Disubstituted pyrimidine compounds for hexanketokinase inhibition

Abstract

Disclosed herein are methods of inhibiting hexanketokinase (KHK) in a biological sample or subject, and methods of treating or preventing a disease or condition (e.g., a disease or condition associated with KHK dysregulation), the method comprises administering to the biological sample or the subject an effective amount of a compound represented by Formula I or a compound of Table A or a pharmaceutically acceptable salt thereof. (I)

Description

Technical Field

[0001] This disclosure generally relates to novel hexokinase (KHK) inhibitor compounds, pharmaceutical compositions thereof, and the use of these compounds for treating conditions such as nonalcoholic fatty liver disease (NAFLD), metabolic dysfunction-associated steatohepatitis (MASH), metabolic dysfunction-associated steatohepatitis (MASLD), nonalcoholic steatohepatitis (NASH), hypertriglyceridemia, hypercholesterolemia, type 2 diabetes (T2D), diabetic kidney disease (DKD), alcoholic steatohepatitis (ASH), addictive cravings, including cravings for sugar or alcohol 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 hexokinase or fructokinase) catalyzes the first step in fructose metabolism, phosphorylating fructose to fructose-1-phosphate (F1P) and consuming intracellular ATP. There is no negative feedback mechanism by which F1P inhibits KHK metabolism of fructose; therefore, F1P accumulation is directly related to the amount of fructose, which is either (1) transported into the cell via the GLUT transporter or (2) formed intracellularly from glucose via the polyol pathway and metabolized by KHK. The accumulation of F1P and the consumption of ATP have detrimental consequences in cells and tissues, including oxidative stress, osmotic stress, endothelial dysfunction, and metabolic disorders. Responses to these impairments include lipogenesis, hyperuricemia, and gluconeogenesis, which trigger metabolic diseases, including metabolic syndrome and its complications.

[0003] Two isoforms of KHK exist: KHK-A, which is widely expressed but has a low affinity for fructose, and KHK-C, which is preferentially expressed in the liver, kidney, brain, and gut and has a much higher affinity for fructose. While KHK-C drives most of the physiological flow of fructose to F1P, KHK-A can compensate for the downregulation, inhibition, or absence of KHK-C, especially with elevated intracellular fructose concentrations. KHK Humans with this genetic polymorphism exhibit a benign phenotype of essential fructoseuria and do not accumulate F1P; while the more severe symptom, hereditary fructose intolerance, is caused by enzymes encoding aldolase B. ALDOB Polymorphisms in the gene cause the second step of fructose metabolism catalyzed by the aldolase B, leading to the harmful accumulation of F1P and an increase in oxidative stress due to the 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 hepatocellular stress, including alpha-1 antitrypsin deficiency and hemochromatosis. Targeting this first rate-limiting step in fructose metabolism, namely phosphorylation via KHK, is considered a promising therapeutic strategy for these metabolic disorders and other diseases caused by fructose metabolism, including cancer, neurodegenerative diseases (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] To date, attempts to inhibit KHK have stalled in preclinical or clinical development. In Phase 1 and 2 clinical studies, the most advanced compound developed, PF-06835919, showed only mild inhibition of fructose uptake and metabolism. While this molecule exhibits potent inhibition of the KHK-C enzyme alone, its potency was reduced by approximately 10-fold in cell-based assays of KHK activity, meaning a 300 mg dose twice daily was required in the Phase 2 clinical study. Even with these high doses, only minor effects on hepatic steatosis and markers of liver damage were observed. As a carboxylic acid, PF-06835919 is a substrate for organic anion transporters, resulting in increased accumulation of it in the liver relative to other tissues and organs. Although KHK-C is preferentially expressed in the liver, it is also highly expressed in the kidneys, intestines, and some brain centers (other sites of fructose metabolism to F1P), potentially leading to metabolic disorders, endothelial dysfunction, metabolic diseases, or desire disorder. Furthermore, PF-06835919 is a KHK-C biased inhibitor with much weaker inhibition of KHK-A. PF-06835919 is disclosed in US 2017 / 0183328A1. See also *Journal of Medicinal Chemistry*, 2020, 63, 13546-13560. Additionally, US Patent No. 11,124,500 discloses certain disubstituted pyrazole KHK inhibitor compounds. Furthermore, Durham et al., *Journal of Medicinal Chemistry*, 2023, 66, 15960-15976, “Identification of LY3522348: A highly selective and orally effective pentoxifyllokinase inhibitor”. Identification of LY3522348:A Highly Selective and Orally Efficacious Ketohexokinase Inhibitor The study disclosed a hexylose kinase inhibitor. Although KHK-C drives fructose metabolism in several tissues at physiological fructose concentrations, the contribution of KHK-A to the metabolism of fructose into F1P becomes more significant with increasing fructose concentrations (due to diet, metabolic diseases, or inhibition by KHK-C).

[0006] Therefore, there is an urgent need to develop a KHK inhibitor that is potent in both enzyme and cell-based assays; provides balanced inhibition of KHK-C and KHK-A to minimize escape flow to F1P via KHK-A or compensation via KHK-A; and is distributed systemically to inhibit KHK-C and KHK-A not only in the liver but also in the kidneys, intestines, and all other tissues. Summary of the Invention

[0007] This disclosure generally relates to methods for inhibiting hexokinase (KHK), methods for treating or preventing diseases or conditions in subjects (e.g., said diseases or conditions are related to KHK dysregulation or fructose metabolism, including those secondary to excessive fructose and / or alcohol intake), and compounds and compositions that can be used in such methods.

[0008] This disclosure provides compounds of formula I and their pharmaceutically acceptable salts: (I) Where R 1 For H or OH; R 2 C 1-6 Alkyl or C 1-6 Halogenated alkyl; R 3 C 1-6 Alkyl or C 1-6 Halogenated alkyl; R 4 H, halogenated, CN, C 1-6 Alkyl, C 1-6 Alkoxy or C 3-5 Cycloalkyl; A is a 5-membered heteroaryl group containing 2-3 nitrogen ring atoms; X is a bond or C 1-6 alkylene-C(O); and R 5 It is a 4- to 6-membered heterocyclic alkyl group having 1 or 2 cyclic nitrogen atoms, wherein the heterocyclic alkyl group is optionally surrounded by 1 or 2 C atoms. 1-6 Alkyl substitution, provided that in R 4 When it is H, exclude R. 2 It is methyl and R 3 It is a trifluoromethyl compound. In the examples, A is a pyrazolyl group. In the examples, the compound has the structure of formula Ix: (Ix).

[0009] Furthermore, methods are provided for administering safe and effective amounts of compounds as disclosed herein, such as those represented by Formula I or those listed in Table A, to biological samples or patients.

[0010] This document also provides a method for inhibiting hexylose kinase (KHK) in a biological sample or in a patient (e.g., in cells) by administering an effective amount of such a compound as disclosed herein, such as a compound represented by Formula I or a compound in Table A.

[0011] Further, methods for treating or preventing a disease or condition in a subject (e.g., said disease or condition is related to KHK disorder or fructose metabolism, including those secondary to excessive fructose and / or alcohol intake) are provided, said methods comprising administering to said subject an effective amount of a compound as disclosed herein, such as a compound represented by Formula I or a compound in Table A.

[0012] Pharmaceutical compositions are also provided comprising, as disclosed herein, for example, compounds represented by Formula I or those in Table A, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable excipients, carriers, adjuvants, or mediators.

[0013] The use of the compounds described herein for inhibiting, for example, hexyl kinase (KHK) in cells, and for treating or preventing diseases or conditions in subjects (e.g., said diseases or conditions are related to KHK dysregulation or fructose metabolism, including those secondary to excessive fructose and / or alcohol intake).

[0014] This document further provides the use of the compounds described herein in the preparation of a medicament for inhibiting hexokinase (KHK), and for the treatment or prevention of a disease or condition in a subject (e.g., said disease or condition is related to KHK dysregulation or fructose metabolism, including via excessive fructose and / or alcohol intake). Attached Figure Description

[0015] Figure 1 The XPRD diffraction patterns of A21 free base (top trace) and A21 HCl salt (bottom trace) are shown for comparison.

[0016] Figure 2 The relationship between A21 free base and HCl salt (DMSO-d6) is shown. 1 Comparison of H NMR spectra. Detailed Implementation

[0017] This document provides the use of compounds, and their use for inhibiting, for example, hexokinase (KHK) in cells, and for treating or preventing a disease or condition in a subject (e.g., said disease or condition is related to KHK dysregulation or fructose metabolism, including those secondary to excessive fructose and / or alcohol intake). It also provides the use of the compounds described herein, or pharmaceutically acceptable salts thereof, or pharmaceutically acceptable compositions comprising such compounds or pharmaceutically acceptable salts thereof, for inhibiting, for example, hexokinase (KHK) in cells, and for treating or preventing a disease or condition in a subject (e.g., said disease or condition is related to KHK dysregulation).

[0018] Unless otherwise stated, the structures described herein also imply all isomers (e.g., enantiomers, diastereomers, cis-trans, conformations, and rotations) of the structure. For example, 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 specifically indicated as only one of the isomers. Therefore, single stereochemical isomers of the compounds of the present invention, as well as enantiomers, diastereomers, cis / trans, conformations, and rotational mixtures, are all within the scope of this disclosure. In some cases, the compounds disclosed herein are stereoisomers. "Stereoisomer" means a compound that differs in chirality at one or more stereocenters. Stereoisomers include enantiomers and diastereomers. The compounds disclosed herein may exist as single stereoisomers or as mixtures of multiple stereoisomers. The stereochemistry of the compounds shown herein indicates relative stereochemistry, not absolute stereochemistry, unless otherwise discussed. As indicated herein, a single stereoisomer, diastereomer, or enantiomer means a compound containing 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.

[0019] Unless otherwise stated, all tautomer forms of the compounds disclosed herein are within the scope of this disclosure.

[0020] Furthermore, unless otherwise stated, the structures described herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, those where hydrogen is replaced by deuterium or tritium, or carbon by... 13 C or 14 Compounds having the structure of this invention, other than those enriched with carbon, are within the scope of this disclosure. Such compounds can be used as analytical tools or probes, for example, in bioassays. Such compounds, especially deuterium analogs, can also be useful therapeutically.

[0021] The compounds disclosed herein are defined by their chemical structure and / or chemical name. When a compound is referred to by both its chemical structure and chemical name, and there is a conflict between the chemical structure and chemical name, the chemical structure determines the properties of the compound.

[0022] compound This article provides compounds of formula I and their pharmaceutically acceptable salts: (I) in R 1 It is H or OH; R 2 C 1-6 Alkyl or C 1-6 Halogenated alkyl groups; R 3 C 1-6 Alkyl or C 1-6 Halogenated alkyl groups; R 4 H, halogenated, CN, C 1-6 Alkyl, C 1-6 Alkoxy or C 3-5 cycloalkyl; A is a 5-membered heteroaryl group containing 2-3 nitrogen ring atoms; X is a key or C 1-6 alkylene-C(O); and R 5 It is a 4- to 6-membered heterocyclic alkyl group having 1 or 2 cyclic nitrogen atoms, wherein the heterocyclic alkyl group is optionally surrounded by 1 or 2 C atoms. 1-6 Alkyl substitution, The condition is that in R 4 When it is H, exclude R. 2 It is methyl and R 3 It is a trifluoromethyl compound.

[0023] In some cases, A is a 5-membered heteroaryl group containing 2 nitrogen ring atoms. In some cases, A is a 5-membered heteroaryl group containing 3 nitrogen ring atoms. In some cases, A is a pyrazolyl group. In some cases, the compound or salt has the structure of formula Ix: (Ix).

[0024] In some cases, X is the key. In other cases, X is the C key. 1-6 Alkylene-C(O). In some cases, X is CH2-C(O). In some cases, XR 5 C(O)-C 1-6 Alkylene-R 5 .

[0025] In some cases, R 5 It is a 4-membered heterocyclic alkyl group having one cyclic nitrogen atom, wherein the heterocyclic alkyl group is optionally surrounded by one or two C atoms. 1-6 Alkyl substitution. In some cases, R 5 It is a 5-membered heterocyclic alkyl group having 1 or 2 cyclic nitrogen atoms, wherein the heterocyclic alkyl group is optionally surrounded by 1 or 2 C atoms. 1-6 Alkyl substitution. In some cases, R 5 It is a 6-membered heterocyclic alkyl group having 1 or 2 cyclic nitrogen atoms, wherein the heterocyclic alkyl group is optionally surrounded by 1 or 2 C atoms. 1-6 Alkyl substitution. In some cases, R 5 It is a 4-membered heterocyclic alkyl group having one cyclic nitrogen atom, wherein the heterocyclic alkyl group is optionally surrounded by one or two C atoms. 1-6 Alkyl substitution. In some cases, R 5 It is a 5-membered heterocyclic alkyl group having one cyclic nitrogen atom, wherein the heterocyclic alkyl group is optionally surrounded by one or two C atoms. 1-6 Alkyl substitution. In some cases, R 5 It is a 5-membered heterocyclic alkyl group having 2 cyclic nitrogen atoms, wherein the heterocyclic alkyl group is optionally surrounded by 1 or 2 C atoms. 1-6 Alkyl substitution. In some cases, R 5 It is a 6-membered heterocyclic alkyl group having 2 cyclic nitrogen atoms, wherein the heterocyclic alkyl group is optionally surrounded by 1 or 2 C atoms. 1-6 Alkyl substitution. In some cases, R 5 It is an aza-butane or piperazine group, and optionally surrounded by one or two carbon atoms. 1-6 Alkyl substitution. In some cases, R 5 It is a nitrogen-containing heterocyclic butyl group, and optionally surrounded by 1 or 2 carbon atoms. 1-6 Alkyl substitution. In some cases, R 5 It is piperazine-based and optionally surrounded by 1 or 2 carbon atoms. 1-6 Alkyl substitution. In some cases, R 5 It is unreplaced. In some cases, R 5 A C 1-6 Alkyl substitution. In some cases, R 5 It is replaced by one methyl group. In some cases, R 5 Two Cs 1-6 Alkyl substitution.

[0026] In some cases, the compound has the structure of formula Ia or Ib: (Ia) or (Ib). In some cases, the compound has the structure of formula Ia: (Ia). In some cases, the compound has the structure of Ib: (Ib).

[0027] In some cases, compounds of formula I have the structure of formula II: (II), where C A and C B This indicates carbon stereocenters with the same or opposite stereochemistry. For C A In the case of a carbon stereocenter, R 1 It cannot be H. Therefore, for compounds of formula (II), R 1 It is OH. In some cases, the compound has the structure of formula (IIx): (IIx). In some cases, compounds of formula I have the structure of formula IIa or (IIb): (IIa) or (IIb). In some cases, C A and C B This indicates carbon stereocenters with the same stereochemistry. In some cases, C... A and C B This indicates a carbon stereocenter with opposite stereochemistry. In some cases, C... A It is carbon with an R configuration, and C B It is carbon with an S-configuration. In some cases, C A It is carbon with an S-configuration, and C B It is carbon with an R configuration.

[0028] In some cases, R 1 For H. In some cases, R 1 It is OH.

[0029] In some cases, R 2 C 1-6 Alkyl group. In some cases, R 2 It is a methyl group. In some cases, R... 2 C 1-6 Haloalkyl. In some cases, R 2 It is a C1 haloalkyl group. In some cases, R 2 It is CHF2 or CF3. In some cases, R 2 For CHF2. In some cases, R 2 For CF3. In some cases, R 2 It can be methyl, CHF2 or CF3.

[0030] In some cases, R3 C 1-6 Alkyl group. In some cases, R 3 It is a methyl group. In some cases, R... 3 C 1-6 Haloalkyl. In some cases, R 3 It is a C1 haloalkyl group. In some cases, R 3 It is CHF2 or CF3. In some cases, R 3 For CHF2. In some cases, R 3 For CF3. In some cases, R 3 It can be methyl, CHF2 or CF3.

[0031] In some cases, R 4 For H. In some cases, R 4 Halogenation. In some cases, R 4 For F or Cl. In some cases, R 4 For F. In some cases, R 4 For Cl. In some cases, R 4 C 1-6 Alkyl group. In some cases, R 4 It is methyl or ethyl. In some cases, R 4 It is a methyl group. In some cases, R... 3 It is CHF2, and R 4 It is a methyl group. In some cases, R... 4 It is an ethyl group. In some cases, R... 4 C 1-6 Alkyl group. In some cases, R 4 It is methoxylated. In some cases, R 4 C 3-5 Cycloalkyl. In some cases, R 4 It is cyclopropyl. In some cases, R 4 It can be methyl, ethyl, methoxy, F, Cl, CHF2, CF3 or cyclopropyl.

[0032] In some cases, R 1 H or hydroxyl, R 2 It is a methyl group, X is a bond, and R 5 It is a 4- to 6-membered heterocyclic alkyl group having one cyclic nitrogen atom, wherein the heterocyclic alkyl group is optionally separated by a C14 group. 1-2 Alkyl substitution. In some cases, X is C. 1-6 Alkylene-C(O), R 5 To be optionally bounded by 1 or 2 C 1-6 Alkyl-substituted nitrogen-containing heterocyclic butyl groups, R 1 It is H or hydroxyl, and R 2 C1-3 alkyl.

[0033] The preferred compound is R. 3 For CHF2, R 4 It is methyl, R 1 It is a hydroxyl group and R 2 Compounds containing methyl groups.

[0034] “In R 4 When it is H, exclude R. 2 It is methyl and R 3 The condition "a compound of trifluoromethyl" means that R is a trifluoromethyl compound. 2 It is methyl and R 3 No single compound that is trifluoromethyl is included in the claims.

[0035] As used herein, the term "alkyl" or "alkylene" refers to a saturated straight-chain or branched hydrocarbon. The term C... n This indicates that the alkyl group has "n" carbon atoms. For example, a C4 alkyl group refers to an alkyl group with 4 carbon atoms. 1-6 Alkyl refers to an alkyl group having a number of carbon atoms covering the entire range (i.e., 1 to 6 carbon atoms) and all subranges (e.g., 1-6, 2-6, 1-5, 2-6, 1-4, 2-5, 1, 2, 3, 4, 5, and 6 carbon atoms). Specific examples include, but are not limited to, methyl, ethyl, isopropyl, n-propyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl. (Claim term C) 1-6 Alkylene-C(O) is defined as being attached to R via the C(O) moiety. 5 .

[0036] As used herein, the terms “halogen” and “halogenated” refer to F, Cl, Br, or I.

[0037] As used herein, the term "haloalkyl" refers to an alkyl group substituted with one or more halogen substituents. For example, C1-C6 haloalkyl refers to a C1-C6 alkyl group substituted with one or more halogen atoms, such as 1, 2, 3, 4, 5, or 6 halogen atoms. Non-limiting examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, and trichloromethyl. Similarly, haloalkoxy refers to an alkoxy group substituted with one or more halogen atoms, such as 1, 2, 3, 4, 5, or 6 halogen atoms.

[0038] As used in this article, the term "alkoxy" refers to –O-alkyl.

[0039] The term "cycloalkyl" refers to a non-aromatic monocyclic, fused, bridged, or spirocyclic system in which the ring atom is carbon, and the cycloalkyl group may be saturated or have one or more unsaturated units. A cycloalkyl group may have three to five ring carbon atoms. Specific examples include, but are not limited to, cyclopentyl, cyclopropyl, and cyclobutyl. The cycloalkyl ring may be unsubstituted or substituted, as described herein.

[0040] As used herein, the term "heterocyclic alkyl" refers to a non-aromatic monocyclic, fused, spiro, or bridged ring system that may be saturated or contain one or more unsaturated units having five to eight ring atoms, wherein 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 heterocyclic alkyl comprises 4 to 6 ring members. In some embodiments, the heterocycle comprises 4 ring members. In some embodiments, the heterocyclic alkyl comprises 6 ring members. Examples of heterocyclic alkyl groups include, but are not limited to, oxacyclobutane, azacyclobutane, thiaanthryl, piperidinyl, piperazinyl, pyrrolyl, pyrazolidine, imidazoalkyl, oxazolyl, isoxazolyl, thiazoalkyl, isothiazolyl, tetrahydrofuranyl, tetrahydrothiophenyl, morpholino (including, for example, 3-morpholino, 4-morpholino), 2-thiomorpholino, 3-thiomorpholino, 4-thiomorpholino, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, pyrrolidine-2-one, 1-tetrahydropiperazinyl, 2-tetrahydropiperazinyl, 3-tetrahydropiperazinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 1-pyrazole The heterocyclic alkyl rings are linyl, 3-pyrazolinyl, 4-pyrazolinyl, 5-pyrazolinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2-thiazolyl, 3-thiazolyl, 4-thiazolyl, 1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, dihydrofuranyl, 1,3-dioxolanecycloyl, 1,4-dioxacyclohexyl, 1,3-oxathiol, oxathiyl, 1,3-dithienyl, 1,4-oxathiylhexyl, 1,4-oxathiyl, 1,4-dithiyl, thiomorpholinyl, tetrahydropyranyl, dihydropyranyl, and 1,3-dihydro-imidazolin-2-one. The heterocyclic alkyl rings are either unsubstituted or substituted, as described herein.

[0041] The term "heteroaryl" refers to an aromatic heterocycle having five members. A heteroaryl group has two or three cyclic 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). The heteroaryl ring can be unsubstituted or substituted, as described herein.

[0042] As described herein, the compounds of this disclosure may optionally be substituted with one or more substituents, as generally stated or exemplified by a particular class, subclass, and species of this disclosure. It should be understood that the phrase “optionally substituted” is used interchangeably with the phrase “substituted or unsubstituted.” Generally, the term “substituted” (whether or not preceded by the term “optionally”) means that one or more hydrogen groups in a given structure are replaced by a group with a specified substituent. Unless otherwise stated, an optionally substituted group may have a substituent at each substituted position of the group. When more than one position in a given structure can be substituted by more than one substituent selected from a particular group, the substituents may be the same or different at each position.

[0043] The specific compounds under consideration include those listed in the table below. Compounds exhibiting a specific stereocenter indicate at least relative stereoisomerism. Compounds having a chiral center but without a specified stereoisomerism indicate a mixture of stereocenters at said chiral center.

[0044] The compound may be one of the compounds listed in Table A or a pharmaceutically acceptable salt thereof. 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.

[0045] Table A In some cases, the compound is selected from compounds A3, A9, A12, A19, A21, A24, A35, and their pharmaceutically acceptable salts.

[0046] abbreviations LC / MS = Liquid Chromatography-Mass Spectrometry TFA = Trifluoroacetic acid min = minutes mL = milliliters FA = Formic acid DTBPF = 1,1'-bis(di-tert-butylphosphine)ferrocene Oxone = Tetrabutylammonium monopersulfate DMF = dimethylformamide DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene BOP = benzotriazol-1-yl-oxy-tris-(dimethylamino)-hexafluorophosphate Ts = 4-Toluenesulfonate ACN and MeCN = Acetonitrile DIEA = N,N-diisopropylethylamine DCM = dichloromethane TLC = Thin-layer chromatography HATU = 1-[bis(dimethylamino)methylene]-1 H -1,2,3-triazolo[4,5- b Pyridine cation 3-oxide hexafluorophosphate NMR = Nuclear Magnetic Resonance NMP = 1-Methyl-2-pyrrolidone RT = Retention Time TEA = Triethylamine THF = Tetrahydrofuran TBDMS = tert-butyldimethylsilyl aq = aqueous solution eq = equivalent dppf = 1,1'-ferrocene-bis(diphenylphosphine) DAST = (diethylamino)sulfur trifluoride FCC = Fast Column Chromatography HPLC = High Performance Liquid Chromatography Bpin = 4,4,5,5-Tetramethyl-1,3,2-dioxacyclopentaborane-2-yl Tf = trifluoromethanesulfonyl group PMB = 4-methoxybenzyl Bn = benzyl LAH = Lithium Aluminum Hydrogen General Synthesis Method Certain methods for preparing the compounds of this disclosure are provided as an additional feature of this disclosure and are illustrated by the following exemplary reaction schemes. Those skilled in the art will understand that other synthetic routes can be used to synthesize the compounds of this invention. Detailed descriptions of the various reaction steps are provided in the Examples section herein. Although specific starting materials and reagents are described in the schemes and discussed herein, other starting materials and reagents can be readily substituted to provide a variety of derivatives and / or reaction conditions. Furthermore, many compounds prepared according to this disclosure by the methods described herein can be further modified using conventional chemical methods well known to those skilled in the art. Specifically, it is noteworthy that compounds prepared according to these schemes can be further modified to provide new examples within the scope of this disclosure. Additionally, as will be apparent from the detailed description provided in the Experimental section, the preparation methods employed are more extensive than the general procedures described herein. In addition, 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 edition, edited by Smith, MB, John Wiley & Sons, New York: 2019, the entire contents of which are hereby incorporated by reference.

[0047] Starting materials are typically available from commercial sources such as Merck Sigma-Aldrich Inc. and Enamine Ltd. Aldrich Chemicals (Milwaukee, Wisconsin), or readily prepared using methods known to those skilled in the art (e.g., by methods generally described in the following references: Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, 1-19 editions, Wiley, New York (1967-1999), or Beilstein's Handbuch derorganischenChemie, 4, Aufl (ed.), Springer-Verlag, Berlin, including supplements (also available via the Beilstein online database)).

[0048] As a preliminary observation, it is noteworthy that some methods of preparing the compounds described herein may require protection of long-range functionalities (e.g., primary amines, secondary amines, carboxyl groups in intermediates). The need for such protection will vary depending on the nature of the long-range functionality 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 within the scope of those skilled in the art. For a general description of protecting groups and their uses, see TW Greene, *Protective Groups in Organic Synthesis*, John Willie & Son Publishing, New York, 1991, and Greene, *Protective Groups in Organic Synthesis*, John Willie & Son Publishing, New York, 2006.

[0049] For example, some compounds contain primary amine or carboxylic acid functional groups, which, if not protected, may interfere with reactions at other sites on the molecule. Therefore, such functional groups can be protected with appropriate protecting groups that can be removed in subsequent steps. Suitable protecting groups for amines and carboxylic acids include those commonly used in peptide synthesis (such as N-tert-butoxycarbonyl, benzyloxycarbonyl, and 9-fluorenylmethylethoxycarbonyl for amines, and lower alkyl esters or benzyl esters for carboxylic acids), which are generally chemically inactive under the described reaction conditions and can usually be removed without chemically altering other functionalities in the Formula I compound.

[0050] Compounds of Formula I or their salts can be prepared by a variety of methods known to those skilled in the art. The following schemes, preparations, and examples outline non-limiting instances of these methods. Unless otherwise stated, all substituents are as defined herein. Reagents, solvents, and starting materials are commercially available, known in the literature, or readily accessible to those skilled in the art. The products of each synthetic procedure can be recovered and separated 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 into racemic, enantiomers, or diastereomers using well-known techniques such as crystallization, chiral chromatography, or supercritical fluid chromatography. These techniques can be applied at appropriate stages of synthesis. The diastereomers / enantiomers of Formula I compounds can be prepared as racemic mixtures, followed by appropriate chiral separation as described herein, or subsequently reacted, for example, with a desired chiral substituted nitrogen-containing butane compound.

[0052] Those skilled in the art will understand that compounds of Formula I or their salts can also be synthesized by means similar to those described herein, with appropriate modifications, such as: a) using appropriate protecting / deprotecting strategies; b) using appropriate substituted starting materials and reactants; c) making appropriate changes to the order of the synthetic steps; d) converting one functional group to another; e) using alternative stereochemical configurations.

[0053] Option 1: Scheme 1 provides a general strategy for the preparation of compounds of formula I. LG is defined as the "leaving group" and is used in certain reactions to synthesize compounds of formula I. Pathway A shows the conversion of pyrimidine 1 to pyrazole compound 3 via Suzuki coupling between borate 2 and LG2 of 1. Compound 3 (LG1) is then subjected to nucleophilic aromatic substitution (SN) with azacyclobutane 4. Ar The reaction provides compound I. Pathway B shows the SN reaction between LG1 and aziridine 4 via 1. Ar The reaction is an alternative strategy to convert pyrimidine 1 into azacyclic butane compound 5. Subsequently, Suzuki coupling of borate 2 with 5 (LG2) provides compound I. The synthesis of compounds according to these strategies uses suitable substituted reactants, suitable protecting / deprotecting steps, and other functional group transformations, if necessary.

[0054] Option 2: Scheme 2 illustrates 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, such as BOC, which can be removed during synthesis or at an appropriate time at the end.

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

[0056] In step F, compound 1A also undergoes a Suzuki reaction with borate 2A to give pyrazole 8. The reaction conditions are similar to those in step C, using DTBPF PdCl2 as a palladium catalyst. Then, in step G, compound 8 undergoes an SN reaction with azacyclobutane 4. Ar The reaction yields compound 9. Typical reaction conditions include a base (e.g., triethylamine) in an organic solvent (such as THF) at high temperature. In step H, compound 9 is deprotected, for example using TFA to remove the BOC group, to yield compound 10, which is then methylated in step I to yield compound (1a). Typical reaction conditions include the use of formaldehyde and a reducing agent, such as NaBH(OAc)3, in an organic solvent (such as dichloromethane) at room temperature.

[0057] Option 3: Scheme 3 shows 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, such as BOC, which can be removed during synthesis or at an appropriate time at the end.

[0058] In step J, using similar conditions from Scheme 2, chloropyrimidine 1B undergoes a Suzuki reaction with borate 2B to give pyrazole compound 11. Then, in step K, compound 11 is oxidized to give sulfone 12. Typical reaction conditions include the use of an oxidizing agent, such as Oxone, in an organic solvent (e.g., dimethylformamide) at room temperature. In step L, compound 12 undergoes an SN reaction with aziridine 4. ArThe reaction yields compound 7. Typical reaction conditions include a base (e.g., triethylamine) in an organic solvent (e.g., NMP) at high temperature. Compound 7 is deprotected as described in step E of scheme 2 to provide compound (1b).

[0059] In step M, compound 1B also undergoes a Suzuki reaction with borate 2A to give pyrazole 13. The reaction conditions are similar to those in step C from scheme 2, using DTBPF PdCl2 as a palladium catalyst. Then, in step K (as described above), compound 13 is oxidized to give sulfone 14. In step N, using conditions similar to those in step L, compound 14 then undergoes an SN reaction with azacyclobutane 4. Ar The reaction yields compound 9, which is then converted into compound of formula (1a) as described in scheme 2.

[0060] Option 4: Scheme 4 illustrates an alternative strategy for synthesizing a subset of compounds of formula I, namely compounds of formula Ia. PG is defined as a nitrogen protecting group, such as BOC, which can be removed during or at the appropriate point at the end of the synthesis. Tf is defined as a trifluoromethanesulfonate, which provides -OTf as a reactive group that can be used for Suzuki coupling.

[0061] In step O, dichloropyrimidine 1A is hydrolyzed in an aqueous THF solution using sodium hydroxide at high temperature to give compound 15. In step P, compound 15 is reacted with aziridine 4 via a SN reaction. Ar The reaction yields compound 16. Typical reaction conditions include a base (e.g., diisopropylethylamine) in an organic solvent (e.g., acetonitrile) at high temperature in a microwave environment. Then, in step Q, compound 16 is converted to trifluoromethanesulfonate 17 at room temperature in an organic solvent (e.g., dimethylformamide) in the presence of a base (e.g., diisopropylethylamine). In step R, trifluoromethanesulfonate 17 undergoes a Suzuki reaction with borate 2A to yield compound 9. This reaction is carried out at high temperature in an organic solvent (e.g., 1,4-dioxane) with a base (e.g., K3PO4) in the presence of a palladium catalyst (e.g., Pd(dppf)Cl2). Compound 9 is then converted to compound (1a) as described in scheme 2.

[0062] As will be understood by those skilled in the art, the foregoing synthetic schemes and representative examples (described herein) are not intended to constitute a comprehensive list of all means by which the compounds described and claimed in this application can be synthesized. Other methods will be readily apparent to those skilled in the art. Furthermore, the various synthetic steps described herein may be performed in an alternating sequence or order to obtain the desired compound. This disclosure further covers “intermediate” compounds, including structures produced from the described synthetic procedures prior to obtaining the final desired compound, whether isolated, in-situ generated, or not isolated. These intermediates are included within the scope of this disclosure. Exemplary embodiments of such intermediate compounds are shown in the following examples.

[0063] Pharmaceutically acceptable salts The compounds described herein may exist in free form or, where appropriate, as salts. Pharmaceutically acceptable salts are of particular interest because they can be used for medical purposes to administer the compounds described herein. Non-pharmaceutically acceptable salts may be used in manufacturing processes, for separation and purification purposes, and, in some cases, for the separation of stereoisomers of the disclosed compounds or their intermediates.

[0064] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound that, within reasonable medical judgment, is suitable for contact with the tissues of humans and lower animals without excessive side effects such as toxicity, irritation, allergic reactions, etc., and is commensurate with a reasonable benefit / risk ratio.

[0065] Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. described pharmaceutically acceptable salts in detail in the *Journal of Pharmaceutical Sciences*, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds described herein include salts 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] In cases where the compounds described herein contain a basic group or a sufficiently basic bioisostere, acid addition salts can be prepared by: 1) reacting the purified compound in its free basic form with a suitable organic or inorganic acid, and 2) separating the resulting salt. In practice, acid addition salts may be a more convenient form to use, and using the salt is equivalent to using the free basic form.

[0067] Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts formed by amino groups with inorganic acids (such as hydrochloric acid, hydrogen bromate, 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 salts prepared by other methods used in the art (such as ion exchange). Other pharmaceutically acceptable salts include adipates, alginates, ascorbic acid salts, aspartate salts, benzenesulfonates, benzoates, hydrogen sulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, gluconate-heptate, glycerophosphates, glycolates, gluconate, glycolates, hemisulfates, heptahydrates, heptahydrates, hexanoates, hydrobromates, hydroiodates, 2-hydroxy- Ethyl sulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, pyroglutamate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc.

[0068] Although other acids and bases are not pharmaceutically acceptable on their own, they can be used to prepare salts that serve as intermediates in 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 compounds in their free form and pharmaceutically acceptable salts are also contemplated.

[0070] prodrug This disclosure also includes prodrugs of Formula I compounds (or any of the embodiments described herein) and / or pharmaceutically acceptable salts thereof. The term prodrug is intended to represent a covalently bonded carrier capable of releasing the active ingredient of Formula I (or any of the embodiments described herein) when administered to a mammalian subject. Release of the active ingredient occurs in vivo. Prodrugs can be prepared using techniques known to those skilled in the art. These techniques typically modify appropriate functional groups in an established compound. However, these modified functional groups can be regenerated in vivo or through conventional operations. Prodrugs of Formula I compounds (or any of the embodiments described herein) include compounds in which hydroxyl, amino, carboxyl, or similar groups are modified. Examples of prodrugs include, but are not limited to, esters (e.g., acetates, formates, and benzoate derivatives), urethanes (e.g., those with hydroxyl or amino functional groups in Formula I compounds), and other similar compounds. N,N- Dimethylaminocarbonyl), amides (e.g., trifluoroacetamido, acetamido, etc.), etc. Prodrugs of Formula I (or any of the embodiments described herein) and / or pharmaceutically acceptable salts thereof are also within the scope of this disclosure.

[0071] Metabolites This disclosure also includes metabolites of compounds of formula I, i.e., compounds formed in the body upon administration of the drug. Examples of metabolites according to this disclosure include: (i) In the case that the compound of formula I contains a methyl group, its hydroxymethyl derivative (-CH3->-CH2OH); (ii) In the case that the compound of formula I contains an alkoxy group, its hydroxyl derivative (-OR ->-OH); (iii) When the compound of formula I contains a tertiary amino group, its secondary amino derivatives (-NRR -> -NHR or -NRH). (iv) When the compound of formula I contains a secondary amino group, its main derivatives (-NHR->-NH2). (v) In the case that the compound of formula I contains a phenyl moiety, its phenolic derivative (-Ph -> -PhOH); and (vi) When 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 compounds or pharmaceutically acceptable salts thereof. The term solvate is used herein to describe molecular complexes comprising compounds of this disclosure and one or more pharmaceutically acceptable solvent molecules (e.g., ethanol). Such solvent molecules are commonly used in the pharmaceutical industry and are known to be harmless to the recipient, such as water, ethanol, etc. Other solvents may be used as intermediate solvates, such as methanol, methyl tert-butyl ether, ethyl acetate, methyl acetate, (S)-propylene glycol, (R)-propylene glycol, 1,4-butynediol, etc., when preparing more desired solvates.

[0073] When the solvent is water, the term hydrate is used. Pharmaceutically acceptable solvates include hydrates and other solvates, wherein the crystalline solvent may be isotopically substituted, for example, D₂O, d-acetone, d-DMSO. The solvates and / or hydrates are preferably in crystalline form. The classification system for organic hydrates is a system that defines segregating sites, channels, or metal ion coordination hydrates—see KR Morris, *Polymorphism in Pharmaceutical Solids* (edited by HG Brittain, Marcel Dekker, 1995). Segregating site hydrates are hydrates in which water molecules are separated by intercalation into organic molecules and do not directly contact each other. In channel hydrates, water molecules are located within lattice channels, adjacent to other water molecules. In metal ion coordination hydrates, water molecules are bonded to metal ions.

[0074] The scope of this disclosure also includes multi-component complexes (excluding salts and solvates) in which the drug and at least one other component are present in stoichiometric or non-stoichiometric amounts. The compounds of this disclosure may also exist as complexes, such as cages or drug-host inclusion complexes, in which the drug and host are present in stoichiometric or non-stoichiometric amounts compared to the aforementioned solvates. Complexes of pharmaceutical products containing two or more organic and / or inorganic components are also included, which may be in stoichiometric or non-stoichiometric amounts. The resulting complexes may be ionized, partially ionized, or non-ionized. For a review of such complexes, see Haleblian's *Journal of Pharmaceutical Sciences*, 64(8), 1269-1288 (August 1975).

[0075] The compounds disclosed herein can be combined with soluble macromolecular entities, such as cyclodextrins and their suitable derivatives, or polyethylene glycol-containing polymers, to improve their pharmacokinetic properties, solubility, dissolution rate, taste masking, bioavailability, and / or stability for use in any of the aforementioned routes of administration. For example, drug-cyclodextrin complexes have been found to be generally useful for most dosage forms and routes of administration. Encapsulated and non-encapsulated complexes can be used. As an alternative to direct complexation with drugs, cyclodextrins can be used as excipients, i.e., as carriers, diluents, or solubilizers. The most commonly used cyclodextrins for these purposes are α-, β-, and γ-cyclodextrins.

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

[0077] The compounds disclosed herein exist as a continuum ranging from completely amorphous to completely crystalline solid states. The term 'amorphous' refers to a state in which the material lacks long-range order at the molecular level and can exhibit solid or liquid physical properties depending on temperature. Typically, such materials do not produce characteristic X-ray diffraction patterns and, although exhibiting solid properties, are more formally described as liquids. Upon heating, a change in properties from solid to liquid occurs, characterized by a change of state, typically a second-order change ('glass transition'). The term 'crystalline' refers to a solid phase in which the material has a regular, ordered internal structure at the molecular level and produces a characteristic X-ray diffraction pattern with defined peaks. Such materials will also exhibit liquid properties upon sufficient heating, but the change from solid to liquid is characterized by a phase transition, typically a first-order phase transition ('melting point').

[0078] Some compounds or combinations of pharmaceutical preparations disclosed herein may exist in more than one crystalline form (commonly referred to as "polymorphs"). Polymorphs can be prepared by crystallization under different conditions, for example, recrystallization using different solvents or mixtures of different solvents; crystallization at different temperatures; and / or various cooling methods during crystallization, from very rapid cooling to very slow cooling. Polymorphs can also be obtained by heating or melting the compounds of this disclosure, followed by gradual or rapid cooling. The presence of polymorphs can 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 pharmaceutically acceptable carriers, diluents, adjuvants, or catalysts. 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 catalyst. In embodiments, the pharmaceutical composition comprises a safe and effective amount of a compound as disclosed herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, adjuvant, or catalyst. Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients, or carriers, appropriately selected according to the intended form of administration and consistent with conventional pharmaceutical practice.

[0080] "Effective dose" includes "therapeutic effective dose" and "preventive effective dose." The term "therapeutic effective dose" refers to the amount that effectively treats and / or improves a patient's condition or illness related to KHK disorder. The term "preventive effective dose" refers to the amount that is effective in preventing and / or significantly reducing the chance of developing a condition or illness related to KHK disorder.

[0081] Pharmaceutically acceptable carriers may contain inert components that do not unduly inhibit the biological activity of the compound. Pharmaceutically acceptable carriers should be biocompatible, for example, non-toxic, non-inflammatory, non-immunogenic, or without other undesirable reactions or side effects when administered to subjects. Standard pharmaceutical reconstitution techniques can be used.

[0082] As used herein, pharmaceutically acceptable carriers, adjuvants, or mediators include any solvent, diluent or other liquid mediator, dispersant or suspending agent, surfactant, isotonic agent, thickener or emulsifier, preservative, solid binder, lubricant, etc., suited to a desired specific dosage form. Remington's Pharmaceutical Sciences, 16th edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers and known techniques for the formulation of pharmaceutically acceptable compositions. Unless any conventional carrier medium is incompatible with the compounds described herein, such as by producing any undesirable biological effect or otherwise interacting in a harmful manner with any other component of the pharmaceutically acceptable composition, its use within the scope of this disclosure is contemplated. As used herein, the phrase "side effect" encompasses an unwanted and adverse effect of a therapy (e.g., a prophylactic or therapeutic agent). Side effects are always undesirable, but an undesirable effect is not necessarily adverse. Adverse effects of therapies (such as prophylactic or therapeutic agents) may be harmful, uncomfortable, or risky. Side effects include, but are not limited to, fever, chills, drowsiness, gastrointestinal toxicity (including gastric and intestinal ulcers and erosions), nausea, vomiting, neurotoxicity, nephrotoxicity, kidney toxicity (including symptoms such as papillary necrosis and chronic interstitial nephritis), hepatotoxicity (including elevated serum liver enzyme levels), bone marrow toxicity (including leukopenia, bone marrow suppression, thrombocytopenia, and anemia), dry mouth, metallic taste, prolonged pregnancy, weakness, drowsiness, pain (including muscle pain, bone pain, and headache), hair loss, fatigue, dizziness, extrapyramidal symptoms, akathisia, cardiovascular disturbances, and sexual dysfunction.

[0083] Examples of materials that can be used 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), mixtures of saturated vegetable fatty acid metaglycerides, water, salts, or 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, and hydroxypropyl methylcellulose. Lanolin, sugars (such as lactose, glucose, and sucrose); starches (such as corn starch and potato starch); cellulose and its derivatives (such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate); powdered tragacanth; malt; gelatin; talc; excipients (such as cocoa butter and suppository waxes); oils (such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil, and soybean oil); glycols; such propylene glycols or polyethylene glycols; esters (such as ethyl oleate and ethyl laurate); agar; buffers (such as magnesium hydroxide and aluminum hydroxide); alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol and phosphate buffer solutions and other non-toxic and compatible lubricants (such as sodium lauryl sulfate and magnesium stearate); as well as colorants, release agents, coating agents, sweeteners, flavoring agents, and aromatic groups. Preservatives and antioxidants may also be present in the composition at the discretion of the formulator.

[0084] In some embodiments, the pharmaceutical compositions disclosed herein may be formulated with an adjunct active ingredient.

[0085] The carrier can be a solvent or a 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 flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. The prevention of microorganisms in the compositions disclosed herein is achieved by adding antibacterial and / or antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents (e.g., sugars or sodium chloride) are preferred. Extended absorption of the injectable composition can be provided by using delayed-absorption agents in the composition, such as aluminum monostearate and gelatin.

[0086] In some embodiments, the pharmaceutical composition may be present in a matrix that controls the release of the composition. In some embodiments, the matrix may comprise lipids, polyvinyl alcohol, polyvinyl acetate, polycaprolactone, poly(glycolic acid), poly(lactic acid), polycaprolactone, polylactic acid, polyanhydride, polylactic acid-co-glycolic acid, polyamino acids, polyethylene oxide, acrylate-terminated polyethylene oxide, polyamide, polyethylene, polyacrylonitrile, polyphosphazene, poly(oxocyanate), isobutyl sucrose acetate (SAIB), combinations thereof, and other polymers, as described in U.S. Patent No. 6,667,371; No. 6, Polymers disclosed in patents No. 613,355; No. 6,596,296; No. 6,413,536; No. 5,968,543; No. 4,079,038; No. 4,093,709; No. 4,131,648; No. 4,138,344; No. 4,180,646; No. 4,304,767; and No. 4,946,931 are hereby expressly incorporated herein by reference in their entirety. In these embodiments, the matrix sustains drug release.

[0087] Pharmaceutically acceptable carriers and / or diluents may also include any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, etc. The use of such media and reagents for pharmaceutically active substances is well known in the art. Unless any conventional media or reagent is incompatible with the active ingredient, its use in a pharmaceutical composition should be considered.

[0088] In some embodiments, the pharmaceutical composition is in the form of an aqueous suspension, which may be prepared from a solution or suspension. Regarding solutions or suspensions, dosage forms may include micelles of lipophilic substances, liposomes (phospholipid capsules / membranes), and / or fatty acids (e.g., palmitic acid). In specific embodiments, the pharmaceutical composition is a solution or suspension capable of dissolving in the mucosal secretions of the epithelium of the tissue to which it is applied and / or delivered, which may advantageously enhance absorption.

[0089] The pharmaceutical composition may be an aqueous solution, a non-aqueous solution, or a combination of aqueous and non-aqueous solutions. Suitable aqueous solutions include, but are not limited to, aqueous gels, aqueous suspensions, aqueous microsphere suspensions, aqueous microsphere dispersions, aqueous liposome dispersions, liposome aqueous micelles, aqueous microemulsions, and any combination thereof, or any other aqueous solution that can be dissolved in a fluid secreted through the mucous membranes of the nasal cavity. Exemplary non-aqueous solutions include, but are not limited to, non-aqueous gels, non-aqueous suspensions, non-aqueous microsphere suspensions, non-aqueous microsphere dispersions, non-aqueous liposome dispersions, non-aqueous emulsions, non-aqueous microemulsions, and any combination thereof, or any other non-aqueous solution that can be dissolved or mixed in a fluid secreted through the mucous membranes.

[0090] Examples of powder formulations include, but are not limited to, simple powder mixtures, micronized powders, freeze-dried powders, lyophilized powders, powder microspheres, coated powder microspheres, liposome dispersions, and any combination thereof. Powder microspheres can be formed from various polysaccharides and celluloses, including but not limited to starch, methylcellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, alginate polyvinyl alcohol, gum arabic, chitosan, and any combination thereof.

[0091] Pharmaceutical compositions may optionally include absorption enhancers, such as agents that inhibit enzyme activity, reduce mucus viscosity or elasticity, reduce mucociliary clearance, open tight junctions, and / or dissolve the active compound. Chemical enhancers are known in the art and include chelating agents (e.g., EDTA), fatty acids, bile salts, surfactants, and / or preservatives. Permeation enhancers can be particularly useful when formulating compounds that exhibit poor membrane permeability, lack lipophilicity, and / or are degraded by aminopeptidase. The concentration of absorption enhancers in a pharmaceutical composition will vary depending on the selected pharmaceutical agent and formulation.

[0092] To extend shelf life, preservatives may optionally be 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 will vary depending on the preservative used, the compound being formulated, the formulation, etc. In representative embodiments, the preservative is present in an amount of about 2% by weight or less.

[0093] Alternatively, the composition may contain 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 orally, rectally, parenterally, intracerebrospinally, vaginally, intraperitoneally, topically (e.g., by powder, ointment, or drops), or buccally to humans and other animals. In some embodiments, the compounds or compositions disclosed herein may be 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, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions may also include adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and aromatizers.

[0096] Injectable formulations, such as sterile injectable aqueous or oily suspensions, can be formulated using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable formulations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic, parenteral-acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable mediators and solvents that can be used are water, Ringer's solution, USP, and isotonic sodium chloride solution. Additionally, sterile fixed oils are routinely used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Furthermore, fatty acids such as oleic acid are used to prepare injectables.

[0097] Injectable formulations can be sterilized, for example, by filtering through a bacterial trap or by incorporating a sterilizing agent in the form of a sterile solid composition, which can be dissolved or dispersed in sterile water or other sterile injectable media before use.

[0098] To prolong the effects of the compounds described herein, it is generally desirable to slow the absorption of compounds administered subcutaneously or intramuscularly. This can be achieved by using liquid suspensions of crystalline or amorphous materials with poor water solubility. The absorption rate of the compound then depends on its dissolution rate, which in turn can depend on crystal size and morphology. Alternatively, delayed absorption of parenteral-administered compounds can be achieved by dissolving or suspending the compound in an oily medium. Injectable reservoir forms are prepared by forming microencapsulation matrices of the compound within biodegradable polymers, such as poly(lactide-polyglycolic acid). The release rate of the compound can be controlled depending on the ratio of compound to polymer and the properties of the specific polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Reservoir-type injectable formulations are also prepared by trapping the compound in liposomes or microemulsions compatible with human tissue.

[0099] The composition for rectal or vaginal application is specifically a suppository, which can be prepared by mixing the compound described herein with a suitable non-irritating excipient or carrier (such as cocoa butter, polyethylene glycol, or suppository wax) that is solid at ambient temperature but liquid at body temperature and thus melts in the rectal or vaginal cavity and releases the active compound.

[0100] Solid dosage forms for oral administration include buccal 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, such as sodium citrate or dicalcium phosphate, and / or a) fillers or enriching agents, such as starch, lactose, sucrose, glucose, mannitol, and silica; b) binders, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; c) humectants, such as glycerin; d) disintegrants, such as agar-agar, calcium carbonate, potato or cassava starch, alginate, certain silicates, and sodium carbonate; e) solution blockers, such as paraffin; f) absorption enhancers, such as quaternary ammonium compounds; g) wetting agents, such as cetyl alcohol and glyceryl monostearate; h) adsorbents, such as kaolin and bentonite; and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain a buffer. In the case of buccal films, the film may use a water-soluble polymer, which allows the film to rapidly hydrate, adhere, and dissolve when placed on the tongue or in the mouth, thereby achieving systemic drug delivery.

[0101] Similar solid compositions can also be used as fillers in soft-filled and hard-filled gelatin capsules, which use excipients such as lactose or toffee and high molecular weight polyethylene glycol. Solid dosage forms such as tablets, sugar-coated pills, capsules, pellets, and granules can be prepared using coatings and shells (such as enteric coatings and other coatings well known in the field of pharmaceutical formulation). These dosage forms may optionally contain emulsifiers, and their composition may also allow the active ingredient to be released, either exclusively or preferentially, in a specific portion of the intestine in a delayed manner. Examples of encapsulation compositions that can be used include polymeric substances and waxes. Similar solid compositions can also be used as fillers in soft-filled and hard-filled gelatin capsules, which use excipients such as lactose or toffee and high molecular weight polyethylene glycol.

[0102] The active compound can also be in microencapsulated form with one or more excipients as described herein. Solid dosage forms such as tablets, sugar-coated pills, capsules, pellets, and granules can be prepared using coatings and shells (such as enteric coatings, release-controlled coatings, and other coatings well known in the field of pharmaceutical formulation). In such solid dosage forms, the active compound can be mixed with at least one inert diluent (such as sucrose, lactose, or starch). Under normal circumstances, in addition to inert diluents, such dosage forms may also contain other substances, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pellets, the dosage forms may also contain buffers. The dosage forms may optionally contain emulsifiers, and their composition may also allow the dosage forms to release the active ingredient, either only or preferentially, in a specific portion of the intestine in a delayed manner. Examples of encapsulation compositions that can be used include polymers and waxes.

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

[0104] The sterile injectable form of the compositions described herein can be an aqueous or oily suspension. These suspensions can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. Sterile injectable formulations can also be sterile injectable solutions or suspensions in non-toxic, parenteral-acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable mediators and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile fixed oils are routinely used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids (such as oleic acid) and their glycerol derivatives can be used to prepare injectables, as well as natural, pharmaceutically acceptable oils (such as olive oil or castor oil), especially in their polyoxyethyleneized form. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersants commonly used to formulate pharmaceutically acceptable dosage forms, including emulsions and suspensions. For formulation purposes, other commonly used surfactants (such as Tween and Span) and other emulsifiers or bioavailability enhancers commonly used in the preparation of pharmaceutically acceptable solid, liquid or other dosage forms may also be used.

[0105] The pharmaceutical compositions described herein can be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions, or solutions. In the case of tablets for oral administration, common carriers include, but are not limited to, lactose and corn starch. Lubricants such as magnesium stearate are typically also added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When an aqueous suspension for oral administration is required, the active ingredient is combined with an emulsifier and a suspending agent. If desired, certain sweeteners, flavoring agents, or coloring agents may also be added.

[0106] Alternatively, the pharmaceutical compositions described herein may be administered in suppository form for rectal administration. These pharmaceutical compositions can be prepared by mixing the pharmaceutical agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and thus 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 to the lower intestine can be achieved using rectal suppository formulations (as described above) or suitable enema formulations. Topical application may also include the use of transdermal patches.

[0108] For topical application, the pharmaceutical composition can be formulated into a suitable ointment containing an active ingredient suspended or dissolved in one or more carriers. Carriers for topical application of the compounds of this disclosure include, but are not limited to, mineral oil, liquid paraffin oil, white paraffin oil, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsified waxes, and water. Alternatively, the pharmaceutical composition can be formulated into a suitable lotion or cream containing an 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, hexadecyl ester wax, cetearyl alcohol, 2-octyldodecyl alcohol, benzyl alcohol, and water.

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

[0110] The pharmaceutical compositions can also be administered via nasal aerosol or inhaler. Such compositions are prepared according to techniques well-known in the field of pharmaceutical formulation and can be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to improve bioavailability, fluorocarbons, and / or other conventional solubilizers or dispersants.

[0111] Compounds used in the methods of this disclosure can be formulated into unit dosage forms. The term "unit dosage form" refers to a physically discrete unit suitable as a single dose to a subject receiving treatment, wherein each unit contains a predetermined amount of active material, optionally associated with a suitable drug carrier, calculated to produce the desired therapeutic effect. A unit dosage form can be a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form can be the same or different for each dose.

[0112] processing method This document provides for the use of the compounds described herein as therapeutic agents. The compounds described herein, or pharmaceutically acceptable salts thereof, may be used to inhibit hexokinase (KHK) and to treat or prevent diseases or conditions in biological samples (e.g., cell cultures) or in humans (e.g., subjects) (e.g., said diseases or conditions are related to KHK dysregulation or fructose metabolism, including those secondary to excessive fructose and / or alcohol intake). Methods for treating or preventing diseases or conditions related to KHK dysregulation or fructose metabolism, including those secondary to excessive fructose and / or alcohol intake, are also provided. The compounds, pharmaceutical compositions, and methods disclosed herein may be used to treat subjects, such as, but not limited to, mammals, humans, non-human mammals, domesticated animals (e.g., laboratory animals, domestic pets, or livestock), non-domesticated animals (e.g., wild animals), dogs, cats, rodents, mice, hamsters, cows, birds, chickens, fish, pigs, horses, goats, sheep, or rabbits, preferably humans.Therefore, the compound can be used to treat any one or more of the following diseases, conditions, symptoms, or related comorbidities (collectively 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-onset diabetes (MODY), malnutrition-associated diabetes, gestational diabetes, hyperglycemia, insulin resistance, hepatic insulin resistance, impaired glucose tolerance, diabetic neuropathy, diabetic nephropathy, diabetic kidney disease (DKD), and kidney disease. Diseases, acute renal dysfunction, renal tubular dysfunction, proximal tubular pro-inflammatory changes, diabetic retinopathy, adipocyte dysfunction, visceral fat deposition, obesity, eating disorders, excessive sugar cravings, excessive alcohol consumption, dyslipidemia, hyperlipidemia, hypertriglyceridemia, elevated total cholesterol, high LDL cholesterol, high non-HDL cholesterol, low HDL cholesterol, hyperinsulinemia, non-alcoholic fatty liver disease (NAFLD), metabolic dysfunction-associated fatty liver disease (MASLD), non-alcoholic steatohepatitis (NASH), metabolic dysfunction-associated steatohepatitis (MASH), MA SLD with increased alcohol intake (MetALD), hepatic steatosis, fibrosis, cirrhosis, hepatocellular carcinoma, hereditary fructose intolerance (HFI), alcoholic steatohepatitis (ASH), viral liver disease, diseases associated with liver fibrosis or cirrhosis such as alpha-1 antitrypsin deficiency, hemochromatosis, pancreatic diseases including pancreatic cancer; gallbladder disease (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, angioplasty Restenosis, intermittent claudication, postprandial hyperlipidemia, 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, restenosis, impaired glucose metabolism, impaired fasting glucose, hyperuricemia, gout, erectile dysfunction, skin and connective tissue diseases, foot ulcers, ulcerative colitis, hyperbeta-lipoproteinemia, Alzheimer's disease, schizophrenia, cognitive impairment, inflammatory bowel disease, ulcerative colitis, Crohn's disease, and irritable bowel syndrome.

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

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

[0115] KHK, or fructokinase, catalyzes the first step in fructose metabolism, phosphorylating fructose to fructose-1-phosphate (F1P) and consuming intracellular ATP and adenine nucleotide pools. There is no negative feedback mechanism by which F1P inhibits KHK metabolism of fructose; therefore, F1P accumulation is directly related to the amount of fructose, which is either (1) transported into the cell via the GLUT transporter or (2) formed intracellularly from glucose via the polyol pathway and metabolized by KHK. The accumulation of F1P and the consumption of ATP and adenine nucleotide pools have detrimental consequences in cells, tissues, and organs, including oxidative stress, endothelial dysfunction, and metabolic disorders. Responses to these impairments include adipogenesis and gluconeogenesis, which trigger metabolic diseases. Patients experiencing KHK metabolism of fructose outside of normal parameters are at risk of developing diseases or conditions due to KHK-mediated dysregulation of fructose metabolism, i.e., diseases or conditions associated with KHK dysregulation.

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

[0117] The terms “disease,” “symptom,” and “symptom” are used interchangeably here to refer to medical or pathological symptoms associated with KHK disorder.

[0118] As used herein, the terms “subject” and “patient” are used interchangeably. The terms “subject” and “patient” refer to an animal (e.g., birds such as chickens, quails, or turkeys) or a mammal, specifically “mammal,” including non-primates (e.g., cows, pigs, horses, sheep, rabbits, guinea pigs, rats, cats, dogs, and mice) and primates (e.g., monkeys, chimpanzees, and humans), and more specifically, humans. Humans can be male or female. In one embodiment, the subject is a non-human animal, such as a farm animal (e.g., a horse, cow, 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 material 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 enzyme isolates) or in humans (e.g., in 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 an appropriate time period. Such assays are known in the art.

[0121] As used herein, the terms “treat,” “treatment,” and “treating” refer to both therapeutic and preventative treatment. For example, therapeutic treatment includes reducing or improving the progression, severity, and / or duration of a disease or condition associated with KHK dysregulation, or improving one or more symptoms (specifically, one or more identifiable symptoms) of a disease or condition associated with KHK dysregulation caused by the administration of one or more therapies (e.g., one or more therapeutic agents, such as compounds or compositions of this disclosure). In specific embodiments, therapeutic treatment includes improving at least one measurable physical parameter of a disease or condition associated with KHK dysregulation. In other embodiments, therapeutic treatment includes inhibiting the progression of a disease or condition associated with KHK dysregulation by, for example, by physical means of stabilizing identifiable symptoms, by physiological means of stabilizing physical parameters, or both. In other embodiments, therapeutic treatment includes reducing or stabilizing a disease or condition associated with KHK dysregulation.

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

[0123] As used herein, the terms “prevention” or “preventive use” and “preventive treatment” refer to any medical or public health procedure designed to prevent, rather than treat or cure, a disease. As used herein, the terms “prevent,” “prevention,” and “preventing” refer to reducing the risk of acquiring or developing a given symptom, or reducing or suppressing the recurrence of said symptom in a healthy subject. The term “chemoprevention” refers to the use of a medicine, such as a small molecule drug (not a “vaccine”), to prevent a symptom or disease.

[0124] As used herein, preventative use includes use in cases where a disease or condition associated with KHK dysregulation or fructose metabolism is detected, including secondary to excessive fructose and / or alcohol consumption. Preventative use may also include treatment of individuals who do not yet have a disease or condition associated with KHK dysregulation or are not considered to be at high risk of developing such a disease or condition, in order to reduce the chance of developing such a disease or condition.

[0125] In some embodiments, the methods disclosed herein are preventive or “preventive” measures taken against patients (specifically, individuals) who are prone to complications due to diseases or conditions associated with KHK dysregulation.

[0126] As used herein, “effective amount” means an amount sufficient to elicit the desired biological response. In this disclosure, the desired biological response is the inhibition of KHK in a biological sample or subject, or the reduction or improvement of the severity, duration, progression, or onset of a disease or condition associated with KHK dysregulation, prevention of the progression of a disease or condition associated with KHK dysregulation, prevention of the recurrence, development, onset, or progression of symptoms associated with a disease or condition associated with KHK dysregulation, or enhancement or improvement of the preventive or therapeutic effect of another therapy for a disease or condition associated with KHK dysregulation. The precise amount of the compound administered to the subject will depend on the route of administration, the type and severity of the disease or condition, and the characteristics of the subject, such as general health status, age, sex, weight, and tolerance to the drug. A skilled technician will be able to determine the appropriate dosage based on these and other factors. When co-administered with other agents, such as when co-administered with another drug, the “effective amount” of the second agent will depend on the type of drug used. Appropriate dosages are known for approved agents and can be adjusted by a skilled technician based on the subject’s condition, the type of condition being treated, and the amount of the compound described herein. Where no specific dosage is specified, a safe and effective dose should be assumed. For example, the compounds described herein may be administered to subjects at doses ranging from approximately 0.01 to 100 mg / kg body weight / day for therapeutic or prophylactic treatment.

[0127] Dosing regimens are typically selected based on a number of factors, including the disease or condition being treated and its severity; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health status, sex, and diet; the timing, route of administration, and excretion rate of the specific compound used; the subject's renal and hepatic function; the specific compound used or its salts, the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors well-known in the medical field. A skilled technician can readily determine and prescribe the effective amount of the compound described herein required to treat, prevent, inhibit (completely or partially) or halt the progression of a disease or condition.

[0128] The dosage range of the compounds described herein for use may be from about 0.01 to about 100 mg / kg body weight / day, from about 0.01 to about 50 mg / kg body weight / day, from about 0.1 to about 50 mg / kg body weight / day, or from about 1 to about 25 mg / kg body weight / day. It should be understood that the total daily dose may be administered as a single dose or may be administered multiple times, 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 the patient within, for example, 48 hours (or within 40 hours, or less than 2 days, or less than 1.5 days, or 24 hours) of symptom onset. The compounds may also be administered to the patient outside this timeframe, for example, within two weeks, six months, one year, five years, or ten years of symptom onset. Therapeutic treatment may continue for any appropriate duration, such as 5 days, 7 days, 10 days, 14 days, etc. For preventative treatment, the compounds described herein may be administered to the patient for any appropriate duration, such as 7 days, 10 days, 14 days, 20 days, 28 days, 35 days, 42 days, etc.

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

[0131] A safe and effective amount can be achieved in the methods or pharmaceutical compositions of this disclosure using a compound of Formula I or Table A, alone or in combination with another suitable therapeutic agent (e.g., a drug or a vaccine), or a pharmaceutically acceptable salt thereof. When using "combination therapy," a safe and effective amount can be achieved using a first amount of a compound of Formula I or Table A, or a pharmaceutically acceptable salt thereof, and a second amount of another suitable therapeutic agent (e.g., a drug or a vaccine).

[0132] In some embodiments, the compound of Formula I or Table A, or its pharmaceutically acceptable salt, and the additional therapeutic agent are each administered in a safe and effective amount (i.e., each in an amount that would be therapeutically effective when administered alone). In other embodiments, the compound of Formula I or Table A, or its pharmaceutically acceptable salt, and the additional therapeutic agent are each administered in an amount that does not provide a therapeutic effect (subtherapeutic dose). In still other embodiments, the compound of Formula I or Table A, or its pharmaceutically acceptable salt, may be administered in a safe and effective amount, while the additional therapeutic agent is administered in a subtherapeutic dose. In still other embodiments, the compound of Formula I or Table A, and its pharmaceutically acceptable salt, may be administered in a subtherapeutic dose, while the additional therapeutic agent is administered in a safe and effective amount.

[0133] Non-limiting examples of other therapeutic agents that may be administered to the subject include antidiabetic drugs, anti-obesity drugs, antihypertensive drugs, anti-anxiety drugs, antidepressants, drugs for the treatment of diabetic nephropathy, drugs for the treatment of diabetic neuropathy, cholesterol / lipid-regulating drugs, calcium channel blockers, cardiac glycosides, diuretics, antiplatelet drugs, anticoagulants, anti-osteoporosis 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 the treatment of bile duct or gallbladder diseases (e.g., primary biliary cholangitis or primary sclerosing cholangitis) and viral hepatitis, drugs for the treatment of AATD and hemochromatosis, drugs for the treatment of heart failure (especially ejection-preserving heart failure), and drugs for the treatment of MASH or NASH. Other non-limiting examples of additional therapeutic agents that may be administered together with compounds disclosed herein (e.g., compounds of Formula I or Table A) include the therapeutic agents 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 drugs 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, etc.). Nagliptin, vildagliptin and saxagliptin, meglitinides, insulin and insulin analogs or mimics, and SGLT1 and / or SGLT2 inhibitors (e.g., dapagliflozin, empagliflozin, tofogliflozin, canagliflozin, ertugliflozin and sotagliflozin).In some cases, compounds of Formula I or Table A, or pharmaceutically acceptable salts, may be co-administered with one or more anti-obesity drugs selected from the group consisting of: amylin analogs (e.g., cangrilintide, pramlintide, AZD6234, LY3841136, amycretin, petrelintide, NN9487, and LY3541105), incretin receptor agonists or modulators (e.g., semaglutide, liraglutide, tirzepatide, survodutide). e), Retatrutide, Pemvidutide, VK2735, RGT-075, Cagrilintide / Semaglutide, Danuglipron, PF-0695422, NN9487, NN9541, CT-388, CT-868, CT-996, Orforglipron, Efinopegdutide, Efocipegtrutide, AZD9550, DR10624, NLY01, Maridebart cavraglutide Cafraglutide, ECC5004, mazdutide, exenatide, dulaglutide, TERN-601, ecnoglutide, and XW-004, melanocortin 4 receptor agonists (e.g., setmelanotide), leptin receptor agonists (e.g., metreleptin and mibavademab), anti-GIPR mAbs, and activin type II receptor antagonists or ligand traps (e.g., bimagrumab, taldefgrobep alfa, trevogrumab, garetosmab, apitegromab, 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 modulators selected from the group consisting of: HMG-CoA reductase inhibitors (e.g., pravastatin, lovastatin, atorvastatin, rosuvastatin, simvastatin, and fluvastatin), cholesterol ester transfer protein inhibitors (e.g., obicetrapib and dalcetrapib), ezetimibe, and PCSK9 inhibitors or modulators (e.g., alirocumab, evolocumab, inclisiran, tafolecimab, recaticimab, AZD-0780, VERVE-102, MK-0616).In some cases, compounds of Formula I or Table A, or pharmaceutically acceptable salts, may be co-administered with one or more agents selected from the group consisting of: FGF21 analogs (e.g., efruxifermin, pegozafermin, BOS-580, B1344, BI3006337, NN9500, NN9499, and HEC8843), thyroid hormone beta receptor agonists (e.g., resimeltiro), and other similar agents. esmetirom), VK2809, ASC41, TERN-501 and ALG-055009, incretin hormone receptor agonists or modulators, PPAR agonists (e.g. pioglitazone, lanifibranor, PXL-065 and saroglitazaar), FASN inhibitors (e.g., denifanstat), acetyl-CoA carboxylase inhibitors (e.g., firsocostat and classtat) Inhibitors or modulators of DGAT1 and / or DGAT2 (e.g., ervogostat, SNP-610, SNP-630, ION224, and PF-07202954), inhibitors or modulators of PNPLA3 (e.g., ALN-PNP, AZD-2693, PF-07853578, LY3849891, JNJ-75220795, and AMG609), α-1 antitrypsin replacement therapy, base editing, or siRNA or R NAi or antisense therapies used to treat alpha-1 antitrypsin deficiency (e.g., fazirsiran, belcesiran, ALN-AAT, NTLA-2003, WVE-006, BEAM-302, and KRRO-110), antiretroviral therapies used to treat HCV or HBV, and inhibitors or modulators of HSD17B13 (e.g., rapirosiran, INI-822, ARO-HSD, and AZD-7503).

[0136] In some cases, compounds of Formula I or Table A, or pharmaceutically acceptable salts, may be co-administered with fructose transporter inhibitors, said fructose transporter inhibitors being 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, gavorestat, ranirestat, epalrestat, fidarestat, imirestat, tolrestat, or risarestat.

[0137] As used herein, the terms “combination” or “co-administration” may be used interchangeably to refer to the use of more than one therapy (e.g., one or more prophylactic and / or therapeutic agents). The use of these terms does not limit the order in which therapies (e.g., prophylactic and / or therapeutic agents) are administered to a subject.

[0138] Co-administration encompasses the co-administration of first and second amounts of compounds in a substantially simultaneous manner, such as as a single pharmaceutical composition, for example, capsules or tablets having first and second amounts in a fixed ratio, or each in multiple separate capsules or tablets. Additionally, such co-administration also encompasses the sequential use of each compound in any order.

[0139] In embodiments, this disclosure relates to methods of combination therapy for inhibiting KHK in biological samples or patients, or methods of combination therapy for treating or preventing diseases or conditions associated with KHK dysregulation in patients using compounds or pharmaceutical compositions described herein, such as compounds of Formula I or Table A, or pharmaceutically acceptable salts thereof. Therefore, pharmaceutical compositions also include pharmaceutical compositions comprising compounds as disclosed herein in combination with one or more additional therapeutic or preventative agents for treating or preventing diseases or conditions associated with KHK dysregulation.

[0140] The methods of using the compounds and compositions disclosed herein also include chemotherapy combined with compounds or compositions of Formula I or Table A or their pharmaceutically acceptable salts, or combinations of the compounds or compositions disclosed herein with another therapeutic or preventative agent.

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

[0142] More specifically, the first therapy (e.g., a preventative or therapeutic agent, such as a compound of this disclosure) may be administered to the subject before (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), simultaneously with, or after (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] It should be understood that 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 can produce an enhanced or synergistic therapeutic effect, wherein the combined effect is greater than the additive effect produced by administering alone 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.

[0144] As used herein, the term "synergistic" refers to a combination of the disclosed compound with another therapy (e.g., a prophylactic or therapeutic agent) that is more effective than the additive effect of the assumed therapy. The synergistic effect of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) may allow for the use of lower doses of one or more therapies and / or a reduced frequency of administration of said therapy to a subject. The ability to use lower doses of therapies (e.g., prophylactic or therapeutic agents) and / or reduce the frequency of administration of said therapy may reduce toxicity associated with administration of said therapy to a subject without reducing the efficacy of said therapy in the prevention, management, or treatment of a condition. Furthermore, synergistic effects may improve the efficacy of the agent in the prevention, management, or treatment of a condition. Finally, the synergistic effect of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) may avoid or reduce adverse or undesirable side effects associated with the use of either therapy alone.

[0145] The presence of synergistic effects can be determined using appropriate methods for assessing drug interactions. Appropriate methods include, for example, the Sigmoid-Emax equation (Holford, NHG, and Scheiner, LB, *Clinical Pharmacokinetics* 6:429-453 (1981)), the Loewe additive equation (Loewe, S, and Muischnek, H., *Arch. Exp. Pathol Pharmacol* 114:313-326 (1926)), and the median effect equation (Chou, TC, and Talalay, P., *Advances in Enzyme Regulation* 22:27-55 (1984)). Each of the equations mentioned above can be applied to experimental data to generate corresponding graphs to assist in assessing the effects of drug combinations. The corresponding graphs associated with the equations mentioned above are concentration-response curves, equivalence curves, and combination exponent curves.

[0146] Chiral separation The compounds described herein may have an asymmetric center and exist as racemates, racemic mixtures, single diastereomers, or enantiomers, and all isomeric forms are included in this disclosure. Compounds of this disclosure having a chiral center can exist and be separated in both optically active and racemic forms. Some compounds may exhibit polymorphism. This disclosure covers the racemic, optically active, polymorphic, or stereoisomeric forms or mixtures thereof of the compounds of this disclosure having the useful properties described herein. The optically active form can be prepared, for example, by resolution of the racemic form using recrystallization techniques, by synthesis from an optically active starting material, by chiral synthesis, or by chromatographic separation using a chiral stationary phase, or by enzymatic resolution. The corresponding compound can be purified first, then derivatized to form the compounds described herein, or the compound itself can be purified.

[0147] Stereotropic stereoisomers or diastereomers are typically prepared by combining a methanesulfonylpyrimidine compound with a stereotropically substituted azahexacyclic butane compound, thereby yielding the desired azahexacyclic butanepyrimidine compound. The methanesulfonylpyrimidine and the stereotropic azahexacyclic butane compound are selected to achieve the desired stereotropic I compound, or the azahexacyclic butanepyrimidine compound can be further derivatized to achieve an alternative I compound. Stereochemical preparation of the hydroxymethyl azahexacyclic butane precursor is known in the literature (e.g., *Journal of Medicinal Chemistry* 2020, 63, 13546-13560). The methyl azahexacyclic butane precursor enantiomers are available from commercial sources. The stereochemistry of the azahexacyclic butane stereocenter is maintained during combination with the methanesulfonylpyrimidine and during further derivatization. Therefore, the stereochemistry of the desired I stereoisomers and diastereomers is known. Alternatively, substituted pyrimidine compounds can be combined with stereooriented substituted azacyclobutane compounds to achieve the desired Formula I compound (or further derivatization), as described in examples (e.g., Example 1).

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

[0149] Examples of methods for obtaining optically active materials include at least the following.

[0150] i) Physical separation of crystals: This technique allows for the manual separation of macroscopic crystals of individual enantiomers. It can be used if individual enantiomers exist, meaning the material is a conglomerate, and the crystals are visually distinct. ii) Simultaneous crystallization: Therefore, the technique of crystallizing individual enantiomers from a racemic solution is only possible when the racemic mixture is a solid aggregate. iii) Co-crystallization This technique enables individual enantiomers to crystallize together from a solution of racemic mixtures. iv) Enzymatic resolution: A technique for partially or completely separating racemic mixtures by utilizing the different reaction rates of enantiomers with enzymes; v) Enzymatic asymmetric synthesis: This technique utilizes at least one enzymatic reaction in the synthesis to obtain an enantiomeric pure precursor or an enriched synthetic precursor of the desired enantiomer. vi) Chemical asymmetric synthesisThis refers to a synthetic technique for synthesizing desired enantiomers from achiral precursors under conditions of asymmetry (i.e., chirality) in the product, which can be achieved using chiral catalysts or chiral auxiliaries. vii) Diastereomer separation: This technique converts individual enantiomers into diastereomers by reacting racemic compounds with an enantiomerically pure reagent (chiral auxiliary agent). The resulting diastereomers are then separated by chromatography or crystallization, based on the more pronounced structural differences exhibited by the diastereomers, and the chiral auxiliary agent is subsequently removed to obtain the desired enantiomer. viii) First-order and second-order asymmetric transformations: This technique allows diastereomers from the racemic mixture to become dominant in a solution of diastereomers from the desired enantiomer, or where preferential crystallization of diastereomers from the desired enantiomer disrupts the equilibrium, ultimately resulting in the conversion of all materials from the desired enantiomer to the crystalline diastereomer in principle. The desired enantiomer is then released from the diastereomers. ix) Dynamic decomposition: This technique refers to the partial or complete resolution (or further resolution of partially resolved compounds) of racemic compounds by means of unequal reaction rates between enantiomers and chiral, non-racemic reagents or catalysts under kinetic conditions. x) Enantiomer-specific synthesis from non-racemic precursors: Thus, a synthetic technique is used to obtain desired enantiomers from achiral starting materials, wherein the stereochemical integrity is not compromised or is only minimally compromised during the synthesis process; xi) Chiral liquid chromatography: This technique separates the enantiomers in a liquid mobile phase by means of the different interactions between the enantiomers of the racemic mixture and the stationary phase (including but not limited to via chiral HPLC). The stationary phase can be made of a chiral material, or the mobile phase can contain other chiral materials to induce different interactions; xii) Chiral gas chromatography: This technique allows the racemic mixture to volatilize and the enantiomers to be separated by the different interactions between the enantiomers in the gaseous flow phase and the column containing a fixed non-racemic chiral adsorbed phase. xiii) Extraction with chiral solvents: This technique separates the enantiomers by preferentially dissolving one enantiomer in a specific chiral solvent; xiv) Transchiral membrane transport: This technique allows a racemic mixture to contact a thin-film barrier. The barrier typically separates two miscible fluids, one containing the racemic mixture, and preferential transmembrane transport is induced by driving forces such as concentration or pressure difference. Separation occurs due to the non-racemic chiral nature of the membrane, which allows only one enantiomer of the racemic mixture to pass through.

[0151] One embodiment uses chiral chromatography, including but not limited to simulated moving bed chromatography. Various chiral stationary phases are commercially available.

[0152] This disclosure will be better understood by referring to the following non-limiting examples.

[0153] Compound Synthesis The following preparation methods for compounds of Formula I and intermediates are provided to enable those skilled in the art to better understand and practice this disclosure. They should not be considered as limiting the scope of this disclosure, but are merely illustrative and representative.

[0154] The starting materials and reagents used in the preparation of these compounds were available from commercial suppliers such as Aldrich Chemical Company (Milwaukee, Wisconsin), Bachem (Torrance, Calif.), or Sigma (St. Louis, Mo.), or were prepared by methods known to those skilled in the art following procedures set forth in the following references: *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 Supplements (Elsevier Science Publishers, 1989); *Organic Reactions*, Volumes 1–40 (John Wiley and Sons, 1991). (1991); *March's Advanced Organic Chemistry* (John Willie & Son Publishing, 4th Edition) and *Larock's Comprehensive Organic Transformations* (VCH Publishers Inc., 1989). These schemes are merely examples of some methods by which the compounds disclosed herein can be synthesized, and various modifications may be made to these schemes, which are recommended for reference by those skilled in the art. If desired, conventional techniques (including but not limited to filtration, distillation, crystallization, chromatography, etc.) can be used to separate and purify the starting materials, intermediates, and final products of the reaction. Such materials can be characterized using conventional means (including physical constants and spectroscopic data).

[0155] The following LC-MS conditions are mentioned in the synthesis examples in this article.

[0156] Method A: 5-95% B - 3.5 min: LC / MS. The column used for chromatography was a 5 μm C18 90A, 30 × 3.0 mm. The detection method was a diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 50-2000. Mobile phase A was 0.04% TFA / water, and mobile phase B was 0.02% TFA / HPLC grade acetonitrile. The gradient was 5-95% B over 3.50 min, 5% B over 0.01 min, 5-95% B (0.01-2.50 min), and held at 95% B for 0.50 min, 95-5% B (3.00-3.01 min), and held at 5% B for 0.49 min. The flow rate was 1 mL / min (0.01-3.00 min) - 1.2 mL / min (3.01-3.50 min).

[0157] Method B: 10-100AB_2 min: LC / MS. The column used for chromatography was C18 5 μm, 3.0 × 30 mm (5 μm particles). The detection method was a diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was 0.04% TFA / water, and mobile phase B was 0.02% TFA / HPLC grade acetonitrile. The gradient was 10-100% B over 1.30 min, 10% B over 0.01 min, 10-100% B (0.01-0.70 min), and held at 100% B for 0.60 min. The flow rate was 1.5 mL / min (0.00-1.30 min).

[0158] Method C: 5-95% B - 2 min: LC / MS. The column used for chromatography was a 5 μm C18 90A, 30 × 3.0 mm. The detection method was a diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 50-2000. Mobile phase A was 0.04% trifluoroacetic acid / water, and mobile phase B was 0.02% trifluoroacetic acid / HPLC-grade acetonitrile. The gradient was 5-95% B over 1.50 min, 5% B over 0.01 min, 5-95% B (0.01-0.70 min), 95% B for 0.46 min, 95-5% B (1.61-1.50 min), and held at 5% B for 0.11 min. The flow rate was 1.5 mL / min.

[0159] Method D: 10-100AB_1 min: LC / MS. The column used for chromatography was C18, 3.0 x 30 mm (5 μm particles). The detection method was a diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 50-2000. Mobile phase A was 0.04% TFA / water, and mobile phase B was 0.02% TFA / HPLC grade acetonitrile. The gradient was 10-100% B over 0.90 min, 10% B over 0.01 min, 10-100% B (0.01-0.50 min), and held at 100% B for 0.40 min. The flow rate was 2.0 mL / min.

[0160] Method E: 5-95AB-6 min-220-254-ELSD: LC / MS. Gradient: 5% B within 0.01 min; 5-95% B (0.01-1.60 min), 95-100% B (1.60-2.50 min), 100-5% (2.50-2.52 min), and maintained at 5% B for 0.48 min. Flow rate: 0.8 mL / min. Mobile phase A: 0.037% trifluoroacetic acid / water; Mobile phase B: 0.018% trifluoroacetic acid / acetonitrile. Column used for chromatography: C18 3.0 × 30 mm, 2.5 μm column (2.5 μm particles). Detection methods: diode array (DAD) and evaporative light scattering (ELSD) detection, as well as positive electrospray ionization. MS range: 100-1000.

[0161] Method F: 5-95% B for 6 min - 220-254-ELSD: LC / MS. The gradient was 5% B over 0.40 min and 5-95% B over 0.40-3.40 min, maintained at 95% B for 0.45 min, and then 95-5% B over 0.01 min, at a flow rate of 0.8 mL / min. Mobile phase A was H₂O + 10 mM NH₄HCO₃; 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). MS mode was positive electrospray ionization. MS range was 100-1000.

[0162] Method G: 5-95% BCD_2 min: LC / MS. The column used for chromatography was C18 2.1 × 50 mm (5 μm particles). The detection method was a diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was 10 mM ammonium bicarbonate / water, and mobile phase B was HPLC-grade acetonitrile. The gradient was 5-95% B over 1.50 min, 5% B over 0.01 min, 5-95% B (0.01-0.70 min), 95% B for 0.46 min, 95-5% B (1.61-1.50 min), and maintained at 5% B for 0.11 min. The flow rate was 1.5 mL / min.

[0163] Method H: 5-95AB_0.8 min: Mobile phase: The flow rate was increased from 5% acetonitrile (0.01875% trifluoroacetic acid) / water (0.0375% trifluoroacetic acid) to 95% acetonitrile / water over 0.60 min at a rate of 2.0 mL / min; then held at 95% acetonitrile for 0.18 min at a rate of 2.0 mL / min; returned to 5% acetonitrile / water and held for 0.02 min at a rate of 2.0 mL / min. The column temperature was 50 °C, and the column was a 2.1 x 30 mm C18 reversed-phase column (5 μm particles).

[0164] Method P: 5-95% B-6 min 220-254-ELSD: LC / MS. Gradients were 5% B over 0.40 min and 5-95% B over 2.60 min, held at 95% B for 1.00 min, and then 95-5% B over 0.01 min, at a flow rate of 1.0 mL / min. Mobile phase A was 0.04% trifluoroacetic acid / water, and mobile phase B was 0.02% trifluoroacetic acid / 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). MS mode was positive electrospray ionization. MS range was 100-1000.

[0165] Method R: 10-80 AB_10 min: LC / MS. The gradient was 10-80% AB over 8.00 min, held at 80% AB for 2.00 min, 80-10% AB over 0.01 min, and then held at 10% AB for 2.99 min (0.5 mL / min flow rate). Mobile phase A was 0.04% trifluoroacetic acid / water, and mobile phase B was 0.02% trifluoroacetic acid / acetonitrile. The column used for chromatography was a HaloAQ-C18 3.0*100 mm column (2.7 μm particles). The detection method was a diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000.

[0166] Method T: Example 1: Synthesis of compound A1 Synthesis of tert-butyl piperazine-1-carboxylate (4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)-1H-pyrazol-1-yl)acetyl)piperazine-1-carboxylate To a solution of 2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)pyrazol-1-yl]acetic acid (8.50 g, 33.72 mmol, 1 equivalent) in DMF (85 mL), tert-butyl piperazine-1-carboxylate (6.28 g, 33.72 mmol, 1 equivalent), DIEA (13.07 g, 101.16 mmol, 17.62 mL, 3 equivalent), and HATU (19.23 g, 50.58 mmol, 1.5 equivalent) were added. The mixture was exchanged three times with N2 and stirred at 25 °C for 1 h under N2 protection. TLC and LCMS showed that all starting materials were consumed and new spots were formed. The reaction mixture was quenched by adding water (50 mL). The mixture was extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate = 3:1 to 0:1 (TLC: petroleum ether:ethyl acetate = 0:1, Rf = 0.7) to give tert-butyl piperazine-1-carboxylate.

[0167] LCMS (ESI+): m / z 421.2 (M+H) + Retention time: 0.689 minutes (Method B) 1 H NMR (400 MHz, DMSO-d6) δ ppm 7.84 (s, 1 H) 7.56 (s, 1 H) 5.16 (s,2 H) 3.35 - 3.54 (m, 8 H) 1.42 (s, 9 H) 1.26 (s, 12 H) Synthesis of tert-butyl piperazine-1-carboxylate To a solution of 4-chloro-5-methyl-2-methylthio-6-(trifluoromethyl)pyrimidine (0.5 g, 2.06 mmol, 1 equivalent) in a mixture of dioxane (4.2 mL) and H₂O (0.8 mL), tert-butyl piperazine-1-carboxylate (952.69 mg, 2.27 mmol, 1.1 equivalents) was added, followed by the addition of 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 exchanged three times with N₂ and stirred at 80 °C for 2 hours under N₂ protection. TLC and LCMS showed that all starting materials were 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 layers were combined and concentrated under reduced pressure to give the residue. The residue was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate = 10:1 to 1:1 (TLC: petroleum ether:ethyl acetate = 1:2, Rf = 0.4) to give tert-butyl 4-[2-[4-[5-methyl-2-methylthio-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylate.

[0168] LCMS (ESI+): m / z 445.3 (M-55) + Retention time: 2.077 minutes (Method A) 1 ¹H NMR (400 MHz, 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) Synthesis of tert-butyl piperazine-1-carboxylate Oxone (4.23 g, 6.87 mmol, 4 equivalents) was added to a solution of 4-[2-[4-[5-methyl-2-methylthio-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylic acid tert-butyl ester (0.86 g, 1.72 mmol, 1 equivalent) in DMF (8.6 mL). The mixture was stirred at 25 °C for 12 h. LCMS showed that the reaction was complete. The reaction mixture was poured into a saturated aqueous solution of NaCl (20 mL) and extracted with ethyl acetate (3 × 10 mL). The combined organic layers were washed with a saturated aqueous solution of Na2SO3 (20 mL) and then dried over Na2SO4 and concentrated to give tert-butyl 4-[2-[4-[5-methyl-2-methylsulfonyl-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylate (which was used directly without further purification).

[0169] LCMS (ESI+): m / z 555.2 (M+Na) + Retention time: 0.569 minutes (Method C) 1 ¹H NMR (400 MHz, 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 = 6.9 Hz, 4H), 3.42–3.36 (m, 2H), 3.33 (s, 3H), 2.62 (d, J = 1.0 Hz, 3H), 1.41 (s, 9H) Synthesis of tert-butyl piperazine-1-carboxylate A solution of 4-[2-[4-[5-methyl-2-methylsulfonyl-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylic acid tert-butyl ester (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 min. LCMS showed a main peak with 8% retention of the starting material and the desired product. (TLC: dichloromethane:methanol = 3:1, Rf = 0.3). The reactants were acidified to pH = 3 with HCl (1 M) and then extracted with EtOAc (3 × 10 mL). The organic compounds were combined and concentrated under reduced pressure to give tert-butyl 4-[2-[4-[2-hydroxy-5-methyl-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylate, which was used directly without further purification.

[0170] LCMS (ESI+): m / z 415.1 (M-55) + Retention time: 0.5-10 minutes (Method C) 1 H NMR (400 MHz, 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 = 5.1 Hz, 2H),3.47 (s, 4H), 3.40 (br d, J = 5.1 Hz, 2H), 2.28 (s, 2H), 2.26 (br s, 1H),1.41 (s, 9H) Synthesis of tert-butyl piperazine-1-carboxylate DBU (242.71 mg, 1.59 mmol, 240 μL, 1.5 equivalents) and BOP (940.13 mg, 2.13 mmol, 2 equivalents) were added to a solution of 4-[2-[4-[2-hydroxy-5-methyl-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylic acid tert-butyl ester (0.5 g, 1.06 mmol, 1 equivalent) in DMF (5 mL). The mixture was stirred at 20 °C for 2 h. TLC and LCMS showed that all starting materials were 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 combined 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 give tert-butyl 4-[2-[4-[2-(benzotriazol-1-yloxy)-5-methyl-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylate.

[0171] LCMS (ESI+): m / z 610.2 (M+Na) + Retention time: 0.651 minutes (Method C) 1 ¹H NMR (400 MHz, chloroform-d) δ = 8.15 (d, J = 8.4 Hz, 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 = 1.1 Hz, 3H), 1.50 (s, 9H) Synthesis of tert-butyl piperazine-1-carboxylate To a solution of tert-butyl piperazine-1-carboxylate (300 mg, 510.59 μmol, 1 equivalent) in acetonitrile (3 mL), DIEA (197.97 mg, 1.53 mmol, 266.81 μL, 3 equivalents) and (2R)-2-(trifluoromethyl)azacyclobutane (CAS No. 2554776-09-7; 258.05 mg, 868.01 μmol, 1.7 equivalents, TsOH) were added. The reaction mixture was stirred at 80 °C for 16 hours. LCMS showed retention of approximately 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, and detection of 30% of the desired product. The reaction mixture was poured into water (5 mL) and extracted with ethyl acetate (2 × 5 mL). The organic layers were combined and concentrated under reduced pressure to obtain a residue, which was purified by preparative TLC (SiO2, petroleum ether: ethyl acetate = 1:5, Rf = 0.5) to give tert-butyl 4-[2-[4-[5-methyl-6-(trifluoromethyl)-2-[(2R)-2-(trifluoromethyl)azacyclobutan-1-yl]pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylate (0.04 g, 69.26 μmol, 13.56% yield) as a pale yellow solid.

[0172] LCMS (ESI+): m / z 600.3 (M+Na) + Retention time: 2.100 minutes (Method A) Synthesis of 2-[4-[5-methyl-6-(trifluoromethyl)-2-[(2R)-2-(trifluoromethyl)azacyclobutan-1-yl]pyrimidin-4-yl]pyrazol-1-yl]-1-piperazin-1-yl-acetone A solution of 4-[2-[4-[5-methyl-6-(trifluoromethyl)-2-[(2R)-2-(trifluoromethyl)azacyclobutan-1-yl]pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylic acid tert-butyl ester (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 h. LCMS showed that all starting materials were consumed and new peaks with the desired Ms were observed. The reaction mixture was purified by preparative HPLC with a Phenomenex Luna C18 column (75 × 30 mm, 3 μm particles) and a mobile phase of [H₂O (0.1% TFA)-ACN]; gradient: 15%–45% B over 8.0 min to give 2-[4-[5-methyl-6-(trifluoromethyl)-2-[(2R)-2-(trifluoromethyl)azacyclobutan-1-yl]pyrimidin-4-yl]pyrazol-1-yl]-1-piperazin-1-yl-acetone.

[0173] LCMS (ESI+): m / z 478.2 (M+H) + Retention time: 2.218 minutes (Method E) 1 H NMR (400 MHz, 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, 8.4 Hz, 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 = 1.5 Hz, 3H) Example 2: Synthesis of compound A2 Synthesis of tert-butyl piperazine-1-carboxylate To a solution of 4-[2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxane-2-yl)pyrazol-1-yl]acetyl]piperazine-1-carboxylic acid tert-butyl ester (5 g, 11.90 mmol, 1 equivalent) in dioxane (45 mL) and H₂O (4.5 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 equivalent), and Pd(PPh₃)₄ (1.37 g, 1.19 mmol, 0.1 equivalent) were added. The mixture was exchanged three times with N₂ and stirred at 80 °C for 2 h under N₂ protection. TLC and LCMS showed that all starting materials were 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 combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate = 1:0 to 1:5 (TLC: petroleum ether:ethyl acetate = 1:1, Rf = 0.6) to give tert-butyl 4-(2-(4-(2-chloro-6-(trifluoromethyl)pyrimidin-4-yl)-1H-pyrazol-1-yl)acetyl)piperazine-1-carboxylate.

[0174] LCMS (ESI+): m / z 419.1 (M+H) + Retention time: 0.757 minutes (Method B) 1 ¹H NMR (400 MHz, chloroform-d) δ ppm 8.34 (s, 1 H) 8.16 (s, 1 H) 7.59 (s, 1 H) 5.09 (s, 2 H) 3.37 - 3.69 (m, 8 H) 1.48 (s, 9 H) Synthesis of (R)-4-(2-(4-(6-(trifluoromethyl)-2-(2-(trifluoromethyl)azacyclobutan-1-yl)pyrimidin-4-yl)-1H-pyrazol-1-yl)acetyl)piperazine-1-carboxylic acid tert-butyl ester To a solution of tert-butyl piperazine-1-carboxylate (290 mg, 610.69 μmol, 1 equivalent) in NMP (2.9 mL), (R)-2-(trifluoromethyl)azacyclobutane 4-toluenesulfonate (181.55 mg, 619.69 μmol, 1 equivalent) and K₂CO₃ (253.20 mg, 1.83 mmol, 3 equivalent) were added. The mixture was stirred at 60 °C for 3 h. LCMS showed that all starting materials were consumed and new peaks with the desired Ms were observed. The reaction mixture was quenched with H₂O (10 mL) and extracted with ethyl acetate (3 × 5 mL). The combined organic layers were washed with brine (5 mL), dried over Na₂SO₄, filtered, and 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 give (R)-4-(2-(4-(6-(trifluoromethyl)-2-(2-(trifluoromethyl)azacyclobutane-1-yl)pyrimidin-4-yl)-1H-pyrazol-1-yl)acetyl)piperazine-1-carboxylic acid tert-butyl ester.

[0175] 1 H NMR (400 MHz, 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 = 7.6 Hz, 2H), 3.55 -3.33 (m,8H), 2.72 - 2.59 (m, 1H), 2.47 - 2.36 (m, 1H), 1.42 (s, 9H) Synthesis of 1-piperazin-1-yl-2-[4-[6-(trifluoromethyl)-2-[(2R)-2-(trifluoromethyl)azacyclobutan-1-yl]pyrimidin-4-yl]pyrazol-1-yl]acetone 4-[2-[4-[6-(trifluoromethyl)-2-[(2R)-2-(trifluoromethyl)azacyclobutan-1-yl]pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylic acid tert-butyl ester (60 mg, 106.48 μmol, 1 equivalent) was added to a solution in DCM (0.5 mL) and TFA (0.1 mL). The mixture was stirred at 25 °C for 1 h. LCMS showed that all starting materials were consumed and new peaks with the desired Ms were observed. The reactants were alkalized to pH 7 with saturated aqueous sodium bicarbonate solution and then extracted with ethyl acetate (3 × 5 mL). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by preparative HPLC (column: Waters Xbridge BEH C18100*30mm*10um; mobile phase: [water(NH4HCO3)-ACN]; gradient: 35%-65%B over 8 minutes) to give 1-piperazin-1-yl-2-[4-[6-(trifluoromethyl)-2-[(2R)-2-(trifluoromethyl)azacyclobutan-1-yl]pyrimidin-4-yl]pyrazol-1-yl]acetone.

[0176] LCMS (ESI+): m / z 464.1 (M+H) + Retention time: 2.178 minutes (Method E) 1 H NMR (400 MHz, 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, 8.8 Hz, 2H), 3.63 -3.53 (m, 4H), 2.93 - 2.78 (m, 4H), 2.67 (dtd, J = 6.1, 9.1, 11.8 Hz, 1H), 2.51 (tdd, J = 6.1, 8.3, 11.9 Hz, 1H) Example 3: Synthesis of compound A3 Synthesis of tert-butyl piperazine-1-carboxylate A mixture of 4-[2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)pyrazol-1-yl]acetyl]piperazine-1-carboxylic acid tert-butyl ester (242.50 mg, 576.97 μmol, 1 equivalent), 4-chloro-5-methyl-2-methylthio-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 equivalent) 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. LCMS showed that 4-[2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)pyrazol-1-yl]acetyl]piperazine-1-carboxylate tert-butyl ester was completely consumed and had a main peak with the desired Ms. The reaction mixture was quenched with water (3 mL) and extracted with ethyl acetate (4 mL × 3). The combined organic layers were washed with brine (4 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1:3, Rf = 0.6) to give 4-[2-[4-[5-methyl-2-methylthio-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylate tert-butyl ester.

[0177] LCMS (ESI+): m / z 445.1 (M-55) + Retention time: 0.837 minutes (Method B) 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.48 (s, 1 H) 8.16 (s, 1 H) 5.29 (s, 2 H) 3.37 - 3.59 (m, 8 H) 2.59 (s, 3 H) 2.46 (d, J=1.31 Hz, 3 H) 1.42 (s, 9H).

[0178] Synthesis of tert-butyl piperazine-1-carboxylate Oxone (982.57 mg, 1.60 mmol, 4 equivalents) was added to a solution of 4-[2-[4-[5-methyl-2-methylthio-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylic acid tert-butyl ester (200 mg, 399.57 μmol, 1 equivalent) in DMF (2 mL). The mixture was stirred at 25 °C for 12 h. LCMS showed that 4-[2-[4-[5-methyl-2-methylthio-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylic acid tert-butyl ester was completely consumed and had a main peak with the desired Ms. The reaction solution was filtered, and the filtrate was quenched with ice water (3 mL) and extracted with ethyl acetate (6 mL × 3). The combined organic layers were washed with brine (10 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue, which was purified by preparative TLC (SiO2, petroleum ether / ethyl acetate = 1:3, Rf = 0.3) to give tert-butyl 4-[2-[4-[5-methyl-2-methylsulfonyl-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylate.

[0179] LCMS (ESI+): m / z 433.1 (M-100) + Retention time: 0.706 minutes (Method B) Synthesis of tert-butyl piperazine-1-carboxylate To (2S)-2-methylazacyclobutane (CAS No. 935669-67-3; 80 mg, 743.61 μmol, 2 equivalents, HCl salt), 4-[2-[4-[5-methyl-2-methylsulfonyl-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylic acid tert-butyl ester (198 mg, 371.81 μmol, 1 equivalentTEA (150.49 mg, 1.49 mmol, 207.00 μL, 4 equivalents) was added to a solution of NMP (2 mL). The mixture was stirred at 60 °C for 3 h. LCMS showed that 4-[2-[4-[5-methyl-2-methylsulfonyl-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylic acid tert-butyl ester was completely consumed and had a main peak with the desired Ms. The reaction mixture was quenched with water (1.5 mL) and extracted with ethyl acetate (2 mL × 3). The combined organic layers were washed with brine (2 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue, which was purified by preparative HPLC (neutral conditions: H2O (0.05% NH3H2O ​​+ 10 mM NH4HCO3); B: ACN) to give 4-[2-[4-[5-methyl-2-[(2S)-2-methylazacyclobutan-1-yl]-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylic acid tert-butyl ester.

[0180] LCMS (ESI+): m / z 524.2 (M+H) + Retention time: 0.898 minutes (Method B) Synthesis of 2-[4-[5-methyl-2-[(2S)-2-methylazacyclobutane-1-yl]-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]-1-piperazin-1-yl-acetone TFA (0.08 mL) was added to a solution of 4-[2-[4-[5-methyl-2-[(2S)-2-methylazacyclobutan-1-yl]-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylate (50 mg, 95.50 μmol, 1 equivalent) in DCM (0.42 mL). The mixture was stirred at 25 °C for 1 h. LCMS showed that 4-[2-[4-[5-methyl-2-[(2S)-2-methylazacyclobutan-1-yl]-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylate was completely consumed and had a main peak with the desired Ms. The pH of the reaction mixture was adjusted to 7-8 with saturated aqueous NaHCO3 solution. The reaction mixture was quenched with water (1 mL) and extracted with ethyl acetate (1.5 mL × 3). The combined organic layers were washed with brine (1.5 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give 2-[4-[5-methyl-2-[(2S)-2-methylazacyclobutan-1-yl]-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]-1-piperazin-1-yl-acetone.

[0181] LC / MS (ESI+): m / z 424.2 (M+H) + Retention time: 2.806 minutes (Method F) 1 H NMR (400 MHz, methanol-d4) δ ppm 8.25 (s, 1 H) 8.09 (s, 1 H) 5.25 (s, 2 H) 4.45 - 4.55 (m, 1 H) 3.96 - 4.10 (m, 2 H) 3.63 (br s, 4 H) 2.85 - 3.01(m, 4 H) 2.42 (br s, 1 H) 2.40 (d, J=1.31 Hz, 3 H) 1.98 - 2.08 (m, 1 H) 1.54(d, J=6.20 Hz, 3 H) The following compounds were prepared using a method similar to that described for compounds A1-A3.

[0182] Example 4: Synthesis of compound A8 Synthesis of ethyl 4,4,4-trifluoro-2-methoxy-3-oxo-butyrate NaH (23.70 g, 592.56 mmol, 1 equivalent) was added to a solution of ethyl 2-methoxyethyl (35 g, 296.28 mmol, 1 equivalent) in THF (175 mL) at 0 °C. The mixture was stirred at 50 °C for 12 h. TLC showed that all starting materials were consumed and new spots were formed. The reaction mixture was poured into a solution of H₂SO₄ / H₂O = 1 / 10 (11 V) and extracted with ethyl acetate (3 × 150 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate = 3:1 to 1:1 (TLC: petroleum ether:ethyl acetate = 3:1, Rf = 0.4) to give ethyl 4,4,4-trifluoro-2-methoxy-3-oxo-butyrate.

[0183] 1 H NMR (400 MHz, DMSO-d6) δ ppm 4.12 - 4.20 (m, 2 H) 3.91 (s, 1 H)3.31 (s, 3 H) 1.20 - 1.24 (m, 3 H) Synthesis of 5-methoxy-2-methylthio-6-(trifluoromethyl)pyrimidin-4-ol To a solution of ethyl 4,4,4-trifluoro-2-methoxy-3-oxo-butyrate (10 g, 46.70 mmol, 1 equivalent) in EtOH (50 mL), 2-methylisothiourea; sulfuric acid (19.50 g, 70.05 mmol, 1.5 equivalent) and Na₂CO₃ (1 M, 107.40 mL, 2.3 equivalent) were added. The mixture was stirred at 25 °C for 12 h. TLC and LC / MS showed that all starting material was consumed and new spots were formed. The reaction mixture was quenched with water (100 mL) and extracted with ethyl acetate (3 × 150 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give 5-methoxy-2-methylthio-6-(trifluoromethyl)pyrimidin-4-ol, which was used directly without further purification.

[0184] LC / MS (ESI+): m / z 240.9 (M+1) + Retention time: 0.429 minutes (Method D) Synthesis of 4-chloro-5-methoxy-2-methylthio-6-(trifluoromethyl)pyrimidine: POCl3 (63.80 g, 41.60 mmol, 1 equivalent) was added to a solution of 5-methoxy-2-methylthio-6-(trifluoromethyl)pyrimidin-4-ol (10 g, 41.60 mmol, 1 equivalent) in ACN (100 mL). The mixture was stirred at 80 °C for 12 h. The reaction was shown to be complete by LC / MS. The reaction mixture was quenched by adding water (50 mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate = 1:1 (TLC: petroleum ether:ethyl acetate = 1:1, Rf = 0.4) to 4-chloro-5-methoxy-2-methylthio-6-(trifluoromethyl)pyrimidin.

[0185] LC / MS (ESI+): m / z 258.8 (M+1) + Retention time: 0.583 minutes (Method D) 1 H NMR (400 MHz, DMSO-d6) δ ppm 3.91 (s, 3 H) 2.55 (s, 3 H) Synthesis of tert-butyl 3-[4-[5-methoxy-2-methylthio-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylate To a solution of 4-chloro-5-methoxy-2-methylthio-6-(trifluoromethyl)pyrimidine (400 mg, 1546.50 μmol, 1 equivalent) and 3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)pyrazol-1-yl]azacyclobutane-1-carboxylic acid tert-butyl ester (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) and di-tert-butyl(cyclopentyl)phosphine; palladium dichloro; 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 materials were 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 combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate = 1:1 (TLC: petroleum ether:ethyl acetate = 1:1, Rf = 0.4) to give tert-butyl 3-[4-[5-methoxy-2-methylthio-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylate.

[0186] LCMS (ESI+): m / z 390 (M-55) + Retention time: 0.995 minutes (Method G) Synthesis of tert-butyl 3-[4-[5-methoxy-2-methylsulfonyl-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylate Oxone (1.66 g, 2.69 mmol, 4 equivalents) was added to a solution of 3-[4-[5-methoxy-2-methylthio-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylic acid tert-butyl ester (300 mg, 673.46 μmol, 1 equivalent) in DMF (3 mL). The mixture was stirred at 30 °C for 24 h. 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 × 20 mL) and NaCl (50 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give 3-[4-[5-methoxy-2-methylsulfonyl-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylic acid tert-butyl ester, which was used directly without further purification.

[0187] LCMS (ESI+): m / z 422.0 (M-55) + Retention time: 0.847 minutes (Method C) Synthesis of tert-butyl 3-[4-[2-[(2S,3R)-3-hydroxy-2-methyl-azacyclobutane-1-yl]-5-methoxy-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylate TEA (317.90 mg, 3.14 mmol, 437.28 μL, 5 equivalents) was added to a solution of 3-[4-[5-methoxy-2-methylsulfonyl-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylic acid tert-butyl ester (300 mg, 628.33 μmol, 1 equivalent) and (2S,3R)-2-methylazacyclobutane-3-ol (200.7 mg, 628.33 mmol, 1 equivalent, R-CSA salt; synthesized as described in the Journal of Medicinal Chemistry 2020, 63, 13546−13560, intermediate 13 / (R)-CSA salt) in NMP (3 mL). The mixture was stirred at 60 °C for 12 h. TLC and LCMS showed that all starting materials were consumed and new spots were formed. The reaction mixture was extracted with ethyl acetate (3 × 10 mL) and a solution of NaCl (20 mL). The residue was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate = 1:1 (TLC: petroleum ether:ethyl acetate = 1:1, Rf = 0.4) to give tert-butyl 3-[4-[2-[(2S,3R)-3-hydroxy-2-methyl-azacyclobutane-1-yl]-5-methoxy-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylate.

[0188] LCMS (ESI+): m / z 485 (M+1) + Retention time: 0.553 minutes (Method D) Synthesis of (2S,3R)-1-[4-[1-(azacyclobutane-3-yl)pyrazol-4-yl]-5-methoxy-6-(trifluoromethyl)pyrimidin-2-yl]-2-methyl-azacyclobutane-3-ol A solution of tert-butyl 3-[4-[2-[(2S,3R)-3-hydroxy-2-methyl-azacyclobutan-1-yl]-5-methoxy-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]azacyclobutan-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 h. TLC and LCMS showed that all starting material was consumed and new spots were formed. The reaction mixture was concentrated under reduced pressure to give (2S,3R)-1-[4-[1-(azacyclobutan-3-yl)pyrazol-4-yl]-5-methoxy-6-(trifluoromethyl)pyrimidin-2-yl]-2-methyl-azacyclobutan-3-ol].

[0189] LCMS (ESI+): m / z 385 (M+1) + Retention time: 0.353 minutes (Method D) Synthesis of (2S,3R)-1-[5-methoxy-4-[1-(1-methylazacyclobutane-3-yl)pyrazol-4-yl]-6-(trifluoromethyl)pyrimidin-2-yl]-2-methyl-azacyclobutane-3-ol 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 to a solution of (2S,3R)-1-[4-[1-(azacyclobutan-3-yl)pyrazol-4-yl]-5-methoxy-6-(trifluoromethyl)pyrimidin-2-yl]-2-methyl-azacyclobutan-3-ol (200 mg, 520.35 μmol, 1 equivalent) in DCM (4 mL). 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 reversed-phase HPLC (column: Pheromones Gemini NX-C18 75×30 mm (3 μm particles); mobile phase: [H2O (0.05% NH3H2O ​​+ 10 mM NH4HCO3)-ACN]; gradient: (25%-50% B over 8.0 min)) to give (2S,3R)-1-[5-methoxy-4-[1-(1-methylazacyclobutan-3-yl)pyrazol-4-yl]-6-(trifluoromethyl)pyrimidin-2-yl]-2-methyl-azacyclobutan-3-ol).

[0190] LC / MS (ESI+): m / z 399 (M+1) + Retention time: 2.636 minutes (Method E) 1 H NMR (400 MHz, methanol-d4) δ ppm 8.46 (s, 1 H) 8.24 (s, 1 H) 5.14 (quin, J=7.12 Hz, 1 H) 4.28 (dd, J=9.23,6.05 Hz, 1 H) 4.07 - 4.22 (m, 2 H)3.88 (t, J=8.07 Hz, 2 H) 3.62 - 3.78 (m, 6 H) 2.49 (s, 3 H) 1.55 (d, J=6.11Hz, 3 H) The following compounds were prepared using a method similar to that described for compound A8.

[0191] Example 5: Synthesis of compounds A21 and A35 Synthesis of ethyl 4,4-difluoro-2-methyl-3-oxobutyrate Sodium hydride (1.96 g, 48.96 mmol, 60% purity, 1 equivalent) was added to a solution of ethyl propionate (5 g, 48.96 mmol, 5.62 mL, 1 equivalent) in tetrahydrofuran (25 mL) at 0 °C. After addition, the mixture was stirred at this temperature for 5 min, and then ethyl 2,2-difluoroacetate (12.15 g, 97.91 mmol, 2 equivalent) was added dropwise at 25 °C. The resulting mixture was stirred at 50 °C for 2 h. TLC indicated that the starting material was consumed and a major new spot was formed. The reaction mixture was quenched at 0 °C by adding a saturated ammonium chloride solution (50 mL), then diluted with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic layers were washed with brine (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give ethyl 4,4-difluoro-2-methyl-3-oxo-butyrate (7.8 g, 88% yield) as a yellow oil, which was used directly in the next step without purification.

[0192] 1 H NMR (400 MHz, chloroform-d) δ ppm 5.75 - 6.08 (m, 1 H), 4.10 (q, J =7.13 Hz, 2 H), 3.88 (q, J = 7.09 Hz, 1H), 1.41 (d, J = 7.25 Hz, 3 H), 1.27 -1.35 (m, 3 H).

[0193] Synthesis of 6-(difluoromethyl)-5-methyl-2-(methylthio)pyrimidin-4-ol 2-Methylisothiourea sulfate (7.73 g, 27.75 mmol, 1 equivalent) and sodium carbonate solution (1 M, 63.84 mL, 2.3 equivalent) were added to a solution of ethyl 4,4-difluoro-2-methyl-3-oxo-butyrate (5 g, 27.75 mmol, 1 equivalent) in ethanol (250 mL) at 25 °C. The resulting mixture was stirred at 25 °C for 12 h. LC-MS showed that the starting material was consumed and the desired compound was detected. The reaction mixture was concentrated under vacuum to give an oil, which was extracted with ethyl acetate (60 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate = 0:1 to 3:1 (TLC: petroleum ether:ethyl acetate = 3:1, R...). f= 0.45) to give 6-(difluoromethyl)-5-methyl-2-methylthio-pyrimidin-4-ol as a white solid (3.7 g, 65% yield).

[0194] LCMS (ESI+): m / z 207.0 (M+H) + Retention time: 0.318 minutes (Method H) 1 H NMR (400 MHz, DMSO-d6) δ ppm 9.85 - 10.59 (m, 1 H), 6.59 - 6.94 (m, 1 H), 2.41 (s, 3 H) 1.95 (t, J = 2.25 Hz, 3 H).

[0195] LCMS (ESI+): m / z 207.0 (M+H) + Retention time: 0.318 minutes (Method H) Synthesis of 4-chloro-6-(difluoromethyl)-5-methyl-2-(methylthio)pyrimidine Phosphorus oxychloride (13.01 g, 84.86 mmol, 7.91 mL, 5 equivalents) was added to a solution of 6-(difluoromethyl)-5-methyl-2-methylthio-pyrimidin-4-ol (3.5 g, 16.97 mmol, 1 equivalent) in acetonitrile (35 mL) at 20 °C. The mixture was stirred at 80 °C for 4 h. LCMS showed that the starting material was completely consumed and the desired mass was detected. The reaction mixture was concentrated under vacuum to give an oil, which was extracted with ethyl acetate (60 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the compound 4-chloro-6-(difluoromethyl)-5-methyl-2-methylthio-pyrimidin (2.5 g, 66% yield) as a gray solid. This was used directly for the next step without purification.

[0196] LCMS (ESI+): m / z 225.0 (M+H) + Retention time: 0.439 minutes (Method H) 1 H NMR (400 MHz, DMSO-d6) δ ppm 6.96 - 7.25 (m, 1 H), 2.54 (d, J=1.38Hz, 3 H), 2.33 (d, J=1.50 Hz, 3 H).

[0197] Synthesis of tert-butyl 3-[4-[6-(difluoromethyl)-5-methyl-2-methylthiopyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylate At 25°C, 4-chloro-6-(difluoromethyl)-5-methyl-2-methylthiopyrimidine (500 mg, 2.19 mmol, 1 equivalent ) and tert-butyl 3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)pyrazol-1-yl]azacyclobutane-1-carboxylate (914.05 mg, 2.62 mmol, 1.2 mmol) equivalent Add Na2CO3 (935.4 mg, 10.95 mmol, 5 mg) to a solution of dioxane (5 mL) and H2O (0.5 mL). equivalent ) and DTPPF PdCl2 (49.8 mg, 111.28 μmol, 0.05 when quantity The mixture was stirred at 85 °C for 2 hours. LCMS showed that the starting material was completely consumed and a main peak with the desired quality was detected. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (40 mL × 3). The combined organic layers were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate = 1:0 to 10:1 (TLC: petroleum ether:ethyl acetate = 10:1, Rf = 0.6) to give tert-butyl 3-[4-[6-(difluoromethyl)-5-methyl-2-methylthiopyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylate (600 mg, 37% yield) as a yellow oil.

[0198] LCMS (ESI+): m / z 412.0 (M+H) + Retention time: 0.495 minutes (Method H) 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.61 (s, 1 H), 8.26 (s, 1 H), 7.11 (t, J = 53.28 Hz, 1 H), 5.30 - 5.39 (m, 1 H), 4.28 - 4.37 (m, 2 H), 4.20 (brs, 2 H), 2.57 (s, 3 H), 2.44 (s, 3 H), 1.41 (s, 9 H).

[0199] Synthesis of tert-butyl 3-[4-[6-(difluoromethyl)-5-methyl-2-methylsulfonyl-pyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylate At 20°C, tert-butyl 3-[4-[6-(difluoromethyl)-5-methyl-2-methylthiopyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylate (500 mg, 1.22 mmol, 1 equivalent Add Oxone (820 mg, 7.29 mmol, 4 mmol) to a solution of DMF (5 mL). equivalent The mixture was stirred at 30°C for 24 hours. LCMS showed that the reactants were completely consumed and a main peak with the desired mass was detected. The reaction mixture was poured into water (15 mL). The solid precipitated and collected by filtration. The filter cake was dried under high vacuum to give tert-butyl 3-[4-[6-(difluoromethyl)-5-methyl-2-methylsulfonyl-pyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylate (400 mg, 74% yield) as a yellow solid. It was used directly in the next step without purification.

[0200] LCMS (ESI+): m / z 387.9 (M-55) + Retention time: 0.422 minutes (Method H) 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.79 (s, 1 H), 8.40 (s, 1 H), 7.35 (t, J = 52.84 Hz, 1 H), 5.32 - 5.42 (m, 1 H), 4.29 - 4.46 (m, 2 H), 4.21 (brs, 2 H), 3.49 (s, 3 H), 2.61 (s, 3 H), 1.42 (s, 9 H).

[0201] Synthesis of tert-butyl 3-[4-[6-(difluoromethyl)-2-[(2S,3R)-3-hydroxy-2-methyl-azacyclobutane-1-yl]-5-methyl-pyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylate At 20°C, tert-butyl 3-[4-[6-(difluoromethyl)-5-methyl-2-methylsulfonyl-pyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylate (450 mg, 1.01 mmol, 1 equivalent Add TEA (513.40 mg, 5.07 mmol, 706.19 μL, 5) to a THF (5 mL) solution. equivalent) and (2S,3R)-2-methylazacyclobutane-3-ol (176.81 mg, 2.03 mmol, 2 Equivalent, R-CSA salt; Synthesized as described in the *Journal of Medicinal Chemistry* 2020, 63, 13546−13560 (intermediate 13 / (R)-CSA salt). The mixture was stirred at 60 °C for 3 h. LCMS showed that the starting material was completely consumed and a main peak with the desired mass was detected. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give tert-butyl 3-[4-[6-(difluoromethyl)-2-[(2S,3R)-3-hydroxy-2-methyl-azacyclobutane-1-yl]-5-methyl-pyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylate (400 mg, 887.94 μmol, 87.50% yield) as a white solid. It was used directly in the next step without purification.

[0202] LCMS (ESI+): m / z 451.1 (M+H) + Retention time: 0.463 minutes (Method H) 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.45 (s, 1 H), 8.15 (s, 1 H), 6.90 (t, J = 53.72 Hz, 1 H), 5.28 - 5.47 (m, 1 H), 4.31 (br t, J = 8.50 Hz, 2 H), 4.16 (brdd, J = 8.50, 6.38 Hz, 3 H), 4.05 (td, J = 10.98, 5.07 Hz, 2 H), 3.61 (dd, J = 8.76, 4.88 Hz, 1 H), 3.49 (s, 1 H), 2.33 (s, 3 H), 1.44 - 1.49 (m, 3H), 1.41(s, 9 H).

[0203] Synthesis of (2S,3R)-1-[4-[1-(azacyclobutane-3-yl)pyrazol-4-yl]-6-(difluoromethyl)-5-methyl-pyrimidin-2-yl]-2-methyl-azacyclobutane-3-ol A35 At 0 °C, tert-butyl 3-[4-[6-(difluoromethyl)-2-[(2S, 3R)-3-hydroxy-2-methyl-azacyclobutane-1-yl]-5-methyl-pyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylate (380 mg, 843.54 μmol, 1 when quantity A solution of the starting material was prepared in DCM (5 mL) and TFA (1 mL). The mixture was stirred at 20 °C for 1 hour. LCMS showed that the starting material was completely consumed and a main peak with the desired mass was detected. The reaction mixture was concentrated under vacuum to give (2S,3R)-1-[4-[1-(azacyclobutan-3-yl)pyrazol-4-yl]-6-(difluoromethyl)-5-methylpyrimidin-2-yl]-2-methyl-azacyclobutan-3-ol (250 mg, 85% yield) as a colorless oil.

[0204] LCMS (ESI+): m / z 351.0 (M+H) + Retention time: 0.281 minutes (Method H) 1 H NMR (400 MHz, methanol-d4) δ 8.31 (s, 1H), 8.14 (s, 1H), 6.58 (t, J =54.3 Hz, 1H), 5.36 (quin, J = 7.5 Hz,1H), 4.28 (dd, J = 6.1, 8.9 Hz, 1H), 4.21 - 4.11 (m, 4H), 4.01 - 3.93 (m, 2H), 3.73 (dd, J= 4.9, 9.0 Hz, 1H), 2.42(s, 3H), 1.55 (d, J = 6.1 Hz, 3H) Synthesis of (2S,3R)-1-[4-(difluoromethyl)-5-methyl-6-[1-(1-methylazacyclobutan-3-yl)pyrazol-4-yl]pyrimidin-2-yl]-2-methyl-azacyclobutan-3-ol A21 To (2S,3R)-1-[4-[1-(azacyclobutane-3-yl)pyrazol-4-yl]-6-(difluoromethyl)-5-methyl-pyrimidin-2-yl]-2-methyl-azacyclobutane-3-ol (250 mg, 710.48 μmol, 1 equivalent Add NaBH(OAc)3 (752.81 mg, 3.55 mmol, 5 mg) to the solution in DCM (10 mL). equivalent ) and formaldehyde (61.02 mg, 2.13 mmol, 30%, 1 when quantity The mixture was stirred at 20°C for 1 hour. LCMS showed that the starting material was completely consumed and a main peak with the desired mass was detected. The reactants were concentrated under vacuum. The residue was purified by preparative HPLC (TFA conditions; column: Welch Ultimate C18 150 × 25 mm × 5 μm; mobile phase: [water (TFA)-ACN]; gradient: 15%-35% B over 2 min) to give (2S,3R)-1-[4-(difluoromethyl)-5-methyl-6-[1-(1-methylazacyclobutan-3-yl)pyrazol-4-yl]pyrimidin-2-yl]-2-methyl-azacyclobutan-3-ol (104 mg, 32% yield) as a white solid.

[0205] LCMS (ESI+): m / z 365.3 (M+H) + Retention time: 1.783 minutes (Method F) 1 H NMR (400 MHz, methanol-d4) δ ppm 8.28 (d, J = 12.38 Hz, 2 H), 6.58 (t,J = 54.34 Hz, 1 H), 5.47 (br t, J =6.75 Hz, 1 H), 4.35 - 4.84 (m, 4 H), 4.27(dd, J = 8.82, 6.44 Hz, 1 H), 4.09 - 4.23 (m, 2 H), 3.72 (dd, J = 8.94, 4.94Hz, 1 H), 3.13 (br d, J = 1.50 Hz, 3H), 2.41 (s, 3 H), 1.55 (d, J = 6.25 Hz, 3 H).

[0206] Example 6: Synthesis of HCl salts of A21 Free A21 base (2.1 mL (10 volumes) of methanol, 2.1 mL (10 volumes) of THF, and 4.2 mL (20 volumes) of acetone) was dissolved at 50 °C and then aliquoted into six HPLC vials, each containing approximately 30 mg of A21 in solution. 1.1 mol of equivalent HCl (91 μL of 1 M THF) was added to the approximately 30 mg A21 in the solution at 50 °C. The sample was stirred at 50 °C for 1 hour before cooling to 5 °C overnight at 0.1 °C / min. After cooling to 5 °C, the resulting clear solution was left open to evaporate at room temperature. The A21 HCl salt suspension was filtered using a sintered PE filter cartridge under positive pressure, and the solid was dried under suction. The A21 HCl salt was separated from 1 g of free A21 base from 5 volumes of methanol using 0.45 μm PTFE filter paper, yielding 86.1%.

[0207] Figure 1 The XPRD diffraction patterns of A21 free base and A21 HCl salt are shown for comparison.

[0208] Figure 2 The relationship between A21 free base and HCl salt (DMSO-d6) is shown. 1 Comparison of H NMR spectra. A summary of peaks is listed.

[0209] A21 free base: 6H peak at 2.33 ppm; A21 HCl salt: 2 x 3H peaks at 2.34 and 2.96 ppm.

[0210] A21 free base: 2H peaks at 3.42 and 3.71 ppm; A21 HCl salt: 2H peaks at 4.41 and 4.59 ppm.

[0211] A21 free base: 1H peak at 5.07 ppm; A21 HCl salt: 1H peak at 5.48 ppm.

[0212] Example 7: Synthesis of compound A41 Synthesis of ethyl 2-ethyl-4,4,4-trifluoro-3-oxo-butyrate Ethyl butyrate (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 indicated that almost all the starting material was consumed, and a major new spot with high polarity was detected. The reaction mixture was adjusted to pH 2 with 3N HCl (100 mL) and extracted with DCM (100 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give ethyl 2-ethyl-4,4,4-trifluoro-3-oxo-butyrate (3.5 g, 39% yield) as a colorless oil.

[0213] 1 ¹H NMR (400 MHz, chloroform-d) δ = 4.27 - 4.18 (m, 2H), 3.80 - 3.69 (m, 1H), 2.02 (quin, J = 7.4 Hz, 2H), 1.30 - 1.23 (m, 3H), 1.02 - 0.97 (m, 3H) Synthesis of 5-ethyl-2-methylthio-6-(trifluoromethyl)pyrimidin-4-ol Na₂CO₃ (1 M, 54.20 mL, 2.3 equivalents) was added to a solution of 2-methylisothiourea sulfate (6.56 g, 23.57 mmol, 1 equivalent) in EtOH (25 mL). The mixture was stirred at 25 °C for 30 min. Then, ethyl 2-ethyl-4,4,4-trifluoro-3-oxo-butyrate (5 g, 23.57 mmol, 1 equivalent) was added. The mixture was stirred at 25 °C for 12 h. 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 combined organic layers were washed with an aqueous solution of NaCl (100 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give 5-ethyl-2-methylthio-6-(trifluoromethyl)pyrimidin-4-ol (4.5 g, 80% yield) as a yellow oil.

[0214] LCMS (ESI+): m / z 238.9 (M+H) + Retention time: 0.477 minutes (Method D) Synthesis of 4-chloro-5-ethyl-2-methylthio-6-(trifluoromethyl)pyrimidine POCl3 (160.91 g, 1.05 mol, 97.82 mL, 10 equivalents) was added to a solution of 5-ethyl-2-methylthio-6-(trifluoromethyl)pyrimidin-4-ol (25 g, 104.94 mmol, 1 equivalent) in ACN (250 mL). The mixture was stirred at 80 °C for 5 h. 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 a residue. The residue was diluted with H2O (500 mL) and extracted with ethyl acetate (200 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by rapid silica gel chromatography (ISCO®; 120 g SepaFlash® silica gel column, 0-5% ethyl acetate / petroleum ether eluent, gradient 80 mL / min) to give 4-chloro-5-ethyl-2-methylthio-6-(trifluoromethyl)pyrimidine (25 g, 93% yield) as a colorless oil.

[0215] LCMS (ESI+): m / z 257.0 (M+H) + Retention time: 0.645 minutes (Method D) 1 ¹H NMR (400 MHz, chloroform-d) δ = 2.85 (q, J = 7.3 Hz, 2H), 2.59 (s, 3H), 1.24 (t, J = 7.5 Hz, 3H) Synthesis of tert-butyl piperazine-1-carboxylate: A mixture of 4-chloro-5-ethyl-2-methylthio-6-(trifluoromethyl)pyrimidine (1 g, 3.90 mmol, 1 equivalent), 4-[2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)pyrazol-1-yl]acetyl]piperazine-1-carboxylic acid tert-butyl ester (3.28 g, 7.79 mmol, 2 equivalents), DTBPFPdCl2 (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 and purged three times with N2. The mixture was then stirred at 80 °C under N2 atmosphere for 2 h. 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 × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 3 / 1) to give tert-butyl 4-(2-(4-(5-ethyl-2-(methylthio)-6-(trifluoromethyl)pyrimidin-4-yl)-1H-pyrazol-1-yl)acetyl)piperazine-1-carboxylate (800 mg, 39% yield) as a white solid.

[0216] 1 H NMR (400 MHz, 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).

[0217] Synthesis of tert-butyl piperazine-1-carboxylate: Oxone (1.88 g, 3.05 mmol, 3 equivalents) was added to a solution of tert-butyl piperazine-1-carboxylate (800 mg, 1.02 mmol, 1 equivalent) in DMF (8 mL). The mixture was stirred at 25 °C for 12 h. 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 combined organic layers were washed with brine (10 mL × 7), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by rapid silica gel chromatography (ISCO®; 4 g SepaFlash® silica gel column, 70-80% ethyl acetate / petroleum ether eluent, gradient 40 mL / min) to give tert-butyl 4-(2-(4-(5-ethyl-2-(methanesulfonyl)-6-(trifluoromethyl)pyrimidin-4-yl)-1H-pyrazol-1-yl)acetyl)piperazine-1-carboxylate (600 mg, yield 71%) as a white solid.

[0218] 1 H NMR (400 MHz, 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) Synthesis of tert-butyl-4-[2-[4-[2-[(2R)-2-(difluoromethyl)azacyclobutane-1-yl]-5-ethyl-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylate: To (2R)-2-(difluoromethyl)azacyclobutane (CAS No. 2231665-58-8; 15.20 mg, 105.88 μmol, 1 equivalent HCl salt), 4-(2-(4-(5-ethyl-2-(methanesulfonyl)-6-(trifluoromethyl)pyrimidin-4-yl)-1H-pyrazol-1-yl)acetyl)piperazine-1-carboxylic acid tert-butyl ester (50 mg, 105.88 μmol, 1 equivalent Add DIEA (68.42 mg, 529.38 μmol, 92.21 μL, 5) to a solution of ACN (1 mL). equivalent) and CsF (32.17 mg, 211.75 μmol, 7.82 μL, 2 equivalent The mixture was stirred at 130 °C for 12 hours. Five additional 50 mg vials were prepared as described above. LC-MS showed complete consumption of the starting material and a 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 × 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give tert-butyl 4-[2-[4-[2-[(2R)-2-(difluoromethyl)azacyclobutane-1-yl]-5-ethyl-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylate (155 mg, 49% yield), which was used directly for the next step.

[0219] 1 H NMR (400 MHz, 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) Synthesis of (R)-2-(4-(2-(2-(difluoromethyl)azacyclobutane-1-yl)-5-ethyl-6-(trifluoromethyl)pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(piperazin-1-yl)ethyl-1-one: 4-[2-[4-[2-[(2R)-2-(difluoromethyl)azacyclobutane-1-yl]-5-ethyl-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]acetyl]piperazine-1-carboxylic acid tert-butyl ester (106 mg, 184.81 μmol, 1 equivalentThe solution of the starting material 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 a main peak with the desired m / z was detected. The reactants were adjusted to pH 7-8 with aqueous NaHCO3 solution and extracted with ethyl acetate (5 mL × 3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by preparative HPLC (neutral conditions: column: Waters XbridgeBEH C18 100*30 mm*10 μm; mobile phase: [H2O (10 mM NH4HCO3)-ACN]; gradient: 35%-65% B over 8.0 min) to give 2-[4-[2-[(2R)-2-(difluoromethyl)azacyclobutane-1-yl]-5-ethyl-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]-1-piperazin-1-yl-acetone (40.1 mg, 45% yield) as a white solid.

[0220] 1H NMR (400 MHz, 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 - 3.51 (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 = 7.4 Hz, 3H) Example 8: Synthesis of compound A46 Synthesis of 4-(difluoromethyl)-5-methyl-2-(methylthio)-6-((trimethylsilyl)ethynyl)pyrimidine To 4-chloro-6-(difluoromethyl)-5-methyl-2-methylthiopyrimidine (1.4 g, 1 equivalent Add 1.35 g of acetylenol (trimethyl)silane (2.2 g) to a solution of THF (14 mL). equivalent) and TEA (1.89 g, 3 equivalents) and Pd(PPh3)2Cl2 (62.49 mg, 89.02 μmol, 0.1 equivalent ) and CuI (16.95 mg, 89.02 μmol, 0.1 equivalent The mixture was stirred at 25 °C for 12 hours. Several new peaks were observed on 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 combined organic layers were washed with aqueous NaCl solution (5 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 0 to 95 / 5) to give 4-(difluoromethyl)-5-methyl-2-(methylthio)-6-((trimethylsilyl)ethynyl)pyrimidine (0.7 g, 32% yield) as a black oil.

[0221] LCMS (ESI+): m / z 287.2 (M+H) + Retention time: 2.629 minutes (Method F) Synthesis of 4-(difluoromethyl)-6-ethynyl-5-methyl-2-(methylthio)pyrimidine KF (116.63 mg, 2.01 mmol, 47.03 μL, 1.15 equivalents) was added 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). The mixture was stirred at 25 °C for 12 h. Several new peaks were observed on 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 × 2). The combined organic layers were washed with aqueous NaCl solution (5 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by preparative TLC (SiO2, PE:MTBE = 3:1) to give 4-(difluoromethyl)-6-ethynyl-5-methyl-2-(methylthio)pyrimidine (0.12 g, 32% yield) as a dark brown solid.

[0222] LCMS (ESI+): m / z 215.1 (M+H) + Retention time: 0.483 minutes (Method D) Synthesis of tert-butyl 3-(4-(6-(difluoromethyl)-5-methyl-2-(methylthio)pyrimidin-4-yl)-1H-1,2,3-triazol-1-yl)azacyclobutane-1-carboxylate CuI (888.98 μg, 4.67 μmol, 0.01 equivalent) and tert-butyl 3-azido-azacyclobutane-1-carboxylate (92.53 mg, 466.78 μmol, 1 equivalent) were added 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). The mixture was stirred at 100 °C for 12 h. Several new peaks were observed on LC-MS, and 80% of the desired compound was detected. The reaction mixture was diluted with H2O (3 mL) and extracted with EA 6 mL (3 mL × 2). The combined organic layers were washed with aqueous NaCl solution (5 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by preparative TLC (SiO2, PE:EA = 1:1) to give tert-butyl 3-(4-(6-(difluoromethyl)-5-methyl-2-(methylthio)pyrimidin-4-yl)-1H-1,2,3-triazol-1-yl)azacyclobutane-1-carboxylate (0.14 g, 73% yield) as a brown solid.

[0223] LCMS (ESI+): m / z 413.4 (M+H)+, retention time: 0.550 minutes (Method R) Synthesis of tert-butyl 3-(4-(6-(difluoromethyl)-5-methyl-2-(methylsulfonyl)pyrimidin-4-yl)-1H-1,2,3-triazol-1-yl)azacyclobutane-1-carboxylate Oxone (402.43 mg, 654.61 μmol, 3 equivalents) was added to a solution of tert-butyl 3-(4-(6-(difluoromethyl)-5-methyl-2-(methylthio)pyrimidin-4-yl)-1H-1,2,3-triazol-1-yl)azacyclobutane-1-carboxylate (0.09 g, 218.20 μmol, 1 equivalent) in DMF (0.5 mL). The mixture was stirred at 25 °C for 12 h. LC-MS showed detection of 90% of the desired compound. The reaction mixture was filtered, and the filtrate was concentrated under high vacuum at 40 °C to give tert-butyl 3-(4-(6-(difluoromethyl)-5-methyl-2-(methanesulfonyl)pyrimidin-4-yl)-1H-1,2,3-triazol-1-yl)azacyclobutane-1-carboxylate (0.07 g, 72% yield), which was used in the next step without further purification.

[0224] LCMS (ESI+): m / z 467.2 (M+Na)+, retention time: 0.456 minutes (Method D) Synthesis of tert-butyl 3-(4-(6-(difluoromethyl)-2-((2S,3R)-3-hydroxy-2-methylazacyclobutane-1-yl)-5-methylpyrimidin-4-yl)-1H-1,2,3-triazol-1-yl)azacyclobutane-1-carboxylate To a solution of tert-butyl 3-(4-(6-(difluoromethyl)-5-methyl-2-(methanesulfonyl)pyrimidin-4-yl)-1H-1,2,3-triazol-1-yl)azacyclobutane-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-methylazacyclobutane-3-ol (39.20 mg, 135.00 μmol, 1 equivalent) were added. The mixture was stirred at 60 °C for 12 h. LC-MS showed detection of 77% of the desired compound. The reaction mixture was diluted with EA (2 mL) and extracted with H₂O (2.5 mL × 2). The organic layer was washed with an aqueous NaCl solution (5 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give tert-butyl 3-(4-(6-(difluoromethyl)-2-((2S,3R)-3-hydroxy-2-methylazacyclobutane-1-yl)-5-methylpyrimidin-4-yl)-1H-1,2,3-triazol-1-yl)azacyclobutane-1-carboxylate (0.09 g, 74% yield), which was used in the next step without further purification.

[0225] LCMS (ESI+): m / z 452.4 (M+H)+, retention time: 0.498 minutes (Method R) Synthesis of (2S,3R)-1-(4-(1-(azacyclobutane-3-yl)-1H-1,2,3-triazol-4-yl)-6-(difluoromethyl)-5-methylpyrimidin-2-yl)-2-methylazacyclobutane-3-ol A mixture of 0.08 g (177.20 μmol, 1 equivalent) of tert-butyl 3-(4-(6-(difluoromethyl)-2-((2S,3R)-3-hydroxy-2-methylazacyclobutan-1-yl)-5-methylpyrimidin-4-yl)-1H-1,2,3-triazol-1-yl)azacyclobutan-1-carboxylic acid in DCM (0.5 mL) and TFA (0.5 mL) was stirred at 25 °C for 12 h. LC-MS showed detection of 83% of the desired compound. The mixture was concentrated under high vacuum to give (2S,3R)-1-(4-(1-(azacyclobutan-3-yl)-1H-1,2,3-triazol-4-yl)-6-(difluoromethyl)-5-methylpyrimidin-2-yl)-2-methylazacyclobutan-3-ol (0.075 g, 96% yield), which was used in the next step without further purification.

[0226] LCMS (ESI+): m / z 352.3 (M+H)+, retention time: 0.304 minutes (Method D) Synthesis of (2S,3R)-1-(4-(difluoromethyl)-5-methyl-6-(1-(1-methylazacyclobutan-3-yl)-1H-1,2,3-triazol-4-yl)pyrimidin-2-yl)-2-methylazacyclobutan-3-ol To a solution of (2S,3R)-1-(4-(1-(azacyclobutan-3-yl)-1H-1,2,3-triazol-4-yl)-6-(difluoromethyl)-5-methylpyrimidin-2-yl)-2-methylazacyclobutan-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 equivalent) 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 h. LC-MS showed detection of the desired compound. The reactants were dried under high vacuum at 40 °C to obtain a residue, which was purified by preparative HPLC (TFA conditions) to give (2S,3R)-1-(4-(difluoromethyl)-5-methyl-6-(1-(1-methylazacyclobutan-3-yl)-1H-1,2,3-triazol-4-yl)pyrimidin-2-yl)-2-methylazacyclobutan-3-ol (13 mg, 62% yield) as a white solid.

[0227] LCMS (ESI+): m / z 366.1 (M+H)+, retention time: 1.823 minutes, Method E 1H NMR (400 MHz, methanol-d4) δ 1.56 (d, J=6.11 Hz, 3H), 2.63 (s, 3H), 3.17 (s, 3H), 3.75 (dd, J=9.11, 4.83 Hz, 1H), 4.11-4.24 (m, 2H), 4.30 (dd, J=9.05, 6.24 Hz, 1H), 4.71-4.85 (m, 4H), 5.75 (br t, J=6.97 Hz, 1H), 6.45-6.95(m, 1H), 8.40-8.77 (m, 1H) Example 9: Synthesis of compounds A31 and A25 Synthesis of (E)-2,2,2-trifluoro-N-[(4-methoxyphenyl)methyl]ethyleneimine (4-methoxyphenyl)methylamine (3.81 g, 27.76 mmol, 3.60 mL, 0.8 equivalent) was added to a solution of 1-ethoxy-2,2,2-trifluoro-ethanol (5 g, 34.70 mmol, 4.10 mL, 1 equivalent) in toluene (50 mL). The reaction mixture was stirred at 110 °C for 36 h. The desired product was detected by TLC. The reaction mixture was concentrated under reduced pressure to give (E)-2,2,2-trifluoro-N-[(4-methoxyphenyl)methyl]ethyleneimine (7.3 g, 97% yield) as a colorless oil, which was used directly in the next reaction.

[0228] 1 H NMR (400 MHz, chloroform-) d ) δ ppm 7.57 - 7.62 (m, 1 H) 7.16 - 7.22 (m, 2H) 6.89 - 6.95 (m, 2 H) 4.78 (s, 2 H) 3.82 - 3.83 (m, 3 H) Synthesis of (rac-3S,4R)-3-(benzyloxy)-1-(4-methoxybenzyl)-4-(trifluoromethyl)azacyclobutane-2-one TEA (16.31 g, 161.15 mmol, 22.43 mL, 5 equivalents) was added to a solution of (E)-2,2,2-trifluoro-N-[(4-methoxyphenyl)methyl]ethyleneimine (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). The reaction mixture was stirred at 40 °C for 48 h. The desired product was detected by TLC. The reaction mixture was quenched by adding water (50 mL) and extracted with DCM (70 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by preparative HPLC (column: Pheromones Luna C18 (250*70 mm, 15 μm); mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 53%-73% B over 20.0 min) to give (rac-3S,4R)-3-(benzyloxy)-1-(4-methoxybenzyl)-4-(trifluoromethyl)azacyclobutane-2-one (0.5 g, 4% yield).

[0229] LCMS (ESI+): m / z 366.1 (M+1) + Retention time: 8.215 minutes (Method R) Synthesis of (rac-2R, 3R)-3-(benzyloxy)-1-(4-methoxybenzyl)-2-(trifluoromethyl)azacyclobutane LAH (2.5 M, 1.31 mL, 3 equivalents) was added to a solution of AlCl3 (437.96 mg, 3.28 mmol, 179.49 μL, 3 equivalents) in THF (4 mL) for 10 minutes at 0 °C. The reaction mixture was stirred at 40 °C for 30 minutes. (rac-3S,4R)-3-benzyloxy-1-[(4-methoxyphenyl)methyl]-4-(trifluoromethyl)azacyclobutane-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. The desired product was detected by TLC. The reaction mixture was quenched with H2O (10 mL) at 0 °C and extracted with DCM (10 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and 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 give (rac-2R, 3R)-3-(benzyloxy)-1-(4-methoxybenzyl)-2-(trifluoromethyl)azacyclobutane (0.28 g, 73% yield) as a colorless oil.

[0230] LCMS (ESI+): m / z 352.1 (M+1) + Retention time: 0.956 minutes (Method G) Synthesis of (rac-2R,3R)-2-(trifluoromethyl)azacyclobutane-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)azacyclobutane (260 mg, 739.98 μmol, 1 equivalent) in MeOH (13 mL) and HCl / MeOH (0.01 mL). The reaction mixture was stirred at 20 °C for 12 h under H₂ (15 psi). LCMS showed the desired product was detected. The resulting product was dissolved in MeOH (30 mL) and filtered. The filtrate was concentrated to give (rac-2R,3R)-2-(trifluoromethyl)azacyclobutane-3-ol (100 mg, 708.76 μmol, 76% yield, HCl salt) as a colorless oil.

[0231] 1 H NMR (400 MHz, acetonitrile- d 3) δ ppm 4.70 - 5.05 (m, 2 H) 4.11 - 4.23 (m, 1H) 4.00 (br dd, J = 10.45, 7.03 Hz, 1 H) Synthesis of tert-butyl 3-[4-[2-chloro-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylate To a solution of 3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)pyrazol-1-yl]azacyclobutane-1-carboxylic acid tert-butyl ester (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 equivalent) were added. The reaction mixture was stirred at 80 °C for 2 h. TLC showed the desired product. The reaction mixture was diluted with H2O (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated to obtain a residue. The residue was purified by preparative TLC (TLC: petroleum ether: ethyl acetate = 1:2, Rf = 0.3) to give tert-butyl 3-[4-[2-chloro-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylate (0.3 g, 32% yield) as a colorless oil.

[0232] LCMS (ESI+): m / z 348.0 (M-55) + Retention time: 0.591 minutes (Method D) Synthesis of tert-butyl 3-(4-(2-((rac-2R,3R)-3-hydroxy-2-(trifluoromethyl)azacyclobutane-1-yl)-6-(trifluoromethyl)pyrimidin-4-yl)-1H-pyrazol-1-yl)azacyclobutane-1-carboxylate TEA (140.34 mg, 1.39 mmol, 193.03 μL, 4 equivalents) was added to a solution of (rac-2R,3R)-2-(trifluoromethyl)azacyclobutane-3-ol (48.92 mg, 346.72 μmol, 1 equivalent) and tert-butyl 3-[4-[2-chloro-6-(trifluoromethyl)pyrimidin-4-yl]pyrazol-1-yl]azacyclobutane-1-carboxylate (140 mg, 346.72 μmol, 1 equivalent) in THF (1.4 mL). The reaction mixture was stirred at 80 °C for 12 h. The desired product was detected by TLC. The reaction mixture was diluted with H2O (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated to obtain a residue. The residue was purified by preparative TLC (TLC: petroleum ether: ethyl acetate = 1:1, Rf = 0.4) to obtain tert-butyl 3-(4-(2-((rac-2R,3R)-3-hydroxy-2-(trifluoromethyl)azacyclobutane-1-yl)-6-(trifluoromethyl)pyrimidin-4-yl)-1H-pyrazol-1-yl)azacyclobutane-1-carboxylate (91 mg, 52% yield) as a colorless oil.

[0233] LCMS (ESI+): m / z 509 (M+H) + Retention time: 8.48 minutes (Method R) Synthesis of (rac-2R,3R)-1-(4-(1-(azacyclobutane-3-yl)-1H-pyrazol-4-yl)-6-(trifluoromethyl)pyrimidin-2-yl)-2-(trifluoromethyl)azacyclobutane-3-ol A31 A solution of tert-butyl 3-(4-(2-((rac-2R,3R)-3-hydroxy-2-(trifluoromethyl)azacyclobutan-1-yl)-6-(trifluoromethyl)pyrimidin-4-yl)-1H-pyrazol-1-yl)azacyclobutan-1-carboxylate (90 mg, 177.02 μmol, 1 equivalent) in TFA (0.3 mL) and DCM (1.5 mL) was stirred for 0.5 h at 20 °C. The desired product was detected by TLC. The reactants were concentrated to give (rac-2R,3R)-1-(4-(1-(azacyclobutan-3-yl)-1H-pyrazol-4-yl)-6-(trifluoromethyl)pyrimidin-2-yl)-2-(trifluoromethyl)azacyclobutan-3-ol (70 mg, 97% yield) as a colorless oil.

[0234] LCMS (ESI+): m / z 409.1 (M+1) + Retention time: 0.373 minutes (Method D) Synthesis of (rac-2R,3R)-1-(4-(1-(1-methylazacyclobutane-3-yl)-1H-pyrazol-4-yl)-6-(trifluoromethyl)pyrimidin-2-yl)-2-(trifluoromethyl)azacyclobutane-3-ol A25 To a solution of (rac-2R,3R)-1-(4-(1-(azacyclobutan-3-yl)-1H-pyrazol-4-yl)-6-(trifluoromethyl)pyrimidin-2-yl)-2-(trifluoromethyl)azacyclobutan-3-ol (60 mg, 146.95 μmol, 1 equivalent) in DCM (1.2 mL), NaBH(OAc)3 (93.43 mg, 440.85 μmol, 3 equivalent) 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. The desired product was detected by TLC. The reaction mixture was concentrated to obtain the residue. The residue was purified by preparative HPLC (column: Pheromon Luna C18 75*30mm*3um; mobile phase: [H2O(0.1%TFA)-ACN]; gradient: 20%-40%B over 8.0 min) to give (rac-2R,3R)-1-(4-(1-(1-methylazacyclobutane-3-yl)-1H-pyrazol-4-yl)-6-(trifluoromethyl)pyrimidin-2-yl)-2-(trifluoromethyl)azacyclobutane-3-ol (40 mg, 64% yield) as a white solid.

[0235] LCMS (ESI+): m / z 423.0 (M+1) + Retention time: 2.779 minutes (Method F) 1 ¹H NMR (400 MHz, methanol-d⁴) δ ppm 8.51 (s, ¹H) 8.22 (s, ¹H) 7.42 (s, ¹H) 5.08 (t, J =7.03 Hz, 1 H) 4.67 - 4.73 (m, 1 H) 4.58 - 4.66 (m, 1 H) 4.42(dd, J =9.17, 6.72 Hz, 1 H) 3.93 (dd, J =9.41, 4.40 Hz, 1 H) 3.86 (br t, J =7.95Hz, 2 H) 3.65 (br t, J =7.64 Hz, 2 H) 2.48 (s, 3 H) The following compounds were prepared using appropriate intermediates and reactants, employing methods similar to those described for the other compounds disclosed herein.

[0236] Tests of compound activity The efficacy of the compounds of this disclosure and salts of such compounds as pharmaceutical preparations in treating the diseases / symptoms described herein in mammals (e.g., humans, men or women) is demonstrated by the activity and efficacy of the compounds of this disclosure in one or more of the conventional assays and in vivo assays described and mentioned herein. In vivo assays (with appropriate modifications that can be made by those skilled in the art) can be used to determine the activity of other pharmaceutical agents as well as the compounds of this disclosure. Therefore, the methods described herein can also be used to demonstrate the efficacy of combinations of the compounds of this disclosure. Assays and models can also confirm specific other property advantages, such as side effect characteristics; half-life. Furthermore, such assays provide a way to compare the activities of the compounds of this disclosure and salts of such compounds (or other pharmaceutical agents described herein) with each other and with the activities of other known compounds. The results of these comparisons are useful for determining dose levels for treating such diseases in mammals (including humans).

[0237] The absorption, distribution, metabolism, and excretion (ADME) and pharmacokinetics (PK) of the compound, as well as exemplary assays, are discussed in the online publication of Thomas DY Chung, David B. Terry, and Layton H. Smith, “InVitro 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 / ).

[0238] Animal models of non-alcoholic fatty liver disease were described in *Nutrients*, October 2017; 9(10): 1072; *International Journal of Molecular Sciences*, 2022, 23, 15791; and *Digestion*, 2020; 101:522-535. An animal model of fructose inhibition was described in *Molecular Metabolism*, 2021; 48:101196.

[0239] Example 10: KHK Inhibition Assay Human and rat recombinant KHK-A and KHK-C isoenzyme IC 50 Determining the value The assay reagent mixture was prepared to the final concentration shown in Table B by combining NADH, water, TEA, KCl, MgCl2, PEP, ATP, DTT, and coupling enzymes (pyruvate kinase and lactate dehydrogenase, LDH).

[0240] Table B Add the relevant KHK isoenzyme to a final concentration of 6 nM. Dilute each inhibitor compound aliquot with a 5-fold serial dilution to produce a final concentration ranging from 1000 nM to 0.064 nM. Add the inhibitor aliquot to the assay reagent mixture containing fructose-containing KHK (2 mM) in a 96-well plate. Measure the absorbance at 340 nm spectrophotometrically and analyze the inhibition using nonlinear regression.

[0241] The results of the measurements are presented in Table C.

[0242] Table C Example 11: Cell-based power assay (HepG2 assay) The inhibitory effect of the compound on human KHK in HepG2 cells was tested. HepG2 cells were grown in RPMI 1640 supplemented medium on 100 mm plates until they were nearly confluent (approximately 5-8 x 10⁻⁶ cells). 6 (1 cell). Remove the culture medium and add 4 mL of trypsin solution (0.25% (w / v) + 0.25% (w / v) EDTA), then incubate at 37°C for 5–10 minutes. Pelletize the cells and resuspend them in 2 mL of complete culture medium.

[0243] The cells were then placed in 96-well plates with 20,000-50,000 cells per well and grown at 37°C for 20-28 hours to allow them to attach.

[0244] The inhibitor compound was diluted to 50 μM in DMSO and then serially diluted with MEM medium (Thermo Fisher Scientific). The final inhibitor concentration ranged from 1000 nM to 0.064 nM (5-fold serial dilution).

[0245] Remove RPMI medium from the confluent cell plate. Incubate the plate at 37°C for 35 minutes, then add 0.1 mL of MEM medium containing a diluted inhibitor compound and fructose, and 0.1 mL of MEM medium without the inhibitor compound, fructose, or trypsin; thus obtaining a final fructose concentration of 20 mM. Incubate the plate at 37°C for 20 minutes, then place it on ice to terminate the reaction.

[0246] Next, 200 μL of cold 80% (v / v) methanol:water was added to all wells, except for the control used to count cells. The plate was vortexed, and cell debris was removed by centrifugation. For the measurement of F1P produced, the resulting supernatant fraction was analyzed by LC / MS.

[0247] The IC50 data is presented in Table D.

[0248] Table D The structures of compounds C1-C3 are provided in paragraph

[00374] .

[0249] Example 12: Cell-based power assay (KHK-C-overexpression HepG2 assay) The inhibitory effect of the compound on human KHK-C in engineered HepG2 cells was tested.

[0250] Engineered HepG2 cells were generated using methods known to those skilled in the art. In short, HepG2 cells were incubated with a lentivirus carrying a transgene encoding human KHK-C and grown under antibiotic selection.

[0251] KHK-C-overexpressing HepG2 cells were grown to near confluence (approximately 25-40 x 10⁻⁶ cells) in RPMI 1640 supplemented medium on T-182 cm² flasks. 6(1 cell). Remove the culture medium, wash the flask with 10 mL PBS, and add 4 mL trypsin solution (0.25% (w / v) + 0.25% (w / v) EDTA), then incubate at 37°C for 3–5 minutes. Pelletize the cells and resuspend them in 2 mL of complete culture medium.

[0252] The cells were then placed in 96-well plates with 20,000-40,000 cells per well and grown at 37°C for 20-28 hours to allow them to attach.

[0253] The inhibitor compounds were diluted to 50 μM in DMSO and then serially diluted using MEM medium (Thermo Fisher Scientific). The final inhibitor concentrations ranged from 5000 nM to 0.32 nM. Control compounds C1, C2, and C3 were tested for comparison with the compounds disclosed herein.

[0254] Remove RPMI medium from the confluent cell plate. Then incubate the plate at 37°C with 0.1 mL of MEM containing a diluted inhibitor compound for 35 minutes. Next, add another 0.1 mL of MEM containing a diluted inhibitor compound and fructose, or 0.1 mL of MEM without the inhibitor compound and / or fructose; to obtain a final fructose concentration of 30 mM. Incubate the plate at 37°C for 5 minutes, then place it on ice to terminate the reaction.

[0255] Next, 200 μL of cold 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. To measure the generated F1P, the resulting supernatant was analyzed by LC / MS.

[0256] The IC50 data is presented in Table E.

[0257] Table E Example 13: CYP Inhibition Assay in Human Liver Microsomes Cytochrome P450 inhibition profile analysis A potential limitation of novel drugs for metabolic diseases lies in the risk of drug-drug interactions mediated by the inhibition of cytochrome P450 (CYP) enzymes involved in the metabolism of exogenous substances, including CYP1A2, CYP2C9, CYP2C8, CYP2D6, and CYP3A4. Commonly used concomitant medications in patients with metabolic diseases, type 2 diabetes, obesity, hypertension, and / or MASLD / MASH include various 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. If these drugs are co-administered with inhibitors of CYP enzymes, the total exposure to these drugs may be affected. Therefore, a desirable characteristic for clinical candidates is the absence of significant inhibition of CYP enzymes while maintaining potency as an inhibitor of KHK-C and KHK-A. Preferred compounds of this disclosure possess the beneficial characteristic of no significant CYP inhibition.

[0258] Test compounds for inhibition of CYP enzymes Human liver microsomes (HLMs) were incubated with known substrates of 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 at a high concentration of 50 μM and serially diluted approximately 3-fold to a low concentration of 50 nM.

[0259] Each well contained 0.2 mg / mL HLM, 1 mM NADPH, a substrate-dependent final concentration of each substrate, and a specified concentration of a positive control inhibitor or test compound. The wells were incubated at 37°C for 10 min, and the reaction was terminated by adding a cold stop solution (e.g., acetonitrile containing 200 ng / mL tolbutamide), followed by centrifugation at 4000 rpm for 20 min to precipitate the protein. The supernatant was added to 0.5 volumes of HPLC-water, vortexed for 10 min, and analyzed by LC-MS-MS.

[0260] Example 14: hERG suppression determination using an automated patch clamp method Another potential limitation of the new drug lies in the risk of cardiotoxicity mediated by binding to or inhibiting cardiac ion channels, including, for example, hERG (human ether-a-go-go related gene). hERG is a subunit of a potassium channel that mediates cardiac repolarization and is inhibited by a variety of small molecules, which can cause arrhythmias. Therefore, maximizing the window between the potency as an inhibitor of the target enzyme (hexylose kinase) and the potency as an inhibitor of hERG is a desirable characteristic of clinical candidates. The preferred compounds of this disclosure have the advantageous property of no significant hERG inhibition and / or a large window between the potency of KHK inhibitors and the potency of hERG inhibitors.

[0261] The tested compound inhibited hERG (human ether-a-go-go related gene). CHO cells stably expressing the hERG channel were seeded on a Nanion SyncroPatch 384PE in approximately 298 mOsm 10 mM HEPES buffer at pH 7.4 containing 140 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 5 mM glucose for spectroscopic analysis using an automated patch clamp method.

[0262] The positive control (amitriptyline) and the test compound were tested on a 5-point dose-response curve, starting at a high concentration of 30 μM and serially diluted approximately 3-fold to a low concentration of 300 nM.

[0263] The current was induced using a voltage command protocol consisting of the following: a continuous hold potential of -80 mV, followed by an initial transition to -50 mV for 80 ms for leakage reduction, then a transition to +20 mV for 4,800 ms to open the hERG channel, followed by a transition to -50 mV for 5,000 ms to generate an hERG "tail current," which was measured and collected for analysis, and then a hold potential of -80 mV for 1,000 ms. This command protocol was repeated every 20 seconds during the assay (300 seconds before and 300 seconds after the addition of the control or test compound).

[0264] Example 15: Steady-state dynamics Another potential limitation of novel drugs that inhibit hexylose kinase subtypes is the risk that elevated substrate (e.g., fructose and / or ATP) concentrations could reduce drug efficacy due to competitive inhibition. Inhibition of hexylose kinase to metabolize fructose to fructose-1-phosphate is expected to increase the concentrations of both ATP and fructose, potentially to a degree sufficient to counteract the effects of a novel drug as a competitive inhibitor. Therefore, a non-competitive inhibition mode of the target enzyme, where the efficacy as an enzyme inhibitor is unaffected by the concentration of any enzyme substrate, is likely a desirable characteristic for clinical candidates. The inhibition mode can be determined by a series of steady-state kinetic experiments, as known to those skilled in the art, for example, as described in Copeland, RA, *Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacists* (2013), in which the potency of a compound as an inhibitor of hexokinase is determined at a range of substrate concentrations, i.e., by altering the concentration of ATP under conditions of fructose excess; or by altering the concentration of fructose under conditions of ATP excess. Preferred compounds of this disclosure possess beneficial properties in terms of inhibition mode, thus increasing ATP and / or fructose concentrations does not affect their potency as inhibitors of hexokinase.

[0265] All references provided herein are incorporated herein by reference in their entirety. As used herein, all abbreviations, symbols, and conventions are consistent with those used in contemporary 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.

[0266] It should be understood that although this disclosure has been described in conjunction with its specific description, the foregoing description is intended to illustrate and not limit the scope of this disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A compound having the structure of Formula I or a pharmaceutically acceptable salt thereof: （I） in R 1 It is H or OH; R 2 C 1-6 Alkyl or C 1-6 Halogenated alkyl groups; R 3 C 1-6 Alkyl or C 1-6 Halogenated alkyl groups; R 4 H, halogenated, CN, C 1-6 Alkyl, C 1-6 Alkoxy or C 3-5 cycloalkyl; A is a 5-membered heteroaryl group containing 2-3 nitrogen ring atoms; X is a key or C 1-6 alkylene-C(O); and R 5 It is a 4- to 6-membered heterocyclic alkyl group having 1 or 2 cyclic nitrogen atoms, wherein the heterocyclic alkyl group is optionally surrounded by 1 or 2 C atoms. 1-6 Alkyl substitution; The condition is that in R 4 When it is H, exclude R. 2 It is methyl and R 3 It is a trifluoromethyl compound.

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

3. The compound or salt according to claim 1 or 2, having the structure of formula Ix: (Ix)。 4. The compound or salt according to claim 1, having the structure of formula II: (II), where C A and C B This indicates carbon stereocenters with the same or opposite stereochemistry.

5. The compound or salt according to claim 2, wherein C A It is carbon with an R configuration, and C B It is carbon with an S-configuration.

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

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

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

9. The compound or salt according to any one of claims 1 to 8, wherein R 5 It is a 4-membered heterocyclic alkyl group having one cyclic nitrogen atom, wherein the heterocyclic alkyl group is optionally surrounded by one or two C atoms. 1-6 Alkyl substitution.

10. The compound or salt according to any one of claims 1 to 8, wherein R 5 It is a 6-membered heterocyclic alkyl group having 2 cyclic nitrogen atoms, wherein the heterocyclic alkyl group is optionally surrounded by 1 or 2 C atoms. 1-6 Alkyl substitution.

11. The compound or salt according to claim 9 or 10, wherein R 5 It is not replaced.

12. The compound or salt according to claim 9 or 10, wherein R 5 A C 1-6 Alkyl substitution.

13. The compound or salt according to claim 1, having a structure of formula Ia or Ib: (Ia) or (Ib).

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

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

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

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

18. The compound or salt according to any one of claims 1 to 15, wherein R 2 C 1-6 Halogenated alkyl groups.

19. The compound or salt according to claim 18, wherein R 2 It is CHF2 or CF3.

20. The compound or salt according to any one of claims 1 to 19, wherein R 3 It is CHF2 or CF3.

21. The compound or salt according to claim 20, wherein R 3 It is CHF2.

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

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

24. The compound or salt according to claim 23, wherein R 4 It can be F or Cl.

25. The compound or salt according to any one of claims 1 to 21, wherein R 4 C 1-6 Alkyl group.

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

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

28. The compound or salt according to claim 27, wherein R 4 It can be methyl or ethyl.

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

30. The compound or salt according to claim 29, wherein R 3 It is CHF2, and R 4 It is a methyl group.

31. The compound or salt according to any one of claims 1 to 21, wherein R 4 C 3-5 Cycloalkyl.

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

33. The compound according to claim 5, wherein R 1 H or hydroxyl, R 2 For methyl, R 3 For CHF2, R 4 It is a methyl group, X is a bond, and R 5 It is a 4- to 6-membered heterocyclic alkyl group having one cyclic nitrogen atom, wherein the heterocyclic alkyl group is optionally C 1-2 Alkyl substitution.

34. The compound according to claim 5, wherein X is C. 1-6 Alkylene-C(O), R 5 To be optionally bounded by 1 or 2 C 1-6 Alkyl-substituted nitrogen-containing heterocyclic butyl groups, R 1 H or hydroxyl, R 2 C 1-3 Alkyl, R 3 It is CHF2, and R 4 It is a methyl group.

35. A compound listed in Table A or a pharmaceutically acceptable salt thereof.

36. A compound selected from: (2S,3R)-1-{6-(difluoromethyl)-5-methyl-4-[1-(1-methyl-3-azacyclobutane)-4-pyrazolyl]-2-pyrimidinyl}-2-methyl-3-azacyclobutanol (A21); 2-(4-{2-[(S)-2-methyl-1-azacyclobutane]-5-methyl-6-(trifluoromethyl)-4-pyrimidinyl}-1-pyrazolyl)-1-(1-piperazinyl)-1-acetone (A3); (2S,3R)-1-{6-(difluoromethyl)-4-[1-(1-methyl-3-azacyclobutane)-4-pyrazolyl]-2-pyrimidinyl}-2-methyl-3-azacyclobutanol (A9); (2S,3R)-2-methyl-1-{5-methyl-4-[1-(1-methyl-3-azacyclobutane)-4-pyrazolyl]-6-(trifluoromethyl)-2-pyrimidinyl}-3-azacyclobutanol (A12); (2S,3R)-1-{6-(difluoromethyl)-5-methoxy-4-[1-(1-methyl-3-azacyclobutane)-4-pyrazolyl]-2-pyrimidinyl}-2-methyl-3-azacyclobutanol (A19); (2S,3R)-1-{4-[1-(3-azacyclobutane)-4-pyrazolyl]-6-(difluoromethyl)-5-methyl-2-pyrimidinyl}-2-methyl-3-azacyclobutanol (A35); and 2-(4-{2-[(S)-2-methyl-1-azacyclobutane]-6-(difluoromethyl)-5-methyl-4-pyrimidinyl}-1-pyrazolyl)-1-(1-piperazinyl)-1-acetone (A24). Or its pharmaceutically acceptable salt.

37. The compound according to claim 36, wherein the compound is selected from: 2-(4-{2-[(S)-2-methyl-1-azacyclobutane]-5-methyl-6-(trifluoromethyl)-4-pyrimidinyl}-1-pyrazolyl)-1-(1-piperazinyl)-1-acetone (A3); and 2-(4-{2-[(S)-2-methyl-1-azacyclobutane]-6-(difluoromethyl)-5-methyl-4-pyrimidinyl}-1-pyrazolyl)-1-(1-piperazinyl)-1-acetone (A24). Or its pharmaceutically acceptable salt.

38. The compound according to claim 36, wherein the compound is selected from: (2S,3R)-1-{6-(difluoromethyl)-5-methyl-4-[1-(1-methyl-3-azacyclobutane)-4-pyrazolyl]-2-pyrimidinyl}-2-methyl-3-azacyclobutanol (A21), and (2S,3R)-1-{4-[1-(3-azacyclobutane)-4-pyrazolyl]-6-(difluoromethyl)-5-methyl-2-pyrimidinyl}-2-methyl-3-azacyclobutanol (A35). Or its pharmaceutically acceptable salt.

39. The compound according to claim 36, wherein the compound is selected from: (2S,3R)-1-{6-(difluoromethyl)-4-[1-(1-methyl-3-azacyclobutane)-4-pyrazolyl]-2-pyrimidinyl}-2-methyl-3-azacyclobutanol (A9); and (2S,3R)-1-{6-(difluoromethyl)-5-methoxy-4-[1-(1-methyl-3-azacyclobutane)-4-pyrazolyl]-2-pyrimidinyl}-2-methyl-3-azacyclobutanol (A19); Or a pharmaceutical salt of any of the compounds.

40. The compound according to claim 36, wherein: (2S,3R)-2-methyl-1-{5-methyl-4-[1-(1-methyl-3-azacyclobutane)-4-pyrazolyl]-6-(trifluoromethyl)-2-pyrimidinyl}-3-azacyclobutanol (A12). Or its pharmaceutically acceptable salt.

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 hexylose kinase (KHK) in cells, the method 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 ailment of a subject, the method 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 of claim 43, wherein the disease or condition is related to KHK disorder.

45. The method according to claim 43 or 44, wherein the disease or condition is metabolic syndrome, hypertriglyceridemia, hypercholesterolemia, nonalcoholic fatty liver disease (NAFLD), metabolic dysfunction-associated fatty liver disease (MASLD), MASLD with increased alcohol intake (MetALD), nonalcoholic steatohepatitis (NASH), metabolic dysfunction-associated steatohepatitis (MASH), type 2 diabetes (T2D), diabetic kidney disease (DKD), alcoholic steatohepatitis (ASH), alcohol-related liver disease (ALD), liver fibrosis or cirrhosis, liver disease caused by hepatocyte stress, hereditary fructose intolerance, hyperuricemia, gout, addictive cravings, neurodegenerative diseases, or cancer.

46. ​​The method of claim 45, wherein the disease or symptom is NASH or MASH.

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

48. The method of 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 receptor agonist or modulator, or a sodium / glucose transporter inhibitor.

49. The method of claim 48, wherein the incretin receptor agonist or modulator is semaglutide, dulaglutide, liraglutide, tirzepatide, survodutide, retatrutide, pemvidutide, VK2735, orforglipron, cagrilintide / semaglutide, or danuginide. Pron), Maridebartcafraglutide, RGT-075, PF-0695422, NN9487, NN9541, CT-388, CT-868, CT-996, Efinopegdutide, Efocipegtrutide, AZD9550, DR10624, NLY01, ECC5004, Mazdutide, Exenatide, TERN-601, Ecnoglutide, or XW-004.

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

51. The method of claim 48, wherein the aldose reductase inhibitor is AT-001, AT-003, gavorestat, ranirestat, epalrestat, fidarestat, imirestat, tolrestat, or risarestat.

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