Long-chain compounds and uses thereof

Abstract

The long-chain compound and its use, the structure of the long-chain compound is represented by formula (I). The compound of the present invention can directly inhibit ACLY, and has a remarkable therapeutic effect on metabolic diseases such as hypercholesterolemia and non-alcoholic fatty liver disease, and is useful for the development of therapeutic drugs for metabolic diseases such as hypercholesterolemia and non-alcoholic fatty liver disease. [Formula 1] JPEG2024536860000049.jpg2076

Description

[Technical Field]

[0001] The present invention relates to novel long-chain fatty acid-based ACLY inhibitors, their pharmaceutically acceptable salts, medicinal esters, crystalline hydrates, solvates or mixtures thereof, as well as pharmaceutical compositions comprising such inhibitors and their use in the manufacture of medicaments for the treatment of metabolic diseases such as hypercholesterolemia, non-alcoholic steatohepatitis (NASH), and liver fibrosis. [Background technology]

[0002] Acetyl coenzyme A, produced in the mitochondrial tricarboxylic acid cycle (TCA cycle), is the starting substrate for the de novo synthesis of endogenous cholesterol and triglycerides. Acetyl coenzyme A produced in the tricarboxylic acid cycle does not normally cross the mitochondrial membrane directly into the cytoplasm. Instead, it is converted into citrate in the mitochondria by the catalytic action of citrate synthase. Citrate crosses the mitochondrial membrane via the citrate transporter and enters the cytoplasm. It is then degraded to acetyl coenzyme A and oxaloacetate by ATP-citrate synthase (adenosine triphosphate citrate lyase, ACLY) located in the endoplasmic reticulum. Oxaloacetate is converted to malate by NADH, which is then further converted to pyruvate by NAPDH. Malate and pyruvate are then transported back to the mitochondria by their respective transporters. Acetyl coenzyme A directly participates in the de novo synthesis of triglycerides and cholesterol as a substrate. Many metabolic diseases, such as nonalcoholic steatohepatitis (NASH), hypercholesterolemia, and atherosclerosis, are all associated with abnormally elevated levels of hepatic lipid synthesis. Therefore, ACLY inhibitory activity significantly reduces de novo lipid synthesis in hepatocytes, and ACLY inhibitors can be used to treat metabolic diseases. Bempedoic acid (ETC-1002) is the world's first commercially available small molecule ACLY inhibitor and is known to be used primarily to treat statin-resistant heterozygous familial hypercholesterolemia. Furthermore, ACLY inhibitors such as benzenesulfonamides have been shown to have lipid-lowering pharmacological activity at the cellular and preclinical animal levels.

[0003] Therefore, ACLY inhibitors are needed for the treatment of metabolic diseases such as hypercholesterolemia, nonalcoholic steatohepatitis (NASH), and liver fibrosis. Summary of the Invention [Problem to be solved by the invention]

[0004] The object of the present invention is to provide the use of long-chain compounds for the treatment of diseases such as hypercholesterolemia, non-alcoholic steatohepatitis (NASH), liver fibrosis, and atherosclerosis. [Means for solving the problem]

[0005] In a first aspect, the present invention provides use of a compound represented by general formula (I), or a deuterium-substituted compound, stereoisomer, enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt thereof, or a mixture of two or more compounds represented by general formula (I), for use in the manufacture of a medicament for treating a metabolic disease or a medicament for treating atherosclerosis. [ka] (however, [ka] represents a double bond or a single bond. m and n each independently represent 1, 2, 3, 4, 5, or 6. R1, R2, R3, R4, R5, and R6 are each independently H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C3-C7 cycloalkyl group, a C3-C7 cycloalkenyl group, a phenyl group, or a benzyl group. Alternatively, R1 and R2 together with the carbon atoms adjacent thereto form a C3-C7 cycloalkyl group or a C3-C7 cycloalkenyl group. Alternatively, R5 and R6 together with the carbon atoms adjacent thereto form a C3-C7 cycloalkyl group or a C3-C7 cycloalkenyl group. Alternatively, R3 and R4 together with the carbon atoms adjacent thereto form a C3-C7 cycloalkyl group or a C3-C7 cycloalkenyl group. Y1 and Y2 are each independently selected from -OH, -COOH, -COCoA, and -COOR7, where R7 is a methyl group, an ethyl group, or [ka] The structure of CoA is: [ka] .)

[0006] In another preferred embodiment, [ka] represents a double bond, and the configuration of the double bond is cis or trans.

[0007] In another preferred embodiment, the compound has a structure represented by general formula II: [ka]

[0008] In another preferred embodiment, the compound has a structure represented by general formula III: [ka]

[0009] In another preferred embodiment, the compound has a structure represented by general formula IV: [ka] (In the formula, p is 0, 1, 2, or 3.)

[0010] In another preferred embodiment, R1, R2, R3, R4, R5, and R6 are each independently H, a C1-C4 alkyl group, a deuterated C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C3-C7 cycloalkyl group, a deuterated C3-C7 cycloalkyl group, a C3-C7 cycloalkenyl group, a phenyl group, or a benzyl group; or R1 and R2 together with the adjacent carbon atoms form a deuterated C3-C7 cycloalkyl group, a C3-C7 cycloalkyl group, or a C3-C7 cycloalkenyl group; or R5 and R6 together with the adjacent carbon atoms form a deuterated C3-C7 cycloalkyl group, a C3-C7 cycloalkyl group, or a C3-C7 cycloalkenyl group; Alternatively, R3 and R4 together with the carbon atoms adjacent thereto form a deuterated C3-C7 cycloalkyl group, a C3-C7 cycloalkyl group, or a C3-C7 cycloalkenyl group.

[0011] In another preferred embodiment, R1 and R2 are each independently H, a methyl group, an ethyl group, a deuterated methyl group, a deuterated ethyl group, a deuterated propyl group, or a propyl group, and preferably R1 and R2 are simultaneously H or simultaneously a methyl group, or R1 and R2 together with the carbon atom adjacent thereto form a C3-C6 cycloalkyl group, a C3-C5 cycloalkyl group, or a C3-C4 cycloalkyl group.

[0012] In another preferred embodiment, R3 and R4 are each independently H, a methyl group, an ethyl group, a deuterated methyl group, a deuterated ethyl group, a deuterated propyl group, or a propyl group, and preferably R3 and R4 are simultaneously H or simultaneously a methyl group, or R3 and R4 together with the carbon atom adjacent thereto form a C3-C6 cycloalkyl group, a C3-C5 cycloalkyl group, or a C3-C4 cycloalkyl group.

[0013] In another preferred embodiment, R5 and R6 are each independently H, a methyl group, an ethyl group, a deuterated methyl group, a deuterated ethyl group, a deuterated propyl group, or a propyl group, and preferably R5 and R6 are simultaneously H or simultaneously a methyl group, or R5 and R6 together with the carbon atoms adjacent thereto form a C3-C6 cycloalkyl group, a C3-C5 cycloalkyl group, or a C3-C4 cycloalkyl group.

[0014] In another preferred embodiment, Y1 and Y2 are each independently -COOH, -COCoA, or -COOR7, where R7 is a methyl group, an ethyl group, or [ka] The structure of CoA is as follows: [ka]

[0015] In another preferred embodiment, the compound is selected from the group consisting of: [ka]

[0016] In another preferred embodiment, the compound is further selected from the following group: [ka]

[0017] In another preferred embodiment, the metabolic disease is selected from hypercholesterolemia, non-alcoholic steatohepatitis (NASH), and liver fibrosis. In another preferred embodiment, the atherosclerosis is caused by hypercholesterolemia.

[0018] In a second aspect, the present invention provides a compound represented by general formula (I), or a deuterium-substituted compound, stereoisomer, enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt thereof, or a mixture of two or more compounds represented by general formula (I). [ka] (however, [ka] represents a double bond or a single bond. m and n each independently represent 1, 2, 3, 4, 5, or 6. R1, R2, R3, R4, R5, and R6 are each independently H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C3-C7 cycloalkyl group, a C3-C7 cycloalkenyl group, a phenyl group, or a benzyl group. Alternatively, R1 and R2 together with the carbon atoms adjacent thereto form a C3-C7 cycloalkyl group or a C3-C7 cycloalkenyl group. Alternatively, R5 and R6 together with the carbon atoms adjacent thereto form a C3-C7 cycloalkyl group or a C3-C7 cycloalkenyl group. Alternatively, R3 and R4 together with the carbon atoms adjacent thereto form a C3-C7 cycloalkyl group or a C3-C7 cycloalkenyl group. Y1 and Y2 are each independently selected from -OH, -COOH, -COCoA, and -COOR7, where R7 is a methyl group, an ethyl group, or [ka] The structure of CoA is: [ka] .)

[0019] In another preferred embodiment, the compound has a structure represented by general formula II, III, or IV: [ka] (In the formula, p is 0, 1, 2, or 3.)

[0020] In another preferred embodiment, R1, R2, R3, R4, R5, and R6 are each independently H, a C1-C4 alkyl group, a deuterated C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, a C3-C7 cycloalkyl group, a C3-C7 cycloalkenyl group, a phenyl group, or a benzyl group; or R1 and R2 together with the adjacent carbon atoms form a deuterated C3-C7 cycloalkyl group, a C3-C7 cycloalkyl group, or a C3-C7 cycloalkenyl group; or R5 and R6 together with the adjacent carbon atoms form a deuterated C3-C7 cycloalkyl group, a C3-C7 cycloalkyl group, or a C3-C7 cycloalkenyl group; Alternatively, R3 and R4 together with the carbon atoms adjacent thereto form a deuterated C3-C7 cycloalkyl group, a C3-C7 cycloalkyl group, or a C3-C7 cycloalkenyl group.

[0021] In another preferred embodiment, the compound is selected from the group consisting of: [ka]

[0022] In another preferred embodiment, the compound is further selected from the following group: [ka]

[0023] In a third aspect of the present invention, there is provided a pharmaceutical composition comprising a compound according to the first aspect, or a deuterium-substituted compound thereof, a stereoisomer, an enantiomer, a diastereomer, a racemate or a pharmaceutically acceptable salt thereof, or a mixture of two or more compounds of the compound represented by general formula (I), Pharmaceutically acceptable salts are also provided. The dosage forms of the drug combinations described in this invention may be various and include, but are not limited to, tablets, capsules, granules, syrups, solutions, suspensions or aerosols.

[0024] In a fourth aspect of the present invention, there is provided a method of treating a metabolic disease, comprising the step of administering to a subject in need thereof a compound according to the second aspect, or a deuterium-substituted compound, stereoisomer, enantiomer, diastereomer, racemate or pharmaceutically acceptable salt thereof, or a mixture of two or more compounds represented by general formula (I), or a pharmaceutical composition according to the third aspect. In another preferred embodiment, the metabolic disease is selected from hypercholesterolemia, non-alcoholic steatohepatitis (NASH), and liver fibrosis.

[0025] In a fifth aspect of the present invention, there is provided a method of treating atherosclerosis, comprising the step of administering to a subject in need thereof a compound according to the second aspect, or a deuterium-substituted compound, stereoisomer, enantiomer, diastereomer, racemate or pharmaceutically acceptable salt thereof, or a mixture of two or more compounds of general formula (I), or a pharmaceutical composition according to the third aspect. In another preferred embodiment, the atherosclerosis is caused by hypercholesterolemia.

[0026] Beneficial effects Chinese Patent Application No. 201610105067 (CN107118098) discloses fatty acid compounds, their preparation methods and their uses, which have the activity of activating APMK and the ability to suppress glucose supply in mouse primary hepatocytes, and are useful for the preparation of drugs for treating obesity or diabetes.

[0027] The inventors unexpectedly found that such compounds have the activity of inhibiting ACLY. Further evaluation of the pharmacological activity in vivo and in vivo revealed that representative compound A3 inhibited the de novo synthesis of lipids in primary hepatocytes and inhibited the ApoE expression in golden hamsters. - / -It was found to improve hyperlipidemia in mice and significantly ameliorate the symptoms of NASH in ob / ob mice and cynomolgus monkeys induced by a high-lipid, high-sugar, high-cholesterol diet. Furthermore, research results showed that compounds with trans double bonds have better in vivo activity than compounds with cis double bonds. Furthermore, at equivalent oral doses, compound A3 demonstrated a plasma exposure level more than four times higher than that of bempedoic acid, indicating that compound A3 has a better therapeutic effect.

[0028] As described above, this series of compounds disclosed in this application possesses good ACLY inhibitory activity, inhibits de novo synthesis of triglycerides and cholesterol in primary hepatocytes, significantly improves spontaneous hyperlipidemia in golden hamsters and aged rhesus monkeys, and exhibits remarkable therapeutic effects in both rodent and non-primate NASH models.

[0029] Such novel ACLY small molecule inhibitors have significant therapeutic effects on metabolic diseases such as hypercholesterolemia and non-alcoholic steatohepatitis, and are useful for the development of therapeutic agents for metabolic diseases such as hypercholesterolemia and non-alcoholic steatohepatitis.

[0030] Of course, it is understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions. Each feature disclosed in the specification can be freely replaced with any homologous, equivalent, or similar purpose-serving alternative feature. Due to space limitations, a detailed description will not be given here. [Brief explanation of the drawings]

[0031] [Figure 1]Figure 1 shows the inhibitory effect of compound A3 on de novo lipid synthesis in primary hepatocytes. (A) shows the inhibitory effect of compound A3 on de novo triglyceride synthesis in hepatocytes from WT and ACLY knockout mice. (B) shows the inhibitory effect of compound A3 on de novo triglyceride synthesis in hepatocytes from WT and ACLY knockout mice. *P<0.05, **P<0.01, ***P<0.001 compared to the control group. #P<0.05, ##P<0.01, ###P<0.001 compared to the corresponding dose groups. [Figure 2] Figure 2 shows the evaluation of the serum total cholesterol and low-density lipoprotein cholesterol content lowering effect of multiple administrations of compounds A3 and A4 in golden hamsters, where * is P<0.05 compared to the model control group, ** is P<0.01 compared to the model control group, and *** is P<0.001 compared to the model control group. [Figure 3] Figure 3 shows the effect of long-term administration of the test compound Compound A3 on the formation of major arterial plaque in ApoE- / - mice, where * indicates P<0.05 compared to the model control group, ** indicates P<0.01 compared to the model control group, and *** indicates P<0.001 compared to the model control group. [Figure 4] Figure 4 shows the therapeutic effect of compound A3 on NASH in ob / ob mice. A shows the appearance of the liver in ob / ob mice and the pathological analysis of the liver after long-term administration of the compound (n=8). B shows the statistical results of the pathological analysis using CD68 immunohistochemistry. C shows the statistical results of the pathological analysis using picrosirius red staining. * indicates P<0.05 compared to the model control group, ** indicates P<0.01 compared to the model control group, and *** indicates P<0.001 compared to the model control group. [Figure 5] Figure 5 shows the changes in liver NAS scores after long-term administration of compound A3, where * indicates P<0.05 compared to the model control group, ** indicates P<0.01 compared to the model control group, and *** indicates P<0.001 compared to the model control group. DETAILED DESCRIPTION OF THE INVENTION

[0032] The inventors of the present application have conducted extensive and in-depth research and have found that the compounds of the present application have ACLY inhibitory activity, that such compounds inhibit de novo lipid synthesis in primary hepatocytes, and that they exhibit therapeutic effects in animal models of various metabolic diseases. Furthermore, they have been found to be capable of being developed as therapeutic agents for metabolic diseases such as hypercholesterolemia and non-alcoholic steatohepatitis, and are also useful in the treatment of atherosclerosis. Based on this, the present invention has been completed.

[0033] term In the present invention, unless otherwise specified, the terms used have the ordinary meanings known to those skilled in the art. In the present invention, the term "C1-C6" means having 1, 2, 3, or 4 carbon atoms. The term "C3-C7" means having 3, 4, 5, 6, or 7 carbon atoms. The same notation applies as above.

[0034] As used herein, the term "alkyl group" refers to a saturated linear or branched hydrocarbon moiety, for example, the term "C1-C4 alkyl group" refers to a linear or branched alkyl group having from 1 to 4 carbon atoms, including, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl groups.

[0035] As used herein, the term "alkenyl group" refers to a straight or branched chain hydrocarbon moiety containing at least one double bond; for example, the term "C2-C4 alkenyl group" refers to a straight or branched chain alkenyl group having 2 to 4 carbon atoms and containing one double bond, including, but not limited to, vinyl, propenyl, butenyl, and isobutenyl groups.

[0036] In the present invention, the term "alkynyl group" refers to a straight or branched chain alkynyl group containing one triple bond, including, but not limited to, ethynyl, propargyl, butynyl, isobutynyl, and the like.

[0037] In the present invention, the term "cycloalkyl group" refers to a saturated cyclic hydrocarbon group moiety, for example, the term "C3-C7 cycloalkyl group" refers to a cyclic alkyl group having 3 to 7 carbon atoms in the ring, including, but not limited to, cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group, etc.

[0038] In the present invention, the term "cycloalkenyl group" refers to a cyclic hydrocarbon moiety containing at least one double bond, for example, the term "C3-C7 cyclic alkenyl group" includes, but is not limited to, cyclic alkenyl groups having 3 to 7 carbon atoms in the ring, such as cyclopropenyl groups, cyclobutenyl groups, cyclopentenyl groups, and cyclohexenyl groups.

[0039] Pharmaceutically acceptable salts according to the present invention may be salts of an anion with a positively charged group in the compound of Formula I. Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, malate, toluenesulfonate, tartrate, fumarate, glutamate, glucuronate, lactate, glutarate, or maleate. Similarly, pharmaceutically acceptable salts may be salts of a cation with a negatively charged group in the compound of Formula I. Suitable cations include sodium, potassium, magnesium, calcium, and ammonium ions, such as tetramethylammonium.

[0040] The present invention will be further described below with reference to specific examples. It should be understood that these examples are used only to illustrate the present invention and do not limit the scope of the present invention. Experimental methods for which specific conditions are not specified in the following examples generally follow conventional conditions, such as those described in Sambrook et al., "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989), or manufacturer recommendations. Unless otherwise specified, percentages and parts are by weight.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are for illustrative purposes only.

[0042] Example 1: Compound synthesis and characterization Synthesis of compound A4 [ka]

[0043] 1. Compound 5-hexyn-1-ol (9 g, 91 mmol) and pyridinium p-toluenesulfonate (25.1 g, 100 mmol) were dissolved in 100 mL of DCM, tetrahydropyran (12.5 mL, 126 mmol) was added, and the mixture was stirred at room temperature overnight. The reaction was monitored for completeness by TLC. Most of the DCM was removed, leaving approximately 30 mL. Approximately 100 mL of ethyl ether was added and the mixture was stirred for 0.5 h. The solid was removed by filtration, washed once with ethyl ether, and the filtrate was concentrated and applied to a column to obtain 18 g of product B1 as an oil in a PE / EA (volume ratio) of 50 / 1, with a yield of >100%.

[0044] 2. Compound 5-bromo-1-pentanol (15.1 g, 91 mmol) and pyridinium p-toluenesulfonate (25.1 g, 100 mmol) were dissolved in 100 mL of DCM, tetrahydropyran (12.5 mL, 126 mmol) was added, and the mixture was stirred at room temperature overnight. The reaction was monitored for completeness by TLC. Most of the DCM was removed, leaving approximately 30 mL. Approximately 100 mL of ethyl ether was added, and the mixture was stirred for 0.5 h. The solid was removed by filtration, washed once with ethyl ether, and the filtrate was concentrated and applied to a column. 18 g of compound B2 was obtained in a 79% yield using a PE / EA (volume ratio) of 50 / 1.

[0045] 3. Compound B2 (5.4 g, 30 mmol) was dissolved in 40 mL of anhydrous THF and cooled to -40 °C. Lithium n-butoxide (19 mL, 30.4 mmol) was added dropwise (addition completed in approximately 0.5 h). 10 mL of HMPA was added and the mixture was stirred at the same temperature for 1 h. Then, a 10 mL THF solution of compound B2 (7.78 g, 31 mmol) was added. The mixture was allowed to react at the same temperature for 1 h, then slowly warmed to room temperature and stirred overnight. The reaction was quenched by adding saturated ammonium chloride solution, and the layers were separated. The aqueous phase was further extracted once with ethyl acetate. The organic phases were combined and washed with water and saturated brine. The mixture was dried, concentrated, and loaded onto a column. 8.5 g of product B3 was obtained in 80% yield using a PE / EA (volume ratio) of 20 / 1, 10 / 1, and 5 / 1.

[0046] 4. Compound B3 (3 g, 8.5 mmol) was dissolved in 100 mL of methanol and 10 mL of p-toluenesulfonic acid (300 mg) and stirred at room temperature overnight. The reaction was monitored for completeness by TLC. The mixture was concentrated to remove most of the methanol, diluted with water, and extracted twice with ethyl ether. The organic phases were combined, washed with water and saturated brine, dried, concentrated, and loaded onto a column. PE / EA (volume ratio) was changed from 2 / 1 to 1 / 1 to 1 / 2 to give product B4 (1.1 g, 70% yield).

[0047] 5. Compound B4 (1.1 g, 5.9 mmol) and carbon tetrabromide (5.95 g, 17.9 mmol) were dissolved in 30 mL of anhydrous DCM and cooled to 0 °C. A solution of triphenylphosphine (4.7 g, 17.9 mmol) in 10 mL of DCM was added dropwise and stirred at room temperature for 1 h to allow the reaction to complete. The mixture was concentrated to approximately 15 mL, and 50 mL of ethyl ether was added. The mixture was stirred for 0.5 h. The solid was removed by filtration. The filtrate was concentrated and applied to a column. 1.84 g of bromide B5 was obtained in 100% yield using PE / EA = 30 / 1.

[0048] 6. Compound B5 (1.84 g, 5.9 mmol) and ethyl isobutyrate (2.73 g, 23.6 mmol) were dissolved in 50 mL of anhydrous THF and cooled to 0 °C. LDA (15.7 mL, 23.6 mmol) was added dropwise (addition completed in approximately 0.5 h). The mixture was maintained at the same temperature for 1 h, then warmed to room temperature and stirred overnight. The reaction was monitored for completeness by TLC. The reaction was quenched with saturated ammonium chloride solution, the layers were separated, and the aqueous phase was extracted twice with EA. The organic phases were combined, washed with water and saturated brine, dried, and concentrated. The mixture was applied to a column using a PE / EA (volume ratio) of 50 / 1 to 20 / 1 to obtain 1.8 g of product B6 in 80% yield.

[0049] Nickel acetate (749 mg, 3 mmol) was suspended in 20 mL of absolute ethanol under 7.1 atmospheres of H2, and sodium borohydride (114 mg, 3 mmol) was quickly added. The mixture was purged with hydrogen gas twice, resulting in a dark solution. After stirring at room temperature for 15 minutes, ethylenediamine (0.45 mL, 6 mmol) was added, followed by a solution of compound B6 (1.8 g, 4.7 mmol) in 10 mL of absolute ethanol. After stirring at room temperature for 2 hours, the reaction was monitored for completeness by TLC. The mixture was diluted with 50 mL of ethyl ether, filtered through diatomaceous earth, and the filtrate was concentrated and loaded onto a column to give product B7 (1.7 g, 95% yield) in a PE / EA (volume ratio) of 50 / 1.

[0050] 8. Compound B7 (1.7 g, 4.45 mmol) was dissolved in 50 mL of ethanol and added with 10 mL of aqueous KOH (2 g, 35 mmol). The mixture was heated to reflux for 6 h and monitored for complete reaction by TLC. After cooling, most of the ethanol was removed by rotary evaporation, diluted with water (40 mL), and extracted twice with ethyl ether to remove impurities. The aqueous layer was acidified with 2N NCl and extracted four times with DCM. The organic layers were combined, washed with water and saturated brine, dried, and concentrated. The mixture was then loaded onto a column using DCM / MeOH (volume ratio) = 100 / 1 to give 1.1 g of A4.

[0051] A4 1 H NMR (600 MHz, DMSO) δ5.31-5.32(m, 2H), 1.96-1.98 (m, 4H), 1.40-1.44(m, 4H), 1.22-1.30 (m, 4H), 1.18-1.22 (m, 6H), 1.06 (s, 12H).

[0052] Using different substrates and a similar synthetic scheme to A4, the following compounds were obtained. A2 1 H NMR (300 MHz, CD3Cl) δ5.31-5.32(m, 2H), 1.96-1.98 (m, 4H), 1.40-1.44(m, 4H), 1.18-1.22 (m, 8H), 1.06 (s, 12H)

[0053] A6 1 H NMR (300 MHz, CD3Cl) δ5.30-5.32(m, 2H), 1.96-1.98 (m, 4H), 1.40-1.45(m, 4H), 1.18-1.22 (m, 12H), 1.06 (s, 12H)

[0054] A8 1H NMR (400 MHz, CDCl3) δ 5.40 - 5.29 (m, 2H), 2.05 - 1.93 (m, 4H), 1.57 - 1.48 (m, 4H), 1.39 - 1.22 (m, 14H), 1.19 (s, 6H), 1.18 (s, 6H).

[0055] A10 1 H NMR (300 MHz, CD3Cl) δ5.30-5.32(m, 2H), 1.98-2.02(m, 4H), 1.48-1.53(m, 4H), 1.18-1.22 (m, 16H), 1.06 (s, 12H)

[0056] Synthesis of compound A3 [ka]

[0057] 1. Under nitrogen gas protection, 350 mg of lithium aluminum hydride (9.1 mmol) was dissolved in 10 mL of diethylene glycol dimethyl ether, and 410 mg of B2 (1.1 mmol) was dissolved in 2 mL of diethylene glycol dimethyl ether. The mixture was reacted at 140°C for 18 hours, and the reaction was monitored by TLC until the reaction was complete. The mixture was diluted with 30 mL of ethyl ether, cooled in an ice-water bath, and sodium sulfate decahydrate was added in portions until no more bubbles were generated. The mixture was filtered through diatomaceous earth to remove the solid, washed with anhydrous ethyl ether, dried over anhydrous sodium sulfate, concentrated, and applied to a PE:EA=20:1 column to obtain 250 mg of product B8 as a colorless liquid in a 62% yield.

[0058] 2. Compound B8 (250 mg, 0.71 mmol) was dissolved in 10 mL of methanol and 11 mL of p-toluenesulfonic acid (25 mg) and stirred at room temperature overnight. The reaction was monitored for completeness by TLC. The mixture was concentrated to remove most of the methanol, diluted with water, and extracted twice with ethyl ether. The organic phases were combined, washed with water and saturated brine, dried, concentrated, and applied to a column to obtain product B9 (100 g, 75% yield) using PE / EA (volume ratio) = 4 / 1.

[0059] 3. Compound B9 (100 mg, 0.53 mmol) and carbon tetrabromide (360 mg, 1.1 mmol) were dissolved in 10 mL of anhydrous DCM and cooled to 0 °C. A solution of triphenylphosphine (256 g, 0.98 mmol) in 2 mL of DCM was added dropwise and stirred at room temperature for 1 h to allow the reaction to complete. The mixture was concentrated and loaded onto a column, and 142 mg of bromide B10 was obtained in a 87% yield using a PE / EA (volume ratio) of 20 / 1.

[0060] 4. Compound B10 (142 mg, 0.46 mmol) and ethyl isobutyrate (320 mg, 2.76 mmol) were dissolved in 10 mL of anhydrous THF and cooled to 0 °C. LDA (1.84 mL, 2.76 mmol) was added dropwise (addition completed in approximately 0.5 h). The mixture was maintained at the same temperature for 1 h, then warmed to room temperature and stirred overnight. The reaction was monitored for completeness by TLC. The reaction was quenched with saturated ammonium chloride solution, the layers were separated, and the aqueous phase was extracted twice with EA. The organic phases were combined, washed with water and saturated brine, dried, and concentrated. The mixture was applied to a column using a PE / EA (volume ratio) of 50 / 1 to obtain 140 mg of product B11 in 80% yield.

[0061] 5. Compound B11 (107 mg, 0.28 mmol) was dissolved in 10 mL of ethanol, added with 2 mL of aqueous KOH (200 mg, 3.5 mmol), and heated to reflux for 6 h. The reaction was monitored for completeness by TLC. After cooling, most of the ethanol was removed by rotary evaporation, diluted with water (10 mL), and extracted twice with ethyl ether to remove impurities. The aqueous layer was acidified with 2N NCl and extracted four times with DCM. The organic layers were combined, washed with water and saturated brine, dried, and concentrated. The column was loaded with DCM / MeOH (volume ratio) = 100 / 1 to obtain 70 mg of compound A3.

[0062] A3 1 H NMR (600 MHz,DMSO) δ5.35-5.36(m, 2H), 1.92-1.94 (m, 4H), 1.40-1.43 (m, 4H), 1.27-1.29 (m, 4H), 1.17-1.19 (m, 6H),1.18 ppm (s, 12H).

[0063] Using different substrates and a similar synthetic scheme to A3, the following compounds were obtained. A1 1 H NMR (400 MHz, CDCl3) δ 5.37 - 5.23 (m, 2H), 2.07 - 1.90 (m, 4H), 1.60 - 1.44 (m, 4H), 1.34 - 1.21 (m, 8H), 1.16 (s, 12H).

[0064] A5 1 H NMR (400 MHz, CDCl3) δ 5.41 - 5.30 (m, 2H), 2.09 - 1.87 (m, 4H), 1.60 - 1.46 (m, 4H), 1.41 - 1.32 (m, 4H), 1.31 - 1.23 (m, 8H), 1.22 - 1.07 (s, 12H).

[0065] A7 1H NMR (400 MHz, CDCl3) δ 5.40 - 5.28 (m, 2H), 2.06 - 1.88 (m, 4H), 1.57 - 1.48 (m, 4H), 1.40 - 1.20 (m, 14H), 1.18 (s, 12H).

[0066] A9 1 H NMR (400 MHz, CDCl3) δ 5.33 - 5.24 (m, 2H), 2.00 - 1.92 (m, 4H), 1.54 - 1.47 (m, 4H), 1.31 - 1.17 (m, 28H).

[0067] Compound A11 [ka]

[0068] 1. Bis(pinacolato)diboron (483 mg, 1.9 mmol) and tetrakis(triphenylphosphine)platinum (70 mg) were placed in a 25 mL round-bottom flask. After flushing with argon gas three times, a 10 mL DMF solution of B6 (660 mg, 1.74 mmol) was added dropwise and reacted at 80 °C overnight. The mixture was diluted with water and extracted with ethyl ether. The organic phases were combined, washed with water and saturated brine, dried, and concentrated. The mixture was then loaded onto a column using a PE / EA (volume ratio) of 10 / 1 to obtain 1 g of product B12 in 90% yield.

[0069] 2. Compound B12 (150 mg, 0.23 mmol), iodomethane (0.1 mL), and triphenylphosphine (10 mg) were dissolved in 1 mL of dioxane. The mixture was purged with argon gas three times. Palladium acetate (20 mg) and 0.2 mL of KOH (40 mg) were added and reacted at 90°C for 12 h. The mixture was diluted with water and ethyl acetate and extracted three times with ethyl acetate. The organic phases were combined, washed with water and saturated brine, dried, and concentrated. The mixture was then loaded onto a column using PE / EA = 50 / 1 to obtain product B13 (30 mg, 31% yield).

[0070] 3. Compound B13 (30 mg, 0.07 mmol) was dissolved in 5 mL of ethanol, added with 1 mL of aqueous KOH (50 mg, 0.89 mmol), and heated to reflux for 6 h. The reaction was monitored for completeness by TLC. After cooling, most of the ethanol was removed by rotary evaporation, diluted with water (4 mL), and extracted twice with ethyl ether to remove impurities. The aqueous layer was acidified with 2N NCl and extracted four times with DCM. The organic layers were combined, washed with water and saturated brine, dried, and concentrated. The mixture was loaded onto a column using DCM / MeOH (volume ratio) = 100 / 1 to give 15 mg of A11.

[0071] A11 1 H NMR (300 MHz, CDCl3) δ 1.94-1.96 (m, 4H), 1.58(s, 6H), 1.48-1.50 (m, 4H), 1.25-1.33(m, 12H), 1.13 ppm (s, 12H)

[0072] Synthesis of compound A12 [ka]

[0073] 1. Under nitrogen gas protection, diethylzinc reagent (1 mL, 1 mmol) was added to 1 mL of DCM and cooled to -40 °C. After 5 min, a solution of diiodomethane (0.54 g, 2 mmol) in 0.5 mL of DCM was added dropwise. The mixture was then incubated at -40 °C for 1 h. Trichloroacetic acid (16 mg, 0.1 mmol) and DME (45 mg, 0.5 mmol) were added. After the addition was complete, the mixture was warmed to -15 °C and incubated for 1 h. A solution of B7 (193 mg, 0.5 mmol) in 0.5 mL of DCM was added. After the addition was complete, the mixture was stirred at room temperature overnight. The reaction was quenched with saturated ammonium chloride solution, extracted three times with ethyl acetate, and the organic phases were combined, washed with water and saturated brine, dried, and concentrated. The product B14 (120 mg) was obtained in 60% yield using a PE / EA (volume ratio) of 50 / 1.

[0074] 2. Compound B14 (100 mg, 0.25 mmol) was dissolved in 5 mL of ethanol, added with 1 mL of aqueous KOH (10 mg, 0.18 mmol), and heated to reflux for 6 h. The reaction was monitored for completeness by TLC. After cooling, most of the ethanol was removed by rotary evaporation, diluted with water (4 mL), and extracted twice with ethyl ether to remove impurities. The aqueous layer was acidified with 2N NCl and extracted four times with DCM. The organic layers were combined, washed with water and saturated brine, dried, and concentrated. The column was loaded with DCM / MeOH (volume ratio) = 100 / 1 to give 65 mg of A12.

[0075] A12 1 H NMR (300 MHz, CDCl3) δ 1.1.45-1.52 (m, 4H), 1.21-1.43(m, 16H), 1.18 (s, 12H), 0.65-0.70ppm (m, 2H)

[0076] Synthesis of compound A13 [ka]

[0077] 1. Compound A3 (30 mg) was dissolved in CHCl (5.0 mL), and 1,1'-carbonyldiimidazole (50 mg, 30 mmol) was added. The mixture was stirred at room temperature for 2 h. The mixture was diluted with 5.0 mL of CHCl and washed with water (10 mL each time, twice). The mixture was dried over anhydrous magnesium sulfate and then directly used in the next step.

[0078] 2. Under argon gas protection, the above imidazole intermediate was dissolved in dry THF (8.5 mL) and a solution of trilithium coenzyme A (20 mg, 0.026 mmol) in 0.1 M NaHCO3 (3 mL) was added dropwise. The reaction mixture was stirred at room temperature for 24 h. The reaction mixture was extracted with ethyl acetate (2 × 3 mL). The aqueous layer was adjusted to pH 2 with 1 M HCl. The aqueous layer (~1 mL) was directly loaded onto a column (SPE, Agilent HF Bond Elur C18 column, size: 5 gm / 20 mL, USA) using 0%, 5%, 10%, 20%, 30%, 40%, 50%, and 100% MeCN (10 mL each time). A13 was obtained, the structure of which is 1 The 1 H NMR spectrum was confirmed by LR-ESI-MS.

[0079] Compound A13: Solvent system on a Zorbax-SB C18 column, 4.6 × 50 mm, 3.5 μm: gradient, A: 10 mM NH4COOH in H2O, B: CH3OH, 0 min: 50% A and 50% B; 0–30 min: A 50%→0%, B 50%→100%, 0.5 mL / min. HPLC analysis: tr = 14.777 min and 16.074 min; white solid (13 mg, 42%); 1H NMR (400 MHz, D2O) δ 8.65 (s, 1H) , 8.58(s, 1H), 8.30(s, 1H), 6.01(d, J = 5.4 Hz, 1H), 5.45-5.42(m, 2H), 4.75-4.74 (m, 2H), 4.48 (bs, 1H), 4.28(bs, 2H), 3.98 (s, 1H), 3.82-3.80 (m, 1H), 3.56-3.52(m, 1H), 3.40-3.38 (m, 2H), 3.20-3.18(m, 2H), 2.25-2.22(m, 2H), 2.33-2.30 (m, LR-ESI-MS m / z, C 40 H 68 N7O 19 P3S [MH] - Calculated value: 1074.3, Measured value: 1074.3.

[0080] Synthesis of compound A14 [ka] Using A4 as a substrate, A14 was obtained according to the synthesis method of compound A13: 1H NMR (400 MHz, D2O) δ 8.65 (s, 1H) , 8.57(s, 1H), 8.30(s, 1H), 6.02(d, J = 5.4 Hz, 1H), 5.47-5.42(m, 2H), 4.75-4.73 (m, 2H), 4.49 (bs, 1H), 4.27(bs, 2H), 3.98 (s, 1H), 3.82-3.81 (m, 1H), 3.56-3.52(m, 1H), 3.40-3.38 (m, 2H), 3.20-3.18(m, 2H), 2.25-2.20(m, 2H), 2.33-2.29(m, LR-ESI-MS m / z, C 40 H 68 N7O 19 P3S [MH] - Calculated value: 1074.3, Measured value: 1074.3.

[0081] Synthesis of compound A15 [ka]

[0082] 1. Compound A3 (326 mg, 1 mmol) and (2S,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)-tetrahydro-2H-pyran-3,4,5-triyl triacetate (476 mg, 1.20 mmol) were dissolved in 10 mL of toluene, cooled to 0 °C, and silver oxide (925 mg, 4 mmol) and pyridine (0.35 mL, 4 mmol) were added. The mixture was stirred overnight at room temperature. The mixture was filtered through diatomaceous earth, washed with toluene, concentrated, and then loaded onto a column. 350 mg of B15 was obtained by elution with dichloromethane / methyl t-butyl ether (volume ratio) = 10 / 1.

[0083] 2. B15 (350 mg, 0.54 mmol) was dissolved in 5 mL of methanol, cooled to 0°C, and diisopropylethylamine (0.3 mL) was added. The mixture was stirred at room temperature for 2 days, concentrated, and then loaded onto a column. 100 mg of compound B16 was obtained by elution with petroleum ether / ethyl acetate / isopropanol (volume ratio) = 50 / 50 / 6.

[0084] 3. B16 (60 mg, 0.17 mmol) was dissolved in isopropanol (0.5 mL), pH 7 phosphate buffer solution (5 mL) was added, and CAL-B (100 mg) was added. The mixture was stirred at room temperature for 3 hours, a small amount of isopropanol was removed, the mixture was filtered, washed with water, and extracted twice with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, concentrated, and loaded onto a reverse-phase column. 15 mg of A15 was obtained using a methanol / water (volume ratio) of 90 / 10.

[0085] Compound A15 1 H NMR (400 MHz, CD3OD) δ 5.46 (d, J = 7.9 Hz, 1H), 5.40 - 5.35 (m, 2H), 3.80 (d, J = 9.5 Hz, 1H), 3.57 - 3.34 (m, 3H), 2.01 - 1.94 (m, 4H), 1.60 - 1.47 (m, 4H), 1.39 - 1.23 (m, 10H), 1.22 - 1.17 (m, 6H), 1.17 - 1.11 (m, 6H) ppm. LR-ESI-MS m / z, C 25 H 42 O 10 [MH] - Calculated value: 501.2, Measured value: 501.2.

[0086] Synthesis of compound A16 [ka]

[0087] 1. NaH (672 mg, 16.8 mmol) was suspended in 10 mL of THF and cooled to 0 °C. Dimethyl methylmalonate (2.35 g, 16 mmol) was added dropwise (addition completed in approximately 0.5 h). The mixture was warmed to room temperature and stirred for 0.5 h, after which compound B10 (730 mg, 2.3 mmol) in 10 mL of THF was slowly added dropwise. After addition was complete, the mixture was stirred at 60 °C for 7 h and monitored by TLC for complete reaction. The reaction was quenched with saturated ammonium chloride solution, the layers were separated, and the aqueous phase was extracted twice with EA. The organic phases were combined, washed with water and saturated brine, dried, and concentrated. The mixture was applied to a column using a PE / EA (volume ratio) of 50 / 1 to obtain 510 mg of product B17 in 50% yield.

[0088] 2. Compound B17 (300 mg, 0.68 mmol) was dissolved in 5 mL of DMSO, and LiCl (286 mg, 6.8 mmol) and HO (0.12 mL) were added. The mixture was heated to 180 °C in a microwave oven for 5 minutes, and the reaction was monitored by TLC until the reaction was complete. After cooling, the mixture was diluted with water, and the aqueous phase was extracted four times with EA. The organic phases were combined, washed with water and saturated brine, dried, and concentrated. The mixture was applied to a column using a PE / EA (volume ratio) of 50 / 1 to obtain 166 mg of product B18, a yield of 75%.

[0089] 3. Compound B17 (140 mg, 0.43 mmol) was dissolved in 5 mL of anhydrous THF and cooled to -78 °C. LDA (1.84 mL, 2.76 mmol) and HMPA (0.4 mL) were added dropwise. The mixture was maintained at the same temperature for 1 h, followed by the dropwise addition of CD3I (0.16 mL, 2.58 mmol). The mixture was warmed to room temperature and stirred overnight. The reaction was monitored for completeness by TLC. The reaction was quenched with saturated ammonium chloride solution, the layers were separated, and the aqueous phase was extracted twice with EA. The organic phases were combined, washed with water and saturated brine, dried, and concentrated. The mixture was applied to a column using a PE / EA (volume ratio) of 50 / 1 to obtain 77 mg of product B19 in 49% yield.

[0090] 4. Compound B19 (77 mg, 0.21 mmol) was dissolved in ethanol (2 mL) and added with 2 mL of aqueous NaOH (120 mg, 3 mmol). The mixture was heated to reflux for 6 h and monitored for complete reaction by TLC. After cooling, most of the ethanol was removed by rotary evaporation, diluted with water (10 mL), and extracted twice with ethyl ether to remove impurities. The aqueous layer was acidified with 2N NCl and extracted four times with DCM. The organic layers were combined, washed with water and saturated brine, dried, and concentrated. The mixture was loaded onto a column and purified with DCM / MeOH (volume ratio) = 50 / 1 to obtain 50 mg of compound A16.

[0091] A16 1 H NMR (400 MHz,CD3Cl) δ5.33-5.34(m, 2H), 1.96-1.99 (m, 4H), 1.51-1.55 (m, 4H), 1.35-1.42 (m, 4H), 1.22-1.28 (m, 6H),1.18 (s, 3H) , 1.17 ppm (s, 3H).

[0092] Using compound A16 as a starting material, compound A17 below was obtained by a synthesis scheme similar to that for compound A15. [ka]

[0093] Compound A17 1 H NMR (400 MHz, CD3OD) δ 5.48 (d, J = 7.9 Hz, 1H), 5.37 - 5.41 (m, 2H), 3.82 (d, J = 9.4 Hz, 1H), 3.58 - 3.55 (t, 1H), 3.48 - 3.44 (t, 1H), 3.42-3.38(m, 1H), 2.01 - 1.94 (m, 4H), 1.60 - 1.44 (m, 4H), 1.39 - 1.21 (m, 10H), 1.19 (s, 3H), 1.15 (s, 3H) ppm. LR-ESI-MS m / z, C 25 H 36D6O 10 [MH] - Calculated value: 507.2, Measured value: 507.2.

[0094] Example 2: Biological in vitro activity evaluation (1) ACLY citrate synthase inhibitory activity of the compounds During the catalytic process of citrate decomposition by ACLY, one molecule of ATP is consumed for the activation of citrate. Based on this reaction principle, the inventors used the ADP-Glo ​​detection kit to measure the amount of ATP converted to ADP, which reflected the catalytic activity of ACLY. After establishing an activity detection system and optimizing the reaction conditions, the inventors evaluated the inhibitory activity of the obtained novel ACLY small molecule inhibitors against ACLY.

[0095] Activity detection protocol: Human ACLY protein was purchased from Beijing Sino Biological Co., Ltd. (catalog number: 11769-H07B), and the ADP-Glo ​​detection kit was purchased from Promega (catalog number: V9102). The final reaction system, as shown in Table 1, consisted of a 5 μL total volume, consisting of 1 μL of 2% DMSO, 2 μL of substrate mixture, and 2 μL of enzyme (final concentration: 25 nM). An additional 1 μL of compounds at different concentrations was added and incubated. Reaction conditions were 30 min at 37°C. 2.5 μL of the ADP Reagent from the ADP-Glo ​​kit was added and mixed thoroughly. After incubation for 2 hours, 5 μL of ADP Detection was added, centrifuged, and mixed thoroughly. The values ​​at all wavelengths were then read using an Envision multilabel plate reader. [Table 1]

[0096] Test results: First, the cumulative fluorescence intensity (RFU / min) within the 30-minute reaction time of the enzyme was calculated, and this was taken as the initial enzyme velocity. The ratio of the enzyme velocity after compound addition (vsample) to the enzyme velocity without compound addition (vDMSO) was calculated, and this was taken as the activity percentage (%activity) for each concentration group of the sample. The formula was %activity = (V(sample)) / (v(DMSO)) × 100%. A graph was created of the logarithm of the concentration versus the activity percentage, and a regression curve was calculated using nonlinear regression. Curve fitting was performed using the formula log(inhibitor) vs. response -- Variable slope in GraphPad Prism 8.0 software, and the IC was calculated. 50 The values ​​were calculated and the results are shown in the table below. [Table 2]

[0097] [ka] The above is the structural formula of the positive control ETC-1002-CoA.

[0098] (2) Evaluation of the inhibitory activity of compounds on de novo lipid synthesis in primary hepatocytes ACLY plays a role in decomposing citrate from mitochondria into acetyl-coenzyme A, the latter being a major raw material for cellular lipid synthesis. Suppression of ACLY activity significantly reduces the de novo synthesis of lipids (triglycerides and cholesterol). To further evaluate the functional changes caused by the inhibition of ACLY by test substances, the inventors isolated primary mouse hepatocytes and [1,2- 14 The ability to synthesize lipids from scratch was evaluated by tracking [C]-sodium acetate as a substrate, while the compound ETC-1002 was used as a positive control.

[0099] Test Protocol: Isolation and attachment of primary hepatocytes: After perfusion of mouse liver with calcium-free perfusion solution, the mouse liver was digested with collagenase and subjected to density gradient centrifugation to obtain primary mouse hepatocytes. 3 × 10 5 The cells were seeded at a density of 1000 / mL into 6-well plates (plates were underlaid with 0.2% gelatin before seeding). After seeding, the cells were allowed to attach for 6 h, then replaced with serum-free LG-DMEM (containing 2× double antibodies) and starved overnight.

[0100] Isotope tracing: On day 2, serum-free LG-DMEM containing 10 nM insulin and the corresponding concentrations of compounds (1950 μL, four replicate wells per group) were added to each well. 14 C]-acetate serum-free medium was added and placed in an incubator for 4 hours.

[0101] Lipid saponification: Each well was washed three times with 1.6 mL of cold PBS. The plate was then dried and 600 μL of 0.5 M KOH was added to each well for 1 hour. 480 μL of lipid was aspirated and placed in a glass test tube containing 400 μL of 20% KOH (methanol as solvent). The tube was shaken to homogenize and then saponified in a 95°C water bath for 3 hours. The excess saponification solution was used to measure protein concentration. 400 μL of petroleum ether was added to each tube, pipetted 5-6 times, and centrifuged at 2500 rpm for 5 minutes. 350 μL of the supernatant was aspirated into an EP tube, and 400 μL of petroleum ether was added twice for a total of three extractions. After the supernatant was completely transferred, each tube was acidified with 200 μL of water and 400 μL of 5 N sulfuric acid and shaken to homogenize. Each tube was filled with 400 μL of petroleum ether, pipetted 5-6 times, and then centrifuged at 2500 rpm for 5 minutes. 350 μL of the supernatant was aspirated into an EP tube, and 400 μL of petroleum ether was added, repeating this process twice (a total of three extractions). The tubes were then placed in a fume hood to evaporate the petroleum ether overnight.

[0102] Isotope formulation and quantification: Each tube was filled with 1700 μL of scintillation fluid and read.

[0103] Data processing and analysis: First, the isotope content of free fatty acids and cholesterol (R control, unit: CCPM / mg protein) in the control group of untreated mouse primary hepatocytes was calculated within 4 hours and used as the basal synthesis level. Then, the percentage of lipid synthesis inhibition was calculated for each concentration of sample. The inhibition rates of de novo synthesis of triglycerides and cholesterol after treating primary hepatocytes with some compounds (12.5 μM) are shown in the table below. [Table 3]

[0104] (3) Dependence of the lipid synthesis inhibitory activity of compound A3 on ACLY Test protocol: To further examine the dependence of the inhibitory effect of a series of compounds on de novo lipid synthesis on ACLY, the inventors used ACLY knockout primary hepatocytes to evaluate the de novo lipid synthesis, and also used compound ETC-1002 as a positive control. The specific test protocol was as described above, with 0.1 μCi / well of [ 14 C]-citric acid.

[0105] Test results: In WT mouse primary hepatocytes, both ACLY small molecule inhibitor ETC-1002 and compound A3 significantly increased cell 14 These compounds were able to inhibit the de novo synthesis of triglycerides (Fig. 1A) and cholesterol (Fig. 1B) using [C]-citrate as a substrate. However, in ACLY-knockout hepatocytes, the inhibitory effects of these two compounds on the de novo synthesis of triglycerides and cholesterol were significantly reduced or even lost (Fig. 1).

[0106] Example 3: Drug effects of compounds A3 and A4 on improving hypercholesterolemia (1) The ameliorative effect of compounds A3 and A4 on hyperlipidemia in golden hamsters Dosing Plan: The golden hamster hyperlipidemia model is an ideal animal model for clinically recognized hyperlipidemia drug therapy (pharmacodynamic evaluation). To initially investigate the potential regulatory effects of candidate compounds on the balance of hepatic lipid metabolism, a preliminary pharmacological evaluation was conducted using a golden hamster hyperlipidemia model induced by a high-fat, high-cholesterol diet. Specifically, 56 male golden hamsters were fed a high-fat, high-cholesterol diet (Research diet, catalog number C11953) for two weeks to establish the model, leaving eight animals as a blank control group. Subsequently, the animals were divided into seven groups (n=8): a model control group, a positive control group (ETC-1002 30 mg / kg), compound A3 groups (10 mg / kg and 30 mg / kg), compound A4 groups (10 mg / kg and 30 mg / kg), and a blank control group. Each group was treated with compounds in saline (0.9% NaCl) once daily for a seven-day dosing cycle.

[0107] Indicator detection: Changes in body weight and food intake of golden hamsters were closely monitored during the administration period. Each group was administered with the compound for 7 days, and blood was collected from the eye margin (after overnight starvation for approximately 16 hours). Serum was centrifuged and stored at low temperature for detection of total cholesterol and low-density lipoprotein cholesterol. The relative amount of the relevant indicators was calculated as follows: relative amount (100%) = content in the treatment group / content in the model control group × 100%.

[0108] Results: Compared with the control group, after one week of compound administration, 1) compound A3 significantly reduced serum total cholesterol and low-density lipoprotein cholesterol in both dose groups in golden hamsters, with the magnitude of reduction in the high-dose group being comparable to the effect of the positive control ETC-1002 at 30 mg / kg; 2) compound A4 significantly reduced serum total cholesterol and low-density lipoprotein cholesterol in the 10 mg / kg group but not in the 30 mg / kg group (Figure 2). Furthermore, the study found that compound A4 significantly reduced body weight, food intake, and intolerance, such as slowness of activity, during the administration process. These results suggest that compound A3 is more effective and safer in treating hypercholesterolemia than compound A4. Furthermore, the trans configuration of the double bond is important for its therapeutic effect on hypercholesterolemia.

[0109] (2) ApoE by Compound A3 - / - Amelioration of atherosclerosis in mice Dosage Plan: Hypercholesterolemia is an important trigger of the development of atherosclerosis. On the premise that Compound A3 has a significant improvement in hyperlipidemia, the inventors further found that Compound A3 significantly inhibits ApoE. - / - The researchers found that the administration of riboflavin to male ApoE mice can alleviate the development of atherosclerosis in mice. - / -Sixty mice were divided into six groups (n=10) on average. Five groups were fed a high-cholesterol diet (Research diet, catalog number D12079B) to establish the model, and one group served as a blank control. The remaining groups were also administered long-term treatments: a model control group, a positive control group (ETC-1002 30 mg / kg), compound A3 15 mg / kg, 30 mg / kg, and 60 mg / kg, and a blank control group. Each group was treated with compounds in saline (0.9% NaCl) once daily for 24 weeks.

[0110] Indicator detection: ApoE closely during administration period - / - The mice were monitored for changes in body weight and food intake. After 24 weeks of treatment with the compounds in each group, the mice's arteries were dissected and the arterial plaques were stained with Sudan IV staining. The plaque areas were then calculated using Image-Pro Plus software.

[0111] Test results: As shown in Figure 3, after 24 weeks of compound administration, all three dose groups of compound A3 showed significant ApoE reduction compared with the control group. - / - It was found that the plaque area in the main arteries of the mice was reduced (the figure on the right is a statistical diagram of the figure on the left), demonstrating that compound A3 has a significant effect in improving the development of atherosclerosis induced by hypercholesterolemia.

[0112] Example 4: Evaluation of efficacy of compound A3 in improving non-alcoholic steatohepatitis induced by dietary intake Much research has been done on the pathogenesis of nonalcoholic steatohepatitis (NASH). It can be summarized that lipid deposition is the foundation, inflammation is the core, and fibrosis is a factor that worsens the disease or is difficult to control. On the one hand, hepatocyte ACLY inhibitory activity can effectively reduce hepatic lipid synthesis and reduce factors that drive NASH development. On the other hand, ACLY mediates epigenetic modifications of histones (e.g., acetylation, succinylation), leading to metabolic reprogramming of immune cells, response to inflammatory stimuli, and ROS generation. Furthermore, inhibiting Ito cell lipid synthesis can effectively reduce Ito cell (HSC) activation. Based on the potential involvement of the three major features of NASH development (lipid deposition, inflammation, and fibrosis), the inventors further evaluated the feasibility of compound A3 for improving NASH.

[0113] (1) Compound A3 ameliorates NASH induced by a high-fat, high-sugar, high-cholesterol diet in ob / ob mice Dosing regimen: Sixteen male ob / ob mice, 7-8 weeks old, were divided into two groups (n=8) and fed a high-fat, high-sugar, high-cholesterol diet (Research diet, catalog number D09100310) for 8 weeks to establish the model. Another group of wild-type mice of the same origin served as a blank control. After 8 weeks of model establishment, long-term administration treatment was conducted. Each group consisted of a model control group, a Compound A3 30 mg / kg group, and a blank control group. The compounds in each group were prepared in saline (0.9% NaCl) and administered once daily for a 10-week administration cycle.

[0114] Indicator detection: Changes in body weight and food intake were closely monitored in ob / ob mice and blank control mice during the treatment period. Each group of compounds was administered for 10 weeks, and then the livers were excised and subjected to detection of corresponding indicators of NASH (e.g., HE staining, picrosirius red staining, and immunohistochemistry for inflammatory signals).

[0115] Test results: ob / ob mice developed a pale, granular appearance due to excessive lipid deposition. Administration of Compound A3 significantly reversed this phenomenon (Figure 4A). Furthermore, the inventors performed pathological analysis of the livers: H&E staining revealed significantly reduced hepatic steatosis and ballooning in the Compound A3-treated group compared with the control group (fatty liver, Figure 4A). Macrophage CD68 staining revealed significantly reduced inflammatory cell infiltration in the liver in the Compound A3-treated group (inflammation, Figures 4A and 4B). Picrosirius red staining revealed significantly reduced hepatic collagen deposition in the Compound A3-treated group (fibrosis, Figures 4A and 4C). These results demonstrate that Compound A3 has pharmacological activity in significantly ameliorating diet-induced NASH in ob / ob mice.

[0116] (2) The effect of compound A3 on NASH induced by a high-fat, high-sugar, high-cholesterol diet in cynomolgus monkeys Furthermore, the inventors further evaluated the therapeutic effect of long-term administration of Compound A3 on non-alcoholic steatohepatitis in cynomolgus monkeys in a non-human primate animal model. Treatment regimen: Eighteen male cynomolgus monkeys with nonalcoholic steatohepatitis were divided into two groups (n = 8-10), one as a model control group and the other as a treatment group. They were fed a high-fat, high-sugar, and high-cholesterol diet for 14 weeks and simultaneously received treatment with 20 mg / kg of steroid hormone (20 mg / kg) once daily for a 14-week cycle.

[0117] Detection of indicators: The changes in body weight and food intake of cynomolgus monkeys were closely monitored during the administration period. After 14 weeks of compound administration, liver puncture was performed for pathological analysis and detection of corresponding indicators of NASH. Test results: Liver puncture results showed that long-term administration of compound A3 significantly reduced lipid deposition in the liver of cynomolgus monkeys, and reduced the incidence of inflammation and fibrosis, and the non-alcoholic fatty liver disease activity score (NAS) significantly decreased after administration (Figure 5). These results demonstrate that compound A3 has the pharmacological activity to significantly improve non-alcoholic steatohepatitis in cynomolgus monkeys.

[0118] Example 5: Discussion of pharmacokinetic parameters of oral administration of Compound A3 The previous study found that compound A3 was more effective than ETC-1002 in improving atherosclerosis, and this part of the study will compare the oral drug exposure and related pharmacokinetic parameters of the two compounds.

[0119] Dosage regimen: Six male normal ICR mice were divided into two groups (n=3). One group was orally administered the positive control ETC-1002, and the other group was orally administered the test compound A3 at a dose of 30 mg / kg. Blood was collected from the eye margin at different time points before and after administration to detect plasma drug concentrations.

[0120] Test results: Plasma drug concentrations at different time points were detected, and the pharmacokinetic parameters of the two compounds were calculated (Table 4). From the results, it was found that the plasma exposure of the test compound Compound A3 was significantly higher than that of the positive control drug ETC-1002 (C max : 13.4 μg / mL vs 3.81 μg / mL;AUC 0-t :63.7 h*μg / mL vs 14.4 h*μg / mL, the former being compound A3). [Table 4]

[0121] The above test results show that the compounds of the present invention have the activity of inhibiting ACLY. Further in vitro and in vivo pharmacological activity evaluation showed that the representative compound A3 inhibited the de novo synthesis of lipids in primary hepatocytes, and inhibited the de novo synthesis of lipids in golden hamster and ApoE cells. - / - It was found to improve hyperlipidemia in mice and significantly ameliorate the symptoms of NASH in ob / ob mice and cynomolgus monkeys induced by a high-lipid, high-sugar, high-cholesterol diet. Furthermore, research results showed that compounds with trans double bonds have better in vivo activity than compounds with cis double bonds. Furthermore, at equivalent oral doses, compound A3 demonstrated a plasma exposure level more than four times higher than that of bempedoic acid, indicating that compound A3 has a better therapeutic effect.

[0122] As described above, the compounds of the present application have excellent ACLY inhibitory activity and show good therapeutic effects in various metabolic disease models. They can be further developed as therapeutic agents for metabolic diseases such as hypercholesterolemia and non-alcoholic steatohepatitis.

Claims

1. Use of a compound represented by general formula (I), or a stereoisomer, enantiomer, diastereomer, racemate or pharmaceutically acceptable salt thereof, for the manufacture of a drug for treating a metabolic disease or a drug for treating atherosclerosis. wherein the metabolic disease is selected from the group consisting of hypercholesterolemia, non-alcoholic steatohepatitis (NASH), and liver fibrosis; The above use. 【Chemistry 1】 (however, 【Chemistry 2】 represents a double bond or a single bond. m and n each independently represent 1, 2, 3, 4, 5, or 6; R １ and R2 are each independently H or a C1-C4 alkyl group. Or, R １ and R ２ together with the carbon atom adjacent thereto form a C3-C7 cycloalkyl group. R 3 , R 4 , R 5 and R 6 are each independently H or C1-C4 alkyl. Y １ and Y ２ are each independently —COOH, —COCoA, and 【Transformation 3】 The structure of CoA is as follows: 【Chemistry 4】 。）

2. The use according to claim 1, characterized in that the compound has a structure represented by general formula II: 【Transformation 5】 During the ceremony, 【Transformation 6】 represents a double bond, m and n are each independently 1, 2, 3, 4, 5, or 6; R 1 and R 2 are each independently H or C1-C4 alkyl; R 3 , R 4 , R 5 and R 6 are each independently H or C1-C4 alkyl; R 7 is —OH, —CoA, or 【Transformation 7】 and - CoA is 【Transformation 8】 is.

3. The use according to claim 1, characterized in that the compound has a structure represented by general formula III: 【Chemistry 9】 During the ceremony, m and n are each independently 1, 2, 3, 4, 5, or 6; R 3 , R 4 , R 5 and R 6 are each independently H or C1-C4 alkyl; R 7 is —OH, —CoA, or 【Chemistry 10】 and - CoA is 【Chemistry 11】 is.

4. The use according to any one of claims 1 to 3, characterized in that the compound is a deuterated compound.

5. Use of a compound, or a stereoisomer, enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt thereof, comprising: Used in the manufacture of a drug for treating metabolic diseases or for the manufacture of a drug for treating atherosclerosis, wherein the metabolic disease is selected from the group consisting of hypercholesterolemia, nonalcoholic steatohepatitis (NASH), and liver fibrosis; The compound 【Chemistry 12】 selected from the group consisting of The above use.

6. A compound represented by general formula (I), or a stereoisomer, enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt thereof: 【Chemistry 13】 (however, 【Chemistry 14】 represents a double bond. m and n each independently represent 1, 2, 3, 4, 5, or 6; R １ and R2 are each independently H or a C1-C4 alkyl group. R 3 , R 4 , R 5 , and R 6 are each independently a C1-C4 alkyl group. Y 1 is —COOH; Y2 is -COCoA or 【Chemistry 15】 The structure of CoA is as follows: 【Chemistry 16】 。）

7. 7. The compound according to claim 6, wherein the compound is a deuterated compound, or a stereoisomer, enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt thereof.

8. A compound selected from the group consisting of the following, or a stereoisomer, enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt thereof: 【Chemistry 17】

9. A pharmaceutical composition comprising the compound of any one of claims 6 to 8, or a stereoisomer, enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof.

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