Disubstituted pyrimidine compounds for ketohexokinase inhibition

Abstract

Disclosed herein are methods for inhibiting ketohexokinase (KHK) in a biological sample or subject, and methods for treating or preventing a disease or disorder (e.g., a disease or disorder related to KHK dysregulation), comprising administering to the biological sample or subject an effective amount of a compound represented by formula I or a compound in Table A, or a pharmaceutically acceptable salt thereof. JPEG2026514128000048.jpg5955

Description

[Technical Field]

[0001] This disclosure generally relates to novel ketohexokinase (KHK) inhibitor compounds, their pharmaceutically acceptable compositions, and the use of these compounds for the treatment of conditions such as non-alcoholic fatty liver disease (NAFLD), metabolic dysfunction-associated steatohepatitis (MASH), metabolic dysfunction-associated steatohepatitis (MASLD), non-alcoholic steatohepatitis (NASH), hypertriglyceridemia, hypercholesterolemia, type 2 diabetes (T2D), diabetic nephropathy (DKD), alcoholic steatohepatitis (ASH), addictive cravings including sugar or alcohol craving or alcohol use disorder, neurodegenerative diseases such as Parkinson's disease or Alzheimer's disease, hyperuricemia, gout, or cancer. [Background technology]

[0002] KHK (also known as ketohexokinase or fructokinase) catalyzes the first step in fructose metabolism, phosphorylating fructose to fructose-1-phosphate (F1P) and depleting intracellular ATP. Since there is no negative feedback mechanism in which F1P inhibits KHK metabolism of fructose, F1P accumulation is directly related to the amount of fructose either (1) transported into the cell via GLUT transporters, or (2) formed into the cell from glucose via the polyol pathway and metabolized via KHK. F1P accumulation and ATP depletion lead to adverse consequences in cells and tissues, including oxidative stress, osmotic stress, endothelial dysfunction, and metabolic dysregulation. Responses to these stimuli include adipogenesis, hyperuricemia, and gluconeogenesis, which drive metabolic disorders, including metabolic syndromes and their sequelae.

[0003] There are two isoforms of KHK: KHK-A, which is ubiquitously expressed but has low affinity for fructose, and KHK-C, which has a much higher affinity for fructose and is preferentially expressed in the liver, kidneys, brain, and intestines. KHK-C drives the majority of the physiological flux from fructose to F1P, while KHK-A can compensate for the downregulation, inhibition, or deletion of KHK-C, especially as intracellular fructose concentrations increase. Humans with genetic polymorphisms in KHK have a benign phenotype of essential fructosuria and do not accumulate F1P, but a more severe condition, hereditary fructose intolerance, is caused by a polymorphism in the ALDOB gene, which encodes aldolase B. This catalyzes the second step of fructose metabolism, resulting in increased oxidative stress caused by the harmful accumulation of F1P and depletion of the intracellular adenine nucleotide pool.

[0004] Increased intake of fructose and fructose-containing polysaccharides such as sucrose and high-fructose corn syrup is associated with increased metabolic disorders, including obesity, insulin resistance, type 2 diabetes, dyslipidemia, metabolic liver diseases including NASH, and other liver diseases associated with increased hepatocyte stress, including alpha-1 antitrypsin deficiency and hemochromatosis. Targeting this initial rate-limiting step of fructose metabolism, namely phosphorylation by KHK, has been suggested as a promising therapeutic strategy for these metabolic disorders as well as other diseases driven by fructose metabolism, namely cancer, neurodegenerative disorders including Parkinson's disease and Alzheimer's disease, addictive cravings for sugar and alcohol, hyperuricemia, and other complications of insulin resistance, including diabetic retinopathy and diabetic nephropathy.

[0005] Attempts to inhibit KHK have so far stalled in preclinical or clinical development, with only moderate inhibition of fructose uptake and metabolism demonstrated in Phase 1 and Phase 2 clinical trials by PF-06835919, the most advanced compound developed. While this molecule showed potent inhibition of the isolated KHK-C enzyme, its potency was approximately 10-fold in cell-based assays of KHK activity, requiring a dose of 300 mg twice daily in Phase 2 clinical trials. Even at these high doses, only minimal effects on markers of hepatic fat and liver damage were observed. As a carboxylic acid, PF-06835919 is a substrate for organic anion transporter proteins, leading to increased accumulation in the liver compared to other tissues and organs. While KHK-C is preferentially expressed in the liver, it is also highly expressed in the kidneys, intestines, parts of the brain center, and other sites of fructose metabolism to F1P, potentially leading to metabolic dysregulation, endothelial dysfunction, metabolic disorders, or craving disorders. Furthermore, PF-06835919 is a KHK-C bias inhibitor with much weaker inhibition of KHK-A. PF-06835919 is disclosed in US2017 / 0183328A1. See also J.Med.Chem.2020,63,13546-13560. In addition, U.S. Patent No. 11,124,500 discloses a specific disubstituted pyrazole KHK inhibitor compound. Also, Durham et al. J.Med Chem.2023,66,15960-15976 “Identification of LY3522348: A Highly Selective and Orally Efficacious Ketohexokinase Inhibitor” discloses a ketohexokinase inhibitor. While KHK-C drives fructose metabolism in some tissues at physiological fructose concentrations, as fructose concentrations increase (due to diet, metabolic disorders, or inhibition of KHK-C), the contribution of KHK-A metabolism of fructose to F1P becomes more substantial.

[0006] Therefore, there is a significant need to develop a KHK inhibitor that is potent in both enzyme assays and cell-based assays, provides balanced inhibition of KHK-C and KHK-A, minimizes escape flux to F1P via KHK-A or compensation by KHK-A, and is systemically distributed to inhibit KHK-C and KHK-A not only in the liver but also in the kidney, intestine, and all other tissues. SUMMARY OF THE INVENTION

[0007] The present disclosure generally relates to methods of inhibiting ketohexokinase (KHK), methods of treating or preventing a disease or disorder in a subject (e.g., the disease or disorder is secondary to overconsumption of fructose and / or alcohol and involves KHK dysregulation or is related to fructose metabolism), and compounds and compositions that can be used in such methods.

[0008] The present disclosure provides compounds of formula I, and pharmaceutically acceptable salts thereof,

Chemical formula

[0009] A method is further provided for administering a safe and effective amount of a compound disclosed herein, such as one represented by formula I or one of the compounds in Table A, to a biological sample or patient.

[0010] Methods for inhibiting ketohexokinase (KHK) in a biological sample or patient (for example, in cells) by administering an effective amount of a compound disclosed herein, such as one represented by formula I or one of the compounds in Table A, to the biological sample or patient.

[0011] Further provided are methods for treating or preventing a disease or disorder in a subject (for example, a disease or disorder related to KHK dysregulation or fructose metabolism, including secondary ones following excessive intake of fructose and / or alcohol), the method comprising administering to the subject an effective amount of a compound disclosed herein, for example, one represented by formula I or one of the compounds in Table A.

[0012] Pharmaceutical compositions comprising compounds disclosed herein, such as those represented by formula I or the compounds in Table A, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable excipients, carriers, adjuvants, or vehicles are also provided.

[0013] For example, the use of the compounds described herein is also provided for inhibiting ketohexokinase (KHK) in cells and for treating or preventing a disease or disorder in a subject (e.g., a disease or disorder related to KHK dysregulation or fructose metabolism, including secondary effects following excessive intake of fructose and / or alcohol).

[0014] The use of the compounds described herein for the manufacture of pharmaceuticals for inhibiting ketohexokinase (KHK) and for treating or preventing diseases or disorders in a subject (for example, diseases or disorders related to KHK dysregulation or fructose metabolism, including secondary ones following excessive intake of fructose and / or alcohol). [Brief explanation of the drawing]

[0015] [Figure 1] This shows a comparison of the XPRD diffraction patterns of A21 free base (upper trace) and A21 HCl salt (lower trace). [Figure 2] A comparison of the 1H NMR spectra of A21 free base and its HCl salt (DMSO-d6) is shown. [Modes for carrying out the invention]

[0016] For example, compounds and their uses are provided herein for inhibiting ketohexokinase (KHK) in cells and for treating or preventing diseases or disorders in subjects (e.g., diseases or disorders related to KHK dysregulation or fructose metabolism, including secondary ones following excessive intake of fructose and / or alcohol). Also provided are the use of compounds described herein or pharmaceutically acceptable salts thereof, or pharmaceutically acceptable compositions containing such compounds or pharmaceutically acceptable salts thereof, for inhibiting ketohexokinase (KHK) in cells and for treating or preventing diseases or disorders in subjects (e.g., diseases or disorders related to KHK dysregulation).

[0017] Unless otherwise indicated, the structures shown herein also mean that all isomeric forms of the structure (e.g., enantiomers, diastereomers, cis / trans, conformational, and rotational forms) are included. For example, the R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers are included in this disclosure unless only one of the isomers is specifically shown. Thus, single stereochemical isomers of the compounds of the present invention, as well as enantiomers, diastereomers, cis / trans, conformational, and rotational mixtures, are within the scope of this disclosure. In some cases, the compounds disclosed herein are stereoisomers. A “stereoisomer” refers to a compound with one or more stereocenters that differ in chirality. Examples of stereoisomers include enantiomers and diastereomers. The compounds disclosed herein may exist as single stereoisomers or as mixtures of stereoisomers. The stereochemistry of the compounds shown herein is relative, not absolute, unless otherwise stated. As shown herein, a single stereoisomer, diastereomer, or enantiomer refers to a compound that comprises at least 50% of the indicated stereoisomer, diastereomer, or enantiomer, and in some cases, at least 90% or 95% of the indicated stereoisomer, diastereomer, or enantiomer.

[0018] Unless otherwise indicated, all tautomers of the compounds disclosed herein are within the scope of this disclosure.

[0019] Furthermore, unless otherwise indicated, the structures shown herein also mean that they contain different compounds only in the presence of one or more isotopically enriched atoms. For example, the substitution of hydrogen with deuterium or tritium, or 13 C or 14 Apart from the substitution of carbon with 13C-enriched carbon, compounds having the structure of the present invention are within the scope of this disclosure. Such compounds are useful, for example, as analytical tools or probes in biological assays. Such compounds, particularly deuterium analogs, may also be therapeutically useful.

[0020] The compounds of this disclosure are defined herein by their chemical structure and / or chemical name. If a compound is referred to by both its chemical structure and / or chemical name, and there is a conflict between the two, the chemical structure shall determine the identity of the compound.

[0021] compound Compounds of formula I, and pharmaceutically acceptable salts thereof, [ka] During the ceremony, R 1 However, it is either H or OH, R 2 However, C 1-6 Alkyl or C 1-6 It is a haloalkyl, R 3 However, C 1-6 Alkyl or C 1-6 It is a haloalkyl, R 4 However, H, Haro, CN, C 1-6 Alkyl, C 1-6 Alkoxy, or C 3-5 It is a cycloalkyl, A is a 5-membered heteroaryl compound containing 2-3 nitrogen ring atoms. X is combined or C 1-6 It is alkylene-C(O), R 5 However, it is a 4-6 membered heterocycloalkyl having one or two ring nitrogen atoms, and the heterocycloalkyl has one or two C 1-6 It is optionally substituted with alkyl, However, R 4 If H, then R 2 is methyl, and R 3 Compounds, or pharmaceutically acceptable salts thereof, are provided herein, excluding compounds in which both are trifluoromethyl.

[0022] In some cases, A is a five-membered heteroaryl compound containing two nitrogen ring atoms. In some cases, A is a five-membered heteroaryl compound containing three nitrogen ring atoms. In some cases, A is pyrazolyl. In some cases, the compound or salt has the structure of formula Ix: [ka] It holds.

[0023] In some cases, X is a combination. In some cases, X is C 1-6 It is alkylene-C(O). In some cases, X is CH2-C(O). In some cases, XR 5 is C(O)-C 1-6 Alkilen-R 5 That is the case.

[0024] In some cases, R 5 It is a 4-membered heterocycloalkyl having one ring nitrogen atom, and the heterocycloalkyl has one or two C 1-6 It is optionally substituted with an alkyl group. In some cases, R 5 This is a 5-membered heterocycloalkyl having one or two ring nitrogen atoms, and the heterocycloalkyl has one or two C 1-6 It is optionally substituted with an alkyl group. In some cases, R 5 This is a 6-membered heterocycloalkyl having one or two ring nitrogen atoms, and the heterocycloalkyl has one or two C 1-6 It is optionally substituted with an alkyl group. In some cases, R 5 It is a 4-membered heterocycloalkyl having one ring nitrogen atom, and the heterocycloalkyl has one or two C 1-6 It is optionally substituted with an alkyl group. In some cases, R 5 It is a 5-membered heterocycloalkyl having one ring nitrogen atom, and the heterocycloalkyl has one or two C 1-6 It is optionally substituted with an alkyl group. In some cases, R 5It is a 5-membered heterocycloalkyl having two ring nitrogen atoms, and the heterocycloalkyl has one or two C 1-6 It is optionally substituted with an alkyl group. In some cases, R 5 It is a 6-membered heterocycloalkyl having two ring nitrogen atoms, and the heterocycloalkyl has one or two C 1-6 It is optionally substituted with an alkyl group. In some cases, R 5 is azetidinil or piperazinil, and contains one or two C 1-6 It is optionally substituted with an alkyl group. In some cases, R 5 This is azetidinyl, and contains one or two C 1-6 It is optionally substituted with an alkyl group. In some cases, R 5 It is piperazinyl, and one or two C 1-6 It is optionally substituted with an alkyl group. In some cases, R 5 It is a non-substitution. In some cases, R 5 is one C 1-6 It is substituted with alkyl. In some cases, R 5 It is substituted with one methyl group. In some cases, R 5 This is two C 1-6 It is substituted with alkyl.

[0025] In some cases, the compound has the structure of formula Ia or Ib: [ka] It has. In some cases, the compound has the structure of formula Ia: [ka] It has. In some cases, the compound has the structure of Ib: [ka] It holds.

[0026] In some cases, the compound of formula I has the structure of formula II: [ka] It has, in the formula, C A and C B However, it represents a carbon stereocenter with the same or opposite stereochemistry. A If R is a carbon stereocenter, 1 It cannot be H. Therefore, in the case of the compound of formula (II), R 1 It is OH. In some cases, the compound has the structure of formula (IIx): [ka] It has. In some cases, the compound of formula I has the structure of formula IIa or (IIb): [ka] It has. In some cases, C A and C B This represents a carbon stereocenter with the same stereochemistry. In some cases, C A and C B represents a carbon stereocenter with opposite stereochemistry. In some cases, C A This is a carbon in the R configuration, C B This is a carbon in the S configuration. In some cases, C A This is a carbon in the S configuration, C B This is a carbon atom in the R configuration.

[0027] In some cases, R 1 H is H. In some cases, R 1 It is OH.

[0028] In some cases, R 2 C 1-6 It is alkyl. In some cases, R 2 is methyl. In some cases, R 2 C 1-6 It is a haloalkyl. In some cases, R 2 It is a C1 haloalkyl. In some cases, R2 is CHF2 or CF3. In some cases, R 2 is CHF2. In some cases, R 2 is CF3. In some cases, R 2 is methyl, CHF2, or CF3.

[0029] In some cases, R 3 is C 1-6 alkyl. In some cases, R 3 is methyl. In some cases, R 3 is C 1-6 haloalkyl. In some cases, R 3 is C1 haloalkyl. In some cases, R 3 is CHF2 or CF3. In some cases, R 3 is CHF2. In some cases, R 3 is CF3. In some cases, R 3 is methyl, CHF2, or CF3.

[0030] In some cases, R 4 is H. In some cases, R 4 is halo. In some cases, R 4 is F or Cl. In some cases, R 4 is F. In some cases, R 4 is Cl. In some cases, R 4 is C 1-6 alkyl. In some cases, R 4 is methyl or ethyl. In some cases, R 4 is methyl. In some cases, R 3 is CHF2 and R 4 is methyl. In some cases, R 4 is ethyl. In some cases, R 4 is C 1-6 alkoxy. In some cases, R 4 is methoxy. In some cases, R4 C 3-5 It is a cycloalkyl. In some cases, R 4 It is cyclopropyl. In some cases, R 4 These are methyl, ethyl, methoxy, F, Cl, CHF2, CF3, or cyclopropyl.

[0031] In some cases, R 1 is H or hydroxyl, and R 2 is methyl, X is a bond, and R 5 It is a 4-6 membered heterocycloalkyl having one ring nitrogen atom, and the heterocycloalkyl has one C 1-2 It is optionally substituted with an alkyl group. In some cases, X is C 1-6 It is alkylene-C(O), and R 5 is one or two C 1-6 Azetidinyl is optionally substituted with an alkyl group, and R 1 is H or hydroxyl, and R 2 C 1-3 It is alkyl.

[0032] Preferred compounds are R 3 This is CHF2, and R 4 is methyl, R 1 is hydroxyl, R 2 It is a compound in which methyl is present.

[0033] However, "R 4 If H, then R 2 is methyl, R 3 "Excluding compounds that are both trifluoromethyl" is R 2 is methyl, R 3 This means that any individual compound that is both trifluoromethyl and trifluoromethyl is not included in the claims.

[0034] As used herein, the terms "alkyl" or "alkylene" mean saturated straight-chain or branched-chain hydrocarbons. nThe term "alkyl" means that the alkyl group has "n" carbon atoms. For example, C4 alkyl refers to an alkyl group that has four carbon atoms. 1-6 Alkyl refers to alkyl groups having a large number of carbon atoms, encompassing the entire range (i.e., 1 to 6 carbon atoms) and all subgroups (e.g., 1 to 6, 2 to 6, 1 to 5, 2 to 6, 1 to 4, 2 to 5, 1, 2, 3, 4, 5, and 6 carbon atoms). Specific examples include, but are not limited to, methyl, ethyl, isopropyl, n-propyl, sec-butyl, t-butyl, n-pentyl, and n-hexyl. 1-6 The claim term alkylene-C(O) is R via the C(O) portion. 5 It is defined as something that connects.

[0035] As used herein, the terms "halogen" and "halo" mean F, Cl, Br, or I.

[0036] As used herein, the term "haloalkyl" refers to an alkyl group substituted with one or more halogen substituents. For example, a C1-C6 haloalkyl refers to a C1-C6 alkyl group substituted with one or more halogen atoms, e.g., one, two, three, four, five, or six halogen atoms. Non-limiting examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, and trichloromethyl groups. Similarly, a haloalkoxy refers to an alkoxy group substituted with one or more halogen atoms, e.g., one, two, three, four, five, or six halogen atoms.

[0037] As used herein, the term "alkoxy" refers to an -O-alkyl group.

[0038] The term "cycloalkyl" refers to a non-aromatic monocyclic, condensed, bridging, or spirocyclic system in which the ring atom is carbon and can be saturated or have one or more unsaturated units. Cycloalkyls can have 3 to 5 ring carbon atoms. Specific examples include, but are not limited to, cyclopentyl, cyclopropyl, and cyclobutyl. The cycloalkyl ring is unsubstituted or substituted as described herein.

[0039] As used herein, the term “heterocycloalkyl” refers to a non-aromatic monocyclic, condensed, spiro, or bridging ring system that may be saturated or contain one or more unsaturated units, having five to eight ring atoms, where one or more (e.g., one to three, or one, two, or three) ring atoms are heteroatoms selected from N, S, and O. “N-heterocyclic” indicates that at least one of the ring heteroatoms is a nitrogen atom. In some embodiments, the heterocycloalkyl comprises four to six ring members. In some embodiments, the heterocyclic ring comprises four ring members. In some embodiments, the heterocycloalkyl comprises six ring members. Examples of heterocycloalkyls include oxetanyl, azetidinyl, thietanyl, piperidinyl, piperazinyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, morpholino (e.g., including 3-morpholino and 4-morpholino), 2-thiomorpholino, 3-thiomorpholino, 4-thiomorpholino, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, pyrrolidine-2-one, 1-tetrahydropiperazinyl, 2-tetrahydropiperazinyl, 3-tetrahydropiperazinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 1-pip Examples include, but are not limited to, lazolinyl, 3-pyrazolinyl, 4-pyrazolinyl, 5-pyrazolinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2-thiazolidinyl, 3-thiazolidinyl, 4-thiazolidinyl, 1-imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl, 5-imidazolidinyl, dihydrofuranyl, 1,3-dioxolanyl, 1,4-dioxanyl, 1,3-oxathiol, oxathianyl, 1,3-dithianyl, 1,4-oxathiolanyl, 1,4-oxathianyl, 1,4-dithianyl, thiomorpholinyl, tetrahydropyranyl, dihydropyranyl, and 1,3-dihydro-imidazole-2-onyl. The heterocycloalkyl ring may be unsubstituted or substituted as described herein.

[0040] The term "heteroaryl" refers to a five-membered aromatic heterocycle. A heteroaryl group has two or three ring nitrogen (N) heteroatoms. Examples of heteroaryl groups include imidazolyl, pyrazolyl, and triazolyl (e.g., 1H-1,2,3-triazolyl or 4H-1,2,4-triazolyl). Heteroaryl rings may be unsubstituted or substituted as described herein.

[0041] As described herein, the compounds of this disclosure may, at their discretion, be substituted with one or more substituents, as generally shown or as exemplified by the particular classes, subclasses, and species of this disclosure. The phrase “optionally substituted” will be understood to be used interchangeably with the phrase “substituted or unsubstituted.” In general, the term “substituted” refers to replacing one or more hydrogen radicals of a given structure with radicals of a particular substituent, whether preceded by the term “optionally.” Unless otherwise indicated, an optionally substituted group may have substituents at each of its substitutable positions. If two or more positions in a given structure can be substituted with two or more substituents selected from a given group, the substituents may be identical or different at each position.

[0042] Specific compounds intended for this study include those listed in the table below. Compounds exhibiting a specific stereocenter show at least relative stereoisomerism. Compounds with a chiral center that does not exhibit a specific stereoisomerism show a mixture of stereocenters at that chiral center.

[0043] The compounds may be those listed in Table A, or their pharmaceutically acceptable salts. The compounds in Table A were prepared according to the methods described in the Examples section and other methods known to those skilled in the art. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0044] In some cases, the compound is selected from compounds A3, A9, A12, A19, A21, A24, A35, and their pharmaceutically acceptable salts. In some cases, the compound is selected from compounds A3, A24, and their pharmaceutically acceptable salts. In some cases, the compound is selected from compounds A21, A35, and their pharmaceutically acceptable salts. In some cases, the compound is selected from compounds A9, A12, A19, and their pharmaceutically acceptable salts.

[0045] Abbreviation LC / MS = Liquid Chromatography / Mass Spectrometry TFA = Trifluoroacetic acid min = minutes mL = milliliter FA = Formic Acid DTBPF = 1,1'-bis(di-tert-butylphosphinosene)ferrocene Oxon tetrabutylammonium monoper sulfate DMF = Dimethylformamide DBU = 1,8-diazabicyclo[5.4.0]undeca-7-ene BOP = Benzotriazole-1-yloxytris-(dimethylamino)-phosphonium hexafluorophosphate Ts=4-toluenesulfonate ACN and MeCN = Acetonitrile DIEA = N,N-diisopropylethylamine DCM = Dichloromethane TLC = Thin-Layer Calculation HATU = 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolol[4,5-b]pyridinium 3-oxide hexafluorophosphate NMR=nuclear magnetic resonance NMP = 1-methyl-2-pyrrolidone RT=retention time TEA = Triethylamine THF = Tetrahydrofuran TBDMS = tert-butyldimethylsilyl aq = water-based eq=equivalent dppf = 1,1'-ferrocenediyl-bis(diphenylphosphine) DAST = (Diethylamino) sulfur trifluoride FCC = Flash Column Chromatography HPLC = High-Performance Liquid Chromatography Bpin = 4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl Tf = Trifluoromethylsulfonyl PMB = 4-methoxybenzyl Bn = Benzyl LAH = Aluminum Lithium Hydride

[0046] General synthesis methods Specific processes for the preparation of the compounds of this disclosure are provided as further features of this disclosure and are illustrated by the following exemplary reaction schemes. Those skilled in the art will understand that other synthetic routes may be used to synthesize the compounds of the present invention. For a more detailed description of the individual reaction steps, see the Examples section of this specification. Specific starting materials and reagents are shown in the schemes and described herein, but other starting materials and reagents can be readily substituted to provide a variety of derivatives and / or reaction conditions. In addition, many of the compounds prepared by the methods described herein can be further modified from the viewpoint of this disclosure using conventional chemistry well known to those skilled in the art. In particular, it should be noted that compounds prepared according to these schemes can be further modified to provide new examples within the scope of this disclosure. Furthermore, from the detailed descriptions given in the Experiments section, it will be clear that the preparation methods used extend beyond the general procedures described herein. Furthermore, the general principles of organic chemistry are described in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 2005, and "March's Advanced Organic Chemistry: Reactions Mechanisms and Structure," 8th Ed., Ed.: Smith, MB, John Wiley & Sons, New York: 2019, and their entire contents are incorporated herein by reference.

[0047] Starting materials are generally available from commercially available sources such as Merck Sigma-Aldrich Inc. and Enamine Ltd. Aldrich Chemicals (Milwaukee, Wis.), or can be readily prepared using methods known to those skilled in the art (e.g., prepared by methods generally described in Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v.1-19, Wiley, New York (1967-1999 ed.), or Beilsteins Handbuch der organischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin, including Supplements) (also available from the Beilstein online database).

[0048] As a first point of interest, it should be noted that in the preparation of the compounds of this disclosure, some of the preparation methods useful for the preparation of the compounds described herein may require protection of remote functionalities (e.g., primary amines, secondary amines, carboxyls in intermediates). The need for such protection varies depending on the nature of the remote functionalities and the conditions of the preparation method, and can be readily determined by those skilled in the art. The use of such protection / deprotection methods is also applicable to those skilled in the art. For a general description of protecting groups and their use, see TW Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991 and Greene's Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 2006.

[0049] For example, certain compounds contain primary amine or carboxylic acid functional groups that, if left unprotected, may interfere with reactions at other parts of the molecule. Therefore, such functional groups can be protected by suitable protecting groups that can be removed in subsequent steps. Suitable protecting groups for amine and carboxylic acid protection include those commonly used in peptide synthesis (such as Nt-butoxycarbonyl, benzyloxycarbonyl, and 9-fluorenylmethylenoxycarbonyl for amines, and lower alkyl esters or benzyl esters for carboxylic acids), which are generally non-reactive under the reaction conditions described and can typically be removed without chemically altering other functional groups in the formula I compound.

[0050] Compounds of formula I, or salts thereof, can be prepared by various methods known to those skilled in the art. Non-limiting examples of these methods are outlined in the following schemes, preparations, and examples. All substituents are as defined herein unless otherwise indicated. Reagents, solvents, and starting materials are commercially available, known in the literature, or readily available to those skilled in the art. The products of each synthetic procedure can be recovered and isolated by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, grinding, and crystallization.

[0051] Compounds of formula I can be isolated as racemates, enantiomers, or diastereomers using well-known techniques such as crystallization, chiral chromatography, or supercritical fluid chromatography. These techniques can be applied at appropriate points in the synthesis. Diastereomers / enantiomers of compounds of formula I can be prepared by racemic mixtures followed by appropriate chiral separation as described herein, or by reaction with, for example, a desired chiral-substituted azetidine compound.

[0052] Those skilled in the art will understand that compounds of formula I or salts thereof can also be synthesized by following methods similar to those described herein, for example, by modifying a) the use of appropriate protection / deprotection strategies, b) the use of appropriately substituted starting materials and reactants, c) appropriate changes in the order of the synthesis steps, d) the conversion of one functional group to another, or e) the use of alternative stereochemical configurations.

[0053] Scheme 1: [ka] Scheme 1 provides a general strategy for the preparation of compounds of formula I. LG is defined as a “leaving group” used in specific reactions for the synthesis of compounds of formula I. Route A shows the conversion of pyrimidine 1 to pyrazole compound 3 by Suzuki coupling between boronate 2 and LG2 of 1. Nucleophilic aromatic substitution (SN) between compound 3 (LG1) and azetidine 4. Ar The reaction yields the compound of formula I. Route B is the SN between LG1 of 1 and azetidine 4. Ar An alternative strategy is presented which converts pyrimidine 1 to azetidine compound 5 by reaction. Subsequent Suzuki coupling of boronate 2 and 5 (LG2) yields the compound of formula I. The synthesis of compounds following these strategies utilizes appropriately substituted reactants, appropriate protection / deprotection steps, and other functional group transformations as needed.

[0054] Scheme 2: [ka] Scheme 2 presents a strategy for synthesizing a subset of compounds of formula I, namely compounds of formulas Ia and Ib. PG is defined as a nitrogen protecting group, e.g., BOC, which can be removed at an appropriate moment during synthesis or at the end.

[0055] In step C, dichloropyrimidine 1A is subjected to the Suzuki reaction with boronate 2B to obtain pyrazole compound 6. This reaction is carried out at high temperature in the presence of a palladium catalyst such as tetrakis(triphenylphosphine)palladium(0) and in an organic solvent, such as a base in 1,4-dioxane, such as Na2CO3. Compound 6 is then subjected to the SN reaction with azetidine 4 in step D. Ar The reaction yields compound 7. Typical reaction conditions include a base in an organic solvent such as NMP at high temperature, e.g., K2CO3. In step E, compound 7 is deprotected, for example, by removing the BOC group using TFA to obtain the compound of formula (1b).

[0056] Compound 1A also undergoes a Suzuki reaction with boronate 2A in step F to obtain pyrazole 8. The reaction conditions are the same as in step C, using DTBPF PdCl2 as the palladium catalyst. Compound 8 is then subjected to an SN reaction with azetidine 4 in step G. Ar The compounds react to obtain compound 9. Typical reaction conditions include a base in an organic solvent such as THF at high temperature, e.g., triethylamine. In step H, compound 9 is deprotected, for example, by removing the BOC group using TFA to obtain compound 10, which is then methylated in step I to obtain the compound of formula (1a). Typical reaction conditions include the use of formaldehyde in an organic solvent such as dichloromethane at room temperature and a reducing agent, e.g., NaBH(OAc)3.

[0057] Scheme 3: [ka] Scheme 3 presents an alternative strategy for synthesizing a subset of compounds of formula I, namely compounds of formulas Ia and Ib. PG is defined as a nitrogen protecting group, e.g., BOC, which can be removed at an appropriate moment during synthesis or at the end.

[0058] In step J, chloropyrimidine 1B is reacted with boronate 2B in a Suzuki reaction under conditions similar to those in step C of scheme 2 to obtain pyrazole compound 11. Compound 11 is then oxidized in step K to obtain sulfone 12. Typical reaction conditions include the use of an oxidizing agent in an organic solvent such as dimethylformamide at room temperature, e.g., oxone. In step L, compound 12 is reacted with azetidine 4 in an SN reaction. Ar The reaction yields compound 7. Typical reaction conditions include a base in an organic solvent such as NMP at high temperature, for example, triethylamine. In step E, compound 7 is deprotected as described in scheme 2 to provide the compound of formula (1b).

[0059] Compound 1B is also subjected to a Suzuki reaction with boronate 2A in step M to obtain pyrazole 13. The reaction conditions are the same as those in step C of scheme 2, using DTBPF PdCl2 as the palladium catalyst. Compound 13 is then oxidized in step K (described above) to obtain sulfone 14. In step N, using the same conditions as in step L, compound 14 is then subjected to an SN reaction with azetidine 4. Ar The reaction is carried out to obtain compound 9, which is converted to the compound of formula (1a) as shown in scheme 2.

[0060] Scheme 4: [ka] Scheme 4 presents a further strategy for synthesizing a subset of compounds of formula I, i.e., compounds of formula Ia. PG is defined as a nitrogen protecting group, e.g., BOC, which can be removed at an appropriate moment during synthesis or at the end. Tf is defined as a trifluoromethylsulfonate, providing -OTf as a reactive group that can be used in Suzuki coupling.

[0061] In step O, dichloropyrimidine 1A is hydrolyzed at high temperature using sodium hydroxide in an aqueous THF solution to obtain compound 15. In step P, compound 15 is converted to azetidine 4 and SN. ArThe reaction yields compound 16. Typical reaction conditions include a base in an organic solvent such as acetonitrile at high temperature using microwaves, e.g., diisopropylethylamine. Compound 16 is then converted to triflate 17 in step Q using N-phenyltrifluoromethanesulfonimide in the presence of a base in an organic solvent such as dimethylformamide at room temperature, e.g., diisopropylethylamine. In step R, triflate 17 undergoes a Suzuki reaction with boronate 2A to obtain compound 9. This reaction is carried out at high temperature in the presence of a palladium catalyst such as Pd(dppf)Cl2, using an organic solvent such as 1,4-dioxane with a base, e.g., K3PO4. Compound 9 is then converted to the compound of formula (1a) as described in scheme 2.

[0062] As can be understood by those skilled in the art, the aforementioned synthesis schemes and representative examples (as described herein) are not intended to include a comprehensive list of all means by which the compounds described and claimed in this application may be synthesized. Further methods will be obvious to those skilled in the art. Furthermore, the various synthesis steps described herein may be carried out in alternative or sequential order to obtain the desired compounds. This disclosure further encompasses “intermediate” compounds, including structures produced from the described synthesis procedures, whether isolated or generated in situ and not isolated, before finally obtaining the desired compounds. These intermediates are within the scope of this disclosure. Exemplary embodiments of such intermediate compounds are described in the examples herein.

[0063] Pharmaceutically acceptable salts The compounds described herein may exist in free form or, where appropriate, as salts. Those pharmaceutically acceptable salts are of particular interest because they are useful for administering the compounds described herein for medical purposes. Unpharmaceutically acceptable salts are useful in manufacturing processes for isolation and purification purposes, and, in some examples, for the separation of stereoisomers of the compounds or intermediates thereof.

[0064] As used herein, the term “pharmaceutically acceptable salt” means a salt of a compound that, within the bounds of sound medical judgment, is suitable for use in contact with human and lower animal tissues without excessive side effects such as toxicity, irritation, or allergic reactions, and that is commensurate with a reasonable benefit-to-risk ratio.

[0065] Pharmacopoeia-acceptable salts are well known in the art. For example, SMBerge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Examples of pharmaceutically acceptable salts of the compounds described herein include those derived from suitable inorganic and organic acids and bases. These salts can be prepared in situ during the final isolation and purification of the compounds.

[0066] If the compounds described herein contain a basic group or a sufficiently basic bioisostea, the acid addition salt can be prepared by 1) reacting the purified compound in its free basic form with a suitable organic or inorganic acid, and 2) isolating the salt thus formed. In practice, the acid addition salt may be a more convenient form for use and for the amount of salt used for the free basic form.

[0067] Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed by using inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipine, alginate, ascorbate, aspartic acid, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, glycolate, gluconate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, and 2-hydroxyethanesulfate. Examples include nitrates, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malons, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, palmoates, pectins, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, pyroglutamates, salicylates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, and valersates.

[0068] Other acids and bases, while not pharmaceutically acceptable in themselves, may be used in the preparation of salts that are useful as intermediates for obtaining the compounds described herein and their pharmaceutically acceptable acid or base addition salts.

[0069] It should be understood that the compounds disclosed herein may exist as mixtures / combinations of different pharmaceutically acceptable salts. Mixtures / combinations of the free form of the compounds with pharmaceutically acceptable salts are also intended.

[0070] Prodrug This disclosure also includes prodrugs and / or pharmaceutically acceptable salts thereof of compounds of formula I (or any of its embodiments described herein). The term prodrug is intended to represent a covalently bonded carrier that can release the active ingredient of formula I (or any of its embodiments described herein) when administered to a mammalian subject. The release of the active ingredient occurs in vivo. Prodrugs can be prepared by techniques known to those skilled in the art. These techniques generally involve modifying a suitable functional group in a given compound. However, these modified functional groups regenerate the original functional group in vivo or by routine operations. Prodrugs of compounds of formula I (or any of their embodiments described herein) include compounds modified with hydroxy, amino, carboxylic acid, or similar groups. Examples of prodrugs include, but are not limited to, esters (e.g., acetates, formates, and benzoate derivatives), carbamates of the hydroxy or amino functional group in the compound of formula I (e.g., N,N-dimethylaminocarbonyl), and amides (e.g., trifluoroacetylamino, acetylamino, etc.). Prodrugs of the compound of formula I (or any of its embodiments described herein) and / or pharmaceutically acceptable salts thereof are also within the scope of this disclosure.

[0071] metabolite Furthermore, the scope of this disclosure also includes metabolites of the compounds of formula I, i.e., compounds formed in vivo upon administration of the drug. Some examples of metabolites in accordance with this disclosure include: (i) If the compound of formula I contains a methyl group, its hydroxymethyl derivative (-CH3->CH2OH) (ii) If the compound of formula I contains an alkoxy group, its hydroxy derivative (-OR->OH) (iii) Compounds of formula I containing a tertiary amino group, and their secondary amino derivatives (-NRR->NHR or -NRH), (iv) If the compound of formula I contains a secondary amino group, its primary derivative (-NHR->NH2) (v) If the compound of formula I contains a phenyl moiety, its phenol derivative (-Ph->PhOH), and (vi) If the compound of formula I contains an amide group, its carboxylic acid derivative (-CONH2->COOH).

[0072] Hydrates and solvates The compounds described herein include hydrates and solvates of the compounds, or pharmaceutically acceptable salts thereof. The term solvate is used herein to describe molecular complexes comprising the compounds of this disclosure with one or more pharmaceutically acceptable solvent molecules, such as ethanol. Such solvent molecules are commonly used in the pharmaceutical field and are known to be harmless to recipients, such as water and ethanol. Other solvents, such as methanol, methyl t-butyl ether, ethyl acetate, methyl acetate, (S)-propylene glycol, (R)-propylene glycol, and 1,4-butynediol, may be used as intermediate solvates in the preparation of more desirable solvates.

[0073] The term hydrate is used when the solvent is water. Pharmaceutically acceptable solvates include hydrates and other solvates, and the crystallization solvent may be isotopically substituted, e.g., D2O, d-acetone, d-DMSO. Solvates and / or hydrates preferably exist in crystalline form. The classification system for organic hydrates defines isolation site, channel, or metal ion coordinated hydrates—see Polymorphism in Pharmaceutical Solids by KRMorris (Ed. HGBrittain, Marcel Dekker, 1995). Isolation site hydrates are isolated via organic molecules so that water molecules do not directly contact each other. In channel hydrates, water molecules are in lattice channels and adjacent to other water molecules. In metal ion coordinated hydrates, water molecules are bound to a metal ion.

[0074] The scope of this disclosure also includes multicomponent complexes (excluding salts and solvates) in which the drug and at least one other component exist in stoichiometric or non-stoichiometric amounts. The compounds of this disclosure may also exist as complexes such as inclusion compounds and drug-host-containing complexes, in which, in contrast to the solvates described above, the drug and host exist in stoichiometric or non-stoichiometric amounts. The disclosure also includes drug complexes containing two or more organic and / or inorganic components, which may be in stoichiometric or non-stoichiometric amounts. The resulting complexes may be ionized, partially ionized, or non-ionized. For an overview of such complexes, see Haleblian, J Pharm Sci, 64(8), 1269-1288 (August 1975).

[0075] The compounds of this disclosure may be used in any of the aforementioned modes of administration in combination with cyclodextrins and their suitable derivatives, or soluble macromolecular compounds such as polyethylene glycol-containing polymers, to improve their pharmacokinetic profiles, solubility, dissolution rate, taste masking, bioavailability, and / or stability. For example, drug-cyclodextrin complexes are generally found to be useful in most dosage forms and routes of administration. Both inclusive and non-inclusive complexes may be used. As an alternative to direct complex formation with drugs, cyclodextrins may be used as auxiliary additives, i.e., carriers, diluents, or solubilizers. Alpha-, beta-, and gamma-cyclodextrins are most commonly used for these purposes.

[0076] polymorph This disclosure also includes polymorphic forms (amorphous and crystalline).

[0077] The compounds of this disclosure may exist on a continuum of solid states ranging from completely amorphous to completely crystalline. The term "amorphous" refers to a state in which a material lacks long-range order at the molecular level and may exhibit solid or liquid physical properties depending on the temperature. Typically, such materials do not give a distinctive X-ray diffraction pattern and exhibit solid properties, but are more formally described as liquids. Upon heating, a change from solid to liquid properties occurs, typically characterized by a phase change ("glass transition"), usually a second-order change. The term "crystalline" refers to a solid phase in which a material has an internal structure that is regularly ordered at the molecular level and gives a distinctive X-ray diffraction pattern with defined peaks. When sufficiently heated, such materials also exhibit liquid properties, but the change from solid to liquid is characterized by a phase change ("melting point"), usually a first-order change.

[0078] Certain compounds or combinations of the present disclosure may exist in two or more crystalline forms (generally referred to as “polymorphs”). Polymorphs may be prepared by crystallization under various conditions, for example, by crystallization at different temperatures using different solvents or mixtures of different solvents for recrystallization, and / or by various cooling modes during crystallization, from very rapid to very slow cooling. Polymorphs may also be obtained by heating or melting the compounds of the present disclosure and then gradually or rapidly cooling them. The presence of polymorphs may be determined by solid-state probe NMR spectroscopy, IR spectroscopy, differential scanning calorimetry, powder X-ray diffraction, or other such techniques.

[0079] Pharmaceutical composition The compounds described herein can be formulated into pharmaceutical compositions further comprising a pharmaceutically acceptable carrier, diluent, adjuvant, or vehicle. In embodiments, this disclosure relates to a pharmaceutical composition comprising a compound described herein or a salt thereof, and a pharmaceutically acceptable carrier, diluent, adjuvant, or vehicle. In embodiments, the pharmaceutical composition comprises a safe and effective amount of a compound disclosed herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, adjuvant, or vehicle. Examples of pharmaceutically acceptable carriers include pharmaceutically acceptable diluents, excipients, or carriers that are suitably selected with respect to the intended dosage form and consistent with conventional pharmaceutical practice.

[0080] The term "effective dose" includes "therapeutically effective dose" and "preventive effective dose." The term "therapeutically effective dose" refers to the amount effective in treating and / or improving a disease or disorder associated with KHK dysregulation in a patient. The term "preventive effective dose" refers to the amount effective in preventing and / or substantially reducing the likelihood of developing a disease or disorder associated with KHK dysregulation.

[0081] A pharmaceutically acceptable carrier may contain inactive components that do not excessively inhibit the biological activity of the compound. A pharmaceutically acceptable carrier must be biocompatible, for example, non-toxic, non-inflammatory, non-immunogenic, or free from other undesirable reactions or side effects upon administration to the subject. Standard pharmaceutical formulation techniques can be used.

[0082] The pharmaceutically acceptable carriers, adjuvants, or vehicles used herein include any solvent, diluent, or other liquid vehicle suitable for the desired specific dosage form, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, and the like. Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in the formulation of pharmaceutically acceptable compositions and known techniques for their preparation. Any use of any conventional carrier medium is intended to be within the scope of this disclosure, provided that it is incompatible with the compounds described herein, for example, by producing any undesirable biological effect or otherwise interacting in a detrimental manner with any other component of the pharmaceutically acceptable composition. Where used herein, the term “adverse effect” encompasses any undesirable or harmful effect of a therapy (e.g., a prophylactic or therapeutic agent). Adverse effects are not always undesirable, but undesirable effects are not necessarily harmful. Side effects from therapies (e.g., prophylactic or therapeutic drugs) may be harmful, unpleasant, or dangerous. Side effects include, but are not limited to, fever, chills, lethargy, gastrointestinal toxicity (including gastric and intestinal ulcers and erosions), nausea, vomiting, neurotoxicity, nephrotoxicity, nephrotoxicity (including conditions such as papillary necrosis and chronic interstitial nephritis), hepatotoxicity (including elevated serum liver enzyme levels), myelotoxicity (including leukopenia, myelosuppression, thrombocytopenia and anemia), dry mouth, metallic taste, prolonged pregnancy, weakness, sleep, pain (including muscle pain, bone pain and headache), alopecia, asthenia, dizziness, extrapyramidal symptoms, sitting incapacitation, cardiovascular disorders, and sexual dysfunction.

[0083] Some examples of materials that can function as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffers (such as tween® 80, phosphates, glycine, sorbic acid, or potassium sorbate), saturated vegetable fatty acids, water, salts, or partial glyceride mixtures of electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, methylcellulose, hydroxypropyl methylcellulose, wool fats, sugars such as lactose, glucose, and sucrose, corn starch, and potato starch. Ingredients such as starch, cellulose and its derivatives including sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate, excipients such as tragacanth powder, malt, gelatin, talc, cocoa butter, and suppository wax, oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil, esters such as glycol, propyl glycol, and polyethylene glycol, ethyl oleate, and laurate, buffers such as agar, magnesium hydroxide, and aluminum hydroxide, alginic acid, non-pyrogenic water, isotonic saline, Ringer's solution, ethyl alcohol, and phosphate buffer, as well as other non-toxic, compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and colorants, release agents, coating agents, sweeteners, flavoring agents, and fragrances, preservatives, and antioxidants may also be present in the composition, depending on the judgment of the ingredients.

[0084] In some embodiments, the pharmaceutical compositions disclosed herein can be formulated together with auxiliary active ingredients.

[0085] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, maintaining the required particle size in the case of a dispersion, and by the use of a surfactant. Prevention of microbial action in the compositions disclosed herein is achieved by adding antimicrobial and / or antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it would be preferable to include an isotonic agent, such as sugar or sodium chloride. Long-term absorption of the injectable composition can be achieved by the use of absorption-delaying agents, such as aluminum monostearate and gelatin in the composition.

[0086] In some embodiments, the pharmaceutical composition may be contained within a matrix that controls the release of the composition. In some embodiments, the matrix may contain lipids, polyvinyl alcohol, polyvinyl acetate, polycaprolactone, poly(glycol) acid, poly(lactic) acid, polycaprolactone, polylactic acid, polyane anhydride, polylactide-coglycolide, polyamino acids, polyethylene oxide, acrylic-terminated polyethylene oxide, polyamide, polyethylene, polyacrylonitrile, polyphosphazene, poly(orthoester), sucrose isobutyrate acetate (SAIB), and combinations thereof. Other polymers (each of which is expressly incorporated herein by reference in whole) may also be included, such as those disclosed in U.S. Patents No. 6,667,371, 6,613,355, 6,596,296, 6,413,536, 5,968,543, 4,079,038, 4,093,709, 4,131,648, 4,138,344, 4,180,646, 4,304,767, and 4,946,931. In these embodiments, the matrix continuously releases the drug.

[0087] Pharmaceutically acceptable carriers and / or diluents may also include any solvent, dispersion medium, coating agent, antimicrobial and / or antifungal agent, isotonic agent, and absorption retarder, etc. The use of such media and agents with pharmaceutically active substances is well known to those skilled in the art. Any conventional media or agent is intended for use in a pharmaceutical composition unless it is incompatible with the active ingredient.

[0088] In some embodiments, the pharmaceutical composition is in the form of an aqueous suspension that can be prepared from a solution or suspension. With respect to a solution or suspension, the dosage form may consist of micelles, liposomes (phospholipid vesicles / membranes) of lipophilic substances, and / or fatty acids (e.g., palmitic acid). In certain embodiments, the pharmaceutical composition is a solution or suspension that can dissolve in a fluid secreted by the mucous membrane of the epithelium of the tissue to which it is administered, applied, and / or delivered, and can be advantageously enhanced in absorption.

[0089] Pharmaceutical compositions may be aqueous solutions, non-aqueous solutions, or combinations of aqueous and non-aqueous solutions. Preferred aqueous solutions include, but are not limited to, aqueous gels, aqueous suspensions, aqueous microparticle suspensions, aqueous microparticle dispersions, aqueous liposome dispersions, aqueous micelles of liposomes, aqueous microemulsions, and any combination of the above, or any other aqueous solutions that can be dissolved in fluids secreted by the mucous membrane of the nasal cavity. Exemplary non-aqueous solutions include, but are not limited to, non-aqueous gels, non-aqueous suspensions, non-aqueous microparticle suspensions, non-aqueous microparticle dispersions, non-aqueous liposome dispersions, non-aqueous emulsions, non-aqueous microemulsions, and any combination of the above, or any other non-aqueous solutions that can be dissolved or mixed in fluids secreted by the mucous membrane.

[0090] Examples of powder formulations include, but are not limited to, simple powder mixtures, micronized powders, lyophilized powders, powdered microparticles, coated powdered microparticles, liposome dispersions, and any combination thereof. Powdered microparticles can be formed from a variety of polysaccharides and celluloses, including, but not limited to, starch, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, polyvinyl alcohol alginate, acacia, chitosan, and any combination thereof.

[0091] The pharmaceutical composition may also optionally contain absorption enhancers, such as agents that inhibit enzyme activity, reduce mucous viscosity or elasticity, decrease the mycocele clearance effect, open tight junctions, and / or solubilize active compounds. Chemical enhancers are known in the art and include chelating agents (e.g., EDTA), fatty acids, bile salts, surfactants, and / or preservatives. Penetration enhancers can be particularly useful when formulating compounds that have low membrane permeability, are non-lipophilic, and / or are degraded by aminopeptidases. The concentration of absorption enhancers in the pharmaceutical composition will vary depending on the selected agent and formulation.

[0092] To extend the shelf life, preservatives can be optionally added to the pharmaceutical composition. Suitable preservatives include, but are not limited to, benzyl alcohol, parabens, thimerosal, chlorobutanol, and benzalkonium chloride, as well as combinations thereof. The concentration of the preservative varies depending on the preservative used, the compound being formulated, and the formulation. In typical embodiments, the preservative is present in an amount of about 2% by weight or less.

[0093] Alternatively, the composition may include flavoring agents, for example, to enhance the taste and / or acceptability of the composition to the subject.

[0094] Route of administration and dosage The compounds and pharmaceutically acceptable compositions described herein may be administered to humans and other animals orally, rectally, parenterally, intracisionally, intravaginally, intraperitoneally, topically (by powder, ointment, or drops), or intranasally. In some embodiments, the compounds or compositions disclosed herein are administered orally, by inhalation, or intravenously.

[0095] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid dosage form may include, for example, water or other solvents, inert diluents commonly used in the art, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, fatty acid esters of polyethylene glycol and sorbitan, and mixtures thereof. In addition to inert diluents, the oral composition may also include adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrance agents.

[0096] Injectable preparations, such as sterile injectable aqueous or oily suspensions, can be formulated according to known techniques using suitable dispersants or wetting agents and suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, or emulsions, such as solutions in non-toxic, parenterally acceptable diluents or solvents, for example, in 1,3-butanediol. Acceptable vehicles and solvents that may be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. In addition, sterile fixatives have traditionally been used as solvents or suspension media. For this purpose, any mild fixative containing synthetic mono- or diglycerides can be used. Furthermore, fatty acids, such as oleic acid, are used in the preparation of injectable preparations.

[0097] Injectable formulations can be sterilized, for example, by filtration using a bacterial-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0098] To extend the effects of the compounds described herein, it is often desirable to delay the absorption of the compounds from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous material with low water solubility. The absorption rate of the compound then depends on its dissolution rate, which may depend on the crystal size and crystalline form. Alternatively, delayed absorption of parenterally administered compound forms is achieved by dissolving or suspending the compound in an oily vehicle. Injectable depot formulations are prepared by forming a microencapsulated matrix of the compound in a biodegradable polymer such as polylactide-polyglycolide. The compound release rate can be controlled depending on the compound-to-polymer ratio and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoester) and poly(anhydrous). Depot injectable formulations are also prepared by encapsulating the compound in liposomes or microemulsions compatible with body tissues.

[0099] The compositions for rectal or vaginal administration are specifically suppositories that can be prepared by mixing the compounds described herein with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol, or suppository wax, which is solid at ambient temperature but liquid at body temperature and therefore dissolves in the rectal or vaginal cavity, releasing the active compound.

[0100] Solid dosage forms for oral administration include oral films, capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier, e.g., sodium citrate or dicalcium phosphate, and / or a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) wetting agents such as glycerol; d) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarders such as paraffin; f) absorption enhancers such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl acid, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain a buffer. In the case of oral films, the film may use a water-soluble polymer that allows the film to rapidly hydrate, adhere, and dissolve when placed on the tongue or in the oral cavity, resulting in systemic drug delivery.

[0101] Similar types of solid compositions can also be used as fillers in soft and rigid gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycol, etc. Solid dosage forms of tablets, sugar-coated tablets, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical field. They may optionally contain opacifiers and may be compositions that release only the active ingredient, or preferably, optionally, compositions that release in a delayed manner to a specific part of the intestinal tract. Examples of embedding compositions that can be used include polymers and waxes. Similar types of solid compositions can also be used as fillers in soft and rigid gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycol, etc.

[0102] The active compound may also be in a microencapsulated form with one or more excipients, as described herein. Solid dosage forms of tablets, sugar-coated tablets, capsules, pills, and granules may be prepared with coatings and shells such as enteric coatings, controlled-release coatings, and other coatings well known in the pharmaceutical field. In such solid dosage forms, the active compound may be mixed with at least one inert diluent, such as sucrose, lactose, or starch. Such dosage forms may also, as is common practice, contain additional substances other than the inert diluent, e.g., tableting lubricants and other tableting aids, e.g., magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage form may also contain buffers. These may optionally contain opacifiers and may be compositions that release only the active ingredient, or preferably, optionally, compositions that release in a delayed manner to a specific portion of the intestinal tract. Examples of embedding compositions that can be used include polymers and waxes.

[0103] Dosage forms for topical or transdermal administration of the compounds described herein include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and, if necessary, any required preservatives or buffers. Furthermore, this disclosure envisions the use of transdermal patches, which have the additional advantage of providing controlled delivery of the compound to the body. Such dosage forms can be prepared by dissolving or distributing the compound in a suitable medium. Absorption enhancers can also be used to increase the flow of the compound across the skin. The rate can be controlled either by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel.

[0104] The sterile injectable forms of the compositions described herein may be aqueous or oily suspensions. These suspensions may be formulated according to the art known techniques using suitable dispersants or wetting agents and suspending agents. The sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic, parenterally acceptable diluents or solvents, for example, solutions in 1,3-butanediol. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixative oils have traditionally been used as solvents or suspension media. For this purpose, any mild fixative oil, including synthetic mono- or di-glycerides, may be used. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, and naturally pharmaceutically acceptable oils such as olive oil or castor oil, particularly their polyoxyethylated versions, are useful in the preparation of injectables. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants such as carboxymethylcellulose, or similar dispersants commonly used in pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tweens®, Span, and other emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solids, liquids, or other dosage forms, may also be used for formulation purposes.

[0105] The pharmaceutical compositions described herein may be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions, or solutions. For tablets for oral use, commonly used carriers include, but are not limited to, lactose and corn starch. Lubricants such as magnesium stearate are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When an aqueous suspension is required for oral use, the active ingredient is combined with an emulsifier and a suspending agent. Specific sweeteners, flavorings, or colorants may be added as needed.

[0106] Alternatively, the pharmaceutical compositions described herein may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the drug with a suitable, non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore melts in the rectum to release the drug. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycol.

[0107] Topical application for the lower bowel can be performed using rectal suppositories (as described herein) or suitable enema formulations. Topical application also includes the use of transdermal patches.

[0108] For topical application, the pharmaceutical composition may be formulated in a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Suitable carriers for topical administration of the compounds of this disclosure include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying waxes, and water. Alternatively, the pharmaceutical composition may be formulated in a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0109] For ophthalmic use, the pharmaceutical composition may be formulated as a finely powdered suspension in isotonic, pH-adjusted sterile saline, or more specifically, as a solution in isotonic, pH-adjusted sterile saline, with or without preservatives such as benzalkonium chloride. Alternatively, for ophthalmic use, the pharmaceutical composition may be formulated in an ointment such as petrolatum.

[0110] Pharmaceutical compositions may also be administered by nasal aerosol or inhalation. Such compositions may be prepared in accordance with well-known techniques in the field of pharmaceutical formulations and may be prepared as a solution in physiological saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons and / or other conventional solubilizers or dispersants.

[0111] Compounds for use in the methods of this disclosure can be formulated in unit dosage forms. The term "unit dosage form" refers to a physically distinct unit suitable as a unit dose for the subject being treated, each unit containing a predetermined amount of the active substance calculated to produce a desired therapeutic effect and optionally associated with a suitable pharmaceutical carrier. A unit dosage form may be a single daily dose or one of several daily doses (e.g., about 1 to 4 times or more per day). If multiple daily doses are used, the unit dosage forms may be the same or different for each dose.

[0112] Treatment methods The use of the compounds described herein as therapeutic agents is provided herein. The compounds described herein or pharmaceutically acceptable salts thereof can be used in biological samples (e.g., cell cultures) or humans (e.g., subjects) to inhibit ketohexokinase (KHK) and to treat or prevent diseases or disorders (e.g., diseases or disorders related to KHK dysregulation or fructose metabolism, including secondary diseases following excessive intake of fructose and / or alcohol). The compounds described herein or pharmaceutically acceptable salts thereof can be used in methods to treat or prevent diseases or disorders related to KHK dysregulation or fructose metabolism, including secondary diseases following excessive intake of fructose and / or alcohol. The compounds, pharmaceutical compositions, and methods of this disclosure may be useful for treating subjects such as mammals, humans, non-human mammals, livestock (e.g., laboratory animals, domestic pets, or livestock), non-livestock (e.g., wild animals), dogs, cats, rodents, mice, hamsters, cattle, birds, chickens, fish, pigs, horses, goats, sheep, or rabbits, preferably humans.Therefore, the compounds can be used to treat one or more of the following diseases, disorders, conditions, or associated comorbidities (generally referred to herein as diseases): type 1 diabetes (T1D), type 2 diabetes (T2D), idiopathic T1D, latent autoimmune diabetes in adults (LADA), early-onset diabetes (EOD), atypical diabetes, juvenile mature diabetes (MODY), malnutrition-related diabetes, gestational diabetes, hyperglycemia, insulin resistance, hepatic insulin resistance, impaired glucose tolerance, diabetic neuropathy, diabetic nephropathy, and diabetic kidney disease (DKD). Kidney disease, acute kidney injury, tubular dysfunction, pro-inflammatory changes in the proximal tubules, diabetic retinopathy, adipocyte dysfunction, visceral fat deposition, obesity, eating disorders, excessive sugar cravings, excessive alcohol intake, dyslipidemia, hyperlipidemia, hypertriglyceridemia, increased total cholesterol, high LDL cholesterol, high non-HDL cholesterol, low HDL cholesterol, hyperinsulinemia, non-alcoholic fatty liver disease (NAFLD), metabolic dysfunction-related fatty liver disease (MASLD), non-alcoholic steatohepatitis (NASH), metabolic dysfunction-related fatty liver disease (MASH) MASLD (MetALD) with increased alcohol intake, hepatic steatohepatopathy, fibrosis, cirrhosis, hepatocellular carcinoma, hereditary fructose intolerance (HFI), alcoholic steatohepatitis (ASH), viral liver disease, diseases associated with hepatic fibrosis or cirrhosis, such as alpha-1 antitrypsin deficiency, hemochromatosis, pancreatic diseases including pancreatic cancer, gallbladder diseases (PBC, PSC), coronary artery disease, peripheral vascular disease, hypertension, endothelial dysfunction, impaired vascular compliance, congestive heart failure, myocardial infarction, stroke, hemorrhagic stroke, ischemic stroke, pulmonary hypertension, vascular Postoperative restenosis, intermittent claudication, postprandial fatty left ventricular hypertrophy, peripheral artery disease, macular degeneration, cataracts, glomerulosclerosis, chronic renal failure, metabolic syndrome, syndrome X, premenstrual syndrome, angina pectoris, thrombosis, atherosclerosis, transient ischemic attack, vascular restenosis, glucose metabolism disorder, impaired fasting plasma glucose, hyperuricemia, gout, erectile dysfunction, skin and connective tissue disorders, foot ulcers, ulcerative colitis, hyperapotetalipoproteinemia, Alzheimer's disease, schizophrenia, cognitive impairment, inflammatory bowel disease, ulcerative colitis, Crohn's disease, irritable bowel syndrome.

[0113] In another embodiment, the present disclosure provides a method for treating any one or a combination of the following diseases: T1D, T2D, insulin resistance, kidney disease, acute kidney injury, tubular dysfunction, pro-inflammatory changes in the proximal tubules, adipocyte dysfunction, visceral fat deposition, obesity, eating disorders, excessive sugar craving, excessive alcohol intake, dyslipidemia, hyperlipidemia, hypertriglyceridemia, increased total cholesterol, high LDL cholesterol, high non-HDL cholesterol, low HDL cholesterol, NAFLD, MASLD, MetALD, hepatic steatosis, NASH, MASH, hepatic fibrosis, cirrhosis, hepatocellular carcinoma, HFK, hypertension, endothelial dysfunction, metabolic syndromes, hyperuricemia, and gout.

[0114] Preferred examples of diseases or disorders associated with KHK dysregulation include metabolic syndromes, NAFLD, NASH, MASLD, MASH, MetALD, T2D, hypertriglyceridemia, hypercholesterolemia, DKD, ASH, liver disease resulting from hepatocyte stress (e.g., alpha-1 antitrypsin deficiency [AATD], viral hepatitis, or hemochromatosis), viral diseases, toxic cravings, alcohol use disorders, hyperuricemia, gout, neurodegenerative diseases, and cancer. In some cases, the disease or disorder is NASH or MASH.

[0115] KHK, or fructokinase, catalyzes the first step in fructose metabolism, phosphorylating fructose to fructose-1-phosphate (F1P) and depleting the intracellular ATP and adenine nucleotide pools. Since there is no negative feedback mechanism in which F1P inhibits KHK metabolism of fructose, the accumulation of F1P is directly related to the amount of fructose either (1) transported into the cell via GLUT transporters, or (2) formed into the cell from glucose via the polyol pathway and metabolized via KHK. The accumulation of F1P, as well as the depletion of ATP and adenine nucleotide pools, leads to adverse consequences in cells, tissues, and organs, including oxidative stress, endothelial dysfunction, and metabolic dysregulation. Responses to these stimuli include adipogenesis and gluconeogenesis, which drive metabolic disorders. Patients experiencing KHK metabolism of fructose occurring outside of normal parameters are at risk of developing a dysregulation of KHK-mediated fructose metabolism, i.e., diseases or disorders resulting from KHK dysregulation.

[0116] Non-limiting examples of diseases or disorders associated with excessive fructose intake, increased fructose formation in hepatocytes via the polyol pathway (e.g., osmotic stress similar to alcohol exposure), or KHK dysregulation include metabolic syndromes and metabolic disorders (including type 2 diabetes mellitus (T2D) or hypertriglyceridemia), liver diseases (resulting from non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), and hepatocyte stress (e.g., alpha-1 antitrypsin deficiency or hematological malignancies)), kidney diseases and disorders including diabetic nephropathy (DKD), toxic cravings, alcohol use disorder, hyperuricemia, gout, neurodegenerative diseases (e.g., Parkinson's disease or Alzheimer's disease), and cancer. In some cases, the disease or disorder is NASH.

[0117] The terms “disease,” “disorder,” and “condition” may be used interchangeably herein to refer to medical or pathological conditions related to KHK dysregulation.

[0118] As used herein, the terms “subject” and “patient” are interchangeable. The terms “subject” and “patient” refer to animals (e.g., birds such as chickens, quail, or turkeys, or mammals), specifically non-primates (e.g., cattle, pigs, horses, sheep, rabbits, guinea pigs, rats, cats, dogs, and mice), primates (e.g., monkeys, chimpanzees, and humans), and more specifically, “mammals,” including humans. Humans may be male or female. In one embodiment, the subject is a non-human animal such as a farm animal (e.g., a horse, cattle, pig, or sheep) or a pet (e.g., a dog, cat, guinea pig, or rabbit). In a preferred embodiment, the subject is a human.

[0119] As used herein, the term “biological sample” includes, but is not limited to, cell cultures or extracts thereof, biopsy materials obtained from mammals or extracts thereof, blood, saliva, urine, feces, semen, tears, or other bodily fluids or extracts thereof.

[0120] KHK inhibition can be measured by any suitable method known in the art. For example, KHK inhibition can be measured in biological samples (e.g., cell cultures or cell-free isolated enzymes) or in humans (e.g., subjects). More specifically, for cell-based assays, in each case, cells are cultured in vitro, the test agent is added to the culture, and the endpoint is evaluated after a suitable length of time. Such assays are known in the art.

[0121] As used herein, the terms “treat,” “treat,” and “to treat” refer to both therapeutic and prophylactic treatments. For example, therapeutic treatment includes reducing or mitigating the progression, severity, and / or duration of a disease or disorder associated with KHK dysregulation, or improving one or more symptoms (specifically, one or more identifiable symptoms) of a disease or disorder associated with KHK dysregulation, resulting from the administration of one or more therapies (e.g., one or more therapeutic agents such as compounds or compositions of the Disclosure). In certain embodiments, therapeutic treatment includes improving at least one measurable physical parameter of a disease or disorder associated with KHK dysregulation. In other embodiments, therapeutic treatment includes inhibiting the progression of a disease or disorder associated with KHK dysregulation, for example, by stabilizing an identifiable symptom, physically, for example, by stabilizing a physical parameter, or both. In other embodiments, therapeutic treatment includes reducing or stabilizing a disease or disorder associated with KHK dysregulation.

[0122] The term "chemotherapy" refers to the use of drugs, such as small molecule drugs (not "vaccines"), to treat a disorder or disease.

[0123] As used herein, the terms “prevention” or “preventive use” and “preventive treatment” refer to any medical or public health treatment aimed at preventing a disease rather than treating or curing it. As used herein, the terms “prevent,” “prevention,” and “preventing” refer to reducing the risk of acquiring or developing a given condition, or reducing or inhibiting the recurrence of such condition in a non-disease subject. The term “chemoprevention” refers to the use of a drug, such as a small molecule drug (but not a “vaccine”), for the prevention of a disorder or disease.

[0124] As used herein, prophylactic use includes use in situations in which the presence of a disease or disorder related to KHK dysregulation or fructose metabolism has been detected, including secondary cases following excessive intake of fructose and / or alcohol. Prophylactic use may also include treating a person who does not have a disease or disorder related to KHK dysregulation or is not considered to be at high risk of complications, in order to reduce the likelihood of developing a disease or disorder related to KHK dysregulation.

[0125] In some embodiments, the methods of the present disclosure are prophylactic or “preventive” measures for patients, particularly humans, who are predisposed to complications resulting from diseases or disorders related to KHK dysregulation.

[0126] As used herein, “effective dose” means an amount sufficient to induce a desired biological response. In this disclosure, the desired biological response is to inhibit KHK in a biological sample or subject, or to reduce or improve the severity, duration, progression, or onset of a disease or disorder associated with KHK dysregulation, to prevent the progression of a disease or disorder associated with KHK dysregulation, to prevent the recurrence, onset, onset, or progression of symptoms associated with a disease or disorder associated with KHK dysregulation, or to enhance or improve the preventive or therapeutic effect of another therapy used for a disease or disorder associated with KHK dysregulation. The exact amount of compound administered to a subject depends on the method of administration, the type and severity of the disease or disorder, and the subject’s characteristics such as general health, age, sex, weight, and drug tolerance. A person skilled in the art will be able to determine an appropriate dose in accordance with these and other factors. When co-administered with other drugs, for example, with another drug, the “effective dose” of the second drug depends on the type of drug used. Suitable dosages are known for approved drugs and can be adjusted by those skilled in the art depending on the condition of the subject, the type of condition being treated, and the amount of the compound described herein used. Where a quantity is not explicitly stated, a safe and effective quantity should be assumed. For example, the compounds described herein can be administered to a subject in a dosage range of about 0.01 to 100 mg / kg body weight / day for therapeutic or prophylactic treatment.

[0127] In general, a dosage regimen can be selected according to a variety of factors, including the disease or disorder being treated, its severity, the activity of the specific compound used, the specific composition used, the patient's age, weight, general health, sex, and diet, the timing of administration, route of administration, and excretion rate of the specific compound used, the renal and hepatic function of the subject, the specific compound or its salt used, the duration of treatment, drugs used in combination with or in conjunction with the specific compound used, and similar factors well known in the medical field. A person skilled in the art can easily determine and prescribe the effective amount of the compounds described herein necessary to treat, prevent, inhibit (completely or partially) or block the progression of the disease or disorder.

[0128] The dosages of the compounds for use described herein may range from about 0.01 to about 100 mg / kg body weight / day, about 0.01 to about 50 mg / kg body weight / day, about 0.1 to about 50 mg / kg body weight / day, or about 1 to about 25 mg / kg body weight / day. It is understood that the total daily dose may be administered as a single dose or in multiple doses, such as twice a day (e.g., every 12 hours), three times a day (e.g., every 8 hours), or four times a day (e.g., every 6 hours).

[0129] For therapeutic treatment, the compounds described herein may be administered to a patient within, for example, 48 hours (or 40 hours, or less than 2 days, or less than 1.5 days, or within 24 hours) of the onset of symptoms. The compounds described herein may also be administered to a patient beyond this timeline, for example, within 2 weeks, 6 months, 1 year, 5 years, or 10 years from the onset of symptoms. Therapeutic treatment may be sustained for any preferred period, for example, 5 days, 7 days, 10 days, 14 days, etc. For prophylactic treatment, the compounds described herein may be administered to a patient for any preferred period, for example, 7 days, 10 days, 14 days, 20 days, 28 days, 35 days, 42 days, etc.

[0130] Combination therapy The compounds described herein can be used in combination therapy, i.e., in combination with drugs or vaccines.

[0131] A safe and effective dose can be achieved by the methods or pharmaceutical compositions of this disclosure using the compound of formula I or Table A, or a pharmaceutically acceptable salt thereof, alone or in combination with an additional suitable therapeutic agent, such as a drug or vaccine. When using “combination therapy,” a safe and effective dose can be achieved using a first amount of the compound of formula I or Table A, or a pharmaceutically acceptable salt thereof, and a second amount of an additional suitable therapeutic agent (e.g., a drug or vaccine).

[0132] In one embodiment, the compound of formula I or Table A, or a pharmaceutically acceptable salt thereof, and the additional therapeutic agent are each administered in a safe and effective amount (i.e., an amount that would be therapeutically effective if each were administered alone). In another embodiment, the compound of formula I or Table A, or a pharmaceutically acceptable salt thereof, and the additional therapeutic agent are each administered in an amount that would not produce a therapeutic effect on its own (less than a therapeutic dose). In yet another embodiment, the compound of formula I or Table A, or a pharmaceutically acceptable salt thereof, may be administered in a safe and effective amount, while the additional therapeutic agent may be administered in a less than therapeutic dose. In yet another embodiment, the compound of formula I or Table A, or a pharmaceutically acceptable salt thereof, may be administered in a less than therapeutic dose, while the additional therapeutic agent is administered in a safe and effective amount.

[0133] Non-limiting examples of additional therapeutic agents that may be administered to the target population include antidiabetic drugs, anti-obesity drugs, antihypertensive drugs, anxiolytics, antidepressants, drugs for treating diabetic nephropathy, drugs for treating diabetic neuropathy, cholesterol / lipid modifiers, calcium channel blockers, cardiac glycosides, diuretics, antiplatelet agents, anticoagulants, antiosteoporosis drugs, anti-inflammatory drugs, mineralocorticoid receptor antagonists, phosphodiesterase inhibitors, anti-ulcer and gastroesophageal reflux disease drugs, hormone replacement therapy, fructose transporter inhibitors, aldose reductase inhibitors, xanthine oxidase inhibitors, drugs for treating biliary or gallbladder diseases (e.g., primary biliary cholangitis or primary sclerosing cholangitis) and viral liver diseases, drugs for treating AATD and thrombosis, drugs for treating preserved ejection heart failure in particular, and drugs for treating MASH or NASH. Other non-limiting examples of additional therapeutic agents that can be administered in conjunction with the compounds disclosed herein (e.g., compounds of Formula I or Table A) include those disclosed in U.S. Patent No. 10,174,007, which is incorporated herein by reference.

[0134] In some cases, compounds of formula I or Table A, or pharmaceutically acceptable salts, may be co-administered with one or more antidiabetic agents selected from the group consisting of metformin, sulfonylureas (e.g., glipizide, glimepiride, glipentide, and tolbutamide), thiazolidinediones or peroxisome proliferator-activated receptor gamma (PPARγ) agonists (e.g., pioglitazone), DPP4 inhibitors (e.g., sitagliptin, linagliptin, vildagliptin, and saxagliptin), meglitinide, insulin and insulin analogs or mimics, and SGLT1 and / or SGLT2 inhibitors (e.g., dapagliflozin, empagliflozin, tofogliflozin, canagliflozin, erutin, and sotagliflozin). In some cases, compounds of formula I or Table A, or pharmaceutically acceptable salts, include amylin analogs (e.g., caglilintide, pramlintide, AZD6234, LY3841136, amicretin, petrelintide, NN9487, and LY3541105), incretin hormone receptor agonists or modulators (e.g., semaglutide, liraglutide, tilzepatide, sulfodutide, letatoltide, pemvidutide, VK2735, RGT-075, caglilintide / semaglutide, danuglyprone, PF- 0695422, NN9487, NN9541, CT-388, CT-868, CT-996, olfogliprone, efinopegdutide, efosipegdutide, AZD9550, DR10624, NLY01, maridebaatkafraglutide, ECC5004, mazdutide, exenatide, dulaglutide, TERN-601, echnoglutide, and XW-004), melanocortin 4 receptor agonists (e.g., cetomelanotide), leptin receptor agonists (e.g., metreptin and mivademab), anti-GIPR It may be co-administered with one or more anti-obesity drugs selected from the group consisting of mAbs and activin type II receptor antagonists or ligand traps (e.g., bimaglumab, tardefglobe alfa, trevoglumab, garetsumab, apiteglumab, and SRK-439).

[0135] In some cases, compounds of formula I or Table A, or pharmaceutically acceptable salts, may be co-administered with one or more cholesterol or lipid modifiers selected from the group consisting of HMG-CoA reductase inhibitors (e.g., pravastatin, lovastatin, atorvastatin, rosuvastatin, simvastatin, and fluvastatin), cholesteryl ester transcription protein inhibitors (e.g., ovicetrapib and darcetrapib), ezetimibe, and PCSK9 inhibitors or modulators (e.g., alirocumab, evolocumab, incurislan, taforemab, ricatisimab, AZD-0780, VERVE-102, MK-0616).In some cases, compounds of formula I or Table A, or pharmaceutically acceptable salts, include FGF21 analogs (e.g., efluxifermin, pegosafermin, BOS-580, B1344, BI3006337, NN9500, NN9499, HEC8843), thyroid hormone beta receptor agonists (e.g., resmethylome, VK2809, ASC41, TERN-501, and ALG-055009), and incretin receptor agonists. Inhibitors or modulators, PPAR agonists (e.g., pioglitazone, lanifranol, PXL-065, and saroglitazal), FASN inhibitors (e.g., denifanstat), acetyl-CoA carboxylase inhibitors (e.g., filsocostat and cresacostat), DGAT1 and / or DGAT2 inhibitors or modulators (e.g., erbogostat, SNP-610, SNP-630, ION224) , PF-07202954), inhibitors or modulators of PNPLA3 (e.g., ALN-PNP, AZD-2693, PF-07853578, LY3849891, JNJ-75220795, and AMG609), alpha-1 antitrypsin enzyme replacement therapy, base editing therapy, or siRNA therapy, or RNAi therapy, or antisense therapy for the treatment of alpha-1 antitrypsin deficiency (e.g., fadilsilan, berce It may be co-administered with one or more drugs selected from the group consisting of silane (e.g., ALN-AAT, NTLA-2003, WVE-006, BEAM-302, KRRO-110), antiretroviral therapy for the treatment of HCV or HBV, and inhibitors or modulators of HSD17B13 (e.g., rapirosilane, INI-822, ARO-HSD, AZD-7503) for the treatment of NAFLD, MASLD, NASH, or MASH.

[0136] In some cases, compounds of formula I or Table A, or pharmaceutically acceptable salts, may be co-administered with fructose transporter inhibitors that are inhibitors of GLUT2, GLUT5, or both. In some cases, compounds of formula I or Table A, or pharmaceutically acceptable salts, may be co-administered with aldose reductase inhibitors AT-001, AT-003, gaborestat, ranilestat, epallestat, fidarestat, imirestat, tollestat, or risalestat.

[0137] As used herein, the terms “combined use” or “concurrent administration” can be used interchangeably to refer to the use of two or more therapies (e.g., one or more prophylactic and / or therapeutic agents). The use of these terms does not restrict the order in which the therapies (e.g., prophylactic and / or therapeutic agents) are administered to the target.

[0138] Concurrent administration includes the simultaneous administration of first and second amounts of a compound in a single pharmaceutical composition, such as a capsule or tablet having a fixed ratio of the first and second amounts, or multiple separate capsules or tablets for each compound. In addition, such concurrent administration also includes the use of each compound sequentially in any order.

[0139] In embodiments, this disclosure relates to methods of combination therapy for inhibiting KHK in a biological sample or patient, or for treating or preventing a disease or disorder related to KHK dysregulation in a patient, using compounds or pharmaceutical compositions described herein, such as compounds of formula I or Table A, or pharmaceutically acceptable salts thereof. Accordingly, pharmaceutical compositions also include those containing compounds disclosed herein in combination with one or more additional therapeutic or prophylactic agents for treating or preventing a disease or disorder related to KHK dysregulation.

[0140] The methods of use of the compounds and compositions disclosed herein also include combinations of chemotherapy with the compounds or compositions of Formula I or Table A, or pharmaceutically acceptable salts thereof, or combinations of chemotherapy with the compounds or compositions disclosed herein and other therapeutic or prophylactic agents.

[0141] When co-administration involves separate administrations of a first dose of Formula I or Table A, or a pharmaceutically acceptable salt thereof, and a second dose of an additional therapeutic agent, the compounds are administered within a time frame close enough to produce the desired therapeutic effect. For example, the interval between each administration that can produce the desired therapeutic effect can range from a few minutes to several hours and can be determined by taking into account the properties of each compound, such as potency, solubility, bioavailability, plasma half-life, and kinetic profile. For example, the compound of Formula I or Table A, or a pharmaceutically acceptable salt thereof, and the second therapeutic agent can be administered in any order within approximately 24 hours, 16 hours, 8 hours, 4 hours, 1 hour, or 30 minutes from each other.

[0142] More specifically, the first therapy (e.g., a prophylactic or therapeutic agent, e.g., a compound of the present disclosure) may be administered simultaneously with or following the second therapy (e.g., a prophylactic or therapeutic agent) (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior) or following (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks later).

[0143] The method of co-administering a first amount of a compound of formula I or Table A, or a pharmaceutically acceptable salt thereof, and a second amount of an additional therapeutic agent may result in an enhanced or synergistic therapeutic effect, and it is understood that the co-administrative effect is greater than the additive effect that would result from separate administrations of the first amount of a compound of formula I or Table A, or a pharmaceutically acceptable salt thereof, and the second amount of the additional therapeutic agent.

[0144] As used herein, the term “synergistic” refers to the combination of a compound disclosed herein with another therapy (e.g., a prophylactic or therapeutic agent) that is more effective than the estimated additive effect of the therapies. The synergistic effect of a combination of therapies (e.g., a combination of a prophylactic or therapeutic agent) can enable the use of lower doses of one or more therapies and / or the administration of such therapies to a subject at a lower frequency. The ability to utilize lower doses of therapies (e.g., a prophylactic or therapeutic agent) and / or the ability to administer such therapies at a lower frequency can reduce the toxicity associated with the administration of such therapies to a subject without reducing the effectiveness of such therapies in the prevention, management, or treatment of a disorder. In addition, the synergistic effect can result in improved efficacy of the agents in the prevention, management, or treatment of a disorder. Finally, the synergistic effect of a combination of therapies (e.g., a combination of a prophylactic or therapeutic agent) can avoid or reduce the adverse or undesirable side effects associated with the use of any of therapies alone.

[0145] The presence of synergistic effects can be determined using preferred methods for evaluating drug interactions. Preferred methods include, for example, the Sigmoid-Emax equation (Holford, NHG and Scheiner, LB, Clin. Pharmacokinet. 6:429-453 (1981)), the Loewe additivity equation (Loewe, S, and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114:313-326 (1926)), and the median effect equation (Chou, T.C. and Talalay, P., Adv. Enzyme Regul. 22:27-55 (1984)). Each of these equations, when applied with experimental data, can generate corresponding graphs useful for evaluating the effects of drug combinations. The corresponding graphs associated with these equations are the concentration-effect curve, the isobologram curve, and the combination exponential curve, respectively.

[0146] Chiral separation The compounds described herein have a chiral center and may arise as racemates, racemic mixtures, individual diastereomers, or enantiomers, with all isomeric forms included in this disclosure. The compounds of this disclosure having a chiral center exist in optically active and racemic forms and can be isolated. Some compounds may exhibit polymorphism. This disclosure encompasses the racemic, optically active, polymorphic, or stereoisomeric forms, or mixtures thereof, of the compounds of this disclosure having the useful properties described herein. Optically active forms can be prepared, for example, by recrystallization of the racemic form, by synthesis from optically active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase or by enzymatic resolution. Each compound may be purified and then derivatized to form the compounds described herein, or the compound itself may be purified.

[0147] Stereospecific stereoisomers or diastereomers are typically conveniently prepared by combining methylsulfonylpyrimidine compounds with stereospecifically substituted azetidine compounds to obtain the desired azetidinylpyrimidine compounds. Selecting a methylsulfonylpyrimidine and a stereospecific azetidine compound can achieve the desired stereospecific formula I compound, or the azetidinylpyrimidiyl compound can be further derivatized to achieve alternative formula I compounds. Hydroxymethylazetidine precursor stereochemical preparations are known from the literature (e.g., J.Med.Chem. 2020, 63, 13546-13560). Methylazetidine precursor enantiomers can be obtained from commercial sources. The stereochemistry of the azetidine stereocenter is preserved during combination with methylsulfonylpyrimidine and during further derivatization. Therefore, the stereochemistry of desired formula I stereoisomers and diastereomers is known. Alternative substituted pyrimidine compounds can be combined with stereospecifically substituted azetidine compounds to achieve the desired formula I compound (or further derivatized compounds) as described in the examples (e.g., Ex. 1).

[0148] The optically active form of the compound can be prepared using any method known in the art, including, but not limited to, separation of the racemic form by recrystallization techniques, synthesis from optically active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase.

[0149] Examples of methods for obtaining optically active materials include, at least, the following: i) Physical separation of crystals: A technique in which macroscopic crystals of individual enantiomers are manually separated. This technique can be used when crystals of distinct enantiomers exist, i.e., the material is an aggregate and the crystals are visually distinct. ii) Simultaneous crystallization: This is a technique in which individual enantiomers are crystallized separately from a racemic solution, and is only possible when the latter is an aggregate in a solid state. iii) Cocrystallization: A technique in which individual enantiomers crystallize together from a racemic solution. iv) Enzyme resolution: A technique for partially or completely separating a racemic mixture by different reaction rates of enantiomers with an enzyme. v) Enzyme-asymmetric synthesis: A synthetic technique in which at least one synthetic step uses an enzymatic reaction to obtain an enantiomerically pure or concentrated synthetic precursor of the desired enantiomer. vi) Chemical asymmetric synthesis: A synthetic technique in which a desired enantiomer is synthesized from an achiral precursor under conditions that result in asymmetry (i.e., chirality) in the product, which can be achieved using a chiral catalyst or chiral auxiliary agent. vii) Diastereomer separation: A technique in which a racemic compound is reacted with an enantiomerically pure reagent (chiral auxiliary) that converts individual enantiomers into diastereomers. The resulting diastereomers are then separated by chromatography or crystallization based on their now more distinct structural differences, and the chiral auxiliary is subsequently removed to obtain the desired enantiomers. viii) Primary and Secondary Asymmetric Transitions: Techniques to equilibrate diastereomers from a racemic mixture to obtain the vast majority of diastereomers in solution from the desired enantiomer, or to preferentially crystallize diastereomers from the desired enantiomer, such that, in principle, all materials are ultimately converted from the desired enantiomer to crystalline diastereomers. Subsequently, the desired enantiomer is released from the diastereomer. ix) Kinetic resolution: This technique refers to the achievement of partial or complete resolution of a racemate (or further resolution of a partially resolved compound) by unequal reaction rates of enantiomers with chiral, non-racemic reagents or catalysts under kinetic conditions. x) Enantiomer-specific synthesis from non-racemic precursors: Synthetic techniques in which the desired enantiomer is obtained from non-chiral starting materials and stereochemical integrity is not lost or is only minimally lost throughout the synthesis process. xi) Chiral liquid chromatography: A technique (including, but not limited to, via chiral HPLC) in which enantiomers of a racemic mixture are separated in a liquid mobile phase by their different interactions with the stationary phase. The stationary phase can be made of a chiral material, or the mobile phase can contain additional chiral materials to induce different interactions. xii) Chiral gas chromatography: A technique in which a racemic mixture is volatilized and enantiomers are separated by the different interactions between a gaseous mobile phase and a column containing a fixed non-racemic chiral adsorbent phase. xiii) Extraction in chiral solvents: A technique in which enantiomers are separated by the preferential dissolution of one enantiomer in a specific chiral solvent. xiv) Transport across chiral membranes: A technique in which racemates are placed in contact with a thin membrane barrier. The barrier typically separates two miscible fluids, one containing a racemate, and a driving force such as concentration or pressure difference results in preferential transport across the membrane barrier. The separation occurs as a result of the non-racemic chiral nature of the membrane, which allows only one enantiomer of the racemate to pass through.

[0150] In one embodiment, chiral chromatography, including but not limited to stimulus-transfer-bed chromatography, is used. A wide variety of chiral stationary phases are commercially available.

[0151] This disclosure will be better understood by referring to the following non-limiting embodiments.

[0152] compound synthesis The following preparations of compounds and intermediates of Formula I are given to enable those skilled in the art to better understand and implement the Disclosure. They should not be considered limiting to the scope of the Disclosure, but merely illustrative and representative.

[0153] The starting materials and reagents used in the preparation of these compounds are either available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wis.), Bachem (Torrance, Calif.), or Sigma (St. Louis, Mo.), or prepared by methods known to those skilled in the art, following the procedures described in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). These schemes are merely illustrative of some methods by which the compounds of this disclosure can be synthesized, and various modifications to these schemes can be made, as will be suggested to those skilled in the art who have referred to this disclosure. Starting materials and intermediates, as well as the final products of the reactions, can be isolated and purified as necessary using conventional techniques, including but not limited to filtration, distillation, crystallization, and chromatography. Such materials can be characterized using conventional means, including physical constants and spectral data.

[0154] The following LC / MS conditions are mentioned in the synthesis examples herein.

[0155] Method A: 5-95AB_3.5 min: LC / MS chromatography. The column used was a 5 μm C18 90A, 30 × 3.0 mm column. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 50–2000. Mobile phase A was 0.04% TFA in water, and mobile phase B was 0.02% TFA in HPLC-grade acetonitrile. The gradients were 5–95%B at 3.50 min, 5%B at 0.01 min, 5–95%B with a hold at 95%B for 0.50 min (0.01–2.50 min), and 95–5%B with a hold at 5%B for 0.49 min (3.00–3.01 min). The flow rate ranged from 1 mL / min (0.01–3.00 mins) to 1.2 mL / min (3.01–3.50 mins).

[0156] Method B: 10-100 AB_2 min: LC / MS chromatography. The column used was C18 5 μm, 3.0 × 30 mm (5 μm particles). The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100 to 1000. Mobile phase A was 0.04% TFA in water, and mobile phase B was 0.02% TFA in HPLC-grade acetonitrile. The gradient was 10-100% B at 1.30 min, 10% B at 0.01 min, and 10-100% B (0.01-0.70 min) after holding at 100% B for 0.60 min. The flow rate was 1.5 mL / min (0.00-1.30 min).

[0157] Method C: 5-95AB_2 min: LC / MS chromatography. The column used was a 5 μm C18 90A, 30 × 3.0 mm column. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 50–2000. Mobile phase A was 0.04% trifluoroacetic acid in water, and mobile phase B was 0.02% trifluoroacetic acid in HPLC-grade acetonitrile. The gradient was 5–95%B at 1.50 min, 5%B at 0.01 min, 5–95%B (0.01–0.70 min), 95%B at 0.46 min, and 95–5%B after holding at 5%B for 0.11 min (1.61–1.50 min). The flow rate was 1.5 mL / min.

[0158] Method D: 10-100AB_1 min: LC / MS chromatography. The column used was C18 3.0 × 30 mm (5 μm particles). The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 50 to 2000. Mobile phase A was 0.04% TFA in water, and mobile phase B was 0.02% TFA in HPLC-grade acetonitrile. The gradient was 10-100%B at 0.90 min, 10%B at 0.01 min, and 10-100%B (0.01-0.50 min) after holding at 100%B for 0.40 min. The flow rate was 2.0 mL / min.

[0159] Method E: 5_95AB_6min-220-254-ELSD:LC / MS. The gradient was 5%B for 0.01 min, held at 5%B for 0.48 min, followed by 5-95%B (0.01-1.60 min), 95-100%B (1.60-2.50 min), and 100-5% (2.50-2.52 min). The flow rate was 0.8 mL / min. Mobile phase A was 0.037% trifluoroacetic acid in water, and mobile phase B was 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was a C18 3.0 × 30 mm, 2.5 μm column (2.5 μm particles). Detection methods included diode array (DAD) and evaporative light scattering (ELSD) detection, as well as positive electrospray ionization. The MS range was 100-1000.

[0160] Method F:5_95CD_6min-220-254-ELSD:LC / MS gradient was 5%B at 0.40 min, 5-95%B from 0.40 to 3.40 min, with 95%B held for 0.45 min, followed by 95-5%B at 0.01 min, with a flow rate of 0.8 mL / min. Mobile phase A was H2O + 10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was a C18 2.1 × 50 mm column (5 μm particles). Detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[0161] Method G: 5-95CD_2 min: LC / MS chromatography. The column used was C18 2.1 × 50 mm (5 μm particles). The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100 to 1000. Mobile phase A was 10 mM ammonium bicarbonate in water, and mobile phase B was HPLC-grade acetonitrile. The gradient was 5–95%B at 1.50 min, 5%B at 0.01 min, 5–95%B (0.01–0.70 min), 95%B at 0.46 min, and 95–5%B after holding at 5%B for 0.11 min (1.61–1.50 min). The flow rate was 1.5 mL / min.

[0162] Method H:5-95AB_0.8 min: Mobile phase: The mobile phase was ramped from 5% acetonitrile (0.01875% trifluoroacetic acid) in water (0.0375% trifluoroacetic acid) to 95% acetonitrile in water over 0.60 min, with the flow rate set to 2.0 mL / min. Then, the flow rate was set to 2.0 mL / min for 0.18 min in 95% acetonitrile, returned to 5% acetonitrile in water, and held for 0.02 min. The flow rate was set to 2.0 mL / min. The column temperature was 50°C, and the column was a C18 reversed-phase column with dimensions of 2.1 × 30 mm (5 μm particles).

[0163] Method P:5_95AB_6min 220-254-ELSD:LC / MS. The gradient was 5%B at 0.40 min, 5–95%B at 2.60 min, held at 95%B for 1.00 min, then 95–5%B at 0.01 min, with a flow rate of 1.0 mL / min. Mobile phase A was 0.04% trifluoroacetic acid in water, and mobile phase B was 0.02% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Luna C18 50×2.0 mm column (5 μm particles). Detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100–1000.

[0164] Method R: The LC / MS gradient for 10 - 80% AB was from 10 - 80% B over 8.00 minutes, held at 80% B for 2.00 minutes, changed from 80 - 10% B in 0.01 minutes, and then held at 10% for 2.99 minutes (flow rate 0.5 mL / min). Mobile phase A was water with 0.04% trifluoroacetic acid, and mobile phase B was acetonitrile with 0.02% trifluoroacetic acid. The column used for chromatography was a Halo AQ-C18 3.0×100 mm column (2.7 um particles). The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100 - 1000.

[0165] Method T:

Table 2

Examples

[0166] Example 1: Synthesis of Compound A1

Chemical Structure

[0167] LCMS(ESI+): m / z 421.2(M+H) + ,RT:0.689 minutes (Method B)

[0168] 1 H NMR(400MHz,DMSO-d6)δ ppm 7.84(s,1H)7.56(s,1H)5.16(s,2H)3.35-3.54(m,8H)1.42(s,9H)1.26(s,12H)

[0169] Synthesis of tert-butyl 4-[2-[4-[5-methyl-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate A solution of 4-chloro-5-methyl-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine (0.5 g, 2.06 mmol, 1 equivalent) in a mixture of dioxane (4.2 mL) and H2O (0.8 mL) was mixed with tert-butyl 4-[2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole-1-yl]acetyl]piperazine-1-carboxylate (952.69 mg, 2.27 mmol, 1.1 equivalents), followed by Na2CO3 (655.20 mg, 6.18 mmol, 3 equivalents). The resulting mixture was degassed, and then DTBPF PdCl2 (67.15 mg, 103.03 μmol, 0.05 equivalents) was added. The mixture was changed with N2 three times and stirred at 80°C for 2 hours under N2 protection. TLC and LC-MS showed that all starting material was consumed and new spots were formed. The reaction mixture was quenched with H2O (10 mL) and then extracted with ethyl acetate (3 × 10 mL). The organic layer was mixed and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography on silica gel and eluted with petroleum ether:ethyl acetate = 10:1 to 1:1, (TLC: petroleum ether:ethyl acetate = 1:2, Rf = 0.4) to obtain tert-butyl 4-[2-[4-[5-methyl-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate.

[0170] LCMS(ESI+): m / z 445.3 (M-55) + ,RT:2.077 minutes (Method A)

[0171] 1 ¹H NMR (400MHz, chloroform-d): δ = 8.11 (s, 1H), 8.06 (s, 1H), 5.01 (s, 2H), 3.60-3.45 (m, 4H), 3.43-3.32 (m, 4H), 2.53 (s, 3H), 2.43 (d, J=1.4Hz, 3H), 1.40 (s, 9H)

[0172] Synthesis of tert-butyl 4-(2-(4-(5-methyl-2-(methylsulfonyl)-6-(trifluoromethyl)pyrimidine-4-yl)-1H-pyrazole-1-yl)acetyl)piperazine-1-carboxylate To a solution of tert-butyl 4-[2-[4-[5-methyl-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate (0.86 g, 1.72 mmol, 1 equivalent) in DMF (8.6 mL), Oxone (4.23 g, 6.87 mmol, 4 equivalents) was added. The mixture was stirred at 25°C for 12 hours. LC-MS indicated that the reaction was complete. The reaction product was poured into saturated aqueous NaCl solution (20 mL) and then extracted with ethyl acetate (3 × 10 mL). The mixed organic layer was washed with saturated aqueous solution of Na2SO3 (20 mL), then the organic layer was dried over Na2SO4 and concentrated to obtain tert-butyl 4-[2-[4-[5-methyl-2-methylsulfonyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate (which was used directly without further purification).

[0173] LCMS(ESI+): m / z 555.2(M+Na) + ,RT:0.569 minutes (Method C)

[0174] 1 H NMR(400MHz,chloroform-d)δ=8.32(s,1H),8.13(s,1H),5.04(s,2H),3.62-3.52(m,2H),3.47(br d,J=6.9Hz,4H),3.42-3.36(m,2H),3.33(s,3H),2.62(d,J=1.0Hz,3H),1.41(s,9H)

[0175] Synthesis of tert-butyl 4-[2-[4-[2-hydroxy-5-methyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate A solution of tert-butyl 4-[2-[4-[5-methyl-2-methylsulfonyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate (0.8 g, 1.50 mmol, 1 equivalent) in dioxane (8 mL) was cooled to 0°C, and then NaOH (1 M, 3.00 mL, 2 equivalents) was added. The mixture was stirred at 0°C for 30 minutes. LC-MS showed a major peak for the desired product with 8% of the starting material remaining (TLC: dichloromethane:methanol = 3:1, Rf = 0.3). The reaction product was acidified to pH = 3 with HCl (1 M) and then extracted with ELISA (3 × 10 mL). The organic matter was mixed and concentrated under reduced pressure to obtain tert-butyl 4-[2-[4-[2-hydroxy-5-methyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate, which was used directly without further purification.

[0176] LCMS(ESI+): m / z 415.1 (M-55) + ,RT:0.510 minutes (Method C)

[0177] 1H NMR (400MHz, chloroform-d) δ=11.20-10.27(m,1H),8.40(s,1H),7.94-7.87(m,1H),5.13(s,2H),3.63(s,4H),3.54(br d,J=5.1Hz,2H),3.47(s,4H),3.40(br d,J=5.1Hz,2H),2.28(s,2H),2.26(br s,1H),1.41(s,9H)

[0178] Synthesis of tert-butyl 4-(2-(4-(2-((1H-benzo[d][1,2,3]triazole-1-yl)oxy)-5-methyl-6-(trifluoromethyl)pyrimidine-4-yl)-1H-pyrazole-1-yl)acetyl)piperazine-1-carboxylate A solution of tert-butyl 4-[2-[4-[2-hydroxy-5-methyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate (0.5 g, 1.06 mmol, 1 equivalent) in DMF (5 mL) was mixed with DBU (242.71 mg, 1.59 mmol, 240 μL, 1.5 equivalents) and BOP (940.13 mg, 2.13 mmol, 2 equivalents). The mixture was stirred at 20°C for 2 hours. TLC and LCMS showed that all the starting material was consumed and new spots were formed. The reaction mixture was quenched with H2O (10 mL). The mixture was extracted with ethyl acetate (3 × 10 mL). The organic layers were mixed and concentrated under reduced pressure to obtain the residue. The residue was purified by preparative TLC (SiO2, petroleum ether:ethyl acetate = 1:5, Rf = 0.3) to obtain tert-butyl 4-[2-[4-[2-(benzotriazole-1-yloxy)-5-methyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate.

[0179] LCMS(ESI+): m / z 610.2(M+Na) + ,RT:0.651 minutes (Method C)

[0180] 1 ¹H NMR (400MHz, chloroform-d): δ=8.15 (d, J=8.4Hz, 1H), 7.91 (s, 1H), 7.77 (s, 1H), 7.62-7.43 (m, 3H), 3.69-3.56 (m, 2H), 3.54-3.41 (m, 6H), 2.57 (d, J=1.1Hz, 3H), 1.50 (s, 9H)

[0181] Synthesis of tert-butyl 4-[2-[4-[5-methyl-6-(trifluoromethyl)-2-[(2R)-2-(trifluoromethyl)azetidine-1-yl]pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate A solution of tert-butyl 4-[2-[4-[2-(benzotriazol-1-yloxy)-5-methyl-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylate (300 mg, 510.59 μmol, 1 eq) in acetonitrile (3 mL) was added with DIEA (197.97 mg, 1.53 mmol, 266.81 μL, 3 eq) and (2R)-2-(trifluoromethyl)azetidine (CAS# 2554776-09-7, 258.05 mg, 868.01 μmol, 1.7 eq, TsOH). The reaction mixture was stirred at 80 °C for 16 h. LCMS showed that about 24% of tert-butyl 4-[2-[4-[2-(benzotriazol-1-yloxy)-5-methyl-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylate remained and 30% of the desired product was detected. The reaction mixture was poured into water (5 mL) and extracted with ethyl acetate (2×5 mL). The organic layers were combined, concentrated under reduced pressure, and the residue was purified by preparative TLC (SiO2, petroleum ether:ethyl acetate = 1:5, Rf = 0.5) to afford tert-butyl 4-[2-[4-[5-methyl-6-(trifluoromethyl)-2-[(2R)-2-(trifluoromethyl)azetidin-1-yl]pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylate (0.04 g, 69.26 μmol, 13.56% yield) as a yellowish solid.

[0182] LCMS (ESI+): m / z 600.3 (M+Na) + , RT: 2.100 min (Method A)

[0183] Synthesis of 2-[4-[5-methyl-6-(trifluoromethyl)-2-[(2R)-2-(trifluoromethyl)azetidin-1-yl]pyrimidin-4-yl]pyrazol-1-yl]-1-piperazin-1-yl-ethanone A solution of tert-butyl 4-[2-[4-[5-methyl-6-(trifluoromethyl)-2-[(2R)-2-(trifluoromethyl)azetidine-1-yl]pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate (40 mg, 69.26 μmol, 1 equivalent) in a mixture of DCM (0.5 mL) and TFA (0.1 mL) was stirred at 25°C for 1 hour. LC-MS showed that all starting material was consumed and a new peak with the desired Ms was observed. The reaction mixture was purified by preparative HPLC (column: Phenomenex Luna C18 75×30 mm (3 μm particles), mobile phase: [H2O(0.1% TFA)-ACN], gradient: 15%-45% B, 8.0 minutes) to obtain 2-[4-[5-methyl-6-(trifluoromethyl)-2-[(2R)-2-(trifluoromethyl)azetidine-1-yl]pyrimidine-4-yl]pyrazole-1-yl]-1-piperazine-1-yl-ethane.

[0184] LCMS(ESI+): m / z 478.2(M+H) + ,RT:2.218 minutes (Method E)

[0185] 1 H NMR(400MHz, methanol-d4)δ=8.31(s,1H),8.18(s,1H),5.32(s,2H),4.95-4.90(m,1H),5.00-4.90(m,1H),4.18(td,J=6.0,8.4Hz,2H),3.89(br s,4H),3.37(br s,2H),3.29(br s,2H),2.74-2.61(m,1H),2.58-2.51(m,1H),2.48(d,J=1.5Hz,3H)

[0186] Example 2: Synthesis of Compound A2 [ka] Synthesis of tert-butyl 4-(2-(4-(2-chloro-6-(trifluoromethyl)pyrimidine-4-yl)-1H-pyrazole-1-yl)acetyl)piperazine-1-carboxylate To a solution of tert-butyl 4-[2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole-1-yl]acetyl]piperazine-1-carboxylate (5 g, 11.90 mmol, 1 equivalent) and H2O (4.5 mL) in dioxane (45 mL), 2,4-dichloro-6-(trifluoromethyl)pyrimidine (2.58 g, 11.90 mmol, 2.58 mL, 1 equivalent), Na2CO3 (2.52 g, 23.79 mmol, 2 equivalents), and Pd(PPh3)4 (1.37 g, 1.19 mmol, 0.1 equivalent) were added. The mixture was changed with N2 three times and stirred at 80°C for 2 hours under N2 protection. TLC and LCMS showed that all the starting material was consumed and new spots were formed. The reaction mixture was quenched with water (25 mL). The mixture was extracted with ethyl acetate (3 × 25 mL). The mixed organic layer was washed with brine (30 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography on silica gel and eluted with petroleum ether:ethyl acetate = 1:0 to 1:5, (TLC: petroleum ether:ethyl acetate = 1:1, Rf = 0.6) to obtain tert-butyl 4-(2-(4-(2-chloro-6-(trifluoromethyl)pyrimidine-4-yl)-1H-pyrazole-1-yl)acetyl)piperazine-1-carboxylate.

[0187] LCMS(ESI+): m / z 419.1(M+H) + ,RT:0.757 minutes (Method B)

[0188] 1 ¹H NMR (400MHz, chloroform-d) δ ppm 8.34 (s,1H) 8.16 (s,1H) 7.59 (s,1H) 5.09 (s,2H) 3.37-3.69 (m,8H) 1.48 (s,9H)

[0189] Synthesis of tert-butyl(R)-4-(2-(4-(6-(trifluoromethyl)-2-(2-(trifluoromethyl)azetidine-1-yl)pyrimidine-4-yl)-1H-pyrazole-1-yl)acetyl)piperazine-1-carboxylate To a solution of tert-butyl 4-(2-(4-(2-chloro-6-(trifluoromethyl)pyrimidine-4-yl)-1H-pyrazole-1-yl)acetyl)piperazine-1-carboxylate (290 mg, 610.69 μmol, 1 equivalent) in NMP (2.9 mL), (R)-2-(trifluoromethyl)azetidine 4-methylbenzenesulfonate (181.55 mg, 619.69 μmol, 1 equivalent) and K2CO3 (253.20 mg, 1.83 mmol, 3 equivalents) were added. The mixture was stirred at 60°C for 3 hours. LCMS showed a new peak with the desired Ms after all the starting material had been consumed. The reaction mixture was quenched with H2O (10 mL) and extracted with ethyl acetate (3 × 5 mL). The mixed organic layer was washed with brine (5 mL), dried over Na2SO4, filtered, concentrated under reduced pressure to obtain the residue. The residue was purified by preparative TLC (SiO2, petroleum ether:ethyl acetate = 1:1, Rf = 0.43) to obtain tert-butyl(R)-4-(2-(4-(6-(trifluoromethyl)-2-(2-(trifluoromethyl)azetidine-1-yl)pyrimidine-4-yl)-1H-pyrazole-1-yl)acetyl)piperazine-1-carboxylate.

[0190] 1 H NMR(400MHz,DMSO-d6)δ=8.45(s,1H),8.23(s,1H),7.59(s,1H),5.27(s,2H),5.12-4.98(m,1H),4.11(br t,J=7.6Hz,2H),3.55-3.33(m,8H),2.72-2.59(m,1H),2.47-2.36(m,1H),1.42(s,9H)

[0191] Synthesis of 1-piperazin-1-yl-2-[4-[6-(trifluoromethyl)-2-[(2R)-2-(trifluoromethyl)azetidine-1-yl]pyrimidine-4-yl]pyrazole-1-yl]ethanone In a solution of tert-butyl 4-[2-[4-[6-(trifluoromethyl)-2-[(2R)-2-(trifluoromethyl)azetidine-1-yl]pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate (60 mg, 106.48 μmol, 1 equivalent) in DCM (0.5 mL) and TFA (0.1 mL), the mixture was stirred at 25°C for 1 hour. LC-MS showed a new peak with the desired Ms after all the starting material had been consumed. The reaction product was alkalized to pH=7 with saturated sodium bicarbonate aqueous solution and then extracted with ethyl acetate (3 × 5 mL). The mixed organic layer was washed with brine (5 mL), dried over Na₂SO₄, filtered, concentrated under reduced pressure to obtain the residue. The residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 100×30mm×10um, mobile phase: [water (NH4HCO3)-ACN], gradient: 35%~65%B, 8 minutes) to obtain 1-piperazin-1-yl-2-[4-[6-(trifluoromethyl)-2-[(2R)-2-(trifluoromethyl)azetidine-1-yl]pyrimidine-4-yl]pyrazole-1-yl]ethanone.

[0192] LCMS(ESI+): m / z 464.1(M+H) + ,RT:2.178 minutes (Method E)

[0193] 1 H NMR (400MHz, methanol-d4)δ=8.36(s,1H),8.17(s,1H),7.36(s,1H),5.23(s,2H),5.03-4.91(m,1H),4.19(td,J=6.4,8. 8Hz,2H),3.63-3.53(m,4H),2.93-2.78(m,4H),2.67(dtd,J=6.1,9.1,11.8Hz,1H),2.51(tdd,J=6.1,8.3,11.9Hz,1H)

[0194] Example 3: Synthesis of Compound A3 [ka] Synthesis of tert-butyl 4-[2-[4-[5-methyl-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate A mixture of tert-butyl 4-[2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole-1-yl]acetyl]piperazine-1-carboxylate (242.50 mg, 576.97 μmol, 1 equivalent), 4-chloro-5-methyl-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine (140 mg, 576.97 μmol, 1 equivalent), Pd(PPh3)4 (66.67 mg, 57.70 μmol, 0.1 equivalent), and Na2CO3 (244.61 mg, 2.31 mmol, 4 equivalents) in dioxane (1.6 mL) and H2O (0.4 mL) was degassed, purged three times with N2, and then stirred at 80°C for 12 hours under N2 atmosphere. LC-MS completely consumed tert-butyl 4-[2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole-1-yl]acetyl]piperazine-1-carboxylate and showed one major peak with the desired Ms. The reaction mixture was quenched with water (3 mL) and extracted with ethyl acetate (4 mL × 3). The mixed organic layer was washed with brine (4 mL × 2), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO₂, petroleum ether / ethyl acetate = 1:3, Rf = 0.6) to obtain tert-butyl 4-[2-[4-[5-methyl-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate.

[0195] LCMS(ESI+): m / z 445.1 (M-55) + ,RT:0.837 minutes (Method B)

[0196] 1H NMR(400MHz,DMSO-d6)δ ppm 8.48(s,1H)8.16(s,1H)5.29(s,2H)3.37-3.59(m,8H)2.59(s,3H)2.46(d,J=1.31Hz,3H)1.42(s,9H).

[0197] Synthesis of tert-butyl 4-[2-[4-[5-methyl-2-methylsulfonyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate To a solution of tert-butyl 4-[2-[4-[5-methyl-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate (200 mg, 399.57 μmol, 1 equivalent) in DMF (2 mL), Oxon (982.57 mg, 1.60 mmol, 4 equivalents) was added. The mixture was stirred at 25°C for 12 hours. LC-MS showed a single major peak with the desired Ms, indicating complete consumption of tert-butyl 4-[2-[4-[5-methyl-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate. The reaction solution was filtered, the filtrate was quenched with ice water (3 mL), and extracted with ethyl acetate (6 mL x 3). The mixed organic layer was washed with brine (10 mL x 2), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by preparative TLC (SiO2, petroleum ether / ethyl acetate = 1:3, Rf = 0.3) to obtain the residue, which yielded tert-butyl 4-[2-[4-[5-methyl-2-methylsulfonyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate.

[0198] LCMS (ESI+): m / z 433.1 (M-100) + ,RT:0.706 minutes (Method B)

[0199] Synthesis of tert-butyl 4-[2-[4-[5-methyl-2-[(2S)-2-methylazetidine-1-yl]-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate TEA (150.49 mg, 1.49 mmol, 207.00 μL, 4 equivalents) was added to a solution of (2S)-2-methylazetidine (CAS#935669-67-3, 80 mg, 743.61 μmol, 2 equivalents, HCl salt) and tert-butyl 4-[2-[4-[5-methyl-2-methylsulfonyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate (198 mg, 371.81 μmol, 1 equivalent) in NMP (2 mL). The mixture was stirred at 60°C for 3 hours. LC-MS completely consumed tert-butyl 4-[2-[4-[5-methyl-2-methylsulfonyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate and showed one main peak with the desired Ms. The reaction mixture was quenched with water (1.5 mL) and extracted with ethyl acetate (2 mL x 3). The mixed organic layer was washed with brine (2 mL x 3), dried over Na2SO4, filtered, concentrated under reduced pressure to obtain the residue, which was purified by preparative HPLC (neutral conditions: H2O (0.05% NH3H2O ​​+ 10 mM NH4HCO3); B: ACN) to obtain tert-butyl 4-[2-[4-[5-methyl-2-[(2S)-2-methylazetidine-1-yl]-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate.

[0200] LCMS(ESI+): m / z 524.2(M+H) + ,RT:0.898 minutes (Method B)

[0201] Synthesis of 2-[4-[5-methyl-2-[(2S)-2-methylazetidine-1-yl]-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]-1-piperazine-1-yl-ethane A solution of tert-butyl 4-[2-[4-[5-methyl-2-[(2S)-2-methylazetidine-1-yl]-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate (50 mg, 95.50 μmol, 1 equivalent) in DCM (0.42 mL) was mixed with TFA (0.08 mL). The mixture was stirred at 25°C for 1 hour. LC-MS showed a single major peak with the desired MS, indicating complete consumption of tert-butyl 4-[2-[4-[5-methyl-2-[(2S)-2-methylazetidine-1-yl]-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate. The pH of the reaction mixture was adjusted to 7-8 with saturated NaHCO3 aqueous solution. The reaction mixture was quenched with water (1 mL) and extracted with ethyl acetate (1.5 mL x 3). The mixed organic layer was washed with brine (1.5 mL x 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain 2-[4-[5-methyl-2-[(2S)-2-methylazetidine-1-yl]-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]-1-piperazine-1-yl-ethanone.

[0202] LC / MS (ESI+): m / z 424.2 (M+H) + ,RT:2.806 minutes (Method F)

[0203] 1 H NMR (400MHz, methanol-d4) δ ppm8.25(s,1H)8.09(s,1H)5.25(s,2H)4.45-4.55(m,1H)3.96-4.10(m,2H)3.63(br s,4H)2.85-3.01(m,4H)2.42(br s,1H)2.40(d,J=1.31Hz,3H)1.98-2.08(m,1H)1.54(d,J=6.20Hz,3H)

[0204] The following compounds were prepared using the same method described for compounds A1 to A3. [Table 3]

[0205] Example 4: Synthesis of Compound A8 [ka] Synthesis of ethyl 4,4,4-trifluoro-2-methoxy-3-oxobutanoate To a solution of ethyl 2-methoxyacetate (35 g, 296.28 mmol, 1 equivalent) and ethyl 2,2,2-trifluoroacetate (84.19 g, 592.56 mmol, 81.34 mL, 2 equivalents) in THF (175 mL), NaH (23.70 g, 592.56 mmol, 60% purity, 2 equivalents) was added at 0°C. The mixture was stirred at 50°C for 12 hours. TLC showed that all the starting material was consumed and new spots were formed. The reaction mixture was poured into a solution of H2SO4 / H2O = 1 / 10 (11V) and extracted with ethyl acetate (3 × 150 mL). The mixed organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography on silica gel and eluted with petroleum ether:ethyl acetate = 3:1 to 1:1 (TLC: petroleum ether:ethyl acetate = 3:1, Rf = 0.4) to obtain ethyl 4,4,4-trifluoro-2-methoxy-3-oxo-butanoate.

[0206] 1 H NMR(400MHz,DMSO-d6)δ ppm 4.12-4.20(m,2H)3.91(s,1H)3.31(s,3H)1.20-1.24(m,3H)

[0207] Synthesis of 5-Methoxy-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine-4-ol To a solution of ethyl 4,4,4-trifluoro-2-methoxy-3-oxobutanoate (10 g, 46.70 mmol, 1 equivalent) in EtOH (50 mL), solutions of 2-methylisothiourea, sulfuric acid (19.50 g, 70.05 mmol, 1.5 equivalents), and Na2CO3 (1 M, 107.40 mL, 2.3 equivalents) were added. The mixture was stirred at 25°C for 12 hours. TLC and LC / MS showed that all the starting material was consumed and new spots were formed. The reaction product was quenched with water (100 mL), extracted with ethyl acetate (3 × 150 mL), the mixed organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain 5-methoxy-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine-4-ol, which was used directly without further purification.

[0208] LC / MS(ESI+): m / z 240.9(M+1) + ,RT:0.429 minutes (Method D)

[0209] Synthesis of 4-chloro-5-methoxy-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine: To a solution of 5-methoxy-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine-4-ol (10 g, 41.60 mmol, 1 equivalent) in ACN (100 mL), POCl3 (63.80 g, 416.3 mmol, 38.80 mL, 10 equivalents) was added. The mixture was stirred at 80°C for 12 hours. LC / MS indicated that the reaction was complete. The reaction mixture was quenched with added water (50 mL) and extracted with ethyl acetate (3 × 100 mL). The mixed organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography on silica gel and eluted for 4-chloro-5-methoxy-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine using petroleum ether:ethyl acetate = 1:1 (TLC: petroleum ether:ethyl acetate = 1:1, Rf = 0.4).

[0210] LC / MS(ESI+): m / z 258.8(M+1) + ,RT:0.583 minutes (Method D)

[0211] 1 H NMR(400MHz,DMSO-d6)δ ppm 3.91(s,3H)2.55(s,3H)

[0212] Synthesis of tert-butyl 3-[4-[5-methoxy-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate To a solution of 4-chloro-5-methoxy-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine (400 mg, 1546.50 μmol, 1 equivalent) and tert-butyl 3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole-1-yl]azetidine-1-carboxylate (540.08 mg, 1546.50 μmol, 1 equivalent) (877399-35-4) in dioxane (4 mL) and H2O (0.4 mL), Na2CO3 (819.56 mg, 7.74 mmol, 5 equivalents), ditert-butyl(cyclolopentyl)phosphan, dichloropalladium, and iron (50.40 mg, 77.32 μmol, 0.05 equivalents) were added. The mixture was stirred at 85°C for 2 hours. TLC and LC / MS showed that all starting material was consumed and new spots were formed. The reaction mixture was extracted with a solution of ethyl acetate (3 × 20 mL) and NaCl (20 mL). The mixed organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography on silica gel and eluted with petroleum ether:ethyl acetate = 1:1 (TLC: petroleum ether:ethyl acetate = 1:1, Rf = 0.4) to obtain tert-butyl 3-[4-[5-methoxy-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate.

[0213] LCMS(ESI+): m / z 390(M-55) + ,RT:0.995 minutes (Method G)

[0214] Synthesis of tert-butyl 3-[4-[5-methoxy-2-methylsulfonyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate To a solution of tert-butyl 3-[4-[5-methoxy-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate (300 mg, 673.46 μmol, 1 equivalent) in DMF (3 mL), Oxon (1.66 g, 2.69 mmol, 4 equivalents) was added. The mixture was stirred at 30°C for 24 hours. TLC and LCMS showed that all the starting material was consumed and new spots were formed. The reaction mixture was extracted with a solution of ethyl acetate (3 × 20 mL) and NaCl (50 mL). The mixed organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain tert-butyl 3-[4-[5-methoxy-2-methylsulfonyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate, which was used directly without further purification.

[0215] LCMS(ESI+): m / z 422.0 (M-55) + ,RT:0.847 minutes (Method C)

[0216] Synthesis of tert-butyl 3-[4-[2-[(2S,3R)-3-hydroxy-2-methylazetidine-1-yl]-5-methoxy-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate TEA (317.90 mg, 3.14 mmol, 437.28 μL, 5 equivalents) was added to a solution of tert-butyl 3-[4-[5-methoxy-2-methylsulfonyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate (300 mg, 628.33 μmol, 1 equivalent) and (2S,3R)-2-methylazetidine-3-ol (200.7 mg, 628.33 mmol, 1 equivalent, R-CSA salt, synthesized as described in J.Med.Chem.2020, 63, 13546-13560, intermediate 13 / (R)-CSA salt) in NMP (3 mL). The mixture was stirred at 60°C for 12 hours. TLC and LCMS showed that all starting material was consumed and new spots were formed. The reaction mixture was extracted with a solution of ethyl acetate (3 × 10 mL) and NaCl (20 mL). The residue was purified by column chromatography on silica gel and eluted with petroleum ether:ethyl acetate = 1:1 (TLC: petroleum ether:ethyl acetate = 1:1, Rf = 0.4) to obtain tert-butyl 3-[4-[2-[(2S,3R)-3-hydroxy-2-methylazetidine-1-yl]-5-methoxy-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate.

[0217] LCMS(ESI+): m / z 485(M+1) + ,RT:0.553 minutes (Method D)

[0218] Synthesis of (2S,3R)-1-[4-[1-(azetidine-3-yl)pyrazole-4-yl]-5-methoxy-6-(trifluoromethyl)pyrimidine-2-yl]-2-methylazetidine-3-ol A solution of tert-butyl3-[4-[2-[(2S,3R)-3-hydroxy-2-methylazetidine-1-yl]-5-methoxy-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate (260 mg, 536.67 μmol, 1 equivalent) in TFA (0.5 mL) and DCM (2.6 mL) was stirred at 25°C for 1 hour. TLC and LCMS showed that all the starting material was consumed and new spots were formed. The reaction mixture was concentrated under reduced pressure to obtain (2S,3R)-1-[4-[1-(azetidine-3-yl)pyrazole-4-yl]-5-methoxy-6-(trifluoromethyl)pyrimidine-2-yl]-2-methylazetidine-3-ol.

[0219] LCMS(ESI+): m / z 385(M+1) + ,RT:0.353 minutes (Method D)

[0220] Synthesis of (2S,3R)-1-[5-methoxy-4-[1-(1-methylazetidine-3-yl)pyrazole-4-yl]-6-(trifluoromethyl)pyrimidine-2-yl]-2-methylazetidine-3-ol To a solution of (2S,3R)-1-[4-[1-(azetidine-3-yl)pyrazole-4-yl]-5-methoxy-6-(trifluoromethyl)pyrimidine-2-yl]-2-methylazetidine-3-ol (200 mg, 520.35 μmol, 1 equivalent) in DCM (4 mL), formaldehyde (42.23 mg, 520.35 μmol, 38.74 μL, 37% purity, 1 equivalent) and NaBH(OAc)3 (330.85 mg, 1.56 mmol, 3 equivalents) were added. The mixture was stirred at 25°C for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain the residue. The crude product was purified by reverse-phase HPLC (column: Phenomenex Gemini NX-C18 75×30 mm (3 μm particles), mobile phase: [H2O (0.05% NH3H2O ​​+ 10 mM NH4HCO3)-ACN], gradient: (25%~50% B for 8.0 minutes) to obtain (2S,3R)-1-[5-methoxy-4-[1-(1-methylazetidine-3-yl)pyrazole-4-yl]-6-(trifluoromethyl)pyrimidine-2-yl]-2-methylazetidine-3-ol).

[0221] LC / MS(ESI+): m / z 399(M+1) + ,RT:2.636 minutes (Method E)

[0222] 1H NMR (400MHz, methanol-d4)δ ppm8.46(s,1H)8.24(s,1H)5.14(quin,J=7.12Hz,1H)4.28(dd,J=9.23,6.05Hz,1H)4.07 -4.22(m,2H)3.88(t,J=8.07Hz,2H)3.62-3.78(m,6H)2.49(s,3H)1.55(d,J=6.11Hz,3H)

[0223] The following compounds were prepared using the same method as described for compound A8. [Table 4]

[0224] Example 5: Synthesis of compounds A21 and A35 [ka] Synthesis of ethyl 4,4-difluoro-2-methyl-3-oxobutanoate To a solution of ethyl propanoate (5 g, 48.96 mmol, 5.62 mL, 1 equivalent) in tetrahydrofuran (25 mL), sodium hydride (1.96 g, 48.96 mmol, 60% purity, 1 equivalent) was added at 0°C. After addition, the mixture was stirred at this temperature for 5 minutes, and then 2,2-ethyl difluoroethyl acetate (12.15 g, 97.91 mmol, 2 equivalents) was added dropwise at 25°C. The resulting mixture was stirred at 50°C for 2 hours. TLC showed that the starting material was consumed and one major new spot was formed. The reaction mixture was quenched by adding saturated ammonium chloride solution (50 mL) at 0°C, then diluted with water (50 mL), and extracted with ethyl acetate (50 mL x 3). The mixed organic layers were washed with brine (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain ethyl 4,4-difluoro-2-methyl-3-oxobutanoate (7.8 g, yield 88%) as a yellow oil, which was used directly in the next step without purification.

[0225] ¹H NMR (400 MHz, chloroform-d) δ ppm 5.75-6.08 (m, ¹H), 4.10 (q, J=7.13 Hz, ²H), 3.88 (q, J=7.09 Hz, ¹H), 1.41 (d, J=7.25 Hz, ³H), 1.27-1.35 (m, ³H).

[0226] Synthesis of 6-(difluoromethyl)-5-methyl-2-(methylthio)pyrimidine-4-ol To a solution of ethyl 4,4-difluoro-2-methyl-3-oxo-butanoate (5 g, 27.75 mmol, 1 equivalent) in ethyl alcohol (250 mL), 2-methylisothiourea sulfate (7.73 g, 27.75 mmol, 1 equivalent) and sodium carbonate solution (1 M, 63.84 mL, 2.3 equivalents) were added at 25°C. The resulting mixture was stirred at 25°C for 12 hours. LC-MS showed that the starting material was consumed and the desired compound was detected. The reaction mixture was concentrated under vacuum to obtain oil, which was extracted with ethyl acetate (60 mL x 3). The mixed organic layer was washed with brine (20 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was then analyzed using petroleum ether:ethyl acetate = 0:1~3:1 (TLC: petroleum ether:ethyl acetate = 3:1, R f The sample was purified by column chromatography on silica gel that elutes at a concentration of 0.45 (6-(difluoromethyl)-5-methyl-2-methylsulfanylpyrimidine-4-ol (3.7 g, yield 65%) as a white solid.

[0227] LCMS(ESI+): m / z 207.0(M+H) + ,RT:0.318 minutes (Method H)

[0228] 1 H NMR(400MHz,DMSO-d6)δ ppm 9.85-10.59(m,1H),6.59-6.94(m,1H),2.41(s,3H)1.95(t,J=2.25Hz,3H).

[0229] LCMS(ESI+): m / z 207.0(M+H) + ,RT:0.318 minutes (Method H)

[0230] Synthesis of 4-chloro-6-(difluoromethyl)-5-methyl-2-(methylthio)pyrimidine To a solution of 6-(difluoromethyl)-5-methyl-2-methylsulfanylpyrimidine-4-ol (3.5 g, 16.97 mmol, 1 equivalent) in acetonitrile (35 mL), phosphorus oxychloride (13.01 g, 84.86 mmol, 7.91 mL, 5 equivalents) was added at 20°C. The mixture was stirred at 80°C for 4 hours. LC-MS showed that the starting material was completely consumed and the desired mass was detected. The reaction mixture was concentrated under vacuum to obtain an oil, which was extracted with ethyl acetate (60 mL x 3). The mixed organic layer was washed with brine (20 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 4-chloro-6-(difluoromethyl)-5-methyl-2-methylsulfanylpyrimidine (2.5 g, yield 66%) as a gray solid. This was used directly in the next step without purification.

[0231] LCMS(ESI+): m / z 225.0(M+H) + ,RT:0.439 minutes (Method H)

[0232] 1 H NMR(400MHz,DMSO-d6)δ ppm 6.96-7.25(m,1H),2.54(d,J=1.38Hz,3H),2.33(d,J=1.50Hz,3H).

[0233] Synthesis of tert-butyl 3-[4-[6-(difluoromethyl)-5-methyl-2-methylsulfanylpyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate To a solution of 4-chloro-6-(difluoromethyl)-5-methyl-2-methylsulfanylpyrimidine (500 mg, 2.19 mmol, 1 equivalent) and tert-butyl 3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole-1-yl]azetidine-1-carboxylate (914.05 mg, 2.62 mmol, 1.2 equivalents) in dioxane (5 mL) and H2O (0.5 mL), Na2CO3 (935.4 mg, 10.95 mmol, 5 equivalents) and DTPBF PdCl2 (49.8 mg, 111.28 μmol, 0.05 equivalents) were added at 25°C. The mixture was stirred at 85°C for 2 hours. LCMS showed that the starting material was completely consumed and a single main peak with the desired mass was detected. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (40 mL x 3). The mixed organic layer was washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel and eluted with petroleum ether:ethyl acetate = 1:0 to 10:1 (TLC: petroleum ether:ethyl acetate = 10:1, Rf = 0.6) to obtain tert-butyl 3-[4-[6-(difluoromethyl)-5-methyl-2-methylsulfanylpyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate (600 mg, yield 37%) as a yellow oil.

[0234] LCMS(ESI+): m / z 412.0(M+H) + ,RT:0.495 minutes (Method H)

[0235] 1 H NMR(400MHz,DMSO-d6)δ ppm 8.61(s,1H),8.26(s,1H),7.11(t,J=53.28Hz,1H),5.30-5.39(m,1H),4.28-4.37(m,2H),4.20(br s,2H),2.57(s,3H),2.44(s,3H),1.41(s,9H).

[0236] Synthesis of tert-butyl 3-[4-[6-(difluoromethyl)-5-methyl-2-methylsulfonylpyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate To a solution of tert-butyl 3-[4-[6-(difluoromethyl)-5-methyl-2-methylsulfanylpyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate (500 mg, 1.22 mmol, 1 equivalent) in DMF (5 mL), Oxon (820 mg, 7.29 mmol, 4 equivalents) was added at 20 °C. The mixture was stirred at 30 °C for 24 hours. LC-MS showed that the reactants were completely consumed and a single main peak with the desired mass was detected. The reaction mixture was poured into water (15 mL). The solid was allowed to precipitate and collected by filtration. The filtration cake was dried under high vacuum to obtain tert-butyl 3-[4-[6-(difluoromethyl)-5-methyl-2-methylsulfonylpyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate (400 mg, yield 74%) as a yellow solid. It was used directly for the next step without purification.

[0237] LCMS (ESI+): m / z 387.9 (M-55) + ,RT:0.422 minutes (Method H)

[0238] 1 H NMR(400MHz,DMSO-d6)δ ppm 8.79(s,1H),8.40(s,1H),7.35(t,J=52.84Hz,1H),5.32-5.42(m,1H),4.29-4.46(m,2H),4.21(br s,2H),3.49(s,3H),2.61(s,3H),1.42(s,9H).

[0239] Synthesis of tert-butyl 3-[4-[6-(difluoromethyl)-2-[(2S,3R)-3-hydroxy-2-methylazetidine-1-yl]-5-methylpyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate To a solution of tert-butyl 3-[4-[6-(difluoromethyl)-5-methyl-2-methylsulfonylpyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate (450 mg, 1.01 mmol, 1 equivalent) in THF (5 mL), TEA (513.40 mg, 5.07 mmol, 706.19 μL, 5 equivalents) and (2S,3R)-2-methylazetidine-3-ol (176.81 mg, 2.03 mmol, 2 equivalents, synthesized as described in R-CSA salt, J.Med.Chem.2020, 63, 13546-13560, intermediate 13 / (R)-CSA salt) were added at 20°C. The mixture was stirred at 60°C for 3 hours. LC-MS showed that the starting material was completely consumed and one main peak with the desired mass was detected. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (20 mL x 3). The mixed organic layer was washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain tert-butyl 3-[4-[6-(difluoromethyl)-2-[(2S,3R)-3-hydroxy-2-methylazetidine-1-yl]-5-methylpyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate (400 mg, 887.94 μmol, yield 87.50%) as a white solid. This was used directly for the next step without purification.

[0240] LCMS(ESI+): m / z 451.1(M+H) + ,RT:0.463 minutes (Method H)

[0241] 1 H NMR(400MHz,DMSO-d6)δ ppm 8.45(s,1H),8.15(s,1H),6.90(t,J=53.72Hz,1H),5.28-5.47(m,1H),4.31(br t,J=8.50Hz,2H),4.16(br dd,J=8.50,6.38Hz,3H),4.05 (td,J=10.98,5.07Hz,2H),3.61(dd,J=8.76,4.88Hz,1H),3.49(s,1H),2.33(s,3H),1.44-1.49(m,3H),1.41(s,9H).

[0242] Synthesis of (2S,3R)-1-[4-[1-(azetidine-3-yl)pyrazole-4-yl]-6-(difluoromethyl)-5-methylpyrimidine-2-yl]-2-methylazetidine-3-ol A35 380 mg, 843.54 μmol, 1 equivalent] tert-butyl 3-[4-[6-(difluoromethyl)-2-[(2S,3R)-3-hydroxy-2-methylazetidine-1-yl]-5-methylpyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate (380 mg, 843.54 μmol, 1 equivalent) and 1 mL of TFA were added to DCM (5 mL) at 0°C. The mixture was stirred at 20°C for 1 hour. LC-MS showed that the starting material was completely consumed and one main peak with the desired mass was detected. The reaction mixture was concentrated under vacuum to obtain (2S,3R)-1-[4-[1-(azetidine-3-yl)pyrazole-4-yl]-6-(difluoromethyl)-5-methylpyrimidine-2-yl]-2-methylazetidine-3-ol (250 mg, yield 85%) as a colorless oil.

[0243] LCMS(ESI+): m / z 351.0(M+H) + ,RT:0.281 minutes (Method H)

[0244] 1 H NMR (400MHz, methanol-d4) δ8.31(s,1H),8.14(s,1H),6.58(t,J=54.3Hz,1H),5.36(quin,J=7.5Hz,1H),4.28(dd,J=6. 1,8.9Hz,1H),4.21-4.11(m,4H),4.01-3.93(m,2H),3.73(dd,J=4.9,9.0Hz,1H),2.42(s,3H),1.55(d,J=6.1Hz,3H)

[0245] Synthesis of (2S,3R)-1-[4-(difluoromethyl)-5-methyl-6-[1-(1-methylazetidine-3-yl)pyrazole-4-yl]pyrimidine-2-yl]-2-methylazetidine-3-ol A21 To a solution of (2S,3R)-1-[4-[1-(azetidine-3-yl)pyrazole-4-yl]-6-(difluoromethyl)-5-methylpyrimidine-2-yl]-2-methylazetidine-3-ol (250 mg, 710.48 μmol, 1 equivalent) in DCM (10 mL), NaBH(OAc)3 (752.81 mg, 3.55 mmol, 5 equivalents) and formaldehyde (61.02 mg, 2.13 mmol, 30%, 1 equivalent) were added. The mixture was stirred at 20°C for 1 hour. LC-MS showed that the starting material was completely consumed and a single main peak with the desired mass was detected. The reaction mixture was concentrated under vacuum. The residue was purified by preparative HPLC (TFA conditions, column: Welch Xtimate C18 150×25mm×5μm, mobile phase: [water (TFA)-ACN], gradient: 15%~35%B, 2 minutes) to obtain (2S,3R)-1-[4-(difluoromethyl)-5-methyl-6-[1-(1-methylazetidine-3-yl)pyrazole-4-yl]pyrimidine-2-yl]-2-methylazetidine-3-ol (104 mg, yield 32%) as a white solid.

[0246] LCMS(ESI+): m / z 365.3(M+H) + ,RT:1.783 minutes (Method F)

[0247] 1 H NMR (400MHz, methanol-d4) δ ppm8.28(d,J=12.38Hz,2H),6.58(t,J=54.34Hz,1H),5.47(br t,J=6.75Hz,1H),4.35-4.84(m,4H),4.27(dd,J=8.82,6.44Hz,1H),4.09-4.23(m,2H),3.72(dd,J=8.94,4.94Hz,1H),3.13(br d,J=1.50Hz,3H),2.41(s,3H),1.55(d,J=6.25Hz,3H).

[0248] Example 6: Synthesis of HCl salt of A21 Free A21 base was dissolved at 50°C in 2.1 mL (10 vols) methanol, 2.1 mL (10 vols) THF, and 4.2 mL (20 vols) acetone. The solution was then divided into six HPLC vials, each containing approximately 30 mg of A21. To the approximately 30 mg of A21 in the solution at 50°C, 1.1 molar equivalents of HCl (91 μL of 1 M HCl in THF) were added. The sample was stirred at 50°C for 1 hour, then cooled overnight to 5°C at 0.1°C / min. After cooling to 5°C, the resulting clear solution was left uncapped and evaporated at room temperature. The A21 HCl salt suspension was filtered using PE frit and positive pressure, and the solid was dried under suction. From 1 g of free A21 base, the A21 HCl salt was isolated from 5 vols methanol using 0.45 μm PTFE filter paper, yielding 86.1%.

[0249] Figure 1 shows a comparison of the XPRD diffraction patterns of A21 free base and A21 HCl salt.

[0250] Figure 2 shows the A21 free base and its HCl salt (DMSO-d6). 1 A comparison of 1H NMR spectra is shown. A summary of the peaks is provided below.

[0251] A21 free base: 6H peak at 2.33 ppm, A21 HCl salt: 2×3H peaks at 2.34 and 2.96 ppm.

[0252] A21 free base: 2H peaks at 3.42 ppm and 3.71 ppm; A21 HCl salt: 2H peaks at 4.41 ppm and 4.59 ppm.

[0253] A21 free base: 1H peak at 5.07 ppm, A21 HCl salt: 1H peak at 5.48 ppm.

[0254] Example 7: Synthesis of Compound A41 [ka] Synthesis of ethyl 2-ethyl-4,4,4-trifluoro-3-oxobutanoate Ethyl butanoate (10.79 g, 92.91 mmol, 12.40 mL, 2.2 equivalents) was added to a dry three-necked flask and cooled to 0°C. Then EtONa (3.16 g, 46.45 mmol, 1.1 equivalents) was added. The mixture was stirred at 0-5°C for 1 hour. Then CF3COOEt (6 g, 42.23 mmol, 5.80 mL, 1 equivalent) was added dropwise. The mixture was stirred at 65°C for 12 hours. TLC showed that most of the starting material had been consumed and one major new spot with greater polarity was detected. The reaction mixture was adjusted to pH=2 with 3N HCl (100 mL) and extracted with DCM (100 mL x 3). The mixed organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain ethyl 2-ethyl-4,4,4-trifluoro-3-oxobutanoate (3.5 g, yield 39%) as a colorless oil.

[0255] 1 ¹H NMR (400MHz, chloroform-d): δ = 4.27-4.18 (m, 2H), 3.80-3.69 (m, 1H), 2.02 (quin, J=7.4Hz, 2H), 1.30-1.23 (m, 3H), 1.02-0.97 (m, 3H)

[0256] Synthesis of 5-ethyl-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine-4-ol To a solution of 2-methylisothiourea sulfate (6.56 g, 23.57 mmol, 1 equivalent) in EtOH (25 mL), Na2CO3 (1 M, 54.20 mL, 2.3 equivalents) was added. The mixture was stirred at 25°C for 30 minutes. Then, 2-ethyl-4,4,4-trifluoro-3-oxo-butanoate (5 g, 23.57 mmol, 1 equivalent) was added. The mixture was stirred at 25°C for 12 hours. LC-MS showed that the starting material was completely consumed and the desired mass was detected. The reaction mixture was extracted with ethyl acetate (50 mL × 3). The mixed organic layer was washed with aqueous NaCl solution (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain 5-ethyl-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine-4-ol (4.5 g, yield 80%) as a yellow oil.

[0257] LCMS(ESI+): m / z 238.9(M+H) + ,RT:0.477 minutes (Method D)

[0258] Synthesis of 4-chloro-5-ethyl-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine To a solution of 5-ethyl-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine-4-ol (25 g, 104.94 mmol, 1 equivalent) in ACN (250 mL), POCl3 (160.91 g, 1.05 mol, 97.82 mL, 10 equivalents) was added. The mixture was stirred at 80°C for 5 hours. LC-MS showed that the starting material was completely consumed and the desired mass was detected. The reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was diluted with H2O (500 mL) and extracted with ethyl acetate (200 mL x 3). The mixed organic layer was dried over Na2SO4, filtered, concentrated under reduced pressure to obtain the residue. The residue was purified by flash silica gel chromatography (ISCO®, 120 g SepaFlash® silica flash column, 0-5% ethyl acetate / petroleum ether eluate, gradient 80 mL / min) to obtain 4-chloro-5-ethyl-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine (25 g, yield 93%) as a colorless oil.

[0259] LCMS(ESI+): m / z 257.0(M+H) + ,RT:0.645 minutes (Method D)

[0260] 1 ¹H NMR (400MHz, chloroform-d): δ=2.85 (q, J=7.3Hz, 2H), 2.59 (s, 3H), 1.24 (t, J=7.5Hz, 3H)

[0261] Synthesis of tert-butyl 4-(2-(4-(5-ethyl-2-(methylthio)-6-(trifluoromethyl)pyrimidine-4-yl)-1H-pyrazole-1-yl)acetyl)piperazine-1-carboxylate: A mixture of 4-chloro-5-ethyl-2-methylsulfanyl-6-(trifluoromethyl)pyrimidine (1 g, 3.90 mmol, 1 equivalent), tert-butyl 4-[2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole-1-yl]acetyl]piperazine-1-carboxylate (3.28 g, 7.79 mmol, 2 equivalents), DTBPF PdCl2 (126.96 mg, 194.80 μmol, 0.05 equivalents), and Na2CO3 (2.06 g, 19.48 mmol, 5 equivalents) in dioxane (10 mL) and H2O (1 mL) was degassed, purged three times with N2, and then stirred at 80°C for 2 hours under N2 atmosphere. LCMS showed that the starting material was consumed and the desired mass was detected. The reaction mixture was quenched by adding water (50 mL) and extracted with ethyl acetate (20 mL x 3). The mixed organic layer was washed with brine (10 mL x 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 3:1) to obtain tert-butyl 4-(2-(4-(5-ethyl-2-(methylthio)-6-(trifluoromethyl)pyrimidine-4-yl)-1H-pyrazole-1-yl)acetyl)piperazine-1-carboxylate (800 mg, yield 39%) as a white solid.

[0262] 1 H NMR(400MHz,DMSO-d6,)δ 7.91(s,1H),7.59(s,1H),5.16(s,2H),3.54-3.32(m,8H),2.82-2.66(m,2H),2.46(s,3H),1.42(s,9H),1.25-1.15(m,3H).

[0263] Synthesis of tert-butyl 4-(2-(4-(5-ethyl-2-(methylsulfonyl)-6-(trifluoromethyl)pyrimidine-4-yl)-1H-pyrazole-1-yl)acetyl)piperazine-1-carboxylate: To a solution of tert-butyl 4-(2-(4-(5-ethyl-2-(methylthio)-6-(trifluoromethyl)pyrimidine-4-yl)-1H-pyrazole-1-yl)acetyl)piperazine-1-carboxylate (800 mg, 1.02 mmol, 1 equivalent) in DMF (8 mL), Oxone (1.88 g, 3.05 mmol, 3 equivalents) was added. The mixture was stirred at 25°C for 12 hours. LC-MS showed that the starting material was completely consumed and the desired mass was detected. The reaction mixture was quenched by adding water (10 mL) and extracted with ethyl acetate (12 mL × 3). The mixed organic layer was washed with brine (10 mL × 7), dried over Na₂SO₄, filtered, concentrated under reduced pressure to obtain the residue. The residue was purified by flash silica gel chromatography (ISCO®, 4g SepaFlash® silica flash column, 70-80% ethyl acetate / petroleum ether eluate, gradient 40 mL / min) to obtain tert-butyl 4-(2-(4-(5-ethyl-2-(methylsulfonyl)-6-(trifluoromethyl)pyrimidine-4-yl)-1H-pyrazole-1-yl)acetyl)piperazine-1-carboxylate (600 mg, yield 71%) as a white solid.

[0264] 1 H NMR(400MHz,DMSO-d6)δ 8.66(s,1H),8.29(s,1H),5.35(s,2H),3.44(br s,11H),3.15-2.98(m,2H),1.44-1.42(m,9H),1.28-1.21(m,3H)

[0265] tert-butyl-4-[2-[4-[2-[(2R)-2-(difluoromethyl)azetidine-1-yl]-5-ethyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate: To a solution of (2R)-2-(difluoromethyl)azetidine (CAS#2231665-58-8, 15.20 mg, 105.88 μmol, 1 equivalent, HCl salt) and tert-butyl 4-(2-(4-(5-ethyl-2-(methylsulfonyl)-6-(trifluoromethyl)pyrimidine-4-yl)-1H-pyrazole-1-yl)acetyl)piperazine-1-carboxylate (50 mg, 105.88 μmol, 1 equivalent) in ACN (1 mL), DIEA (68.42 mg, 529.38 μmol, 92.21 μL, 5 equivalents) and CsF (32.17 mg, 211.75 μmol, 7.82 μL, 2 equivalents) were added. The mixture was stirred at 130°C for 12 hours. Five additional vials of 50 mg scale were prepared as described above. LC-MS showed that the starting material was completely consumed and a single main peak with the desired m / z was detected. The reaction mixture was quenched by adding water (30 mL) and extracted with ethyl acetate (10 mL x 3). The mixed organic layer was washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain tert-butyl 4-[2-[4-[2-[(2R)-2-(difluoromethyl)azetidine-1-yl]-5-ethyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate (155 mg, yield 49%), which was used directly in the next step.

[0266] 1 H NMR(400MHz,DMSO-d6)δ 8.39-8.35(m,1H),8.08(s,1H),6.70-6.18(m,1H),5.28(s,2H),4.08-3.96(m,2H),3. 54-3.34(m,8H),2.87-2.76(m,2H),2.45-2.37(m,3H),1.42(s,9H),1.28-1.22(m,3H)

[0267] Synthesis of (R)-2-(4-(2-(2-(difluoromethyl)azetidine-1-yl)-5-ethyl-6-(trifluoromethyl)pyrimidine-4-yl)-1H-pyrazole-1-yl)-1-(piperazin-1-yl)ethane-1-one: A solution of tert-butyl 4-[2-[4-[2-[(2R)-2-(difluoromethyl)azetidine-1-yl]-5-ethyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]acetyl]piperazine-1-carboxylate (106 mg, 184.81 μmol, 1 equivalent) in dichloromethane (1 mL) and TFA (0.2 mL) was stirred at 25°C for 1 hour. LC-MS showed that the starting material was completely consumed and one main peak with the desired m / z was detected. The reaction mixture was adjusted to pH 7-8 with aqueous NaHCO3 and extracted with ethyl acetate (5 mL x 3). The mixed organic layer was washed with brine (5 mL), dried over Na2SO4, filtered, concentrated under reduced pressure to obtain the residue. The residue was purified by preparative HPLC (neutral conditions: column: Waters Xbridge BEH C18 100×30mm×10μm, mobile phase: [H2O (10mM NH4HCO3)-ACN], gradient: 35%~65%B for 8.0 minutes) to obtain 2-[4-[2-[(2R)-2-(difluoromethyl)azetidine-1-yl]-5-ethyl-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]-1-piperazine-1-yl-ethanone (40.1 mg, yield 45%) as a white solid.

[0268] 1H NMR(400MHz,CD3OD)δ 8.30(s,1H),8.12(s,1H),6.49-6.16(m,1H),5.25(s,2H),4.76-4.58(m,1H),4.17-4.02(m,2H),3.65-3.5 1(m,4H),2.98-2.86(m,4H),2.85-2.80(m,2H),2.65-2.51(m,1H),2.49-2.37(m,1H),1.21(t,J=7.4Hz,3H)

[0269] Example 8: Synthesis of Compound A46 [ka] Synthesis of 4-(difluoromethyl)-5-methyl-2-(methylthio)-6-((trimethylsilyl)ethynyl)pyrimidine To a solution of 4-chloro-6-(difluoromethyl)-5-methyl-2-methylsulfanylpyrimidine (1.4 g, 1 equivalent) in THF (14 mL), ethynyl(trimethyl)silane (1.35 g, 2.2 equivalents), TEA (1.89 g, 3 equivalents), Pd(PPh3)2Cl2 (62.49 mg, 89.02 μmol, 0.1 equivalent), and CuI (16.95 mg, 89.02 μmol, 0.1 equivalent) were added. The mixture was stirred at 25°C for 12 hours. Several new peaks were shown by LC-MS, and 51% of the desired compound was detected. The reaction mixture was diluted with water (10 mL) and extracted with EA (5 mL × 2). The mixed organic layer was washed with aqueous NaCl solution (5 mL × 2), dried over Na2SO4, filtered, concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 0~95 / 5) to obtain 4-(difluoromethyl)-5-methyl-2-(methylthio)-6-((trimethylsilyl)ethynyl)pyrimidine (0.7g, yield 32%) as a black oil.

[0270] LCMS(ESI+): m / z 287.2(M+H) + ,RT:2.629 minutes (Method F)

[0271] Synthesis of 4-(difluoromethyl)-6-ethynyl-5-methyl-2-(methylthio)pyrimidine To a solution of 4-(difluoromethyl)-5-methyl-2-(methylthio)-6-((trimethylsilyl)ethynyl)pyrimidine (0.5 g, 1.75 mmol, 1 equivalent) in THF (1 mL), KF (116.63 mg, 2.01 mmol, 47.03 μL, 1.15 equivalents) was added. The mixture was stirred at 25°C for 12 hours. Several new peaks were shown by LC-MS, and 24% of the desired compound was detected. The reaction mixture was diluted with water (5 mL) and extracted with EA (5 mL x 2). The mixed organic layer was washed with aqueous NaCl solution (5 mL x 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by preparative TLC (SiO2, PE:MTBE=3:1) to obtain 4-(difluoromethyl)-6-ethynyl-5-methyl-2-(methylthio)pyrimidine (0.12 g, yield 32%) as a dark brown solid.

[0272] LCMS(ESI+): m / z 215.1(M+H) + ,RT:0.483 minutes (Method D)

[0273] Synthesis of tert-butyl 3-(4-(6-(difluoromethyl)-5-methyl-2-(methylthio)pyrimidine-4-yl)-1H-1,2,3-triazole-1-yl)azetidine-1-carboxylate To a solution of 4-(difluoromethyl)-6-ethynyl-5-methyl-2-(methylthio)pyrimidine (0.1 g, 466.78 μmol, 1 equivalent) in DMF (0.9 mL) and MeOH (0.1 mL), CuI (888.98 μg, 4.67 μmol, 0.01 equivalent) and tert-butyl 3-azido-azetidine-1-carboxylate (92.53 mg, 466.78 μmol, 1 equivalent) were added. The mixture was stirred at 100 °C for 12 hours. Several new peaks were shown on LC-MS, and 80% of the desired compound was detected. The reaction mixture was diluted with H2O (3 mL) and extracted with 6 mL of EA (3 mL x 2). The mixed organic layer was washed with aqueous NaCl solution (5 mL x 2), dried over Na2SO4, filtered, concentrated under reduced pressure to obtain the residue. The residue was purified by preparative TLC (SiO2, PE:EA = 1:1) to obtain tert-butyl 3-(4-(6-(difluoromethyl)-5-methyl-2-(methylthio)pyrimidine-4-yl)-1H-1,2,3-triazole-1-yl)azetidine-1-carboxylate (0.14 g, yield 73%) as a brown solid.

[0274] LCMS(ESI+): m / z 413.4(M+H)+, RT: 0.550 min (Method R)

[0275] Synthesis of tert-butyl 3-(4-(6-(difluoromethyl)-5-methyl-2-(methylsulfonyl)pyrimidine-4-yl)-1H-1,2,3-triazole-1-yl)azetidine-1-carboxylate To a solution of tert-butyl 3-(4-(6-(difluoromethyl)-5-methyl-2-(methylthio)pyrimidine-4-yl)-1H-1,2,3-triazole-1-yl)azetidine-1-carboxylate (0.09 g, 218.20 μmol, 1 equivalent) in DMF (0.5 mL), Oxon (402.43 mg, 654.61 μmol, 3 equivalents) was added. The mixture was stirred at 25°C for 12 hours. LC-MS showed that 90% of the desired compound was detected. The reaction mixture was filtered, and the filtrate was concentrated under high vacuum at 40°C to obtain tert-butyl 3-(4-(6-(difluoromethyl)-5-methyl-2-(methylsulfonyl)pyrimidine-4-yl)-1H-1,2,3-triazole-1-yl)azetidine-1-carboxylate (0.07 g, yield 72%), which was used in the next step without further purification.

[0276] LCMS(ESI+): m / z 467.2(M+Na)+, RT: 0.456 min (Method D)

[0277] Synthesis of tert-butyl 3-(4-(6-(difluoromethyl)-2-((2S,3R)-3-hydroxy-2-methylazetidine-1-yl)-5-methylpyrimidine-4-yl)-1H-1,2,3-triazole-1-yl)azetidine-1-carboxylate To a solution of tert-butyl 3-(4-(6-(difluoromethyl)-5-methyl-2-(methylsulfonyl)pyrimidine-4-yl)-1H-1,2,3-triazole-1-yl)azetidine-1-carboxylate (0.06 g, 135.00 μmol, 1 equivalent) in THF (1 mL), K2CO3 (37.31 mg, 269.99 μmol, 2 equivalents) and (2S,3R)-2-methylazetidine-3-ol (39.20 mg, 135.00 μmol, 1 equivalent) were added. The mixture was stirred at 60°C for 12 hours. LC-MS showed that 77% of the desired compound was detected. The reaction mixture was diluted with EA (2 mL) and extracted with H2O (2.5 mL x 2). The organic layer was washed with two 5 mL NaCl aqueous solutions, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain tert-butyl 3-(4-(6-(difluoromethyl)-2-((2S,3R)-3-hydroxy-2-methylazetidine-1-yl)-5-methylpyrimidine-4-yl)-1H-1,2,3-triazole-1-yl)azetidine-1-carboxylate (0.09 g, yield 74%), which was used in the next step without further purification.

[0278] LCMS(ESI+): m / z 452.4(M+H)+, RT: 0.498 min (Method R)

[0279] Synthesis of (2S,3R)-1-(4-(1-(azetidine-3-yl)-1H-1,2,3-triazole-4-yl)-6-(difluoromethyl)-5-methylpyrimidine-2-yl)-2-methylazetidine-3-ol A mixture of tert-butyl 3-(4-(6-(difluoromethyl)-2-((2S,3R)-3-hydroxy-2-methylazetidine-1-yl)-5-methylpyrimidine-4-yl)-1H-1,2,3-triazole-1-yl)azetidine-1-carboxylate (0.08 g, 177.20 μmol, 1 equivalent) in DCM (0.5 mL) and TFA (0.5 mL) was stirred at 25°C for 12 hours. LC-MS showed that 83% of the desired compound was detected. The mixture was concentrated under high vacuum to obtain (2S,3R)-1-(4-(1-(azetidine-3-yl)-1H-1,2,3-triazole-4-yl)-6-(difluoromethyl)-5-methylpyrimidine-2-yl)-2-methylazetidine-3-ol (0.075 g, yield 96%), which was used in the next step without further purification.

[0280] LCMS(ESI+): m / z 352.3(M+H)+, RT: 0.304 min (Method D)

[0281] Synthesis of (2S,3R)-1-(4-(difluoromethyl)-5-methyl-6-(1-(1-methylazetidine-3-yl)-1H-1,2,3-triazole-4-yl)pyrimidine-2-yl)-2-methylazetidine-3-ol To a solution of (2S,3R)-1-(4-(1-(azetidine-3-yl)-1H-1,2,3-triazole-4-yl)-6-(difluoromethyl)-5-methylpyrimidine-2-yl)-2-methylazetidine-3-ol (0.02 g, 56.92 μmol, 1 equivalent) in DMF (0.4 mL), NaBH(OAc)3 (60.32 mg, 284.61 μmol, 5 equivalents) and formaldehyde (5.70 mg, 56.92 μmol, 5.23 μL, 30% purity, 1 equivalent) were added. The mixture was stirred at 25°C for 12 hours. LC-MS showed that the desired compound was detected. The reaction product was dried at 40°C under high vacuum to obtain the residue, which was purified by preparative HPLC (TFA conditions) to obtain (2S,3R)-1-(4-(difluoromethyl)-5-methyl-6-(1-(1-methylazetidine-3-yl)-1H-1,2,3-triazole-4-yl)pyrimidine-2-yl)-2-methylazetidine-3-ol (13 mg, yield 62%) as a white solid.

[0282] LCMS(ESI+):m / z 366.1(M+H)+,RT:1.823min Method E

[0283] 1 H NMR (400MHz, methanol-d4) δ1.56(d,J=6.11Hz,3H),2.63(s,3H),3.17(s,3H),3.75(dd,J=9.1 1,4.83Hz,1H),4.11-4.24(m,2H),4.30(dd,J=9.05,6.24Hz,1H),4.71-4.85(m,4H),5.75(b rt,J=6.97Hz,1H),6.45-6.95(m,1H),8.40-8.77(m,1H)

[0284] Example 9: Synthesis of compounds A31 and A25 [ka] Synthesis of (E)-2,2,2-trifluoro-N-[(4-methoxyphenyl)methyl]ethanimine To a solution of 1-ethoxy-2,2,2-trifluoroethanol (5 g, 34.70 mmol, 4.10 mL, 1 equivalent) in toluene (50 mL), (4-methoxyphenyl)methaneamine (3.81 g, 27.76 mmol, 3.60 mL, 0.8 equivalents) was added. The reaction mixture was stirred at 110 °C for 36 hours. TLC showed that the desired product was detected. The reaction mixture was concentrated under reduced pressure to obtain (E)-2,2,2-trifluoro-N-[(4-methoxyphenyl)methyl]ethanimine (7.3 g, 97% yield) as a colorless oil, which was used directly in the next reaction.

[0285] ¹H NMR (400 MHz, chloroform-d) δ ppm 7.57-7.62 (m, ¹H) 7.16-7.22 (m, ²H) 6.89-6.95 (m, ²H) 4.78 (s, ²H) 3.82-3.83 (m, ³H)

[0286] Synthesis of (rac-3S,4R)-3-(benzyloxy)-1-(4-methoxybenzyl)-4-(trifluoromethyl)azetidine-2one To a solution of (E)-2,2,2-trifluoro-N-[(4-methoxyphenyl)methyl]ethanimine (7 g, 32.23 mmol, 1 equivalent) and 2-benzyloxyacetyl chloride (23.80 g, 128.92 mmol, 20.02 mL, 4 equivalents) in DCM (70 mL), TEA (16.31 g, 161.15 mmol, 22.43 mL, 5 equivalents) was added. The reaction mixture was stirred at 40°C for 48 hours. TLC showed that the desired product was detected. The reaction mixture was quenched by adding water (50 mL) and extracted with DCM (70 mL x 3). The mixed organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by preparative HPLC (column: Phenomenex luna C18 (250 × 70 mm, 15 μm), mobile phase: [H2O (0.1% TFA)-ACN], gradient: 53%~73% B, 20.0 min) to obtain (rac-3S,4R)-3-(benzyloxy)-1-(4-methoxybenzyl)-4-(trifluoromethyl)azetidine-2-one (0.5 g, 4% yield).

[0287] LCMS(ESI+): m / z 366.1(M+1) + ,RT:8.215 minutes (Method R)

[0288] Synthesis of (rac-2R,3R)-3-(benzyloxy)-1-(4-methoxybenzyl)-2-(trifluoromethyl)azetidine To a solution of AlCl3 (437.96 mg, 3.28 mmol, 179.49 μL, 3 equivalents) in THF (4 mL), LAH (2.5 M, 1.31 mL, 3 equivalents) was added at 0°C for 10 minutes. The reaction mixture was stirred at 40°C for 30 minutes. (rac-3S,4R)-3-benzyloxy-1-[(4-methoxyphenyl)methyl]-4-(trifluoromethyl)azetidine-2-one (400 mg, 1.09 mmol, 1 equivalent) was added to the reaction mixture. The reaction mixture was stirred at 20°C for 3 hours. TLC showed that the desired product was detected. The reaction mixture was quenched with H2O (10 mL) at 0°C and extracted with DCM (10 mL x 3). The mixed organic layer was dried over Na2SO4, filtered, concentrated under reduced pressure to obtain the residue. The residue was purified by preparative TLC (TLC: petroleum ether: ethyl acetate = 1:1, Rf = 0.4) to obtain (rac-2R,3R)-3-(benzyloxy)-1-(4-methoxybenzyl)-2-(trifluoromethyl)azetidine (0.28 g, yield 73%) as a colorless oil.

[0289] LCMS(ESI+): m / z 352.1(M+1) + ,RT:0.956 minutes (Method G)

[0290] Synthesis of (rac-2R,3R)-2-(trifluoromethyl)azetidine-3-ol Pd(OH)2 / C (562.38 mg, 739.98 μmol, 20% purity, 1 equivalent) was added to a solution of (rac-2R,3R)-3-(benzyloxy)-1-(4-methoxybenzyl)-2-(trifluoromethyl)azetidine (260 mg, 739.98 μmol, 1 equivalent) and HCl / MeOH (0.01 mL) in MeOH (13 mL). The reaction mixture was stirred at 20 °C for 12 hours under H2 (15 psi). LC-MS showed that the desired product was detected. The obtained product was dissolved in MeOH (30 mL) and filtered. The filtrate was concentrated to obtain (rac-2R,3R)-2-(trifluoromethyl)azetidine-3-ol (100 mg, 708.76 μmol, yield 76%, HCl salt) as a colorless oil.

[0291] 1 ¹H NMR (400MHz, acetonitrile-d3) δ ppm 4.70-5.05 (m, 2H) 4.11-4.23 (m, 1H) 4.00 (br dd, J=10.45, 7.03Hz, 1H)

[0292] Synthesis of tert-butyl 3-[4-[2-chloro-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate To a solution of tert-butyl 3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole-1-yl]azetidine-1-carboxylate (804.77 mg, 2.30 mmol, 1 equivalent) and 2,4-dichloro-6-(trifluoromethyl)pyrimidine (500 mg, 2.30 mmol, 500.00 μL, 1 equivalent) in dioxane (5 mL) and H2O (0.5 mL), DTBPF PdCl2 (150.19 mg, 230.44 μmol, 0.1 equivalent) and Na2CO3 (488.48 mg, 4.61 mmol, 2 equivalents) were added. The reaction mixture was stirred at 80°C for 2 hours. TLC showed that the desired product was detected. The reaction mixture was diluted with H2O (10 mL) and extracted with ethyl acetate (20 mL x 3). The mixed organic layers were dried over Na2SO4, filtered, and concentrated to obtain the residue. The residue was purified by preparative TLC (TLC: petroleum ether: ethyl acetate = 1:2, Rf = 0.3) to obtain tert-butyl 3-[4-[2-chloro-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate (0.3 g, yield 32%) as a colorless oil.

[0293] LCMS(ESI+): m / z 348.0 (M-55) + ,RT:0.591 minutes (Method D)

[0294] Synthesis of tert-butyl 3-(4-(2-((rac-2R,3R)-3-hydroxy-2-(trifluoromethyl)azetidine-1-yl)-6-(trifluoromethyl)pyrimidine-4-yl)-1H-pyrazole-1-yl)azetidine-1-carboxylate To a solution of (rac-2R,3R)-2-(trifluoromethyl)azetidine-3-ol (48.92 mg, 346.72 μmol, 1 equivalent) and tert-butyl 3-[4-[2-chloro-6-(trifluoromethyl)pyrimidine-4-yl]pyrazole-1-yl]azetidine-1-carboxylate (140 mg, 346.72 μmol, 1 equivalent) in THF (1.4 mL), TEA (140.34 mg, 1.39 mmol, 193.03 μL, 4 equivalents) was added. The reaction mixture was stirred at 80°C for 12 hours. TLC showed that the desired product was detected. The reaction mixture was diluted with H2O (10 mL) and extracted with ethyl acetate (20 mL x 3). The mixed organic layer was dried over Na2SO4, filtered, concentrated, and purified by preparative TLC (TLC: petroleum ether: ethyl acetate = 1:1, Rf = 0.4) to obtain a residue, from which tert-butyl 3-(4-(2-((rac-2R,3R)-3-hydroxy-2-(trifluoromethyl)azetidine-1-yl)-6-(trifluoromethyl)pyrimidine-4-yl)-1H-pyrazole-1-yl)azetidine-1-carboxylate (91 mg, yield 52%) was obtained as a colorless oil.

[0295] LCMS(ESI+): m / z 509(M+H) + ,RT:8.48 minutes (Method R)

[0296] Synthesis of (rac-2R,3R)-1-(4-(1-(azetidine-3-yl)-1H-pyrazole-4-yl)-6-(trifluoromethyl)pyrimidine-2-yl)-2-(trifluoromethyl)azetidine-3-ol A31 A solution of tert-butyl3-(4-(2-((rac-2R,3R)-3-hydroxy-2-(trifluoromethyl)azetidine-1-yl)-6-(trifluoromethyl)pyrimidine-4-yl)-1H-pyrazole-1-yl)azetidine-1-carboxylate (90 mg, 177.02 μmol, 1 equivalent) in TFA (0.3 mL) and DCM (1.5 mL) was stirred at 20°C for 0.5 hours. TLC showed that the desired product was detected. The reaction product was concentrated to obtain (rac-2R,3R)-1-(4-(1-(azetidine-3-yl)-1H-pyrazole-4-yl)-6-(trifluoromethyl)pyrimidine-2-yl)-2-(trifluoromethyl)azetidine-3-ol (70 mg, yield 97%) as a colorless oil.

[0297] LCMS(ESI+): m / z 409.1(M+1) + ,RT:0.373 minutes (Method D)

[0298] Synthesis of (rac-2R,3R)-1-(4-(1-(1-methylazetidine-3-yl)-1H-pyrazole-4-yl)-6-(trifluoromethyl)pyrimidine-2-yl)-2-(trifluoromethyl)azetidine-3-ol A25 To a solution of (rac-2R,3R)-1-(4-(1-(azetidine-3-yl)-1H-pyrazole-4-yl)-6-(trifluoromethyl)pyrimidine-2-yl)-2-(trifluoromethyl)azetidine-3-ol (60 mg, 146.95 μmol, 1 equivalent) in DCM (1.2 mL), NaBH(OAc)3 (93.43 mg, 440.85 μmol, 3 equivalents) and formaldehyde (11.93 mg, 146.95 μmol, 10.94 μL, 37% purity, 1 equivalent) were added. The reaction mixture was stirred at 25°C for 1 hour. TLC showed that the desired product was detected. The reaction mixture was concentrated to obtain the residue. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 75×30mm *3um, mobile phase: [H2O(0.1%TFA)-ACN], gradient: 20%~40%B over 8.0 minutes) to obtain (rac-2R,3R)-1-(4-(1-(1-methylazetidine-3-yl)-1H-pyrazole-4-yl)-6-(trifluoromethyl)pyrimidine-2-yl)-2-(trifluoromethyl)azetidine-3-ol (40 mg, yield 64%) as a white solid.

[0299] LCMS(ESI+): m / z 423.0 (M+1) + ,RT:2.779 minutes (Method F)

[0300] 1 H NMR(400MHz, methanol-d4)δ ppm8.51(s,1H)8.22(s,1H)7.42(s,1H)5.08(t,J=7.03Hz,1H)4.67-4.73(m,1H)4. 58-4.66(m,1H)4.42(dd,J=9.17,6.72Hz,1H)3.93(dd,J=9.41,4.40Hz,1H)3.86(br t,J=7.95Hz,2H)3.65(br t,J=7.64Hz,2H)2.48(s,3H)

[0301] The following compounds were prepared using appropriate intermediates and reactants, in the same manner as described for the other compounds disclosed herein. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5]

[0302] Testing of compound activity The medicinal utility of the compounds disclosed herein and salts of such compounds in the treatment of the diseases / conditions described herein in mammals (e.g., male or female humans) is demonstrated by the activity and benefits of the compounds disclosed herein in one or more conventional assays and in vivo assays described herein. In vivo assays (with appropriate modifications within the scope of the art) can be used to determine the activity of other drugs as well as the compounds disclosed herein. Therefore, the protocols described herein can also be used to demonstrate the usefulness of combination therapy of the compounds disclosed herein. Assays and models may also demonstrate the benefits of certain other properties, e.g., side effect profiles, half-lives. In addition, such assays provide a means by which the activity of the compounds disclosed herein and salts of such compounds (or other drugs described herein) can be compared with each other and with the activity of other known compounds. The results of these comparisons are useful in determining dose levels in mammals, including humans, for the treatment of such diseases.

[0303] The absorption, distribution, metabolism, and excretion (ADME) and pharmacokinetics (PK) of compounds and exemplary assays are described in the online publication by Thomas DYChung, David B. Terry, and Layton H. Smith, “In Vitro and In Vivo Assessment of ADME and PK Properties During Lead Selection and Lead Optimization—Guidelines, Benchmarks, and Rules of Thumb—”(https: / / www.ncbi.nlm.nih.gov / books / NBK326710 / ).

[0304] Animal models of non-alcoholic fatty liver disease are described in Nutrients, 2017 Oct;9(10):1072;Int.J.Mol.Sci.2022,23,15791 and Digestion 2020;101:522-535. Animal models of fructose inhibition are described in Molecular Metabolism, 2021;48:101196.

[0305] Example 10: KHK Inhibition Assay Human and rat recombinant KHK-A and KHK-C isozymes IC 50 Determining the value

[0306] Assay reagent cocktails were prepared by adjusting the final concentrations of NADH, water, TEA, KCl, MgCl2, PEP, ATP, DTT, and coupling enzymes (pyruvate kinase and lactate dehydrogenase, LDH) as shown in Table B. [Table 6]

[0307] To this, the relevant KHK isozyme was added to a final concentration of 6 nM. Aliquots of each inhibitor compound were diluted by 5-fold serial dilution to produce final concentrations ranging from 1000 nM to 0.064 nM. The inhibitor aliquots were added to an assay reagent cocktail containing KHK with fructose (concentration of 2 mM) in a 96-well plate. Absorbance at 340 nm was measured by spectrophotometer, and inhibition was analyzed using nonlinear regression.

[0308] The assay results are shown in Table C. [Table 7]

[0309] Example 11: Cell-based efficacy assay (HepG2 assay) Testing the inhibitory effect of compounds on human KHK in HepG2 cells

[0310] HepG2 cells were raised to near confluence (approximately 5-8 × 10⁴) in RPMI1640 supplement medium on a 100 mm plate. 6 The cells were grown to the specified size. The growth medium was aspirated, 4 mL of trypsin solution (0.25% (w / v) + 0.25% (w / v) EDTA) was added, and the mixture was incubated at 37°C for 5-10 minutes. The cells were pelleted and resuspended in 2 mL of complete medium.

[0311] Next, the cells were placed in 96-well plates with 20,000 to 50,000 cells per well and grown at 37°C for 20 to 28 hours to allow for adhesion.

[0312] The inhibitor compound was diluted to 50 μM in DMSO, and then serially diluted in MEM medium (ThermoFisher). The final inhibitor concentration ranged from 1000 nM to 0.064 nM (5-fold dilution series).

[0313] The RPMI medium was removed from the plate of confluent cells. The plate was then incubated at 37°C for 35 minutes, after which 0.1 mL of diluted inhibitor compound in MEM, or 0.1 mL of MEM medium without the inhibitor compound or fructose, or without trypsin, was added with fructose to obtain a final concentration of 20 mM fructose. The plate was then incubated at 37°C for 20 minutes, and then placed on ice to stop the reaction.

[0314] Next, 200 μL of chilled 80% (v / v) methanol:water was added to all wells except for the control used to enumerate cell counts. The plates were vortexed and cell debris was removed by centrifugation. The resulting supernatant fraction was analyzed by LC / MS to measure the generated F1P.

[0315] The IC50 data is presented in Table D. [Table 8]

[0316] The structures of compounds C1 to C3 are provided in paragraph

[0321] .

[0317] Example 12: Cell-based efficacy assay (KHK-C overexpression HepG2 assay) Testing the inhibitory effect of compounds on human KHK-C in genetically modified HepG2 cells.

[0318] Manipulated HepG2 cells were generated using methods known to those skilled in the art. Briefly, HepG2 cells were incubated with a lentivirus carrying a transgene encoding human KHK-C and grown under antibiotic selectivity.

[0319] KHK-C overexpressing HepG2 cells were raised to T-182cm 2 Near confluence (approximately 25-40 × 10) in RPMI1640 supplement medium on a flask. 6The cells were grown to the specified size. The growth medium was aspirated, the flask was washed with 10 mL of PBS, 4 mL of trypsin solution (0.25% (w / v) + 0.25% (w / v) EDTA) was added, and the mixture was incubated at 37°C for 3–5 minutes. The cells were pelleted and resuspended in 2 mL of complete medium.

[0320] Next, the cells were placed in 96-well plates at a rate of 20,000 to 40,000 cells per well and grown at 37°C for 20 to 28 hours to allow for adhesion.

[0321] The inhibitor compounds were diluted to 50 μM in DMSO and then serially diluted in MEM medium (ThermoFisher). The final inhibitor concentrations ranged from 5000 nM to 0.32 nM. Control compounds C1, C2, and C3 were tested in comparison to the compounds disclosed herein. [Table 9]

[0322] The RPMI medium was removed from the plate of confluent cells. The plate was then incubated in MEM with 0.1 mL of diluted inhibitor compound at 37°C for 35 minutes. Subsequently, an additional 0.1 mL of diluted inhibitor compound in MEM containing fructose, or 0.1 mL of MEM medium without the inhibitor compound and / or fructose, was added to obtain a final fructose concentration of 30 mM. The plate was then incubated at 37°C for 5 minutes and then placed on ice to stop the reaction.

[0323] Next, 200 μL of chilled 80% (v / v) methanol:water was added to all wells, and the plates were sealed with adhesive and placed in a -80°C freezer for 1 hour. The plates were vortexed, and cell debris was removed by centrifugation. The resulting supernatant fraction was analyzed by LC / MS to measure the generated F1P.

[0324] The IC50 data is presented in Table E. [Table 10]

[0325] Example 13: CYP inhibition assay in human liver microsomes Cytochrome P450 inhibition profiling A potential limitation of novel drugs for metabolic disorders is the risk of drug-drug interactions mediated by the inhibition of cytochrome P450 (CYP) enzymes that contribute to heterologous metabolism, including CYP1A2, CYP2C9, CYP2C8, CYP2D6, and CYP3A4. Frequent concomitant medications in patients with metabolic disorders, type 2 diabetes, obesity, hypertension, and / or MASLD / MASH include numerous substrates, inhibitors, or inducers of CYP enzymes, including HMG-CoA reductase inhibitors (statins), thiazolidinediones, fibrates, sulfonylureas, selective serotonin reuptake inhibitors, and angiotensin II receptor blockers. Total exposure to these drugs can be affected when they are co-administered with CYP enzyme inhibitors. Therefore, maintaining efficacy as a KHK-C and KHK-A inhibitor while lacking meaningful inhibition of CYP enzymes is a desirable characteristic for clinical candidates. The preferred compounds of this disclosure lack beneficial properties, namely, significant CYP inhibition.

[0326] Testing of compound inhibition against CYP enzymes Human liver microsomes (HLMs) were incubated with known substrates for specific CYP enzymes (e.g., phenacetin for CYP1A2, diclofenac for CYP2C9, S-mephenytoin for CYP2C19, dextromethorphan for CYP2D6, and midazolam for CYP3A4). Substrate metabolism to known metabolites was monitored by LC-MS-MS. Positive control inhibitors were tested at single concentrations, and test compounds were tested using a 7-point dose-response curve, starting from a high concentration of 50 μM in an approximately 3-fold dilution series down to a low concentration of 50 nM.

[0327] Each assay contained 0.2 mg / mL of HLM, 1 mM NADPH, the substrate-dependent final concentration of each substrate, and a specific concentration of either a positive control inhibitor or the test compound. The wells were incubated at 37°C for 10 minutes, the reaction was stopped by adding a cold stop solution (e.g., 200 ng / mL of tolbutamide in acetonitrile), and the proteins were precipitated by centrifugation at 4000 rpm for 20 minutes. The supernatant was added to 0.5 volume of HPLC water, shaken for 10 minutes, and analyzed by LC-MS-MS.

[0328] Example 14: hERG inhibition assay using automated patch clamp method A further potential limitation of novel drugs is the risk of cardiotoxicity mediated by binding to or inhibition of cardiac ion channels, including, for example, hERGs (human ether-a-go-go related genes). hERGs are subunits of potassium channels that mediate cardiac repolarization and can be inhibited by a diverse class of small molecules, potentially leading to arrhythmias. Therefore, maximizing the window between the efficacy as an inhibitor of the target enzyme, ketohexokinase, and the efficacy as an inhibitor of hERGs is a desirable feature for clinical candidates. Preferred compounds of this disclosure possess beneficial properties (i.e., no significant hERG inhibition and / or a large window between the efficacy of a KHK inhibitor and the efficacy of an hERG inhibitor).

[0329] Testing of compound inhibition against hERG (human ether-a-go-go related gene) To profile CHO cells that stably express hERG channels using an automated patch-clamp method, approximately 298 mOsm of 10 mM HEPES buffer (pH 7.4) containing 140 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 5 mM glucose was seeded on Nanion SyncroPatch 384PE.

[0330] The positive control (amitriptyline) and the test compound were tested using a 5-point dose-response curve, ranging from a high concentration of 30 μM to a series of approximately 3-fold dilutions, and finally to a low concentration of 300 nM.

[0331] The current was drawn using a voltage command protocol consisting of a continuous holding potential of -80mV, first stepping 80 milliseconds to -50mV for leakage subtraction, then stepping 4,800 milliseconds to open the hERG channel, then stepping 5,000 milliseconds to -50mV to induce the hERG "tail current" measured and collected for analysis, and then stepping 1,000 milliseconds to the holding potential of -80mV. This command protocol was repeated every 20 seconds continuously during the assay (300 seconds before and 300 seconds after the addition of the control or test compound).

[0332] Example 15: Steady-state dynamics A further potential limitation of novel drugs inhibiting ketohexokinase isoforms is the risk that increased concentrations of substrates (e.g., fructose and / or ATP) will reduce the drug's potency due to a competitive mode of inhibition. Inhibition of the ketohexokinase metabolism of fructose to fructose-1-phosphate is expected to increase the concentrations of both ATP and fructose, potentially to a degree that could outperform the novel drug as a competitive inhibitor. Therefore, a non-competitive mode of target enzyme inhibition, i.e., where the potency as an enzyme inhibitor is unaffected by the concentration of any enzyme substrate, could be a desirable feature for clinical candidates. The mode of inhibition can be determined, as is known to those skilled in the art, through a series of steady-state dynamics experiments performed, for example, as described in Copeland, RA, Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacologists (2013), and the potency of a compound as a ketohexokinase inhibitor is determined over a range of substrate concentrations, i.e., changes in ATP concentration above fructose, or changes in fructose concentration above ATP. Preferred compounds of this disclosure have beneficial properties with respect to the mode of inhibition, thereby increasing ATP and / or fructose concentrations does not affect the potency of the compound as a ketohexokinase inhibitor.

[0333] All references provided herein are incorporated herein in their entirety by reference. Where used herein, all abbreviations, symbols, and notations are consistent with those used in modern scientific literature. See, for example, Janet S. Dodd, ed., *The ACS Style Guide: A Manual for Authors and Editors*, 2nd Ed., Washington, DC: American Chemical Society, 1997.

[0334] This disclosure is described in conjunction with its detailed description, but it should be understood that the foregoing description is intended to illustrate, and not limit, the scope of this disclosure as defined by the attached claims. Other aspects, advantages, and modifications are within the following claims.

Claims

1. A compound having the structure of formula I, or a pharmaceutically acceptable salt thereof, 【Chemistry 1】 During the ceremony, R １ However, it is either H or OH, R ２ However, C １－６ Alkyl or C １－６ It is a haloalkyl, R ３ However, C １－６ Alkyl or C １－６ It is a haloalkyl, R ４ is H, halo, CN, C １－６ alkyl, C １－６ alkoxy, or C ３－５ cycloalkyl, and A is a five-membered heteroaryl compound containing two to three nitrogen ring atoms. X is combined or C １－６ It is alkylene-C(O), R ５ However, it is a 4-6 membered heterocycloalkyl having one or two ring nitrogen atoms, and the heterocycloalkyl has one or two C １－６ It is optionally substituted with alkyl, However, R ４ If H, then R ２ is methyl, R ３ Compounds, or pharmaceutically acceptable salts thereof, excluding compounds in which both are trifluoromethyl.

2. The compound or salt according to claim 1, wherein A is pyrazolyl.

3. Structure of formula Ix: 【Chemistry 2】 A compound or salt according to claim 1 or 2, having the properties of the compound or salt described in claim 1 or 2.

4. Structure of Equation II: 【Transformation 3】 It has, in the formula, C Ａ and C Ｂ The compound or salt according to claim 1, wherein the compound represents a carbon stereocenter having the same or opposite stereochemistry.

5. C Ａ However, it is a carbon in the R configuration, C Ｂ The compound or salt according to claim 2, wherein the carbon is in an S configuration.

6. The compound or salt according to claim 4 or 5, wherein A is pyrazolyl.

7. A compound or salt according to any one of claims 1 to 6, wherein X is a bond.

8. X is C １－６ The compound or salt according to any one of claims 1 to 6, which is alkylene-C(O).

9. R ５ However, it is a four-membered heterocycloalkyl having one ring nitrogen atom, and the heterocycloalkyl has one or two C １－６ A compound or salt according to any one of claims 1 to 8, which is optionally substituted with an alkyl group.

10. R ５ However, it is a six-membered heterocycloalkyl having two ring nitrogen atoms, and the heterocycloalkyl has one or two C １－６ A compound or salt according to any one of claims 1 to 8, which is optionally substituted with an alkyl group.

11. R ５ The compound or salt according to claim 9 or 10, which is unsubstituted.

12. R ５ However, one C １－６ The compound or salt according to claim 9 or 10, which is substituted with an alkyl group.

13. Structure of formula Ia or Ib: 【Chemistry 4】 A compound or salt according to claim 1, having the following characteristics.

14. R １ The compound or salt according to any one of claims 1 to 13, wherein H is present.

15. R １ The compound or salt according to any one of claims 1 to 13, wherein the compound is an OH group.

16. R ２ However, C １－６ A compound or salt according to any one of claims 1 to 15, wherein the compound is alkyl.

17. R ２ The compound or salt according to claim 16, wherein the compound is methyl.

18. R ２ However, C １－６ A compound or salt according to any one of claims 1 to 15, which is a haloalkyl compound.

19. R ２ However, CHF ２ or CF ３ The compound or salt according to claim 18.

20. R ３ However, CHF ２ or CF ３ The compound or salt according to any one of claims 1 to 19.

21. R ３ However, CHF ２ The compound or salt according to claim 20.

22. R ４ The compound or salt according to any one of claims 1 to 21, wherein H is present.

23. R ４ The compound or salt according to any one of claims 1 to 21, wherein the compound is a halo.

24. R ４ The compound or salt according to claim 23, wherein the compound is F or Cl.

25. R ４ However, C １－６ A compound or salt according to any one of claims 1 to 21, which is an alkoxy.

26. R ４ The compound or salt according to claim 25, wherein the compound is methoxy.

27. R ４ However, C １－６ A compound or salt according to any one of claims 1 to 21, wherein the compound is alkyl.

28. R ４ The compound or salt according to claim 27, wherein the compound is methyl or ethyl.

29. R ４ The compound or salt according to claim 27 or 28, wherein the compound is methyl.

30. R ３ However, CHF ２ And R ４ The compound or salt according to claim 29, wherein the compound is methyl.

31. R ４ However, C ３－５ A compound or salt according to any one of claims 1 to 21, which is a cycloalkyl compound.

32. R ４ The compound or salt according to claim 31, wherein the compound is cyclopropyl.

33. R １ However, H is hydroxyl, and R ２ However, it is methyl, and R ３ However, CHF ２ And R ４ However, X is methyl, X is a bond, and R ５ However, it is a 4-6 member heterocycloalkyl having one ring nitrogen atom, and the heterocycloalkyl is C １－２ The compound according to claim 5, which is optionally substituted with an alkyl group.

34. X is C １－６ It is alkylene-C(O), R ５ However, one or two C １－６ Azetidinyl is optionally substituted with an alkyl group, and R １ However, H is hydroxyl, and R ２ However, C １－３ It is alkyl, R ３ However, CHF ２ And R ４ The compound according to claim 5, wherein the compound is methyl.

35. Compounds listed in Table A, or their pharmaceutically acceptable salts.

36. (2S,3R)-1-{6-(difluoromethyl)-5-methyl-4-[1-(1-methyl-3-azetidinyl)-4-pyrazolyl]-2-pyrimidinyl}-2-methyl-3-azetidinol (A21), 2-(4-{2-[(S)-2-methyl-1-azetidinyl]-5-methyl-6-(trifluoromethyl)-4-pyrimidinyl}-1-pyrazolyl)-1-(1-piperazinyl)-1-ethanone (A3), (2S,3R)-1-{6-(difluoromethyl)-4-[1-(1-methyl-3-azetidinyl)-4-pyrazolyl]-2-pyrimidinyl}-2-methyl-3-azetidinol (A9), (2S,3R)-2-methyl-1-{5-methyl-4-[1-(1-methyl-3-azetidinyl)-4-pyrazolyl]-6-(trifluoromethyl)-2-pyrimidinyl}-3-azetidinol (A12), (2S,3R)-1-{6-(difluoromethyl)-5-methoxy-4-[1-(1-methyl-3-azetidinyl)-4-pyrazolyl]-2-pyrimidinyl}-2-methyl-3-azetidinol (A19), (2S,3R)-1-{4-[1-(3-azetidinyl)-4-pyrazolyl]-6-(difluoromethyl)-5-methyl-2-pyrimidinyl}-2-methyl-3-azetidinol (A35), and A compound selected from 2-(4-{2-[(S)-2-methyl-1-azetidinyl]-6-(difluoromethyl)-5-methyl-4-pyrimidinyl}-1-pyrazolyl)-1-(1-piperazinyl)-1-ethanone (A24), or a pharmaceutically acceptable salt thereof.

37. 2-(4-{2-[(S)-2-methyl-1-azetidinyl]-5-methyl-6-(trifluoromethyl)-4-pyrimidinyl}-1-pyrazolyl)-1-(1-piperazinyl)-1-ethanone (A3), and A compound according to claim 36, selected from 2-(4-{2-[(S)-2-methyl-1-azetidinyl]-6-(difluoromethyl)-5-methyl-4-pyrimidinyl}-1-pyrazolyl)-1-(1-piperazinyl)-1-ethanone (A24), or a pharmaceutically acceptable salt thereof.

38. (2S,3R)-1-{6-(difluoromethyl)-5-methyl-4-[1-(1-methyl-3-azetidinyl)-4-pyrazolyl]-2-pyrimidinyl}-2-methyl-3-azetidinol (A21) and A compound according to claim 36, selected from (2S,3R)-1-{4-[1-(3-azetidinyl)-4-pyrazolyl]-6-(difluoromethyl)-5-methyl-2-pyrimidinyl}-2-methyl-3-azetidinol (A35), or a pharmaceutically acceptable salt thereof.

39. (2S,3R)-1-{6-(difluoromethyl)-4-[1-(1-methyl-3-azetidinyl)-4-pyrazolyl]-2-pyrimidinyl}-2-methyl-3-azetidinol (A9), and A compound according to claim 36, selected from (2S,3R)-1-{6-(difluoromethyl)-5-methoxy-4-[1-(1-methyl-3-azetidinyl)-4-pyrazolyl]-2-pyrimidinyl}-2-methyl-3-azetidinol (A19), or a pharmaceutical salt of any of the above compounds.

40. The compound according to claim 36 is (2S,3R)-2-methyl-1-{5-methyl-4-[1-(1-methyl-3-azetidinyl)-4-pyrazolyl]-6-(trifluoromethyl)-2-pyrimidinyl}-3-azetidinol (A12), or a pharmaceutically acceptable salt thereof.

41. A pharmaceutical composition comprising a compound or salt according to any one of claims 1 to 40 and a pharmaceutically acceptable excipient.

42. A method for inhibiting ketohexokinase (KHK) in cells, comprising contacting the cells with a compound or salt according to any one of claims 1 to 40, or a pharmaceutical composition according to claim 41.

43. A method for treating or preventing a disease or disorder in a subject, comprising administering to the subject a therapeutic amount of a compound or salt according to any one of claims 1 to 40, or a pharmaceutical composition according to claim 41.

44. The method according to claim 43, wherein the disease or disorder is related to KHK dysregulation.

45. The method according to claim 43 or 44, wherein the disease or disorder is a metabolic syndrome, hypertriglyceridemia, hypercholesterolemia, non-alcoholic fatty liver disease (NAFLD), metabolic dysfunction-related fatty liver disease (MASLD), MASLD with increased alcohol intake (MetALD), non-alcoholic steatohepatitis (NASH), metabolic dysfunction-related fatty liver disease (MASH), type 2 diabetes mellitus (T2D), diabetic nephropathy (DKD), alcoholic steatohepatitis (ASH), alcohol-related liver disease (ALD), hepatic fibrosis or cirrhosis, liver disease caused by hepatocyte stress, hereditary fructose intolerance, hyperuricemia, gout, toxic craving, neurodegenerative disease, or cancer.

46. The method according to claim 45, wherein the disease or disorder is NASH or MASH.

47. The method according to any one of claims 43 to 46, further comprising administering to the subject an additional therapeutically effective amount of a therapeutic agent.

48. The method according to claim 47, wherein the additional therapeutic agent comprises metformin, a fructose transporter inhibitor, an aldose reductase inhibitor, a xanthine oxidase inhibitor, a thyroid hormone beta receptor agonist, an incretin hormone receptor agonist or modulator, or a sodium / glucose transporter inhibitor.

49. The method according to claim 48, wherein the incretin hormone receptor agonist or modulator is semaglutide, dulaglutide, liraglutide, tilzepatide, sulvodutide, letatoltide, pembidutide, VK2735, olfoglipron, caglilintide / semaglutide, danuglipron, maridebaatcafraglutide, RGT-075, PF-0695422, NN9487, NN9541, CT-388, CT-868, CT-996, efinopegdutide, efosipegdoltide, AZD9550, DR10624, NLY01, ECC5004, mazdutide, exenatide, TERN-601, echnoglutide, or XW-004.

50. The method according to claim 48, wherein the fructose transporter inhibitor is an inhibitor of GLUT2, GLUT5, or both.

51. The method according to claim 48, wherein the aldose reductase inhibitor is AT-001, AT-003, gaborestat, ranirestat, epalrestat, fidarestat, imirestat, tollestat, or risalestat.

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