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl, 4-(5,6-difluoro-4-(1-fluoroethyl)pyridin-3-yl)-2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl (S)-4-(5,6-difluoro-4-((R)-1-fluoroethyl)pyridin-3-yl)-2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl (S)-4-(5,6-difluoro-4-((S)-1-fluoroethyl)pyridin-3-yl)-2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl Methyl (R)-4-(5,6-difluoro-4-((R)-1-fluoroethyl)pyridin-3-yl)-2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, and Methyl (R)-4-(5,6-difluoro-4-((S)-1-fluoroethyl)pyridin-3-yl)-2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate.

[0040] Depending on the selection of starting materials and procedures, the compounds can exist in one of the possible stereoisomers or as mixtures thereof, for example as pure optical isomers or as stereoisomeric mixtures, such as racemic and diastereoisomeric mixtures, depending on the number of asymmetric carbon atoms. The present disclosure is meant to include all such possible stereoisomers, including racemic mixtures, diastereomeric mixtures, and optically pure forms. Optically active (R)- and (S)-stereoisomers can be prepared using chiral synthons or chiral reagents or resolved using conventional techniques. When the compounds contain double bonds, the substituents can be in the E or Z configuration. When the compounds contain disubstituted cycloalkyl, the cycloalkyl substituents can have cis or trans configuration. All tautomeric forms are also intended to be included.

[0041] As used herein, the term "salt" or "salts" refers to acid addition salts or base addition salts of the compounds of the present disclosure. "Salt" specifically includes "pharmaceutically acceptable salts". The term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the compounds of the present disclosure and that are typically not biologically or otherwise unsuitable. In many cases, the compounds of the present disclosure are capable of forming acid and / or base salts by virtue of the presence of amino and / or carboxyl groups or groups similar thereto.

[0042] Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids.

[0043] Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

[0044] Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, salicylic acid, and the like.

[0045] Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.

[0046] Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the periodic table, in certain embodiments, salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper, with particularly suitable salts including ammonium, potassium, sodium, calcium, and magnesium salts.

[0047] Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, etc. Certain organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0048] In another aspect, the present disclosure provides an agonist, an anti-inflammatory, and / or anti-inflammatory agent, comprising an acetate, an ascorbate, an adipate, an aspartate, a benzoate, a besylate, a bromide / hydrobromide, a bicarbonate / carbonate, a bisulfate / sulfate, a camphorsulfonate, a caprate, a chloride / hydrochloride, a chlortheophyllonate, a citrate, an ethanedisulfonate, a fumarate, a gluceptate, a gluconate, a glucuronate, a glutamate, a glutarate, a glycolate, a hippurate, a hydroiodide / iodide, an isethionate, a sulph ... In one embodiment, the compounds of the present disclosure are provided in the form of a phosphate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methylsulfate, mucate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, polygalacturonate, propionate, sebacate, stearate, succinate, sulfosalicylate, sulfate, tartrate, tosylate, triphenylacetate, trifluoroacetate, or xinafoate salt.

[0049] The compounds of the present disclosure, i.e., compounds of formula (I) containing groups capable of acting as hydrogen bond donors and / or acceptors, may be capable of forming co-crystals with suitable co-crystal formers. These co-crystals may be prepared from compounds of formula (I) by known co-crystal formation procedures. Such procedures include grinding, heating, co-subliming, co-melting, or contacting compounds of formula (I) and co-crystal formers in solution under crystallization conditions and isolating the co-crystals formed thereby. Suitable co-crystal formers include those described in WO2004 / 078163. Thus, the present disclosure further provides co-crystals comprising compounds of formula (I).

[0050] Furthermore, the compounds of the present disclosure, including their salts, may also be obtained in the form of their hydrates or may include other solvents used in their crystallization. The compounds of the present disclosure may inherently or by design form solvates with pharma- ceutically acceptable solvents (including water), and therefore the present disclosure is intended to encompass both solvated and unsolvated forms. The term "solvate" refers to a molecular complex of the compounds of the present disclosure (including their pharma-ceutically acceptable salts) with one or more solvent molecules. Such solvent molecules are those commonly used in the pharmaceutical arts and known to be harmless to the recipient, e.g., water, ethanol, etc. The term "hydrate" refers to a complex in which the solvent molecule is water.

[0051] The present disclosure includes unlabeled and isotopically labeled forms of the compounds of formula (I).Isotopically labeled compounds have the structure shown by the formula given herein, except that one or more atoms are replaced by an atom having a selected atomic mass or mass number.The isotopes that can be incorporated in the compounds of the present disclosure include, for example, hydrogen isotopes.

[0052] Additionally, certain isotopes, particularly deuterium (i.e. 2Incorporation of H or D) may result in certain therapeutic advantages due to greater metabolic stability, such as increased half-life in vivo or reduced required dosage, or improved therapeutic index or tolerability. It is understood that deuterium in this context is considered a substituent of the compounds of the present disclosure. The concentration of deuterium can be defined by the isotopic enrichment factor. As used herein, the term "isotopic enrichment factor" refers to the ratio between the isotopic abundance and the natural abundance of a designated isotope. When a substituent in a compound of this disclosure is designated as deuterium, such compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation). It is to be understood that the term "isotopic enrichment factor" can be applied to any isotope in the same manner as described for deuterium.

[0053] In one embodiment of any aspect of the disclosure, the hydrogen in the compound of formula (I) is present in their normal isotopic abundance. In another embodiment, the hydrogen is isotopically enriched with deuterium (D), and in certain embodiments of the disclosure, formula (I) is deuterated or deuterated as shown in formula (II): [ka] or a pharma- ceutically acceptable salt thereof, wherein R 1 , R 2 and R 3 is as defined in formula (I) according to any aspect or embodiment of the present disclosure; D1 ~R D7are each independently H or D. Compounds of formula (II) can be synthesized according to any one of general schemes 1-2 by using an appropriately deuterated substrate in place of the non-deuterated embodiment.

[0054] Other examples of isotopes that may be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, and fluorine, e.g., 3 H, 11 C. 13 C. 14 C. 15 N, 18 F, and 18 O. Thus, the present disclosure relates to, for example, 3 H and 14 Radioisotopes such as C, or 2 H and 13 It should be understood to include compounds incorporating one or more of any of the foregoing isotopes, including those in which non-radioactive isotopes such as C are present. Such isotopically labeled compounds may be useful for metabolic studies ( 14 C), reaction rate studies (e.g. 2 H or 3 H), detection or imaging techniques including drug or substrate tissue distribution assays, such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), or radiotherapy of patients. 18 F or labeled compounds may be particularly desirable for PET or SPECT studies. Isotopically labeled compounds of the present disclosure may generally be prepared by conventional techniques known to those skilled in the art, or by processes similar to those described in the accompanying examples and preparations, substituting the appropriate isotopically labeled reagent for the previously used non-labeled reagent.

[0055] Any asymmetric atom (e.g., carbon, etc.) of the compound(s) of the disclosure can be present in a racemic or enantiomerically enriched state, for example, in the (R)-configuration, the (S)-configuration, or the (R,S)-configuration. In certain embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess in the (R)-configuration or the (S)-configuration.

[0056] Thus, as used herein, the compounds of the present disclosure may be in the form of one of the possible stereoisomers, rotamers, atropisomers, tautomers, or mixtures thereof, for example, as substantially pure diastereomers, optical isomers (enantiomers), racemates, or mixtures thereof.

[0057] Thus, the compounds of the present disclosure may exist in a racemic mixture or in an enantiomerically enriched form, or in an enantiomerically pure form, or as a mixture of diastereoisomers.

[0058] In any one of the compound formulas in the aspects, embodiments or claims of the present application, C-sp 3 Above The term TIFF2024545200000008.tif1429 denotes either (R) or (S) absolute stereochemistry. In the compound formulas in any one of the aspects, embodiments or claims of this application, C-sp 3 Above The term TIFF2024545200000009.tif1429 denotes either (R) or (S) absolute stereochemistry. In the compound formulas in any one of the aspects, embodiments or claims of this application, C-sp 3 Above The term TIFF2024545200000010.tif1429 refers to a covalent bond where the absolute stereochemistry of the bond is not defined. This means that C-sp 3 Above The term TIFF2024545200000011.tif1429 is meant to include either the (S) or (R) configuration of each chiral center or mixtures thereof. Thus, mixtures of stereoisomers, e.g., mixtures of enantiomers such as racemates, and / or mixtures of diastereoisomers are encompassed by the present disclosure.

[0059] Any resulting mixture of stereoisomers can be separated on the basis of the physical chemical differences of the components into pure or substantially pure enantiomers, diastereomers, racemates, for example, by chromatography and / or fractional crystallization.

[0060] Any resulting racemates of the compounds or intermediates of the present disclosure can be resolved into their optical antipodes by known methods, for example by separation of their diastereomeric salts obtained with optically active acids or bases, by liberating the optically active acidic or basic compounds. In particular, basic moieties can thus be used to resolve the compounds of the present disclosure into their optical isomers, for example by fractional crystallization of salts formed with optically active acids, for example tartaric acid, dibenzoyltartaric acid, diacetyltartaric acid, di-O,O'-p-toluoyltartaric acid, mandelic acid, malic acid, or camphor-10-sulfonic acid. The racemates or racemic intermediates of the present disclosure can also be resolved by chiral chromatography, for example by high pressure liquid chromatography (HPLC) using a chiral adsorbent.

[0061] 5.3.How to make The compounds of the present disclosure can be prepared in a number of ways well known to those skilled in the art of organic synthesis. As an example, the compounds of the present disclosure can be synthesized using the methods described below, together with synthetic methods known in the art of synthetic organic chemistry, or by variations thereof as would be understood by those skilled in the art.

[0062] In general, compounds of formula (I) may be prepared according to the schemes provided below. [ka]

[0063] The starting materials for the above reaction schemes are either commercially available or can be prepared according to methods known to those skilled in the art or by methods disclosed herein. In general, the compounds of the present disclosure are prepared as follows in General Scheme 1 above. Compound (Y) is reacted with intermediates A and B under condensation reaction conditions to give compound E. Compound (R) of formula (I) is obtained by intramolecular lactonization with potassium carbonate and methanol. 1 =CH2F). For General Scheme 1, R 2 and R 3 is as defined herein. [ka]

[0064] Compound (Y) is reacted with intermediate A and methyl (Z)-3-aminobut-2-enoate under condensation reaction conditions to give compound F. Compound (R) of formula (I) is obtained by intramolecular lactonization with potassium carbonate and methanol. 1 =CH3). For General Scheme 2, R 2 and R 3 is as defined herein.

[0065] In one embodiment, a compound of formula (X) or a salt thereof is provided, [ka] R 1 is selected from CH3, CF3, CHF2 and CH2F; R 2 is selected from H and F; R 3 But, C 1~4alkyl, cyclopropyl, and cyclobutyl, each of which is optionally substituted with 1 to 3 F; R x and R y However, each independently, C 1~6 alkyl, e.g., R x is ethyl, and R y is methyl, or a salt thereof.

[0066] In one embodiment, there is provided the use of a compound of formula (X) in the preparation of a compound of formula (I).

[0067] In a further embodiment, there is provided a compound of formula (Y) or a salt thereof, [ka] R 2 is selected from H and F; R 3 But, C 1~4 is selected from alkyl, cyclopropyl and cyclobutyl, each of which is optionally substituted with 1 to 3 F, or a salt thereof.

[0068] In one embodiment there is provided the use of a compound of formula (Y) in the preparation of a compound of formula (I).

[0069] In a further aspect, the disclosure provides a process for preparing a compound of formula (I) in free form or in a pharma- ceutically acceptable salt form, comprising the steps of: 1) reacting a compound of formula (Y) as defined herein with [ka] and TIFF2024545200000017.tif4048(in the formula, R 1 is selected from CH3, CF3, CHF2, and CH2F; R x and R y However, each independently C 1~6alkyl, e.g., R x is ethyl, and R y is methyl) under condensation reaction conditions (e.g., heat) to produce a compound of formula (X) as defined herein.

[0070] In a further embodiment of the process for preparing a compound of formula (I), the process comprises: 2) reacting a compound of formula (X) as defined herein (e.g., prepared according to step 1) under lactonization conditions (e.g., a base and a solvent such as KCO and methanol) to produce a compound of formula (I) as defined herein.

[0071] In a further embodiment, the process of step 2 further comprises: 3) the optional step of purifying the compound of formula (I) as defined herein; and (optionally) 4) the optional step of separating the resulting stereoisomers, for example by chromatography, for example by chiral chromatography.

[0072] Condensation reaction conditions for or below any of the foregoing process steps involve the use of a solvent, e.g., a protic solvent such as an alcohol, e.g., methanol, ethanol, and the like, and heating the substrate mixture. Certain reactions may require the addition of a base, e.g., NaOH, an amine (Knoevenagel condensation). The reaction mixture can be heated to the appropriate temperature required for the reaction to proceed, e.g., room temperature to 100°C, e.g., 70-80°C.

[0073] In a further embodiment, there is provided a process for preparing a compound of formula (I) in free form or in pharma- ceutically acceptable salt form according to any one of general schemes 1-2.

[0074] Any of the process steps above or below lactonization reaction conditions involve the use of an appropriate reagent to generate a lactone group. For example, lactonization conditions may involve the use of a base, e.g., K2CO3, and a solvent, such as a protic solvent, e.g., methanol, ethanol, etc. The reaction may be carried out at room temperature or by heating, e.g., from room temperature to 80°C.

[0075] Compounds of formula (X) and (Y) as defined herein are useful for the preparation of compounds of the present disclosure, such as compounds of (I). Thus, in one aspect, the present disclosure relates to compounds of formula (X) or (Y), or salts thereof. In another aspect, the present disclosure relates to the use of compounds of formula (X) or (Y), or salts thereof, in the manufacture of compounds of formula (I). The present disclosure further includes any variation of the process of the present invention, such as the variation in which an intermediate product obtainable at any stage thereof is used as a starting material and the remaining steps are carried out, or the variation in which the starting material is formed in situ under the reaction conditions, or the variation in which the reaction components are used in the form of their salts or optically pure substances.

[0076] 5.4.How to use The compounds of the present disclosure, in free form or in pharma- ceutically acceptable salt form, have valuable pharmacological properties, e.g., Ca V 1.2 exhibits activating properties and is therefore indicated for use as a tool compound, for example for therapy or as a research chemical.

[0077] The compounds of the present disclosure, for example compounds of formula (I), including (II), (Ia) and (Ib), may be useful for the treatment of an indication selected from the list below. Neuropsychiatric disorders, such as schizophrenia, bipolar disorder, major depressive disorder, and substance use disorders; Neurodevelopmental disorders, such as attention deficit hyperactivity disorder, Phelan-McDermid syndrome, and other autism spectrum disorders; Neurodegenerative disorders, such as multiple sclerosis, frontotemporal dementia and Alzheimer's disease; and Cardiac conditions, such as Brugada syndrome, short QT syndrome, and early repolarization syndrome. In one embodiment, the indication is a neuropsychiatric disorder, such as schizophrenia, bipolar disorder, major depressive disorder, or substance use disorder. In another embodiment, the indication is schizophrenia or bipolar disorder.

[0078] In another aspect, the present disclosure provides a method for the preparation of a Ca V The present invention provides a method for treating a disease or disorder treated by activation of 1.2, comprising administering a therapeutically effective amount of a compound of the present disclosure or a pharma- ceutically acceptable salt thereof. In a further embodiment, the disease is selected from the list of indications above.

[0079] In another aspect, the present disclosure provides a method for the preparation of a Ca V The present invention provides a method of treating a disease or disorder treated by activation of 1.2, comprising administering a compound of the present disclosure or a pharma- ceutically acceptable salt thereof. In a further embodiment, the disease is selected from the list of indications above.

[0080] In another aspect, the disclosure provides a method of treating schizophrenia, bipolar disorder, major depressive disorder, substance use disorder, ADHD, Phelan-McDermid syndrome, autism spectrum disorder, multiple sclerosis, frontotemporal dementia, Alzheimer's disease, Brugada syndrome, short QT syndrome, or early repolarization syndrome in a subject in need thereof, the method comprising administering a therapeutically effective amount of a compound of the disclosure, e.g., a compound of formula (I), or a pharmaceutically acceptable salt thereof.

[0081] In another aspect, the disclosure provides a method of treating schizophrenia, bipolar disorder, major depressive disorder, substance use disorder, ADHD, Phelan-McDermid syndrome, autism spectrum disorder, multiple sclerosis, frontotemporal dementia, Alzheimer's disease, Brugada syndrome, short QT syndrome, or early repolarization syndrome in a subject in need thereof, the method comprising administering a compound of the disclosure, e.g., a compound of formula (I), or a pharmaceutically acceptable salt thereof.

[0082] In a further aspect, the present disclosure provides a compound of the present disclosure, such as a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in therapy. In a further embodiment, the therapy is selected from diseases or disorders that can be treated by activation of Ca V 1.2. In another embodiment, the disease is selected from the list of indications described above.

[0083] In a further aspect, the present disclosure provides a compound of the present disclosure, such as a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of schizophrenia, bipolar disorder, major depressive disorder, substance use disorder, ADHD, Phelan-McDermid syndrome, autism spectrum disorder, multiple sclerosis, frontotemporal dementia, Alzheimer's disease, Brugada syndrome, QT shortening syndrome, or early repolarization syndrome.

[0084] Accordingly, in a further aspect, the present disclosure provides the use of a compound of the present disclosure, such as a compound of formula (I), or a pharmaceutically acceptable salt thereof, in therapy. In a further embodiment, the therapy is selected from diseases that can be treated by activation of Ca V 1.2. In another embodiment, the disease is selected from the list of indications described above.

[0085] In a further aspect, the present disclosure provides the use of a compound of the present disclosure, such as a compound of formula (I), or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament. In a further embodiment, the medicament is for the treatment of diseases that can be treated by activation of Ca V 1.2. In another embodiment, the disease is selected from the list of indications described above.

[0086] In a further aspect, the disclosure provides the use of a compound of the disclosure, e.g., a compound of formula (I), or a pharma- ceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of schizophrenia, bipolar disorder, major depressive disorder, substance use disorder, ADHD, Phelan-McDermid syndrome, autism spectrum disorder, multiple sclerosis, frontotemporal dementia, Alzheimer's disease, Brugada syndrome, short QT syndrome, or early repolarization syndrome.

[0087] Thus, in a further aspect, the disclosure provides the use of a compound of the disclosure, e.g., a compound of formula (I), or a pharma- ceutically acceptable salt thereof, for treating schizophrenia, bipolar disorder, major depressive disorder, substance use disorder, ADHD, Phelan-McDermid syndrome, autism spectrum disorder, multiple sclerosis, frontotemporal dementia, Alzheimer's disease, Brugada syndrome, short QT syndrome, or early repolarization syndrome.

[0088] All of the foregoing and following embodiments relating to uses, methods of treatment, and compounds for use are equally applicable to pharmaceutical compositions comprising a compound of the present disclosure, e.g., a compound of formula (I), or a pharma- ceutically acceptable salt thereof.

[0089] 5.5. Pharmaceutical Compositions In another aspect, the present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure or a pharma- ceutically acceptable salt thereof and a pharma- ceutically acceptable carrier. In a further embodiment, the composition comprises at least two pharma- ceutically acceptable carriers, such as those described herein. The pharmaceutical composition can be formulated for a particular route of administration, such as oral administration, parenteral administration (e.g., by injection, infusion, transdermal or topical administration), and rectal administration. Topical administration may also involve inhalation or intranasal application. The pharmaceutical composition of the present disclosure may be configured in a solid form (including, but not limited to, capsules, tablets, pills, granules, powders, or suppositories), or in a liquid form (including, but not limited to, solutions, suspensions, or emulsions). Tablets may be film-coated or enteric-coated according to methods known in the art. Typically, pharmaceutical compositions comprise an active ingredient in a form that is: a) diluents, such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine; b) tablets or gelatine capsules containing lubricants such as, for example, silica, talcum, stearic acid, its magnesium or calcium salts, and / or polyethylene glycol, and, in the case of tablets, c) binders, such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone, if desired; d) disintegrants, such as starch, agar, alginic acid or its sodium salt, or effervescent mixtures; and e) Also includes absorbents, colorants, flavorings and sweeteners.

[0090] The pharmaceutical compositions of the present disclosure may be in unit dosages of about 1-1000 mg of active ingredient(s), or about 1-500 mg, or about 1-250 mg, or about 1-150 mg, or about 0.5-100 mg, or about 1-50 mg of active ingredient for a subject of about 50-70 kg. The therapeutically effective dosage of the compounds, pharmaceutical compositions, or combinations thereof, depends on the species, weight, age, and individual condition of the subject, the disorder or disease being treated, or its severity. A physician, clinician, or veterinarian of ordinary skill in the art can readily determine the effective amount of each of the active ingredients required to prevent, treat, or inhibit the progression of the disorder or disease.

[0091] The dosage characteristics cited above are demonstrable in in vitro and in vivo tests, preferably using mammals, such as mice, rats, dogs, monkeys, or isolated organs, tissues, and preparations thereof. The compounds of the present disclosure can be applied in vitro in the form of solutions, e.g., aqueous solutions, and in vivo either enterally or parenterally, preferably intravenously, e.g., as a suspension or in aqueous solution. The dosage in vitro is about 10 -3 Molar concentration ~ 10 -9The therapeutically effective amount in vivo may range from about 0.1 to 500 mg / kg, or from about 1 to 100 mg / kg, depending on the route of administration.

[0092] 6. Compound Preparation The following examples are intended to illustrate the present disclosure and are not to be construed as limiting thereof.

[0093] All examples were separated into their single enantiomers and tested in the Sophion QPatch assay described in the Biological Data section below. The absolute stereochemistry of methyl (R)-4-(5-fluoro-4-((R)-1-fluoroethyl)pyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate of Example 2 was determined by single crystal x-ray crystallography (Figure 2). The stereoisomers of methyl (R)-4-(5-fluoro-4-((R)-1-fluoroethyl)pyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate were found to have higher activity than their corresponding enantiomers. Therefore, the C10 position on the dihydropyridine ring is assumed to adopt the same spatial configuration for the most active stereoisomer in each Example. [ka] (R)-4-(5-fluoro-4-((R)-1-fluoroethyl)pyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl

[0094] Example numbers (Examples 1, 2, 3, etc.) are given for the active enantiomers which are all assumed to have the same configuration around the C10 position on the dihydropyridine ring (Examples can be designated as either R or S depending on the substitution of the ring system). All other isomers isolated from the synthesis have been given example numbers with letters (Examples 1b, 2b, 3b, etc.). Although there is strong evidence to suggest that the C10 position shown is the desired stereochemistry, in some of the examples there remains the possibility that the other configuration may be the active enantiomer.

[0095] Temperatures are given in degrees Celsius. Unless otherwise noted, all evaporations are carried out under reduced pressure, typically at about 15 mmHg to 100 mmHg (=20 to 133 mbar). The structure of final products, intermediates, and starting materials is confirmed by standard analytical methods, e.g., microanalysis and spectroscopic characteristics, e.g., MS, IR, NMR.

[0096] All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents and catalysts utilized to synthesize the compounds of the present disclosure are either commercially available or can be prepared by organic synthesis methods known to those skilled in the art. Additionally, the compounds of the present disclosure can be prepared by organic synthesis methods known to those skilled in the art, as shown in the examples below. Abbreviations used are either conventional in the art or are as follows: [Table 1-1] [Table 1-2]

[0097] Small molecule X-ray crystal structure analysis Data collection Intensity data were collected at 100 K on a Bruker AXS three-circle diffractometer with monochromated Cu(Kα) radiation, a microsource generator, and a Photon III detector using APEX 3 software (Bruker AXS (2016)). 21 ω scans were performed at different 2θ and φ positions to ensure adequate data redundancy (14.9, Friedel pairs not merged). Data processing and global cell fine-tuning were performed using Saint (Bruker AXS (2012)). Semi-empirical absorption corrections based on the intensities of symmetry-related reflections measured at different angular settings were applied using SADABS-2016 / 2 (Krause et al (2015)).

[0098] Structure elucidation and fine tuning The structure was solved by double space recycling methods and subsequent DF synthesis and refined based on full matrix least squares on F2 using the SHELXTL suite of programs (Sheldrick GM (2001)). Anisotropic displacement parameters were used for all non-hydrogen atoms. Hydrogen atoms were placed in the DF map and refined at idealized positions using a riding model. References: Allen FH, Kennard O, Watson D et al (1987) Tables of Bond Lengths determined by X-Ray and Neutron Diffraction.Part 1,Bond Lengths in Organic Compounds.J.Chem.Soc.Perkin Trans II;S1-S19. Bruker AXS (2005) SMART V5.632. Bruker AXS Inc., Madison, WI, USA. Bruker AXS (2012) SAINT V7.36A. Bruker AXS Inc., Madison, WI, USA. Krause L, Herbst-Irmer R, Sheldrick GM et al (2015) Comparison of silver and molybdenum microfocus X-ray sources for single-crystal structure determination.J.Appl.Cryst.;48:3-10. Spek AL(2003)Single-crystal structure validation with the program PLATON.J.Appl. Cryst.;36:7-13. Sheldrick GM (2001) SHELXTL V6.12. Bruker AXS Inc. Madison, WI, USA.

[0099] Analytical LCMS method: Method 1: Column: Synergi 2.5μ MAX-RP 100A Mercury; Mobile phase: 0.1% formic acid in water (A) / ACN (B); Gradient / (time / %B): 0.1 / 5, 0.5 / 5, 1.0 / 95, 1.5 / 95, 2.0 / 5, 3.0 / 5; Flow rate: 2.0 mL / min, Temperature: 40 °C Method 2: Method: API 2000; Gradient: Time / %B: 0 / 30, 0.5 / 30, 1.5 / 95, 2.4 / 95, 2.5 / 30, 3.0 / 30; Mobile phase: 0.1% FA in water (A), ACN (B) Method 3: Column: ZORBAX ECLIPSE XDB C18 1.8μm, 50*4.6mm; Mobile phase: 0.1% formic acid in water (A) / ACN (B) Gradient time for %B: 0.0 / 20, 0.25 / 20, 01.0 / 95.0, 2.5 / 95, 3.0 / 20, 4 / 20 Method 4: Column: Kinetex 2.6 μm, 100A 30x3mm; Mobile phase: 0.1% formic acid in water (A) / ACN (B); Gradient / (time / %B): 0.1 / 20, 0.25 / 20, 0.75 / 95, 1.75 / 95, 2.0 / 20, 2.5 / 20; Flow rate: 1.0 mL / min, Temperature: 40 °C

[0100] Preparative HPLC method for purification: Method 1: HPLC column: XBRIDGE-C18 (19.0×150 mm, 5 micron), Mobile phase-A: 0.1% TFA in H2O, B: CH3CN, Gradient (time / %B): 0 / 20, 2 / 20, 8 / 50) Flow rate: [19 mL / min]. Method 2: HPLC column: ZORBAX ECLIPSE XDB C18 (21.2×150 mm, 5 micron), mobile phase-A: 0.1% TFA in H2O, B: CH3CN, gradient (time / %B): 0 / 10, 2 / 20, 10 / 40 and flow rate: [20 mL / min]. Method 3: HPLC column: XBRIDGE C18 (21.2×150 mm, 5 micron), mobile phase-A: 10 mM NH4HCO3 in water, B: CH3CN, gradient (time / % B): 0 / 10, 2 / 20, 8 / 50 and flow rate: [18 mL / min]. Method 4: HPLC column: Gemini NX C18 (21.2 x 150.00 mm, 5 microns); Mobile phase - (A): 0.1% TFA in water (B): acetonitrile / methanol; Flow rate: 15 ml / min; (Time / %B 0 / 20, 2 / 20, 8 / 20) Method 5: HPLC column: KINETEX EVO 5μ C18 (21.2×150 mm), mobile phase: water (A) CH3CN (B), gradient (time / %B): 0 / 20, 2 / 30, 7 / 70 and flow rate: [18 mL / min]. Method 6: HPLC column: KINETEX C18, (21.2 x 150 mm), mobile phase: A: water, B: CH3CN:MeOH, gradient (time / %B): 0 / 20, 2 / 30, 7 / 70, flow rate: 18 mL / min]. Method 7: HPLC column: KINETEX (21.2 x 150 mm, 5 micron), mobile phase: A = 0.05% TFA in water, B = CH3CN, gradient (time / %B): 0 / 20, 2 / 30, 10 / 60, flow rate: 20 mL / min].

[0101] Chiral preparative HPLC method for separation of isomers: Method 1: Column: CHIRALPAK IC (10 x 250 mm, 5 micron), Mobile phase: Hexane (A) IPA:MeOH, 1:1 (B); Flow rate: 8 mL / min; Isocratic: 96:04 (A:B). Method 2: Column: REGIS WELKO (250 x 10 mm, 5 micron), Mobile phase: Hexane (A):EtOH, 1:1 (B); Flow rate: 9 mL / min; Isocratic: 85:15 (A:B). Method 3: Column: CHIRALPAC IG (250 x 10 mm, 5 micron); Mobile phase: IPA (A):MeOH, 1:1 (B); Flow rate: 6 mL / min; Isocratic: 98:2 (A:B). Method 4: Column: LUX CELLULOSE-4 (10 x 250 mm, 5 micron), Mobile phase: Hexane (A) EtOH:IPA 1:1 (B); Flow rate: 8 mL / min; Isocratic: 90:10 (A:B).

[0102] Preparation of intermediates and general procedures: Formation of Intermediate A: Ethyl 4-acetoxy-3-oxobutanoate [ka] To a solution of ethyl 4-chloro-3-oxobutanoate (200 g, 1215.1 mmol) in acetic acid (1500 mL) was added potassium acetate (357 g, 3645.4 mmol). The resulting solution was stirred at 90° C. for 18 h. The solvent was cooled to room temperature, added to water (2 L) and extracted into ethyl acetate (1 L×4 times). The EtOAc phases were combined, washed with saturated NaHCO3 solution (2 L), brine and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by silica flash chromatography ethyl acetate in petroleum ether (0→15%) to give the title compound as a light brown liquid ethyl 4-acetoxy-3-oxobutanoate. 1 H NMR(400MHz, CDCl3)δ 4.78(s,2H),4.20(q,J=14.1,7.2Hz,2H),3.49(s,2H),2.16(s,3H),1.28(t,J=7.2Hz,3H).

[0103] Formation of intermediate B(Z)-methyl 3-amino-4-fluorobut-2-enoate Method 1: [ka] Under nitrogen atmosphere, a solution of methyl acetate (36.5 g, 492.71 mmol) in tetrahydrofuran (200 mL) was cooled to -78°C and lithium diisopropylamide in THF (246.44 mL, 2.0 M, 492.71 mmol) was added slowly to the reaction over 20 min and the resulting mixture was stirred at -78°C for 1 h followed by the dropwise addition of 2-fluoroacetonitrile (19.4 g, 328.9 mmol) in tetrahydrofuran (150 mL). The reaction mixture was stirred at -78°C for an additional 1 h after which saturated ammonium chloride solution (200 mL) was added and the product was extracted into ethyl acetate (5 L). The EtOAc was washed with saturated brine solution (500 mL) and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by silica flash chromatography with ethyl acetate in petroleum ether (0→10%) to afford the title compound as a white crystalline solid, methyl (Z)-3-amino-4-fluorobut-2-enoate.

[0104] Method 2: [ka] Step 1: Under nitrogen atmosphere, a solution of methyl acetate (83.78 g, 1131.0 mmol) in tetrahydrofuran (800 mL) was cooled to -78 °C, then lithium diisopropylamide in THF (565.5 mL, 2.0 M, 1131.0 mmol) was added slowly to the reaction over 20 min. The resulting mixture was stirred at -78 °C for 1 h, then 2-fluoroethyl acetate (100 g, 942.5 mmol) was added dropwise in a solution of tetrahydrofuran (200 mL), and the reaction mixture was stirred at -78 °C for an additional 1 h. Saturated ammonium chloride solution (200 mL) was added to the reaction mixture, and the product was extracted into ethyl acetate (5 L). The EtOAc was washed with saturated brine solution (500 mL) and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by silica flash chromatography ethyl acetate in petroleum ether (0→10%) to give yellow crystals of methyl 4-fluoro-3-oxobutanoate. 1 H NMR (400MHz, CDCl3) δ 4.97(s,1H),4.85(s,1H),3.76(d,J=2.3Hz,3H),3.62(d,J=3.7Hz,2H).

[0105] Step 2: To methyl 4-fluoro-3-oxobutanoate (from step 1, 60 g) in a sealed tube was added a saturated solution of ammonia in methanol (300 mL) at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure to give the title compound as a white solid, methyl (Z)-3-amino-4-fluorobut-2-enoate. 1 H NMR (300MHz, DMSO-d6) δ4.98(t,J=0.7,0.7Hz,1H),4.82(t,J=0.7,0.7Hz,1H),4.53(q,1H),3.53(d,J=1.2Hz,3H).

[0106] Intermediate C: (Z)-3-amino-4,4-dimethoxybut-2-enoic acid methyl ester [ka] To a stirred solution of methyl 4,4-dimethoxy-3-oxobutanoate (2.00 g, 11.35 mmol) in MeOH (20.00 mL) was added NHOAc (4.37 g, 56.76 mmol) at room temperature and the reaction mixture was heated to 80° C. for 16 h. The solvent was removed under reduced pressure and the residue was diluted with DCM (15 mL), filtered over Celite and the Celite cake was washed with DCM (3×15 mL). The filtrate was concentrated under reduced pressure to give the title compound as a deep wine-red syrup, methyl (Z)-3-amino-4,4-dimethoxybut-2-enoate. 1 H NMR (300MHz, CDCl3) 4.80-4.79 (m, 2H), 3.66 (s, 3H), 3.34 (s, 6H).

[0107] General Procedure I [ka] Step 1: To a solution of the aldehyde (1 eq.) in EtOH (0.2 M) was added (Z)-methyl 3-amino-4-fluorobut-2-enoate (Intermediate B, 1 or 1.2 eq.) and ethyl 4-acetoxy-3-oxobutanoate (Intermediate A, 1 or 1.2 eq.). The resulting solution was stirred at 80° C. for 16 h. The solvent was removed under reduced pressure to give the title compound (crude).

[0108] Step 2: To the crude intermediate from step 1 in methanol (10 mL) was added potassium carbonate (5 mmol). The resulting solution was stirred at room temperature for 2 h. The solvent was removed under reduced pressure and added to water. The product was extracted into ethyl acetate, washed with brine and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by silica flash chromatography.

[0109] General Procedure II: Formation of 2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate compounds [ka] Step 1: To a solution of the aldehyde (1 eq.) and methyl (Z)-3-aminobut-2-enoate (1 eq.) in EtOH or MeOH (0.3 M) was added ethyl 4-acetoxy-3-oxobutanoate (Intermediate A, 1 eq.). The resulting solution was stirred at 80° C. for 14 h. The solvent was removed under reduced pressure to give the crude compound, which was generally carried on to Step 2 without further purification.

[0110] Step 2: To the crude intermediate from step 2 in methanol (0.3 M) was added potassium carbonate (3 equiv.). The resulting solution was stirred at room temperature for 2 h. The solvent was removed under reduced pressure, added to water (500 mL), extracted into ethyl acetate, and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was purified by silica flash chromatography to give the product.

[0111] Example 1: (S)-4-(5-fluoro-4-((R)-1-fluoroethyl)pyridin-3-yl)-2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate or (S)-4-(5-fluoro-4-((S)-1-fluoroethyl)pyridin-3-yl)-2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate [ka] Step 1: 1-(3-bromo-5-fluoropyridin-4-yl)ethan-1-ol [ka] To a cooled stirred solution of 3-bromo-5-fluoropyridine (10 g, 56.82 mmol) in THF (100 mL) at -78°C, LDA (2M in THF) (34.09 mL, 68.18 mmol) was added. The reaction was stirred for 1 h, then CH3CHO (9.56 mL, 170.46 mmol) was added at -78°C, and the reaction mixture was stirred at the same temperature for 1 h. The reaction mixture was poured into 10% ammonium chloride solution, and the product was extracted in EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and the solvent was removed under reduced pressure. The crude product was purified by silica flash chromatography ethyl acetate (20%) in hexane to give the title compound as a pale orange liquid. 1-(3-bromo-5-fluoropyridin-4-yl)ethan-1-ol. LCMS: Rt=0.248 min; MS m / z 219.8[M+H]+; [Method 1]. 1 H NMR(400MHz, CDCl3)δ 8.50(s,1H),8.36(d,J=1.2Hz,1H),5.32-5.28(m,1H),2.61-2.59(m,1H),1.62(d,J=6.4Hz,3H).

[0112] Step 2: 3-Bromo-5-fluoro-4-(1-fluoroethyl)pyridine [ka] DAST (8.3 mL, 62.71 mmol) was added dropwise to a cooled, stirred solution of 1-(3-bromo-5-fluoropyridin-4-yl)ethan-1-ol (from step 1, 11.5 g, 52.26 mmol) in DCM (100 mL) at -78 °C. The reaction mixture was allowed to warm to room temperature and stirred for 5 h. The reaction mixture was quenched with ice water and the product was extracted into DCM. The combined organic layers were washed with 10% NaHCO3 solution, brine, dried over anhydrous Na2SO4, filtered and the solvent removed under reduced pressure at 30 °C. The crude product was purified by silica flash chromatography (7% ethyl acetate in hexanes) to give the title compound as a pale yellow liquid. 3-Bromo-5-fluoro-4-(1-fluoroethyl)pyridine. LCMS: Rt = 1.154 min; MS m / z 223.8 [M + H] +; [Method 2]. 1 H NMR (400MHz, CDCl3) δ 8.55 (s, 1H), 8.42 (s, 1H), 6.10-5.92 (m, 1H), 1.74 (dd, J=22.8, 6.4Hz, 3H).

[0113] Step 3: 3-Fluoro-4-(1-fluoroethyl)-5-vinylpyridine [ka] To a stirred solution of 3-bromo-5-fluoro-4-(1-fluoroethyl)pyridine (from step 2, 8.3 g, 37.38 mmol) in IPA (80.00 mL) was added potassium vinyltrifluoroborate (10.01 g, 74.76 mmol) followed by Et3N (20.8 mL, 149.5 mmol) at room temperature and the reaction was purged with argon gas for 10 min. Pd(dppf)Cl2·DCM (1.53 g, 1.869 mmol) was added at room temperature and the reaction was purged with argon gas for an additional 5 min. The reaction mixture was heated to 80 °C for 10 h. The solvent was removed under reduced pressure. The crude product was purified by silica flash chromatography (7% ethyl acetate in hexanes) to give the title compound as a pale yellow liquid. 3-Fluoro-4-(1-fluoroethyl)-5-vinylpyridine. LCMS: Rt=0.935 min; MS m / z 170.1[M+H]+; [Method 2]. 1 H NMR(400MHz,CDCl3)δ 8.52(s,1H),8.36(s,1H),7.07(dd,J=17.6,11.6Hz,1H),6.12-5.92(m,1H), 5.71(d,J=17.6Hz,1H),5.49(d,J=11.2Hz,1H),1.70(dd,J=23.2,6.8Hz,3H).

[0114] Step 4: 5-Fluoro-4-(1-fluoroethyl)nicotinaldehyde [ka] A cooled stirred solution of 3-fluoro-4-(1-fluoroethyl)-5-vinylpyridine (from step 3, 3.8 g, 22.46 mmol) in DCM (250 mL) was slowly purged with O3 for 6 h at -78°C. After completion of the reaction, it was quenched with DMS (2.49 ml, 33.69 mmol) and the reaction mixture was stirred for 20 min. Ice-cold water was added and the product was extracted into DCM. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and the solvent was removed under reduced pressure at 30°C to give the title compound as a pale orange liquid. 5-Fluoro-4-(1-fluoroethyl)nicotinaldehyde. LCMS: Rt=1.406 min; MS m / z 172.2 [M+H]+; [Method 1]. 1 H NMR(400MHz, CDCl3)δ 10.43(s,1H),8.84(s,1H),8.63(s,1H),6.48-6.30(m,1H),1.80(dd,J=23.2,6.6Hz,3H).

[0115] Steps 5 and 6: Methyl 4-(5-fluoro-4-(1-fluoroethyl)pyridin-3-yl)-2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate [ka] The title compound was synthesized using general method II (using the aldehyde from step 4, 2 g, 11.68 mmol). The crude product was purified by silica flash chromatography (60% ethyl acetate in hexanes) to give the title compound as a brown solid (1.7 g). The diastereomers were separated by preparative HPLC (column: WATERS X BRIDGE (150 mm x 20.0 mm), 5.0 μ; mobile phase: A = water, B = ACN; flow rate: 15 ml / min) to give rac-diastereomer 1 and rac-diastereomer 2. The absolute stereochemistry of the four stereoisomers corresponding to the four product peaks was not determined.

[0116] The first eluting isomer, rac-diastereomer 1, was separated into its enantiomers by chiral HPLC (Column: REGIS WHELK, 250 MM x 21.2 MM x 5 microns; Mobile phase: Hexane (A), EtOH:MeOH, 1:1 (B), Flow rate: 15 mL / min).

[0117] The second eluting isomer, rac-diastereomer 2, was separated into its enantiomers by chiral HPLC (Column: LUX AMYLOSE-2, 250 MM x 21.2 MM x 5 microns; Mobile phase: Hexane (A), EtOH:MeOH, 1:1 (B); Flow rate: 15 mL / min).

[0118] Example 1 is the second eluting enantiomer of the rac-diastereomer 2. Chiral HPLC Rt=6.19 min (Column: I CELLUYLOSE-C (250×4.6 mm, 5 microns); Mobile phase: A: n-Hexane, B=IPA:MEOH (50:50); Flow rate: 1.0 ml / min). LCMS:Rt=0.653 min;MS m / z 351.0[M+H]+;[Method 2] 1 H NMR(400MHz,DMSO-d6)δ 10.01(s,1H),8.38(d,J=2.4Hz,1H),8.30(s,1H),6.43-6.26(m,1H),5.00(s,1H),4 .88(d,J=25.6,16.4Hz,2H),3.44(s,3H),2.33(s,3H),1.80(dd,J=23.6,6.4Hz,3H).

[0119] Example 1a: First-eluting enantiomer of rac-diastereomer 2 Chiral HPLC Rt=5.775 min (Column: I CELLUYLOSE-C (250×4.6 mm, 5 microns); Mobile phase: A: n-Hexane, B=IPA:MEOH (50:50); Flow rate: 1.0 ml / min). LCMS: Rt=0.615 min; MS m / z 351.0[M+H]+; [Method 2]. 1H NMR(400MHz,DMSO-d6)δ 10.01(s,1H),8.38(d,J=2.4Hz,1H),8.30(s,1H),6.43-6.26(m,1H),5.00(s,1H),4 .88(d,J=25.6,16.4Hz,2H),3.44(s,3H),2.33(s,3H),1.80(dd,J=23.6,6.4Hz,3H).

[0120] Example 1b: First-eluting enantiomer of rac-diastereomer 1 Chiral HPLC Rt=2.367 min (Column: LUX, AMYLOSE-1 (150×4.6 mm, 5 microns); Mobile phase: A: n-hexane, B=0.1% DEA in ethanol:methanol (70:30); Flow rate: 1.0 ml / min). LCMS: Rt=0.50 min; MS m / z 351.0[M+H]+; [Method 2]. 1 H NMR(400MHz,DMSO-d6)δ 10.03(s,1H),8.39(d,J=2.8Hz,1H),8.24(s,1H),6.58-6.42(m,1H),4.96(s,1H),4 .86(d,J=25.6,16.4Hz,2H),3.45(s,3H),2.36(s,3H),1.72(dd,J=23.6,6.8Hz,3H).

[0121] Example 1c: Second-eluting enantiomer of rac-diastereomer 1 Chiral HPLC Rt=2.765 min (Column: LUX, AMYLOSE-1 (150×4.6 mm, 5 microns); Mobile phase: A: n-hexane, B=0.1% DEA in ethanol:methanol (70:30); Flow rate: 1.0 ml / min). LCMS: Rt=0.498 min; MS m / z 351.2[M+H]+; [Method 2]. 1H NMR(400MHz,DMSO-d6)δ 10.03(s,1H),8.39(d,J=2.8Hz,1H),8.24(s,1H),6.58-6.42(m,1H),4.96(s,1H),4 .86(d,J=25.6,16.4Hz,2H),3.45(s,3H),2.36(s,3H),1.72(dd,J=23.6,6.8Hz,3H).

[0122] Example 2: (R)-4-(5-fluoro-4-((R)-1-fluoroethyl)pyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl [ka] Steps 1 and 2: Methyl 4-(5-fluoro-4-(1-fluoroethyl)pyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate [ka] The title compound was synthesized using general method I (using the aldehyde from step 4 of example 1, 1.8 g, 10.52 mmol). The crude product was purified by silica flash chromatography (5% MeOH in CHCl3) to give the title compound. The diastereomers were separated by preparative HPLC (column: LUNA C18 (250 mm x 21.2 mm), 5.0μ; mobile phase: A: 0.1% HCOOH: B: acetonitrile; flow rate isocratic: 20 mL / min) to give rac-diastereomer 1 and rac-diastereomer 2. The rac-diastereomer 1 was separated into its enantiomers by chiral HPLC (Column: REGIS WHELK, 250 MM x 21.2 MM x 5 microns; Mobile phase: A: Hexane: B: EtOH: MeOH: 1:1, Flow rate: 15 mL / min). The rac-diastereomer 2 was separated into its enantiomers by chiral HPLC (Column: REGIS WHELK, 250 MM x 21.2 MM x 5 microns; Mobile phase: A: Hexane: B: EtOH: MeOH: 1:1, Flow rate: 15 mL / min).

[0123] Example 2 is the first eluting enantiomer of rac-diastereoisomer 2. Chiral HPLC Rt=5.086 min (Regis, (S,S)Whelk-01-(150×4.60 mm, 5 microns); mobile phase: A=n-hexane, B=0.1% DEA in ethanol:methanol (70:30). The absolute stereochemistry of Example 2, methyl (R)-4-(5-fluoro-4-((R)-1-fluoroethyl)pyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, was determined by single crystal x-ray crystallography (Figure 2). LCMS: Rt=0.723 min; MS m / z 369.1[M+H]+; [Method 2]. 1 H NMR(400MHz,DMSO-d6)δ 10.25(s,1H),8.39(d,J=1.8Hz,1H),8.34(s,1H),6.47-6.23(m,1H),5.68(s,1H),5.56(s, 1H),5.06(s,1H),4.84(dd,J=30.0,16.8Hz,2H),3.43(s,3H),1.80(dd,J=23.6,6.8Hz,3H).

[0124] Example 2a: Second-eluting enantiomer of rac-diastereoisomer 2 Chiral HPLC Rt=5.702 min (Regis, (S,S)Whelk-01-(150×4.60 mm, 5 microns); mobile phase: A=n-hexane, B=0.1% DEA in ethanol:methanol (70:30). LCMS: Rt=0.742 min; MS m / z 369.1[M+H]+; [Method 2]. 1H NMR(400MHz,DMSO-d6)δ 10.23(brs,1H),8.38(d,J=2.2Hz,1H),8.34(s,1H),6.48-6.26(m,1H),5.67(s,1H),5 .55(s,1H),5.05(s,1H),4.95-4.74(m,2H),3.43(s,3H),1.80(dd,J=23.2,6.0Hz,3H).

[0125] Example 2b: First-eluting enantiomer of rac-diastereoisomer 1 Chiral HPLC Rt=5.949 min (Regis, (S,S)Whelk-01-(150×4.60 mm, 5 microns); mobile phase: A=n-hexane, B=0.1% DEA in ethanol:methanol (70:30). LCMS: Rt=0.576 min; MS m / z 369.1[M+H]+; [Method 2]. 1 H NMR(400MHz,DMSO-d6)δ 10.23(s,1H),8.39(d,J=2.7Hz,1H),8.29(s,1H),6.64-6.32(m,1H),5.75(s,1H),4. 60(s,1H),5.00(s,1H),4.92-4.75(m,2H),3.43(s,3H),1.69(dd,J=23.7,6.6Hz,3H).

[0126] Example 2c: Second-eluting enantiomer of rac-diastereoisomer 1 Chiral HPLC Rt=8.470 min (Regis, (S,S) Whelk-01-(150×4.60 mm, 5 microns); mobile phase: A=n-hexane, B=0.1% DEA in ethanol:methanol (70:30). LCMS: Rt=0.584 min; MS m / z 369.2[M+H]+; [Method 2]. 1H NMR(400MHz,DMSO-d6)δ 10.26(s,1H),8.41(s,1H),8.31(s,1H),6.61-6.41(m,1H),5.75(s,1H),4.63(s ,1H),5.02(s,1H),4.94-4.79(s,2H),3.44(s,3H),1.72(dd,J=23.2,6.6Hz,3H).

[0127] Example 3: (R)-4-(4-ethyl-5-fluoropyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl [ka] Step 1: 3-Bromo-4-ethyl-5-fluoropyridine [ka] A stirred solution of 3-bromo-5-fluoropyridine (5.0 g, 28.41 mmol) in THF (60.0 mL) was cooled to -78°C and stirred for 10 min. Lithium diisopropylamide (2M in THF) (21.30 mL, 42.61 mmol) was added dropwise at -78°C and the reaction mixture was stirred at the same temperature for 0.5 h. Iodoethane (2.51 mL, 31.25 mmol) was added at -78°C and the reaction mixture was stirred at room temperature for 2 h and allowed to warm to room temperature. The reaction mixture was quenched with a saturated solution of ammonium chloride, diluted with water and the product was extracted into ethyl acetate. The combined organic layers were washed with a saturated brine solution, dried over anhydrous sodium sulfate, filtered and the solvent was removed under reduced pressure. The crude compound was purified by silica flash chromatography ethyl acetate in hexane (5→6%) to give the title compound as a yellow liquid. 3-Bromo-4-ethyl-5-fluoropyridine. 1 H NMR(300MHz, CDCl3)δ 8.48(d,J=3.0Hz,1H),8.30(s,1H),2.83(dq,J=7.8,1.8Hz,2H),1.20(t,J=7.8Hz,3H).

[0128] Step 2: 4-Ethyl-5-fluoronicotinaldehyde [ka] A stirred solution of 3-bromo-4-ethyl-5-fluoropyridine (from step 1, 1.75 g, 8.576 mmol) in THF (20.0 mL) was cooled to -78 °C, then n-butyllithium (1.6 M in THF) (8.04 mL, 12.86 mmol) was added dropwise at -78 °C and stirred for 10 min. N,N-dimethylformamide (0.66 mL, 8.576 mmol) was added at -78 °C and the reaction mixture was stirred at the same temperature for 20 min. The reaction mixture was quenched with a saturated solution of ammonium chloride at -78 °C, diluted with water and the product was extracted in ethyl acetate. The combined organic layers were washed with saturated brine solution, dried over anhydrous sodium sulfate, filtered and the solvent was removed under reduced pressure. The crude compound was purified by silica flash chromatography ethyl acetate in hexane (8 → 10%) to give the title compound as a yellow liquid. 4-Ethyl-5-fluoronicotinaldehyde. 1 H NMR(300MHz,DMSO-d6)δ 10.28(s,1H)8.85(s,1H),8.77(d,J=1.5Hz,1H),3.04(q,J=7.5Hz,2H),1.17(t,J=7.2Hz,3H).

[0129] Steps 3 and 4: 4-(4-ethyl-5-fluoropyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl [ka] The title compound was synthesized using general method I (using the aldehyde from step 2, 0.2 g, 1.31 mmol). The crude product was purified by preparative HPLC to give the title compound methyl 4-(4-ethyl-5-fluoropyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate. LCMS Rt=1.403 min; MS m / z 350.75 [M+H]+; [Method 10].

[0130] The racemic sample was separated into its enantiomers by chiral HPLC (Column: REGIS WHELK-01, (250 MM x 21.1 MM x 5 microns); Mobile phase: N-Hexane (A) EtOH:MeOH, 1:1 (B), Flow rate: 15 ML). The absolute stereochemistry of the two enantiomers corresponding to the two product peaks was not determined.

[0131] Peak 1: Chiral HPLC Rt=4.963 min (Column: CHIRAL PAK-IG (150×4.6 mm×5μ); Mobile phase: A: n-hexane, B=0.1% DEA in ethanol:methanol (70:30); Flow rate: 1.0 ml / min). LCMS: Rt=1.388 min; MS m / z 351.0[M+H]+; [Method 1]. 1 H NMR(300MHz,CDCl3)δ8.20(s,2H),7.28(brs,1H),5.81(s,1H),5.65(s,1H),5. 11(s,1H),4.80(s,2H),3.56(s,3H),3.14-2.96(m,2H),1.33(t,J=7.5Hz,3H).

[0132] Peak 2: Chiral HPLC Rt=8.881 min (Column: CHIRAL PAK-IG (150×4.6 mm×5μ); Mobile phase: A: n-hexane, B=0.1% DEA in ethanol:methanol (70:30); Flow rate: 1.0 ml / min). LCMS: Rt=1.386 min; MS m / z 351.1[M+H]+; [Method 1]. 1H NMR(300MHz,CDCl3)δ 8.20(s,2H),7.28(brs,1H),5.81(s,1H),5.65(s,1H),5.11(s,1H),4.80(s,2H),3.56(s,3H),3.14-2.96(m,2H),1.33(t,J=7.8Hz,3H).

[0133] Example 4: (S)-4-(4-ethyl-5-fluoropyridin-3-yl)-2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl [ka] Steps 1 and 2: 4-(4-ethyl-5-fluoropyridin-3-yl)-2-methyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl [ka] The title compound was synthesized using general method II (using the aldehyde from step 2 of example 3, 0.2 g, 1.31 mmol). The crude product was purified by preparative HPLC to give the title compound. LCMS: Rt=1.375 min; MS m / z 332.75 [M+H]+; [Method 1].

[0134] The racemic sample was separated into its enantiomers by chiral HPLC (Column: REGIS WHELK-01, (250MM x 21.1MM x 5 microns); Mobile phase: N-Hexane (A) EtOH:MeOH, 1:1 (B), Flow rate: 15ML / min). The absolute stereochemistry of the two enantiomers corresponding to the two product peaks was not determined.

[0135] Peak 1: Chiral HPLC Rt=3.639 min (Column: LUX-AMYLOSE-2 (150×4.6 mm×5μ)); Mobile phase: A=n-hexane, B=0.1% TFA in ethanol:methanol (80:20); Flow rate: 1.0 ml / min). LCMS Rt=1.375 min; MS m / z 332.75[M+H]+; [Method 1]. 1 H NMR(300MHz,DMSO-d6)δ 10.0(brs,1H),8.24(s,1H),8.16(s,1H),4.96(s,1H),4.79-4.76(m,2H),3.43(s,3H),3.08-2.81(m,2H),2.31(s,3H),1.22(t,J=7.4Hz,3H).

[0136] Peak 2: Chiral HPLC Rt=4.78 min (Column: LUX-AMYLOSE-2 (150×4.6 mm×5μ)); Mobile phase: A=n-hexane, B=0.1% TFA in ethanol:methanol (80:20); Flow rate: 1.0 ml / min). LCMS: Rt=1.374 min; MS m / z 330.8 [MH]-; [Method 1]. 1 H NMR(400MHz,DMSO-d6)δ 10.0(brs,1H),8.23(s,1H),8.15(s,1H),4.95(s,1H),4.78(dd,J=28.0,16. 4Hz, 2H), 3.42 (s, 3H), 3.08-2.81 (m, 2H), 2.31 (s, 3H), 1.22 (t, J=7.4Hz, 3H).

[0137] Example 5: (R)-4-(4-ethylpyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl [ka] Step 1: 3-Bromo-4-ethylpyridine [ka] To a stirred solution of 3-bromo-4-methylpyridine (2.0 g, 11.62 mmol) in THF (20 mL) cooled to -78 °C, lithium isopropylamide (2 M) in THF (6.97 mL, 13.95 mmol) was added and the reaction mixture was stirred at the same temperature for 1 h. Then, iodomethane (1.98 g, 13.95 mmol) was added and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was quenched by using ammonium chloride solution and extracted with ethyl acetate (2 x 100 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. The crude product was purified by silica flash chromatography (4-6% ethyl acetate in hexanes) to give the desired product as a pale yellow liquid. 3-Bromo-4-ethylpyridine. 1 H NMR(300MHz, CDCl3)δ 8.64(s,1H),8.41(d,J=5.0Hz,1H),7.17(d,J=4.7Hz,1H),2.75(q,J=7.6Hz,2H),1.25(t,J=7.5Hz,3H).

[0138] Step 2: 4-Ethylnicotinaldehyde [ka] To a cooled stirred solution of 3-bromo-4-ethylpyridine (from step 1, 1.0 g, 5.37 mmol) in THF (10 mL) at -78 °C, n-butyllithium solution in hexanes (1.6 M) (4.03 mL, 6.45 mmol) was added. The reaction mixture was stirred at the same temperature for 25 min. Then, dimethylformamide (0.5 mL, 6.45 mmol) was added and the reaction mixture was stirred at room temperature for 15 min. The reaction was quenched by using ammonium chloride solution and extracted with ethyl acetate (2 x 50 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. The crude product was purified by silica flash chromatography (4-6% ethyl acetate in hexanes) to give the desired product 4-ethylnicotinaldehyde as a pale yellow liquid. 1H NMR (300 MHz, CDCl3) δ 10.29 (s, 1H), 8.94 (s, 1H), 8.67 (d, J = 5.2 Hz, 1H), 7.27-7.26 (m, 1H, merged with CDCl3), 3.09 (q, J = 7.3 Hz, 2H), 1.28 (t, J = 7.6 Hz, 3H).

[0139] Steps 3 and 4: 4-(4-ethylpyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl [ka] The title compound was synthesized using general method I (using the aldehyde from step 2, 0.1 g, 0.74 mmol). The crude product was purified by silica flash chromatography (58-60% EtOAc in hexanes) to give the title compound as an off-white solid. LCMS: Rt=0.268 min; MS m / z 333.0 [M+H]+; [Method 4].

[0140] The racemic compound was separated into its enantiomers by chiral HPLC (column: REGIS(S,S)WHELK-01, 250MM x 21.1MM x 5 microns; mobile phase: N-Hexane (A) EtOH:MeOH, 1:1 (B), flow rate: 15ML / min). The absolute stereochemistry of the two enantiomers corresponding to the two product peaks was not determined.

[0141] Peak 1: Chiral HPLC Rt=14.960 min (Column: REGIS(S,S)WHELK-01 (250×4.6 mm×5μ)); Mobile phase: A: n-hexane, B: 0.1% HCOOH in ethanol:methanol (80:20); Flow rate: 1.0 ml / min). LCMS: Rt=1.253 min; MS m / z 332.80 [M+H]+; [Method 1]. 1H NMR(300MHz,DMSO-d6)δ 10.16(brs,1H),8.33(s,1H),8.28(d,J=4.9Hz,1H),7.20(d,J=4.9Hz,1H),5.75(s,1H),5.60(s ,1H),5.02(s,1H),4.90-4.79(m,2H),3.44(s,3H),2.96(q,J=7.6Hz,2H),1.26(t,J=7.5Hz,3H).

[0142] Peak 2: Chiral HPLC Rt=18.565 min (Column: REGIS(S,S)WHELK-01 (250×4.6 mm×5μ)); Mobile phase: A: n-hexane, B: 0.1% HCOOH in ethanol:methanol (80:20); Flow rate: 1.0 ml / min). LCMS: Rt=1.258 min; MS m / z 332.8 [M+H]+; [Method 1]. 1 H NMR(300MHz,DMSO-d6)δ 10.16(brs,1H),8.35(s,1H),8.30(d,J=4.9Hz,1H),7.23(d,J=4.6Hz,1H),5.76(s,1H),5.60(s ,1H),5.03(s,1H),4.93-4.75(m,2H),3.44(s,3H),2.97(q,J=7.6Hz,2H),1.26(t,J=7.5Hz,3H).

[0143] Example 6: (R)-4-(5-fluoro-4-((R)-1-fluoroethyl)pyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate or (R)-4-(5-fluoro-4-((S)-1-fluoroethyl)pyridin-3-yl)-2-(fluoromethyl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate [ka] Step 1: (E)-Ethyl 4-acetoxy-2-((5-fluoro-4-(1-fluoroethyl)pyridin-3-yl)methylene)-3-oxobutanoate [ka] To a stirred solution of ethyl 4-acetoxy-3-oxabutanoate (1.49 g, 9.34 mmol) in benzene (50.00 mL), 5-fluoro-4-(1-fluoroethyl)nicotinaldehyde (from step 4 of Example 1, 1.60 g, 9.34 mmol), piperidinium acetate (0.135 g, 0.934 mmol) were added at room temperature and the reaction mixture was heated to 90° C. for 12 hours. The solvent was removed under reduced pressure to give the crude compound. The crude product was purified by silica flash chromatography (25% ethyl acetate in hexane) to give the title compound as a pale orange syrup. (E)-ethyl 4-acetoxy-2-((5-fluoro-4-(1-fluoroethyl)pyridin-3-yl)methylene)-3-oxobutanoate. LCMS: Rt = 1.03 min and 1.35 min (cis / trans mixture); MS m / z 342.1 [M+H] + ; [Method 2]. 1 H NMR (300MHz, CDCl3) (cis / trans mixture, 1:1)δ 8.46(s,2H),8.26(s,1H),8.21(s,1H),8.11-8.06(m,2H),6.13-5.89(m,2H),5.06(s,2H),4.83(s,2H),4.35(d,J=7.0H) z,2H),4.13(d,J=6.9Hz,2H),2.18(s,3H),2.10(s,3H),1.76-1.61(m,6H),1.37(d,J=7.2Hz,3H),1.04(d,J=7.2Hz,3H).

[0144] Steps 2 and 3: 2'-(acetoxymethyl)-6'-(dimethoxymethyl)-5-fluoro-4-(1-fluoroethyl)-1',4'-dihydro-[3,4'-bipyridine]-3',5'-dicarboxylate 3'-ethyl 5'-methyl [ka] To a stirred solution of (E)-ethyl 4-acetoxy-2-((5-fluoro-4-(1-fluoroethyl)pyridin-3-yl)methylene)-3-oxobutanoate (610.0 mg, 1.79 mmol) in EtOH (8.00 mL) was added (Z)-methyl 3-amino-4,4-dimethoxybut-2-enoate (313.0 mg, 1.79 mmol) at room temperature and the reaction mixture was heated at 90° C. for 16 h. The solvent was removed under reduced pressure to give the crude compound as a brown syrup (0.96 g) which was used directly in the next step.

[0145] To a solution of 3'-ethyl 5'-methyl 2'-(acetoxymethyl)-6'-(dimethoxymethyl)-5-fluoro-4-(1-fluoroethyl)-1',4'-dihydro-[3,4'-bipyridine]-3',5'-dicarboxylate (0.96 g, 1.92 mmol) in MeOH (10.00 mL) was added K2CO3 (0.53 g, 3.85 mmol) and the reaction mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure, the residue was added to water and the product was extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. The crude product was purified by silica flash chromatography (40-80% ethyl acetate in hexanes) to give the desired product, methyl 2-(dimethoxymethyl)-4-(5-fluoro-4-(1-fluoroethyl)pyridin-3-yl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate, as a brown solid. LCMS: Rt = 1.105 min and 1.116 min (diastereomeric mixture); MS m / z 411.1 [M+H] + ; [Method 1]. 1 H NMR (300MHz, CDCl3) (diastereomer mixture, 1:1)δ 8.39-8.22(m,4H),7.44(brs,2H),6.58-6.19(m,2H),6.14(s,1H),6.05(s,1H),5.17(s,1H),5.12(s,1H),4.85 -4.73(m,4H),3.61(s,3H),3.58(s,3H),3.54(s,3H),3.52(s,3H),3.51(s,3H),3.50(s,3H),1.99-1.77(m,6H).

[0146] Step 4: Methyl 4-(5-fluoro-4-(1-fluoroethyl)pyridin-3-yl)-2-formyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate [ka] To a solution of 2-(dimethoxymethyl)-4-(5-fluoro-4-(1-fluoroethyl)pyridin-3-yl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate (from step 6, 410.0 mg, 1.0 mmol) in dioxane (8.00 mL) was added 6N dioxane HCl (2.00 mL) at 5° C. and the reaction mixture was stirred at room temperature for 3 h. The solvent was removed under reduced pressure and diluted with EtOAc. The EtOAc was washed with NaHCO3 solution, brine, dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure to give the title compound, 4-(5-fluoro-4-(1-fluoroethyl)pyridin-3-yl)-2-formyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate. LCMS: Rt = 1.089 min and 1.097 min (diastereomeric mixture); MS m / z 365.1 [M+H] + ; [Method 1]. 1 H NMR (300MHz, CDCl3) (diastereomer mixture, approximately 1:1)δ 10.58(s,1H),10.42(s,1H),8.34-8.25(m,4H),7.87(s,1H),7.84(s,1H),6.51-6.17 (m,2H),5.35(s,1H),5.25(s,1H),4.91-4.77(m,4H),3.70(s,6H),2.04-1.81(m,6H).

[0147] Step 5: 2-(difluoromethyl)-4-(5-fluoro-4-(1-fluoroethyl)pyridin-3-yl)-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate methyl [ka] To a stirred solution of methyl 4-(5-fluoro-4-(1-fluoroethyl)pyridin-3-yl)-2-formyl-5-oxo-1,4,5,7-tetrahydrofuro[3,4-b]pyridine-3-carboxylate (from step 4, 90.0 mg, 0.247 mmol) in DCM (3.00 mL) was added DAST (0.065 mL, 0.49 mmol) at -78 °C and the reaction was stirred at room temperature for 5 h. The reaction was quenched with water at -30 °C and the product was extracted in DCM (3 x 10 mL). The combined organic layers were washed with NaHCO3 solution, brine, dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure to give the crude compound. The rac-diastereomers were separated by preparative HPLC (column: WATERS X BRIDGE (19 x 150 mm), 5.0μ; mobile phase: A = water, B = ACN; flow rate: 15 ml / min) to give rac-diastereomer 1 and rac-diastereomer 2. The absolute stereochemistry of the four stereoisomers corresponding to the four product peaks was not determined. The rac-diastereomer 1 was separated into its enantiomers by chiral HPLC (column: LUX CELLULOSE-4, 250 MM x 21.2 MM x 5 microns; mobile phase: A: Hexanes: B: EtOH; flow rate isocratic: 15 mL / min). The rac-diastereomer 2 was separated into its enantiomers by chiral HPLC (column: CHIRALPAK IJ, 250 MM x 20 MM x 5 microns; mobile phase: A: Hexane: B: EtOH: MeOH: 1:1; flow rate isocratic: 15 mL / min).

[0148] Example 6 is the first eluting peak from rac-diastereoisomer 2. Chiral HPLC Rt=4.486 min (Column: CHIRAL PAK-IJ (150×4.6 mm×5μ)); Mobile phase: A: n-hexane, B: ethanol:methanol (50:50); Flow rate: 1.0 ml / min). LCMS: Rt=0.4231 min; MS m / z 387.1[M+H]+; [Method 2]. 1H NMR(400MHz,CD3OD)δ 8.31-8.30(m,2H),7.50(t,J=53.7Hz,1H),6.43-6.26(m,1H),5.26(s,1H),4.89(d,J=8.8Hz,2H),3.59(s,3H),1.87(dd,J=23.0,6.3Hz,3H) minus 1H.

[0149] Example 6a: Second eluting peak from rac-diastereoisomer 2 Chiral HPLC Rt=5.064 min (Column: CHIRAL PAK-IJ (150×4.6 mm×5μ)); Mobile phase: A: n-hexane, B: ethanol:methanol (50:50); Flow rate: 1.0 ml / min). LCMS: Rt=1.438 min; MS m / z 386.85[M+H]+; [Method 1]. 1 H NMR(400MHz,CD3OD)δ 8.31(d,J=1.0Hz,1H),8.25(d,J=2.8Hz,1H),7.46(t,J=54.4Hz,1H),6.42-6.26(m,1 H),5.23(s,1H),4.79-4.73(m,2H),3.55(s,3H),1.85(dd,J=23.3,6.6Hz,3H) minus 1H.

[0150] Example 6b: First eluting peak from rac-diastereoisomer 1 Chiral HPLC Rt=5.830 min (Column: LUX CELLULOSE-4 (150×4.6 mm×5μ)); Mobile phase: A: n-hexane, B: ethanol:methanol (50:50); Flow rate: 1.0 ml / min). LCMS: Rt=1.125 min; MS m / z 387.1[M+H]+; [Method 1]. 1H NMR(400MHz,CD3OD)δ 8.31(d,J=2.8Hz,1H),8.24(d,J=1.2Hz,1H),7.46(t,J=53.48Hz,1H),6.56-6.40(m,1H ),5.24(s,1H),4.87(d,J=2.1Hz,2H),3.59(s,3H),1.79(dd,J=23.2,6.6Hz,3H) minus 1H.

[0151] Example 6c: Second eluting peak from rac-diastereoisomer 1 Chiral HPLC Rt=6.761 min (Column: LUX CELLULOSE-4 (150×4.6 mm×5μ)); Mobile phase: A: n-hexane, B: ethanol:methanol (50:50); Flow rate: 1.0 ml / min). LCMS: Rt=1.435 min; MS m / z 386.85[M+H]+; [Method 1]. 1 H NMR(400MHz,CD3OD)δ 8.31(d,J=3.0Hz,1H),8.24(d,J=1.2Hz,1H),7.46(t,J=53.6Hz,1H),6.56-6.40(m,1H) ,5.24(s,1H),4.87(d,J=2.1Hz,2H),3.59(s,3H),1.79(dd,J=23.0,6.6Hz,3H) minus 1H.

[0152] Biological Data Many known calcium channel activators are V These molecules not only increase the peak current, but also have additional mechanisms to increase intracellular calcium concentrations, for example, by shifting the voltage sensitivity of the channel to more negative membrane potentials. Figure 1 shows a simulated cardiac action potential from an epicardial environment, with potential (mV) versus time (ms) and a corresponding increase in Ca V1.2 We demonstrate these additional mechanisms by showing the effect of shifting the voltage of activation to more negative membrane potentials. These additional mechanisms may drive or promote cardiovascular effects such as increased blood pressure, changes in heart rate or contractility, and / or arrhythmias due to QT prolongation. For example, the O'Hara-Rudy model can be used to demonstrate the effect of Ca on action potential duration and arrhythmia burden. V The impact of 1.2 adjustments was investigated. It was identified that a hyperpolarization shift in the activation curve of more than 12 mV could potentially cause a QT prolongation and increased risk of arrhythmias of more than 15%. Therefore, minimizing the shift in voltage sensitivity may lead to compounds that reduce the risk of QT prolongation and cardiac arrhythmias.

[0153] The compounds of formula (I) are highly potent Ca antagonists with a biophysical profile that minimizes the cardiovascular risks outlined above. V 1.2 activators. First, the compounds of formula (I) limit their effect on voltage sensitivity by minimizing the hyperpolarization shift to less than 9 mV to mitigate arrhythmia potentials. Second, the compounds of formula (I) inhibit Ca V Third, the compounds of formula (I) increase the peak current of Ca 1.2 by up to 2.5-fold, thereby limiting channel overactivation. Third, the compounds of formula (I) reduce Ca 1.2, a pathophysiological mechanism underlying the cardiac symptoms of Timothy syndrome. V 1.2 Do not delay channel inactivation Additionally, compounds of formula (I) are designed to maximize brain exposure by not exhibiting significant efflux in the brain.

[0154] Ca V 1.2-Generation and maintenance of HEK293(AUX) cell line Monoclonal Ca V 1.2-HEK293(AUX) cell line expresses human Ca V1.2 constitutively expresses alpha 1 (α1) subunit (CACNA1C) and has doxycycline-inducible expression of alpha 2 delta (α2Δ2) auxiliary subunit (CACNA2D2) and beta 2 (β2) auxiliary subunit (CACNB2). To generate the cell line, expression vectors pcDNA5.0 / FRT-TO-CACNA2D2-FCS-P2A-CACNB2 and pCMV6-Entry-CACNA1C were established via gene synthesis and cloning, where pcDNA5.0 / FRT-TO plasmid is from Invitrogen, pCMV6-Entry is from Origene, FCS stands for Furin Cleavage Site, P2A is a peptide self-cleavage sequence derived from porcine teschovirus-1, and FRT is a flippase recognition target site. Next, the parent line Flp-In™293 T-Rex (Invitrogen) was transfected with pcDNA5.0 / FRT-TO-CACNA2D2-FCS-P2A-CACNB2 and flippase vector pOG44 (Invitrogen) to establish targeted integration of the CACNA2D2-FCS-P2A-CACNB2 expression cassette into the pre-engineered FRT site in Flp-In™293 T-Rex. This intermediate cell line was then transfected with pCMV6-Entry-CACNA1C to establish stable CACNA1C expression. Clonal isolation was achieved under neomycin selection. A cell clone (2-19B) with good voltage-dependent barium current (see electrophysiological methods below) was identified as a Ca V 1.2 Active agents were selected for characterization.

[0155] To maintain the cell line, cells were passaged twice a week. At each passage, the growth medium (Table 1) was completely removed and the cells were rinsed successively with 10 mL of D-PBS and 5 mL of warmed TrypLE™ Express Enzyme (Gibco). Both D-PBS and TrypLE™ Express Enzyme were removed immediately after rinsing. The plate was then placed at room temperature for 3-5 minutes. Next, 10 mL of warmed 37°C complete medium was added to rinse the cell growth surface and collect the dissociated cells. Cells were counted and the T175cm 2 2-3 x 10 per flask 6 A cell density of 1000 cells was targeted and seeded into new flasks. [Table 2]

[0156] Ca V 1.2-Ca using HEK293(AUX) cell line and QPatch V 1.2 Electrophysiological characterization of activators 24 h prior to electrophysiology experiments, doxycycline (1 µg / ml) was added to the growth medium (Table 1) and 25 µM verapamil was applied simultaneously to prevent calcium influx from causing cell death. Cell confluency should reach 70%-80% immediately prior to the experiment.

[0157] To harvest cells (for example, T175cm 2 From the flask), the growth medium was completely removed and the cells were rinsed with 10 mL of D-PBS. The D-PBS was aspirated, 10 mL of Detatin (Genlantis) was added, and the plate was placed in a 37 °C incubator for 10 min. The detached cells were placed into a 15 mL conical tube and spun at 1000 rpm for 2 min. The supernatant was removed and the cells were resuspended in QPatch complete medium (Table 2) to obtain the desired cell density of 1.5-3 million cells per QPatch trial. Each experimental trial uses 1.5 mL of cells.

[0158] The cell suspension was then plated on a single-well QPlate with CaV The cells were then acquired on a Sophion QPatch platform using a whole-cell voltage clamp to measure barium currents conducted through 1.2. Extracellular and intracellular patch clamp solutions are listed in Tables 3 and 4, respectively. A dose-response assay protocol was used to measure the peak inward current (Emax) and potency (EC 50 The maximum fold change in EC was determined. The protocol had eight liquid periods. The first liquid period was to stabilize the current amplitude, which was monitored using repeated 200 ms voltage pulses stepwise varying from -80 mV to 0 mV. The second liquid period was to determine the baseline current amplitude in the presence of vehicle control, using a single 20 ms voltage pulse stepwise varying from -80 mV to 0 mV. The third through eighth liquid periods were used to confirm the 6-dose response to compound treatment, also using a single 20 ms voltage pulse stepwise varying from -80 mV to 0 mV. EC 50 was generated using the following formula: I 濃度 =I ベース +(I フル -I ベース )*c n / (XC 50 n +c n ), where c is the concentration and n is the Hill coefficient constant. フル is the maximum achievable current, and I ベース is 0. Using the channel biophysics assay protocol, the current-voltage relationship (IV curve), the half-way channel activation voltage (V 1 / 2 Channel gating properties, including V, V(tau), V(tau) and V(tau) tail current amplitude, were determined. 1 / 2 is the fitting equation G(V)=G V最小 +(G V最大 -G V最小 ) / (1+exp(-(V-V1 / 2) / V 勾配 )) where G represents the conductance and G V最小 is equal to 0, and G V最大is the maximum conductance, and V 勾配 is the slope coefficient. G(V) was pre-calculated for each experimentally applied depolarizing potential (V) and corresponding current amplitude (I(V)) from the formula G(V)=I(V) / (V-0.06), where 0.06 was the experimentally determined reversal potential in volts. The protocol had four liquid periods. The first liquid period was to stabilize the current amplitude, which was monitored using repeated 200 ms voltage pulses stepwise varying from -80 mV to 0 mV. Once the current amplitude had stabilized, a baseline value of tau of inactivation was determined via monoexponential fitting of the inactivation phase of the current trace. The second liquid period was to measure the baseline value constituting the current-voltage relationship in the presence of vehicle control, and the third liquid period was to measure compound effects on the current-voltage relationship. During each of these two drainage periods, cells were subjected to ten 20 ms voltage pulses, each stepping from -80 mV to incremental values ​​ranging from -55 mV to +35 mV (increment size 10 mV). During the fourth drainage period, a 200 ms voltage pulse from -80 mV to 0 mV was again delivered to measure compound effects on inactivating tau. [Table 3] [Table 4] [Table 5]

[0159] Evaluation of compound exposure-cFos induction (PK-PD) relationships in wild-type mice Animal Care and Ethics. All animals were housed under controlled temperature and light cycles (22°C, 12-h light / 12-h dark cycle) with unrestricted access to food and water. All animal experiments were performed in accordance with the institutional guidelines for the care and use of laboratory animals approved by the Institutional Animal Care and Use Committee (IACUC) of Novartis Institutes for BioMedical Research, Inc. (Cambridge, MA, USA).

[0160] Compound administration and brain tissue collection. Wild-type C57BL / 6J male mice were obtained from Jackson laboratories (Bar Harbor, ME). V 1.2 The effects of acute single doses of active agents were evaluated in male 8-week-old mice (n=6 mice per compound). Each compound was dissolved in 10% PEG300, 10% Solutol, 10% Cremophore EL, and 70% phosphate-buffered saline and administered intraperitoneally (ip) at concentrations ranging from 1 mg / kg up to 30 mg / kg depending on the compound. One hour after compound administration, animals were euthanized by exsanguination under deep anesthesia. Blood was collected in EDTA tubes for downstream drug level analysis. Brains were rapidly removed from the skull and the cerebral cortex and cerebellum were regionally dissected. Cerebellar samples were snap frozen in liquid nitrogen to assess compound exposure. Cortical samples for cFos assessment were placed in 500 μL of RNAlater solution (ThermoFisher) to preserve the integrity of the RNA in the samples. Samples were kept in RNAlater at 4° C. for at least 24 hours and then transferred to −80° C. for storage prior to processing.

[0161] Quantification of cFos mRNA induction. Tissue homogenization was performed using the TissueLyser system for 96-well plates (Qiagen). First, frozen cortical samples were thawed, removed from RNAlater, and placed into a TissueLyser tube with Buffer RLT containing 0.5% Reagent DX and one 5 mm TissueLyser metal bead. The TissueLyser tube was loaded into a TissueLyser II tissue homogenizer for three rounds of homogenization, each round lasting 5 min at a bead-beating frequency of 30 Hz. Total RNA was purified from the homogenates using the RNeasy 96 Plus kit (Qiagen), and RNA concentrations and A260 / A280 ratios were quantified by Nanodrop (ThermoFisher), and all samples were normalized to a concentration of 100 ng / μl. RNA was reverse transcribed into cDNA using the Superscript III First-strand synthesis SuperMix Kit (ThermoFisher). For each sample, 6 μl of RNA (600 ng total) was mixed with 1 μL Oligo dT and 1 μL Annealing Buffer and heated to 65° C. for 5 minutes. Then, 10 μL of 2X First-Strand Reaction Mix and 2 μL of Enzyme mix were added to achieve a total reaction volume of 20 μL. Samples were heated to 50° C. for 50 minutes and then to 85° C. for 5 minutes to complete cDNA synthesis.

[0162] Quantitative PCR was performed on the cDNA samples using the Quantitect Multiplex RT-PCR kit (Qiagen) in a 384-well assay format. Each PCR well contained 2 μL of cDNA (60 ng total), 10 μL of RT-PCR master mix, 1 μL of cFos FAM Taqman probe (Mm00487425_m1(FAM) #4351368), 1 μL of GAPDH VIC Taqman probe (Mm99999915-g1(VIC) #4448486), 0.2 μL of Multiplex RT mix, and 5.8 μl of RNase-free water. Samples were heated to 95°C for 15 min, followed by 45 cycles of 94°C for 45 s and 60°C for 45 s in a ViiA7 Real-Time PCR system (ThermoFisher). cFos Ct values ​​were exported, normalized to GAPDH Ct values, and converted to relative fold change in expression using delta-delta Ct relative quantification. cFos fold change between compound treatment and vehicle was analyzed by one-way ANOVA followed by Tukey's post-hoc comparisons.

[0163] Quantification of compound exposure. Cerebellar tissue samples were homogenized (5-fold dilution) in 4 mL of 20% acetonitrile and 80% phosphate buffered saline per gram of tissue. Tissue was homogenized using one of three methods: Handheld probe system, frequency 30 s for 4 min. -1 TissueLyser system using 5 mm steel beads or an OMNI Bead Ruptor Elite homogenizer for 30 s to 1 min depending on tissue type. Tissue samples were added to a 96-well plate (12.5 μL of sample) and processed for quantification by mass spectrometry. result [Table 6] QPatch was performed as a 6-point dose response with 10 individual determinations at each concentration. Only one experimental replicate (n=1) was performed unless stated. *n=2 nd=undecided

[0164] Comparative Example Other Ca V Although 1.2 activators are known, these compounds are not very potent and / or do not possess the desired biophysical properties necessary to activate the channel and have sufficient brain exposure while minimizing cardiovascular risks such as increased blood pressure, changes in heart rate or contractility, and / or arrhythmias due to QT prolongation. [Table 7-1] TIFF2024545200000056.tif169170 [Table 7-2]

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, 【Chemistry 1】 During the ceremony, R １ But C １～６ Alkyl and C １～６ haloalkyl; R ２ is selected from H and halo; R ３ But C １～６ Alkyl, and C ３～８ cycloalkyl, each of which is optionally substituted with 1 to 3 halo, or a pharmaceutically acceptable salt thereof.

2. A compound of formula (I) or a pharmaceutically acceptable salt thereof, 【Chemistry 2】 During the ceremony, R １ But CH ３ , C.F. ３ , CHF ２ and CH ２ selected from F, R ２ is selected from H and F; R ３ But C １～４ A compound selected from alkyl, cyclopropyl and cyclobutyl, each of which is optionally substituted with 1 to 3 F, or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1 of formula (Ia): 【Transformation 3】 or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 1 of formula (Ib): 【Chemistry 4】 or a pharmaceutically acceptable salt thereof.

5. R １ is CH ３ 2. The compound of claim 1, wherein:

6. R １ is CF ３ 2. The compound of claim 1, wherein:

7. R １ is CHF ２ 2. The compound of claim 1, wherein:

8. R １ is CH ２ The compound of claim 1 , wherein:

9. R ２ The compound of claim 1 , wherein is H.

10. R ２ The compound of claim 1 , wherein is F.

11. R ３ C optionally substituted with 1 to 3 F １～４ Alkyl, for example, CHFCH ３ 2. The compound of claim 1, wherein:

12. R ３ The compound of claim 1, wherein is cyclopropyl optionally substituted with 1 to 3 F.

13. R ３ The compound of claim 1, wherein is cyclobutyl optionally substituted with 1 to 3 F.

14. The compound of claim 1 selected from the group consisting of: Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5 or a pharmaceutically acceptable salt thereof.

15. A pharmaceutical composition comprising the compound of any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

16. A compound of formula (I) according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, for use in therapy.

17. 15. A compound of formula (I), or a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 14, for use in the treatment of a neuropsychiatric disorder, such as schizophrenia, bipolar disorder, major depressive disorder, or substance use disorder; a neurodevelopmental disorder, such as attention deficit hyperactivity disorder (ADHD), Phelan-McDermid syndrome, or other autism spectrum disorder; a neurodegenerative disorder, such as multiple sclerosis, frontotemporal dementia, or Alzheimer's disease; or a cardiac condition, such as Brugada syndrome, short QT syndrome, or early repolarization syndrome.

18. 15. A method of treating a neuropsychiatric disorder, such as schizophrenia, bipolar disorder, major depressive disorder, or substance use disorder; a neurodevelopmental disorder, such as ADHD, Phelan-McDermid syndrome, or autism spectrum disorder; a neurodegenerative disorder, such as multiple sclerosis, frontotemporal dementia, or Alzheimer's disease; or a cardiac condition, such as Brugada syndrome, short QT syndrome, or early repolarization syndrome, in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof.

19. 15. A method of treating a neuropsychiatric disorder, such as schizophrenia, bipolar disorder, major depressive disorder, or substance use disorder; a neurodevelopmental disorder, such as ADHD, Phelan-McDermid syndrome, or autism spectrum disorder; a neurodegenerative disorder, such as multiple sclerosis, frontotemporal dementia, or Alzheimer's disease; or a cardiac condition, such as Brugada syndrome, short QT syndrome, or early repolarization syndrome, in a subject in need thereof, said method comprising administering to said subject a compound of any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof.

20. 15. Use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 14 in the manufacture of a medicament for the treatment of a neuropsychiatric disorder, such as schizophrenia, bipolar disorder, major depressive disorder, or substance use disorder; a neurodevelopmental disorder, such as ADHD, Phelan-McDermid syndrome, or autism spectrum disorder; a neurodegenerative disorder, such as multiple sclerosis, frontotemporal dementia, or Alzheimer's disease; or a cardiac condition, such as Brugada syndrome, short QT syndrome, or early repolarization syndrome.

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