Application of polysubstituted indoline-2-thione compounds in the preparation and treatment of drugs for non-alcoholic fatty liver disease and diabetes

Abstract

The present invention discloses the use of polysubstituted indoline-2-thione compounds in the preparation and treatment of drugs for non-alcoholic fatty liver disease and diabetes, specifically the use of polysubstituted 3,3-diformate-indoline-2-thione compounds and their derivatives, hydrates and pharmaceutically acceptable salts thereof in the preparation of drugs for preventing and / or treating non-alcoholic fatty liver disease and / or type 2 diabetes. The compounds of the present invention can inhibit the typical lipid synthesis pathway in the liver; reduce the accumulation of lipid droplets in the liver, and / or reduce the level of intracellular triglycerides, reduce liver lipid accumulation, alleviate liver fatty degeneration; increase insulin sensitivity, and improve insulin resistance. The present invention has broad prospects in the development of clinical drugs for the treatment of non-alcoholic fatty liver disease and / or type 2 diabetes as a leading small molecule.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the use of polysubstituted 3,3-diformate-indoline-2-thione compounds and derivatives, hydrates and pharmaceutically acceptable salts thereof in preventing and treating non-alcoholic fatty liver disease and / or type 2 diabetes. Background Art

[0002] Non-alcoholic fatty liver disease is currently a global chronic liver disease with a global incidence rate of up to 25%. The mortality rate of cardiovascular complications and malignant tumors in advanced patients increases, but the only reliable clinical treatment is still liver transplantation. Due to the shortage of donors and high costs, drug treatment is urgently needed in the clinic. However, due to the complex pathogenesis and pathological classification of fatty liver and its close relationship with systemic metabolism, there are still no clinical targeted drugs for non-alcoholic fatty liver disease, and the world is still actively exploring effective clinical targeted drugs. There are still no clinical therapeutic drugs targeting fatty liver in the world for the following reasons: 1. The drugs under development are not targeted enough and have side effects. 2. The drugs under development are not effective enough in clinical use. Summary of the invention

[0003] The present invention proposes for the first time the use of polysubstituted 3,3-dicarboxylate-indoline-2-thione compounds and their derivatives (also named as polysubstituted 2-thioxoindoline-2-thione 3,3-dicarboxylate acid esters and their derivatives) represented by formula (1) in the preparation of drugs for preventing and / or treating non-alcoholic fatty liver disease and / or treating type 2 diabetes. The polysubstituted 3,3-dicarboxylate-indoline-2-thione compounds and their derivatives proposed by the present invention can be used as a lead small molecule to develop clinical drugs for treating non-alcoholic fatty liver disease.

[0004] The present invention first uses the high-content screening technology of automatic imaging analysis to screen a large number of drugs, and finds that the small molecule compound shown in formula (1) can reduce the accumulation of fat droplets in the liver and treat non-alcoholic fatty liver disease. After using the drug in in vitro cell experiments and in vivo animal experiments, it is found that non-alcoholic fatty liver disease is alleviated by detecting triglyceride content and tissue section staining. At the same time, in terms of mechanism, the mechanism of small molecule function is explored by qPCR and western blot and other technologies, that is, it depends on inhibiting the typical lipid synthesis pathway in the liver. At the same time, the present invention needs to find a high efficacy and low toxicity by calculating IC50 and CCK8 and other technologies, which can be used as a potential lead compound, and it is worth further research as a lead compound for the treatment of non-alcoholic fatty liver disease.

[0005] The present invention provides the use of a polysubstituted 3,3-diformate-indole-2-thione compound represented by formula (1), its derivatives, hydrates and pharmaceutically acceptable salts thereof in the preparation of a drug for preventing and / or treating non-alcoholic fatty liver disease and / or treating type 2 diabetes.

[0006] The general structural formula of the polysubstituted 3,3-dicarboxylate-indole-2-thione compounds and their derivatives, hydrates and pharmaceutically acceptable salts thereof is as shown in the following formula (1):

[0007]

[0008] in,

[0009] X is selected from hydrogen, alkyl, ester, acyl, halogen, alkoxy, sulfonamide;

[0010] R 1 Selected from alkyl, benzyl, substituted alkyl;

[0011] R 2 , R 3 Alkyl or benzyl alone.

[0012] Furthermore, the polysubstituted 3,3-dicarboxylate-indole-2-thione compounds and their derivatives, hydrates and pharmaceutically acceptable salts thereof include any one or more of the following formulae (1a)-(1z):

[0013]

[0014]

[0015] Furthermore, the polysubstituted 3,3-diformate-indolin-2-thione compounds and their derivatives, hydrates and pharmaceutically acceptable salts thereof include any one or more of formula (1a), formula (1b), formula (1d), formula (1e), formula (1g), formula (1k), formula (1l), formula (1o), formula (1p), formula (1r), formula (1s), formula (1u), formula (1v), formula (1w), formula (1x) and formula (1y):

[0016]

[0017] Furthermore, the polysubstituted 3,3-diformate-indole-2-thione compounds and their derivatives, hydrates and pharmaceutically acceptable salts thereof are selected from formula (1a) or formula (1k):

[0018]

[0019] In the application of the present invention, the polysubstituted 3,3-diformate-indoline-2-thione compounds and their derivatives, hydrates and pharmaceutically acceptable salts thereof inhibit typical lipid synthesis pathways in the liver; reduce the accumulation of lipid droplets in the liver, and / or reduce the level of intracellular triglycerides, reduce liver lipid accumulation, and alleviate liver fatty degeneration.

[0020] In the application of the present invention, the polysubstituted 3,3-diformate-indoline-2-thione compounds and their derivatives, hydrates and pharmaceutically acceptable salts thereof increase insulin sensitivity and improve insulin resistance.

[0021] The application of the present invention includes the application in the preparation of diagnostic reagents for predicting non-alcoholic fatty liver disease resistance and / or recurrence rate and / or survival period, and / or the preparation of non-alcoholic fatty liver disease prognosis evaluation reagents, and / or the preparation of drugs for treating non-alcoholic fatty liver disease or reducing non-alcoholic fatty liver disease resistance or prolonging survival period.

[0022] The application of the present invention also includes the use in the preparation of diagnostic reagents for predicting the drug resistance and / or recurrence rate and / or survival period of type 2 diabetes, and / or the preparation of reagents for evaluating the prognosis of type 2 diabetes, as well as the use in the preparation of drugs for type 2 diabetes or for reducing the drug resistance of type 2 diabetes or prolonging the survival period.

[0023] The present invention also provides a regulator, which includes the polysubstituted 3,3-dicarboxylate-indoline-2-thione compounds represented by formula (1) as described above and their derivatives, hydrates and pharmaceutically acceptable salts.

[0024] The present invention also provides a medicine / pharmaceutical composition, which comprises the polysubstituted 3,3-dicarboxylate-indoline-2-thione compounds represented by formula (1) as described above and their derivatives, hydrates and pharmaceutically acceptable salts thereof.

[0025] The present invention also provides a detection reagent / kit, which comprises the polysubstituted 3,3-diformate-indoline-2-thione compounds represented by formula (1) as described above and their derivatives, hydrates and pharmaceutically acceptable salts thereof.

[0026] The present invention also proposes the use of the regulator, drug / drug composition, reagent / kit in the preparation of diagnostic reagents for predicting non-alcoholic fatty liver disease resistance and / or recurrence rate and / or survival period, and / or the preparation of non-alcoholic fatty liver disease prognosis evaluation reagents, and / or the preparation of drugs for treating non-alcoholic fatty liver disease or reducing non-alcoholic fatty liver disease resistance or prolonging survival period.

[0027] The present invention also proposes the use of the regulator, drug / drug composition, reagent / kit in the preparation of diagnostic reagents for predicting type 2 diabetes drug resistance and / or recurrence rate and / or survival period, and / or in the preparation of type 2 diabetes prognosis evaluation reagents, as well as in the preparation of drugs for type 2 diabetes or for reducing type 2 diabetes drug resistance or prolonging survival period.

[0028] The structural formula (1) described in the present invention can be reacted with an inorganic acid or an organic acid to obtain a "pharmaceutically acceptable salt". The inorganic acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, aminosulfonic acid and phosphoric acid, and the organic acid includes citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalene disulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid and isethionic acid, etc.

[0029] The derivatives, hydrates and pharmaceutically acceptable salts thereof mentioned in the present invention have comparable application effects to the polysubstituted 3,3-dicarboxylate-indole-2-thione compounds represented by formula (1), and can prevent and / or treat non-alcoholic fatty liver disease and / or treat type 2 diabetes.

[0030] The present invention also provides a method for preventing and / or treating non-alcoholic fatty liver disease in a subject in need thereof, or a method for evaluating the severity of a patient suffering from non-alcoholic fatty liver disease, or a method for predicting the recurrence time of non-alcoholic fatty liver disease, or a method for predicting or evaluating the prognosis of whether the patient is suitable for treatment with a polysubstituted 3,3-dicarboxylate-indolin-2-thione compound represented by formula (1), its derivatives, hydrates, and pharmaceutically acceptable salts thereof, the method comprising: determining the polysubstituted 3,3-dicarboxylate-indolin-2-thione compound represented by formula (1) in a blood or liver tissue sample of the patient or subject; The invention relates to a method for measuring the level of polysubstituted 3,3-diformyl-indoline-2-thione compounds and their derivatives, hydrates and pharmaceutically acceptable salts thereof, wherein the level is used to prevent or treat non-alcoholic fatty liver disease in the subject, or to assess the severity of non-alcoholic fatty liver disease in patients, or to predict whether a patient will develop non-alcoholic fatty liver disease, or to predict or evaluate the prognosis of whether the patient is suitable for treatment with polysubstituted 3,3-diformyl-indoline-2-thione compounds and their derivatives, hydrates and pharmaceutically acceptable salts thereof as shown in formula (1).

[0031] The present invention also provides a method for preventing and / or treating type 2 diabetes in a subject in need thereof, or a method for evaluating the severity of type 2 diabetes in a patient, or a method for predicting the recurrence time of type 2 diabetes, or a method for predicting or prognosticating whether the patient is suitable for treatment with a polysubstituted 3,3-dicarboxylate-indoline-2-thione compound and its derivatives, hydrates and pharmaceutically acceptable salts thereof as shown in formula (1), the method comprising: determining the level of polysubstituted 3,3-dicarboxylate-indoline-2-thione compound and its derivatives, hydrates and pharmaceutically acceptable salts thereof as shown in formula (1) in a blood sample of the patient or subject, wherein the level is used to prevent or treat type 2 diabetes in the subject, or to evaluate the severity of type 2 diabetes in a patient, or to predict whether the patient will develop type 2 diabetes, or to predict or prognosticate whether the patient is suitable for treatment with a polysubstituted 3,3-dicarboxylate-indoline-2-thione compound and its derivatives, hydrates and pharmaceutically acceptable salts thereof as shown in formula (1).

[0032] The polysubstituted 3,3-dicarboxylate-indole-2-thione compounds and their derivatives, hydrates and pharmaceutically acceptable salts thereof represented by formula (1) include TOIDC represented by formula (1a):

[0033]

[0034] The present invention also proposes a drug screening method, which includes inducing the accumulation of lipid droplets in hepatocytes in vitro, treating the cells with different drugs and staining them with Nile red, and using high-content technology to simultaneously automatically image and analyze fluorescence parameters to evaluate the effect of the drug in reducing lipid droplets.

[0035] The beneficial effects of the present invention include: the present invention finds polysubstituted 3,3-diformate-indoline-2-thione compounds and their derivatives, hydrates and pharmaceutically acceptable salts with higher efficacy and lower toxicity through calculation of IC50 and CCK8 and other technologies, which are worthy of further study as lead compounds for the treatment of non-alcoholic fatty liver disease and / or type 2 diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Flowchart of drug screening for high-content technology.

[0037] Figure 2 This is the screening result of the small molecule compound TOIDC in a high-content experiment. It was found that compared with the control, TOIDC can weaken the fluorescence intensity, reflecting that TOIDC can reduce the lipid droplets in primary liver cells.

[0038] Figure 3The results of the Nile red staining experiment with the small molecule compound TOIDC showed that TOIDC reduced the fluorescence intensity of primary liver cells in a concentration gradient-dependent manner, indicating that the lipid droplet content in the cells decreased more as the drug concentration increased.

[0039] Figure 4 The small molecule compound TOIDC reduced the fluorescence intensity of primary liver cells in a concentration-dependent manner in the Nile red staining experiment ( Figure 4 A), calculated using Image J software, it was found that the half inhibition rate of TOIDC in inhibiting cellular lipid droplet accumulation was 14.17 μM ( Figure 4 B).

[0040] Figure 5 The small molecule compound TOIDC reduced the triglyceride content in primary liver cells in a concentration gradient-dependent manner in the triglyceride quantification experiment.

[0041] Figure 6 The small molecule compound TOIDC reduces the fluorescence intensity in the HepG2 cell line in the Nile red staining experiment. The left picture shows only DMSO, the middle picture shows DMSO and OA / PA, and the right picture shows TOIDC and OA / PA. Figure 6 A), and after calculation using Image J software, it was found that TOIDC had a significant effect in inhibiting the accumulation of cellular lipid droplets ( Figure 6 B).

[0042] Figure 7 The small molecule compound TOIDC reduces the triglyceride content in the HepG2 cell line in the triglyceride quantification experiment. The left column shows only DMSO added, the middle column shows DMSO and OA / PA added, and the rightmost column shows TOIDC and OA / PA added.

[0043] Figure 8 In order to verify in the qPCR primary liver cell experiment that the small molecule compound TOIDC reduces the gene transcription of SREBP1 and reduces the transcription level of downstream key enzymes of lipid synthesis, in the bar graph of each group, the left side is DMSO, the middle is OA / PA+DMSO, and the right side is OA / PA+TOIDC.

[0044] Fig. 9 In order to verify in the WB liver primary cell experiment that the small molecule compound TOIDC reduces the protein levels of SREBP1 and downstream key enzymes of lipid synthesis, inhibits the lipid synthesis pathway in the liver and relieves fatty liver, Fig. 9 A is the concentration of protein developed by WB band; Fig. 9 B is the quantitative results of grayscale value analysis of wb bands by Image J, in which the bar graph of each group shows DMSO on the left, OA / PA+DMSO in the middle, and OA / PA+TOIDC on the right.

[0045] Fig.10 In the qPCR primary liver cell experiment, the levels of SREBP1 and downstream lipid synthesis pathway genes decreased after TOIDC was administered. After the SREBP1-activating compound T0901317 was added, the levels of SREBP1 and downstream synthesis pathway genes recovered, proving that the small molecule compound TOIDC inhibits the lipid synthesis pathway in the liver and relieves fatty liver by reducing the protein levels of SREBP1 and downstream key enzymes of lipid synthesis. In the bar graph of each group, from left to right are DMSO, OA / PA+DMSO, OA / PA+TOIDC, OA / PA+TOIDC+T0901317.

[0046] Fig.11 In the WB liver primary cell experiment, the levels of SREBP1 and the downstream lipid synthesis pathway protein FASN decreased after TOIDC was administered. After adding drugs that activate SREBP1, the levels of SREBP1 and the downstream synthesis pathway protein FASN recovered, proving that the small molecule compound TOIDC inhibits the lipid synthesis pathway in the liver and relieves fatty liver by reducing the protein levels of SREBP1 and downstream key enzymes of lipid synthesis. Fig.11 A is the concentration of protein developed by WB band; Fig.11 B is the quantitative results of grayscale value analysis of wb bands by Image J, in which the bar graphs of each group are DMSO, OA / PA+DMSO, OA / PA+TOIDC, and OA / PA+TOIDC+T0901317 from left to right.

[0047] Fig.12 In the Nile red staining experiment of primary liver cells, the fluorescence of Nile red weakened after administration of TOIDC, and enhanced after the addition of T0901317, a compound that activates SREBP1 transcription, proving that the small molecule compound TOIDC inhibits the lipid synthesis pathway in the liver and relieves fatty liver by reducing the protein levels of SREBP1 and downstream key enzymes of lipid synthesis.

[0048] Fig.13 In the qPCR HepG2 cell experiment, it was verified that the small molecule compound TOIDC reduced the gene transcription of SREBP1 and reduced the transcription level of downstream key enzymes of lipid synthesis. In the bar graph of each group, the left side is DMSO, the middle is OA / PA+DMSO, and the right side is OA / PA+TOIDC.

[0049] Fig.14In the qPCR HepG2 cell experiment, the levels of SREBP1 and downstream lipid synthesis pathway genes decreased after TOIDC was administered. After the SREBP1-activating compound T0901317 was added, the levels of SREBP1 and downstream synthesis pathway genes recovered, proving that the small molecule compound TOIDC inhibits the lipid synthesis pathway in the liver and relieves fatty liver by reducing the expression levels of SREBP1 and downstream key enzymes of lipid synthesis. In the bar graph of each group, from left to right are DMSO, OA / PA+DMSO, OA / PA+TOIDC, OA / PA+TOIDC+T0901317.

[0050] Fig.15 In order to verify in the WB HepG2 cell experiment that the small molecule compound TOIDC reduces the protein levels of SREBP1 and downstream key enzymes of lipid synthesis, thereby inhibiting the lipid synthesis pathway in the liver and alleviating fatty liver, Fig.15 A is the concentration of protein developed by WB band; Fig.15 B is the quantitative results of grayscale value of wb bands analyzed by Image J, in which the bar graphs of each group are DMSO, OA / PA+DMSO, and OA / PA+TOIDC from left to right.

[0051] Fig.16 In the WB HepG2 cell experiment, after TOIDC was administered, the levels of SREBP1 and the downstream lipid synthesis pathway protein FASN decreased. After adding drugs that activate SREBP1, the levels of SREBP1 and the downstream synthesis pathway protein FASN recovered, proving that the small molecule compound TOIDC inhibits the lipid synthesis pathway in the liver and relieves fatty liver by reducing the protein levels of SREBP1 and downstream key enzymes of lipid synthesis. Fig.16 A is the concentration of protein developed by WB band; Fig.16 B is the quantitative results of grayscale value analysis of wb bands by Image J, in which the bar graphs of each group are DMSO, OA / PA+DMSO, OA / PA+TOIDC, and OA / PA+TOIDC+T0901317 from left to right.

[0052] Fig.17 In animal experiments, after three weeks of the experiment, the average liver weight of mice in the control group (intraperitoneal injection of the same concentration of DMSO) was 1.78g ( Fig.17 A left figure), the average liver weight of mice in the drug group (intraperitoneal injection of the same concentration of TOIDC) was 1.43 g ( Fig.17 A (right picture), Fig.17 B is the corresponding bar graph, which shows that the small molecule compound TOIDC reduces the liver weight of high-fat fed mice, reflecting that TOIDC can improve fatty liver in vivo.

[0053] Fig.18 In the animal experiment, after three weeks of the experiment, the average lipid content in the liver of the mice in the control group was 506.9umol / g, and the average lipid content in the liver of the mice in the drug-treated group was 354.1umol / g, indicating that the small molecule compound TOIDC reduced the lipid content in the liver of high-fat fed mice, reflecting that TOIDC can improve fatty liver in vivo.

[0054] Fig.19 Nile red staining and H&E staining of tissue sections showed that compared with the control group, the lipid droplet fluorescence parameters of the mice in the drug-treated group were reduced, and the fat vacuoles in the tissue sections were reduced, verifying that the small molecule compound TOIDC can improve the fatty degeneration of liver tissue and reduce liver lipid accumulation in high-fat-fed mice, reflecting that TOIDC can improve fatty liver in vivo.

[0055] Fig. 20 In the animal experiment, the body weights of the two groups of mice were tested within three weeks of the experiment, and it was found that the average body weight of the mice in the drug-treated group was 36.0 g, which was significantly lower than the average body weight of the mice in the control group, 40.2 g, indicating that TOIDC can reduce the body weight of DIO mice (C57B6 mice).

[0056] Fig.21 In the animal experiment, the mice were killed three weeks after the experiment. It can be seen that the mice in the drug group were significantly smaller than those in the control group ( Fig.21 A), the fat tissue was taken and the fat tissue volume of the mice in the drug-treated group was significantly ( Fig.21 B right) was significantly smaller than that of the control group mice ( Fig.21 B (left side), reflecting that TOIDC can reduce the body weight of DIO mice (C57B6 mice).

[0057] Fig. 22 In order to verify that the small molecule compound TOIDC reduces the gene transcription of SREBP1 and the transcription level of downstream key enzymes of lipid synthesis in the qPCR experiment of animal tissues, in each group of bar graphs, the left side is Vehicle and the right side is TOIDC.

[0058] Fig.23 In order to verify in the WB experiment of animal tissue that the small molecule compound TOIDC reduces the protein levels of SREBP1 and downstream key enzymes of lipid synthesis, thereby inhibiting the lipid synthesis pathway in the liver and alleviating fatty liver.

[0059] Fig.24 In primary liver cells ( Fig.24 A) and HepG2( Fig.24 B) In the toxicity test, primary liver cells were treated with different concentrations of TOIDC, and it was found that there was no difference in cell activity before the concentration reached 60 μM, indicating that the small molecule TOIDC did not affect cell viability at the working concentration.

[0060] Fig.25 In animal experiments, the average serum ALT aminotransferase levels of high-fat mice three weeks after intraperitoneal injection of TOIDC and high-fat mice three weeks after injection of solvent DMSO were 172.3U / L and 179.1U / L, respectively, and the average serum aminotransferase AST levels were 33.8U / L and 32.7U / L, respectively, indicating that intraperitoneal injection of TOIDC has no liver-damaging toxicity to mice.

[0061] Fig.26 In the insulin tolerance experiment, 2 hours after insulin injection, the blood glucose recovery of the mice in the experimental group was slower than that in the control group, reflecting that TOIDC can improve the sensitivity of high-fat mice to insulin. The small molecule compound TOIDC can improve the insulin resistance of high-fat mice. Fig.26 A is the insulin tolerance test curve; Fig.26 B is glucose.

[0062] Fig. 27 This is the overall mechanism diagram of the small molecule compound TOIDC in alleviating fatty liver. After entering the liver cells, the small molecule compound TOIDC inhibits the transcription of SREBP1, thereby reducing the protein levels of SREBP1 and downstream key enzymes of lipid synthesis such as FASN, thereby inhibiting the liver lipid synthesis pathway to alleviate fatty liver. DETAILED DESCRIPTION

[0063] The present invention is further described in detail in conjunction with the following specific examples and drawings, and the protection content of the present invention is not limited to the following examples. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims are the scope of protection. The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art, and the present invention does not specifically limit the content.

[0064] Example 1 Preparation of Compounds 1a-1z

[0065]

[0066] The synthesis process of compound 1a-1z is as follows: N-aryl-N-alkylthioamide (0.2mmol), cobalt bromide (0.02mmol), DBU (0.4mmol) and Na2S2O8 (0.4mmol) as shown in the above formula 1-0 are added to a flask, and then anhydrous acetonitrile CH3CN (4mL) is added, and the obtained mixture is stirred at 50°C (oil bath temperature) for 1-2 hours until the reaction of N-aryl-N-alkylthioamide shown in the raw material formula 1-0 is complete by TLC detection. Then the reaction mixture is filtered to remove the solid, the filtrate is concentrated under reduced pressure, and the residue is separated by column chromatography using a mixed solvent of petroleum ether / ethyl acetate to obtain a 3,3-dicarboxylate-indoline-2-thione compound as shown in the above formula 1. [1]

[0067] [1] Zhi Yao, Zhongliang Cai, Long Zhen, Liqin Jiang. *Org. Lett. 2020, 22, 4505-4510.

[0068]

[0069] 1-Methyl-3,3-dicarboxylic acid dimethyl ester-indole-2-thione (1a): Mp: 139-140℃. 1 H NMR (500MHz, CDCl3) δ7.53(d,J=7.4Hz,1H),7.44(td,J=7.8,1.0Hz,1H),7.22(t,J=7.6Hz,1H),7.00(d,J=7.9Hz,1H),3.79(s,6H),3.63(s,3H). 13 C NMR(125MHz, CDCl3)δ193.7,165.2,145.9,130.2,128.0,125.7,124.8,109.8,75.1,53.9,31.8.HRMS(EI-TOF)m / z:[M]+calculated for C13H13NO4S 279.0565, found 279.0567.

[0070]

[0071] 1-Methyl-3,3-dicarboxylic acid dimethyl ester-5-methoxy-indole-2-thione (1b): Mp: 135-136℃. 1HNMR (600MHz, CDCl3) δ7.34(s,1H),7.23(d,J=8.0Hz,1H),6.90(d,J=8.1Hz,1H),3.79(s,6H),3.61(s,3H),2.38(s,3H). 13 C NMR(150MHz, CDCl3)δ193.2,165.4,143.6,134.9,130.7,128.0,126.3,109.6,75.1,53.9,31.9,21.2.HRMS(EI-TOF)m / z:[M]+ calculated forC14H15NO5S 309.0671, found 309.0674.

[0072]

[0073] 1,5-Dimethyl-3,3-dicarboxylic acid dimethyl ester-indole-2-thione (1c): Mp: 134-135℃. 1 H NMR (600MHz, CDCl3) δ7.34(s,1H),7.23(d,J=8.0Hz,1H),6.90(d,J=8.1Hz,1H),3.79(s,6H),3.61(s,3H),2.38(s,3H). 13 C NMR(150MHz, CDCl3)δ193.2,165.4,143.6,134.9,130.7,128.0,126.3,109.6,75.1,53.9,31.9,21.2.HRMS(EI-TOF)m / z:[M]+ calculated forC14H15NO4S 293.0722, found 293.0720.

[0074]

[0075] 1-Methyl-3,3-dicarboxylic acid dimethyl ester-5-(N-methyl-p-toluenesulfonylamino)-indole-2-thione (1d): 1 H NMR (600MHz, CDCl3) δ7.33(d,J=7.8Hz,2H),7.21(d,J=9.8Hz,1H),7.18(d,J=7.8Hz,2H) ,7.09(s,1H),6.87(d,J=8.4Hz,1H),3.70(s,6H),3.54(s,3H),3.08(s,3H),2.35(s,3H). 13C NMR (150MHz, CDCl3) δ193.7,164.8,144.8,143.8,138.5,133.0,129.6,129.5,128.1, 127.9,123.7,109.7,74.9,54.0,38.2,31.9,21.6.HRMS(EI-TOF)m / z:[M]+calculated for C21H22N2O6S2 462.0919,found 462.0915.

[0076]

[0077] 1-Methyl-3,3-dicarboxylic acid dimethyl ester-5-fluoro-indoline-2-thione (1e): 1 H NMR (600MHz, CDCl3) δ7.29(dd,J=7.6,2.6Hz,1H),7.16(td,J=8.7,2.6Hz,1H),6.95(dd,J=8.7,4.0Hz,1H),3.81(s,6H),3.62(s,3H). 13 C NMR (151MHz, CDCl3) δ193.11, 164.75, 161.20, 159.58, 142.00, 129.23 (d, J = 9.2Hz), 116.93 ( d,J=24.0Hz),114.01(d,J=26.0Hz),110.39(d,J=8.6Hz),75.02,54.08,31.96.HRMS(EI)m / z calculated for C 13 H 12 FNO4S[M] + 297.0471, found 297.0469.

[0078]

[0079] 1-Methyl-3,3-dicarboxylic acid dimethyl ester-5-chloro-indole-2-thione (1f): 1 H NMR (600MHz, CDCl3) δ7.51(d,J=1.8Hz,1H),7.41(dd,J=8.4,1.9Hz,1H),6.93(d,J=8.4Hz,1H),3.82(s,6H),3.61(s,3H). 13 C NMR (151MHz, CDCl3) δ193.22,164.71,144.47,130.50,130.33,129.16,126.20,110.58,74.89,54.12,31.89.

[0080]

[0081] 1-Methyl-3,3-dicarboxylic acid dimethyl ester-5-bromo-indolin-2-thione (1g): 1 H NMR (600MHz, CDCl3) δ7.65(d,J=1.8Hz,1H),7.56(dd,J=8.4,1.9Hz,1H),6.88(d,J=8.4Hz,1H),3.81(s,6H),3.60(s,3H). 13 C NMR(151MHz, CDCl3)δ193.16,164.71,144.95,133.22,129.45,128.91,117.85,111.02,74.86,54.13,31.86.HRMS(EI)m / z calculated for C 13 H 12 BrNO4S[M] + 356.9670, found 356.9668.

[0082]

[0083] 1-Methyl-3,3-dicarboxylic acid dimethyl ester-5-iodo-indole-2-thione (1h): 1 H NMR (600MHz, CDCl3) δ7.74(d,J=1.4Hz,1H),7.68(dd,J=8.3,1.5Hz,1H),6.69(d,J=8.3Hz,1H),3.74(s,6H),3.52(s,3H). 13 C NMR(151MHz, CDCl3)δ193.15,164.72,145.65,139.12,134.41,129.73,111.44,87.87,74.79,54.06,31.74.HRMS(EI)m / z calculated for C 13 H 12 INO4S[M] + 404.9532, found 404.9534.

[0084]

[0085] 1-Methyl-3,3-dicarboxylic acid dimethyl ester-5-carboxylic acid ethyl ester-indole-2-thione (1i): 1H NMR (600MHz, CDCl3) δ8.10(dd,J=6.0,1.6Hz,2H),6.97(d,J=8.8Hz,1H),4.31(q,J=7.1Hz,2H),3.74(s,6H),3.58(s,3H),1.33(t,J=7.1Hz,3H). 13 C NMR (151MHz, CDCl3) δ194.75,165.74,164.80,149.31,132.59,127.83,127.06,126.83,109.39,74.80,61.33,54.09,31.94,14.39.

[0086] HRMS(EI)m / z calculated for C 16 H 17 NO6S[M] + 351.0777, found 351.0779.

[0087]

[0088] 1-Methyl-3,3-dicarboxylic acid dimethyl ester-5-acetyl-indole-2-thione (1j): 1 H NMR (600MHz, CDCl3) δ8.04(s,1H),8.02(d,J=8.4Hz,1H),7.00(d,J=8.3Hz,1H),3.74(s,6H),3.58(s,3H),2.54(s,3H). 13 C NMR (151MHz, CDCl3) δ196.38,194.85,164.77,149.44,133.82,131.60,128.10,125.71,109.49,74.80,54.13,31.95,26.66.HRMS(EI) m / z calculated for C 15 H 15 NO5S[M] + 321.0671, found 321.0675.

[0089]

[0090] 1-Methyl-3,3-dicarboxylic acid dimethyl ester-7-methoxy-indole-2-thione (1k): 1H NMR (600MHz, CDCl3) δ7.11–7.04(m,2H),6.91(d,J=8.0Hz,1H),3.87(s,3H),3.84(s,3H),3.71(s,6H). 13 C NMR(151MHz, CDCl3)δ193.18,165.37,145.02,133.86,129.55,125.68,117.97,113.72,75.31,56.08,53.87,35.73.HRMS(EI)m / z calculated for C 14 H 15 NO5S[M] + 309.0671, found 309.0668.

[0091]

[0092] 1-7-Dimethyl-3,3-dicarboxylic acid dimethyl ester-indole-2-thione (1l): 1 H NMR (600MHz, CDCl3) δ7.29 (d, J = 7.4Hz, 1H), 7.09 (d, J = 7.6Hz, 1H), 7.02 (t, J = 7.5Hz, 1H), 3.88 (s, 3H), 3.71 (s, 6H), 2.58 (s, 3H). 13 C NMR(151MHz, CDCl3)δ194.46,165.51,143.91,134.26,128.47,124.73,123.59,120.97,74.91,53.86,35.65,19.80.HRMS(EI)m / z calculated forC 14 H 15 NO4S[M] + 293.0722, found 293.0723.

[0093]

[0094] 1-Methyl-3,3-dicarboxylic acid dimethyl ester-6-methoxy-indole-2-thione (1m): 1 H NMR (600MHz, CDCl3) δ7.34(d,J=8.3Hz,1H),6.64(d,J=8.3Hz,1H),6.48(s,1H),3.79(s,3H),3.72(s,6H),3.53(s,3H). 13C NMR(151MHz, CDCl3)δ194.66,165.54,161.70,147.15,126.26,119.71,109.02,97.39,74.52,55.80,53.84,31.83.HRMS(EI)m / z calculated forC 14 H 15 NO5S[M] + 309.0671, found 309.0674.

[0095]

[0096] 1-Methyl-3,3-dicarboxylic acid dimethyl ester 6-chloro-indolin-2-thione (1n): 1 H NMR (500MHz, CDCl3) δ7.37(d,J=8.1Hz,1H),7.12(dd,J=8.1,1.7Hz,1H),6.93(d,J=1.7Hz,1H),3.73(s,6H),3.53(s,3H). 13 C NMR(126MHz, CDCl3)δ194.13,164.85,146.93,136.32,126.54,126.10,124.62,110.47,74.67,54.06,31.85.HRMS(EI)m / z calculated for C 13 H 12 ClNO4S[M] + 313.0176, found 313.0181.

[0097]

[0098] 1-Methyl-3,3-dicarboxylic acid dimethyl ester-4,6-dimethoxy-indole-2-thione (1n): 1 H NMR (500MHz, CDCl3) δ6.21(d,J=1.8Hz,1H),6.15(d,J=1.9Hz,1H),3.79(s,3H),3.75(s,3H),3.66(s,6H),3.50(s,3H). 13 C NMR(126MHz, CDCl3)δ195.57,164.56,163.06,156.64,148.18,107.82,94.45,89.63,73.44,55.92,55.82,53.48,32.07.HRMS(EI)m / zcalculated for C 15 H17 NO6S[M] + 339.0777, found 339.0780.

[0099]

[0100] 1-Methyl-3,3-dicarboxylic acid diethyl ester-indole-2-thione (1p): 1 H NMR (600MHz, CDCl3) δ7.47(d,J=7.5Hz,1H),7.35(t,J=7.8Hz,1H),7.14(t,J=7.6Hz, 1H), 6.93 (d, J = 7.9Hz, 1H), 4.19 (q, J = 7.1Hz, 4H), 3.54 (s, 3H), 1.19 (t, J = 7.1Hz, 6H). 13 C NMR(151MHz, CDCl3)δ193.84,164.75,145.86,130.10,128.15,125.69,124.65,109.81,75.22,62.91,31.76,13.89.HRMS(EI)m / z calculated for C 15 H 17 NO4S[M] + 307.0878, found 307.0875.

[0101]

[0102] 1-Methyl-3,3-diisopropyl-indole-2-thione (1q): 1 H NMR (600MHz, CDCl3) δ7.55(d,J=7.5Hz,1H),7.42(t,J=7.8Hz,1H),7.21(t,J=7.6Hz,1H ),6.99(d,J=7.9Hz,1H),5.16–5.08(m,2H),3.61(s,3H),1.28(dd,J=14.0,6.3Hz,12H). 13 C NMR(151MHz, CDCl3)δ193.94,164.22,145.86,129.97,128.32,125.71,124.49,109.74,75.22,70.65,31.70,21.42,21.40.HRMS(EI)m / z calculated for C 17 H 21 NO4S[M] + 335.1191, found 335.1189.

[0103]

[0104] 1-Methyl-3,3-dicarboxylic acid dibenzyl-indole-2-thione (1r): 1 H NMR(600MHz, CDCl3)δ7.35(d,J=7.5Hz,1H),7.31(t,J=7.8Hz,1H),7.23–7.19(m,6H),7.18 –7.14(m,4H),7.06(t,J=7.6Hz,1H),6.87(d,J=7.9Hz,1H),5.15–5.09(m,4H),3.50(s,3H). 13 C NMR (151MHz, CDCl3) δ193.30,164.50,145.94,134.91,130.26,128.51,128.3 4,128.00,127.75,125.78,124.66,109.88,75.20,68.42,31.79.HRMS(EI)m / z calculated for C 25 H 21 NO4S[M] + 431.1191, found 431.1193.

[0105]

[0106] 1-Isopropyl-3,3-dimethyldicarboxylate-indole-2-thione (1s): 1 H NMR (600MHz, CDCl3) δ7.45 (d, J = 7.5Hz, 1H), 7.32 (t, J = 7.8Hz, 1H), 7.21 (d, J = 8. 0Hz,1H),7.12(t,J=7.5Hz,1H),5.63(s,1H),3.71(s,6H),1.48(d,J=7.1Hz,6H). 13 C NMR (151MHz, CDCl3) δ193.41,165.38,143.96,129.80,128.46,125.77,124.29,111.75,75.25,53.85,48.78,18.51.

[0107]

[0108] 1-n-Butyl-3,3-dimethyl dicarboxylate-indole-2-thione (1t): 1H NMR (600MHz, CDCl3) δ7.45(d,J=7.5Hz,1H),7.35(t,J=7.7Hz,1H),7.13(t,J=7.5Hz,1H),6.93(d,J=7.9Hz,1H),4.10(t ,J=7.5Hz,2H),3.71(s,6H),1.74–1.66(m,2H),1.33(dd,J=13.7,6.6Hz,2H),1.30–1.20(m,4H),0.81(t,J=6.3Hz,3H). 13 C NMR (151MHz, CDCl3) δ193.24,165.30,145.39,130.17,128.18,125.66,124.60 ,110.03,75.23,53.84,44.91,31.45,26.49,26.00,22.54,14.00.HRMS(EI)m / z calculated for C 18 H 23 NO4S[M] + 349.1348, found 349.1351.

[0109]

[0110] 1-(3-Methoxypropyl)-3,3-dicarboxylic acid dimethyl ester-indole-2-thione (1u): 1 H NMR (500MHz, CDCl3) δ7.45 (dd, J=7.5, 0.5Hz, 1H), 7.36 (td, J=7.9, 1.1Hz, 1H), 7.13 (td, J=7.6, 0.7Hz, 1H), 7. 04(d,J=8.0Hz,1H),4.21(t,J=7.0Hz,2H),3.72(s,6H),3.32(t,J=5.8Hz,2H),3.25(s,3H),2.04–1.97(m,2H). 13 C NMR(126MHz, CDCl3)δ193.36,165.29,145.66,130.23,128.00,125.59,124.65,110.18,75.20,69.19,58.61,53.88,42.04,26.27.HRMS(EI)m / z calculated for C 16 H 19 NO5S[M] + 337.0984, found 337.0981.

[0111]

[0112] 1-Benzyl-3,3-dimethyldicarboxylate-indole-2-thione (1v): 1 H NMR (600MHz, CDCl3) δ7.53(d,J=7.5Hz,1H),7.33–7.24(m,6H),7.17(t,J=7.6Hz,1H),6.84(d,J=8.0Hz,1H),5.46(s,2H),3.81(s,6H). 13 C NMR (151MHz, CDCl3) δ194.60,165.27,145.15,133.97,130.22,128.95,127.9 7,127.88,126.91,125.53,124.81,110.79,75.34,53.94,48.15.HRMS(EI)m / z calculated for C 19 H 17 NO4S[M] + 355.0878, found 355.0876.

[0113]

[0114] 1-Benzyl-3,3-dimethyldicarboxylate-5-methoxy-indole-2-thione (1w): 1 H NMR (600MHz, CDCl3) δ7.25–7.16(m,5H),7.04(d,J=2.5Hz,1H),6.73(dd,J=8 .7,2.5Hz,1H),6.65(d,J=8.7Hz,1H),5.35(s,2H),3.74(s,6H),3.69(s,3H). 13 C NMR (151MHz, CDCl3) δ193.41,165.25,157.60,138.65,134.00,129.22,128 .93,127.87,126.92,115.08,112.16,111.25,75.37,55.88,53.95,48.27.

[0115]

[0116] 1-Benzyl-3,3-dimethyldicarboxylate-5-chloro-indoline-2-thione (1x): 1H NMR (600MHz, CDCl3) δ7.44(d,J=2.0Hz,1H),7.23(t,J=7.2Hz,2H),7.21–7.16(m,4H),6.67(d,J=8.5Hz,1H),5.35(s,2H),3.76(s,6H). 13 C NMR (151MHz, CDCl3) δ194.12,164.74,143.72,133.59,130.54,130.29,129.2 2,129.03,128.04,126.85,126.05,111.50,75.10,54.16,48.20.HRMS(EI)m / z calculated for C 13 H 12 ClNO4S[M] + 313.0176, found 313.0172.

[0117]

[0118] 1-Benzyl-3,3-dimethyldicarboxylate-5-bromo-indole-2-thione (1y): 1 H NMR (500MHz, CDCl3) δ7.57(d,J=1.9Hz,1H),7.33(dd,J=8.4,1.9Hz,1H),7.25–7.15(m,5H),6.62(d,J=8.5Hz,1H),5.34(s,2H),3.76(s,6H). 13 C NMR (126MHz, CDCl3) δ194.03,164.73,144.18,133.55,133.16,129.50,129.0 3,128.76,128.03,126.83,117.91,111.93,75.05,54.17,48.15.HRMS(EI)m / z calculated for C 19 H 16 BrNO4S[M] + 432.9983, found 432.9980.

[0119]

[0120] 1-Benzyl-3,3-dimethyl-dicarboxylate-5-ethyl-carboxylate-indole-2-thione (1z): 1H NMR(600MHz, CDCl3)δ8.11(s,1H),7.96(d,J=8.3Hz,1H),7.26–7.22(m,2H),7.21–7.17(m,3H) ,6.80(d,J=8.4Hz,1H),5.40(s,2H),4.28(q,J=7.1Hz,2H),3.77(s,6H),1.30(t,J=7.1Hz,3H). 13 C NMR (151MHz, CDCl3) δ195.57,165.70,164.83,148.61,133.58,132.50,129.03,128.04,1 27.88,127.10,126.82,126.71,110.27,75.01,61.30,54.12,48.19,14.37.HRMS(EI)m / z calculated for C 22 H 21 NO6S[M] + 427.1090, found 427.1094.

[0121] Example 2 Screening of compounds

[0122] The present invention first takes primary liver cells from 6-8 week old C57B6 mice, and after plating at an appropriate density, uses a 0.3 μM mixture of palmitic acid and oleic acid to induce and stimulate the aggregation of lipid droplets in the cells. After the lipid droplets are aggregated, fresh culture medium mixed with small molecule compounds is added to the cells respectively, and the culture medium is removed after culturing for 36 hours. The cells are rinsed with PBS, and after fixation with a fixative, the cells are incubated with a Nile red solution for 15 minutes, then the Nile red solution is removed, and the cells are rinsed with PBS three times and then incubated with a DAPI solution for 5 minutes, and then the solution is removed, and the cells are rinsed three times with PBS, leaving part of the PBS on the cell surface to cover the cells. The cell well plate is placed in a high content instrument, the appropriate parameters are adjusted, and the instrument is waited for to automatically image and take pictures of the wells continuously. Figure 2 After the high-content instrument takes the photo, the parameters are set. After the instrument automatically analyzes the fluorescence parameters, the multi-substituted 3,3-dicarboxylate-indole-2-thione compounds and their derivatives (as shown in Table 1 below) as shown in the above formula (1) with better Nile red fluorescence attenuation effect are screened out compared with the control group. Among them, the small molecule compounds suitable for the present invention include but are not limited to TOIDC shown in formula (1a) and the compound shown in formula (1k).

[0123] Table 1

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130] Example 3 In vitro cell experiment

[0131] In the in vitro cell experiment of the present invention, a mixture of palmitic acid and oleic acid was used to stimulate the accumulation of lipid droplets in liver cells, and then the cells were treated with increasing gradient concentrations of the small molecule drug TOIDC screened in Example 2 of the present invention. Nile red staining was used to find that the accumulated lipid droplets in the cells were gradually reduced. Figure 3 .according to Figure 2 Image J software was used to calculate the fluorescence parameters of Nile red staining after cells were treated with different concentrations of TOIDC. The calculation showed that the half inhibition rate (IC50) of TOIDC in reducing lipid droplet accumulation was 14.17 μM. Figure 4 B. The present invention also used a triglyceride detection kit to find that the level of triglyceride accumulated in primary liver cells treated with TOIDC decreased as the concentration increased. Figure 5 .

[0132] Then, the present invention repeated the experiment in vitro using another cell line HepG2 cell line, and found that the small molecule compound TOIDC also reduced the fluorescence intensity in the HepG2 cell line in the Nile red staining experiment. The left picture shows only DMSO, the middle picture shows DMSO and OA / PA, and the right picture shows TOIDC and OA / PA. After calculation using Image J software, it was found that TOIDC had a significant effect in inhibiting the accumulation of cellular lipid droplets (see Figure 6 ), and the small molecule compound TOIDC also significantly reduced the triglyceride content in the HepG2 cell line in the triglyceride quantification experiment. The left column is only DMSO, the middle column is DMSO and OA / PA, and the rightmost column is TOIDC and OA / PA (see Figure 7 ).

[0133] In the qPCR primary liver cell experiment, the present invention verifies that the small molecule compound TOIDC reduces the gene transcription of SREBP1 and reduces the transcription level of the downstream key enzymes of lipid synthesis. In each group of bar graphs, the left side is DMSO, the middle is OA / PA+DMSO, and the right side is OA / PA+TOIDC (see Figure 8 ).

[0134] In the WB liver primary cell experiment, the present invention verifies that the small molecule compound TOIDC reduces the protein level of SREBP1 and downstream key enzymes of lipid synthesis and inhibits the lipid synthesis pathway in the liver to relieve fatty liver (see Fig. 9 ).

[0135] In the qPCR primary liver cell experiment of the present invention, after TOIDC was administered, the levels of SREBP1 and downstream lipid synthesis pathway genes decreased. After adding the SREBP1-activating compound T0901317, the levels of SREBP1 and downstream synthesis pathway genes recovered, proving that the small molecule compound TOIDC inhibits the lipid synthesis pathway in the liver and relieves fatty liver by reducing the protein levels of SREBP1 and downstream key enzymes of lipid synthesis (see Fig.10 ).

[0136] In the WB liver primary cell experiment of the present invention, after administration of TOIDC, the levels of SREBP1 and downstream lipid synthesis pathway protein FASN decreased. After adding drugs that activate SREBP1, the levels of SREBP1 and downstream synthesis pathway protein FASN recovered, proving that the small molecule compound TOIDC inhibits the lipid synthesis pathway in the liver and relieves fatty liver by reducing the protein levels of SREBP1 and downstream key enzymes of lipid synthesis (see Fig.11 ).

[0137] In the Nile red staining experiment of primary liver cells of the present invention, the fluorescence of Nile red weakened after TOIDC was administered, and the fluorescence of Nile red increased after the addition of T0901317, a compound that activates SREBP1 transcription, which proves that the small molecule compound TOIDC inhibits the lipid synthesis pathway in the liver and relieves fatty liver by reducing the protein levels of SREBP1 and downstream key enzymes of lipid synthesis (see Fig.12 ).

[0138] Similarly, in another cell line qPCR HepG2 cell experiment, it was verified that the small molecule compound TOIDC reduced the gene transcription of SREBP1 and reduced the transcription level of downstream key enzymes of lipid synthesis. In each group of bar graphs, the left side is DMSO, the middle is OA / PA+DMSO, and the right side is OA / PA+TOIDC (see Fig.13 In the WB HepG2 cell experiment, it was verified that the small molecule compound TOIDC reduced the protein levels of SREBP1 and downstream key enzymes of lipid synthesis, thereby inhibiting the lipid synthesis pathway in the liver and alleviating fatty liver (see Fig.14In the qPCR HepG2 cell experiment, the levels of SREBP1 and downstream lipid synthesis pathway genes decreased after TOIDC was administered. After adding the SREBP1-activating compound T0901317, the levels of SREBP1 and downstream synthesis pathway genes recovered, proving that the small molecule compound TOIDC inhibits the lipid synthesis pathway in the liver and relieves fatty liver by reducing the protein levels of SREBP1 and downstream key enzymes of lipid synthesis (see Fig.15 ). In the WB HepG2 cell experiment, the levels of SREBP1 and the downstream lipid synthesis pathway protein FASN decreased after TOIDC was administered. After adding drugs that activate SREBP1, the levels of SREBP1 and the downstream synthesis pathway protein FASN recovered, proving that the small molecule compound TOIDC inhibits the lipid synthesis pathway in the liver and relieves fatty liver by reducing the protein levels of SREBP1 and downstream key enzymes of lipid synthesis (see Fig.16 ).

[0139] The above experimental data show that the small molecule drug TOIDC screened in Example 2 of the present invention can reduce the accumulation of fat droplets in the liver, reduce the content of triglycerides in the liver, and treat non-alcoholic fatty liver disease. The present invention further illustrates through in vitro cell experiments that the small molecule compound screened in Example 2 of the present invention has the potential to treat non-alcoholic fatty liver disease.

[0140] Example 4 In vivo animal experiment

[0141] In the in vivo animal experiment of the present invention, after determining that obese mice fed with high-fat for 16 weeks had fatty liver, they were randomly divided into two groups. One group of mice was intraperitoneally injected with 10 mg / kg of the small molecule TOIDC solution selected in Example 2 of the present invention every day (administration group), and the other group was intraperitoneally injected with the same concentration of DMSO solvent every day (control group). The mice were killed after 3 weeks of the experiment. The gross anatomical diagram shows that the liver of the mice in the administration group is bright red (such as Fig.17 A), the liver of mice in the control group was whiter and larger (as shown on the right side of Fig.17 The results showed that the drug group could alleviate fatty liver (as shown on the left side of A). Fig.17 Then the present invention tested the lipid accumulation in the liver by TG kit and found that the triglyceride level in the liver of the drug administration group decreased (as shown in Fig.18 At the same time, after staining the tissue sections, it was found that the fatty liver of the mice in the drug-treated group was significantly alleviated (as shown in Fig.19 At the same time, the weight of the two groups of mice was measured within three weeks of the experiment. It was found that the average weight of the mice in the drug group was 36.0g, which was significantly lower than the average weight of the mice in the control group (40.2g). Fig. 20 At the same time, the mice were killed three weeks after the experiment. It can be seen that the mice in the drug-treated group were significantly smaller than those in the control group (as shown in Fig.21A), the fat tissue was taken and the fat tissue volume of mice in the drug administration group ( Fig.21 B right) was significantly smaller than that of the control group mice ( Fig.21 B shown on the left).

[0142] The present invention also found that TOIDC inhibited the lipid synthesis pathway in the liver (such as Fig. 22 , Fig.23 shown).

[0143] Next, in the cytotoxicity experiment, the present invention treated primary liver cells with different concentrations of TOIDC and found that there was no difference in cell activity when the concentration was less than 60 μM, indicating that the small molecule TOIDC did not affect cell viability at the working concentration (such as Fig.24 shown).

[0144] In the animal experiment of the present invention, the average serum ALT aminotransferase levels of high-fat mice three weeks after intraperitoneal injection of TOIDC and high-fat mice three weeks after injection of solvent DMSO were 172.3U / L and 179.1U / L, respectively, and the average serum aminotransferase AST levels were 33.8U / L and 32.7U / L, respectively, indicating that intraperitoneal injection of TOIDC has no liver damage toxicity to mice (such as Fig.25 shown).

[0145] The above experimental data show that the small molecule drug TOIDC screened in Example 2 of the present invention can inhibit the lipid synthesis pathway in the liver, reduce the level of triglycerides in the liver, alleviate and relieve fatty liver, and the small molecule drug TOIDC is non-toxic.

[0146] Example 5 Insulin tolerance test

[0147] Five mice in the administration group (10 mg / kg of the small molecule TOIDC screened in Example 2 of the present invention was intraperitoneally injected daily) and five mice in the control group (the same amount of DMSO solvent was intraperitoneally injected daily) were taken respectively. After 3 weeks, the two groups of mice were first starved for 4 hours on an empty stomach. After 4 hours, the tail vein blood of the two groups of mice was taken to test the blood sugar of the mice after starvation with a blood glucose test strip. Then, the two groups of mice were intraperitoneally injected with insulin at a dose of 1.75 IU / kg. The tail vein blood of the two groups of mice was taken to test the blood sugar with a blood glucose test strip and a blood glucose meter at 15 minutes, 30 minutes, 1 hour, 1.5 hours, and 2 hours after the injection of insulin. Finally, it was found that there was no significant difference in blood sugar between the two groups 15 minutes, 1 hour, and 1.5 hours after the injection of insulin. However, 2 hours after insulin injection, the average blood glucose of mice in the TOIDC group (the drug group) was 4.80 mM, and the average blood glucose of mice in the control group was 7.32 mM. There was a significant difference in blood glucose between the two groups of mice, indicating that the blood glucose recovery of mice in the drug group was slower than that of the control group 2 hours after intraperitoneal injection of insulin. In the presence of insulin resistance, intraperitoneal injection of TOIDC can improve insulin sensitivity and improve insulin resistance (such as Fig.26 shown).

[0148] The present invention further illustrates through in vivo cell experiments that the small molecule compounds screened in Example 2 of the present invention have the potential to treat non-alcoholic fatty liver disease and / or type 2 diabetes.

[0149] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the present invention, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. Use of polysubstituted 3,3-diformate-indole-2-thione compounds and pharmaceutically acceptable salts thereof in the preparation of a drug for preventing and / or treating non-alcoholic fatty liver disease and / or type 2 diabetes, characterized in that: The polysubstituted 3,3-diformate-indoline-2-thione compound is selected from the following structural formula:

2. The use according to claim 1, characterized in that The polysubstituted 3,3-dicarboxylate-indole-2-thione compound is selected from Formula 1a or Formula 1k:

3. The use according to claim 1, characterized in that The application is the application in preparing a medicine for treating non-alcoholic fatty liver disease and the application in preparing a medicine for treating type 2 diabetes.

4. Use of the pharmaceutical composition in preparing a drug for treating non-alcoholic fatty liver disease, and in preparing a drug for treating type 2 diabetes, characterized in that: The pharmaceutical composition comprises the polysubstituted 3,3-dicarboxylate-indoline-2-thione compounds and pharmaceutically acceptable salts thereof as claimed in claim 1.

Images