Bile acid derivative and application thereof as FXR antagonist

Abstract

The invention discloses a bile acid derivative and application of the bile acid derivative as an FXR antagonist. The invention discloses a bile acid derivative with a novel structure. The bile acid derivative is a chiral isomer of taurochenodeoxycholic acid (salt). Technicians in the field know that generally, the biological activity of chiral isomers is close, however, the antagonistic activity of the bile acid derivative provided by the invention to farnesoid X receptors is 3-4 times that of the chiral isomers, and the technical effect is unexpected. Therefore, the compound provided by the invention can be used for preparing the farnesoid X receptor antagonist, and has the application prospect of being developed into drugs for preventing, treating or relieving diseases by antagonizing the farnesoid X receptor.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and particularly relates to a bile acid derivative and its use as an FXR antagonist. Background Art

[0002] The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily with a wide range of physiological and pathological functions. It participates in maintaining the homeostasis of bile acids, sugars, and lipids, and is also involved in the development and progression of tumors. Therefore, regulating FXR transcriptional activity is an important therapeutic strategy for a variety of diseases, and breakthroughs have been made in the development of FXR agonists. Recent studies have shown that inhibiting FXR transcriptional activity has a positive impact on a variety of diseases, and significant progress has been made in the development and pharmacological investigation of FXR antagonists.

[0003] According to literature reports (Liu Weiyi et al., China Pharmaceutical University, Research Progress on Farnesoid X Receptor Antagonists and Pharmacological Activities, Chinese Journal of Clinical Pharmacology and Therapeutics, 2020), FXR antagonists can be used to treat a variety of diseases, including:

[0004] 1. FXR antagonists and cholestatic liver injury: FXR's role in maintaining bile acid homeostasis makes FXR antagonists useful for alleviating cholestasis and its induced liver injury;

[0005] 2. FXR antagonists and hypercholesterolemia: Based on the important role of hepatic FXR in cholesterol metabolism, FXR antagonists can be used to treat hypercholesterolemia;

[0006] 3. FXR antagonists and hyperglycemia and diabetes: Inhibition of intestinal FXR transcription can lower blood glucose levels by inhibiting hepatic gluconeogenesis; therefore, intestinal-specific FXR antagonists can be used to treat diabetes.

[0007] 4. FXR antagonists and metabolic syndromes such as obesity / fatty liver disease: FXR antagonists can significantly improve hypercholesterolemia / hyperlipidemia and diabetes. Since dyslipidemia, diabetes, obesity, and non-alcoholic fatty liver disease (NAFLD) are mutually risk factors, FXR antagonists can also be used to improve obesity and NAFLD.

[0008] 5. Cancer: FXR in different tissue locations has distinct effects on tumor development and progression. FXR expression and transcriptional activity are positively correlated with the incidence of esophageal and pancreatic cancer. The FXR antagonist GS can inhibit the development and progression of esophageal and pancreatic cancers by inducing apoptosis and inhibiting tumor cell invasion and metastasis.

[0009] Taurochenodeoxycholic acid (TCDCA, CAS No. 516-35-8) is a conjugated bile acid, and activity studies have shown that it has FXR antagonist activity. A study published in Nature Communications showed that the increase in conjugated bile acids TCDCA and TUDCA after drinking tea significantly inhibited FXR, thereby inhibiting the intestinal FGF15 / FGF19-FGFR4 signaling pathway, activating the activity of hepatic bile acid synthase, and promoting the metabolic process of cholesterol synthesis into bile acids, thereby lowering cholesterol levels (Theabrownin from Pu-erh tea attenuates hypercholesterolemia via modulation of gut microbiota and bile acid metabolism. Nature Communications. Published: 31 October 2019).

[0010]

[0011] The present invention has discovered a new bile acid derivative, which is a chiral isomer of taurochenodeoxycholic acid (salt), but has significantly better activity than taurochenodeoxycholic acid (salt), and the present invention is hereby proposed. Summary of the Invention

[0012] The present invention aims to provide a bile acid derivative and its use as an FXR antagonist.

[0013] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0014] A bile acid derivative having the following chemical structure, or a pharmaceutically acceptable salt thereof:

[0015]

[0016] Use of the bile acid derivatives for preparing farnesoid X receptor antagonists.

[0017] Use of the bile acid derivatives for preparing drugs for preventing, treating or alleviating diseases by antagonizing farnesoid X receptors.

[0018] Preferably, the diseases include cholestatic liver injury, hypercholesterolemia / hyperlipidemia, diabetes, obesity, non-alcoholic fatty liver disease, esophageal cancer and pancreatic cancer.

[0019] The pharmaceutically acceptable salt is used for preparing a farnesoid X receptor antagonist.

[0020] The pharmaceutically acceptable salt is used for preparing a drug for preventing, treating or alleviating a disease by antagonizing the farnesoid X receptor.

[0021] Preferably, the diseases include cholestatic liver injury, hypercholesterolemia / hyperlipidemia, diabetes, obesity, non-alcoholic fatty liver disease, esophageal cancer and pancreatic cancer.

[0022] A sodium salt of the above bile acid derivative.

[0023] Use of the sodium salt of the bile acid derivative for preparing a farnesoid X receptor antagonist.

[0024] The sodium salt of the bile acid derivative is used for preparing cholesterol-lowering drugs.

[0025] Beneficial effects:

[0026] The present invention has discovered a novel bile acid derivative, which is a chiral isomer of taurochenodeoxycholic acid (salt). Those skilled in the art know that chiral isomers generally have similar biological activities. However, the bile acid derivative provided by the present invention exhibits 3-4 times greater farnesoid X receptor antagonist activity than its chiral isomer, a surprising technical effect. Therefore, the compound provided by the present invention can be used to prepare farnesoid X receptor antagonists and has the potential to be developed into drugs that prevent, treat, or alleviate diseases by antagonizing farnesoid X receptors. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the HPLC-ELSD pattern of target compound 1;

[0028] Figure 2 is the mass spectrum of target compound 1;

[0029] Figure 3 is the H NMR spectrum of target compound 1;

[0030] Figure 4 is the NMR carbon spectrum of target compound 1;

[0031] Figure 5 LC-MS comparison of target compound 1 and its chiral isomer TCDCA and isomer TDCA;

[0032] Figure 6 is the percentage of FXR activation in each group (Note: comparison between two groups, **p<0.01);

[0033] Figure 7 Comparison of cholesterol levels in each group (Note: comparison between two groups, **p<0.01). DETAILED DESCRIPTION

[0034] The essential contents of the present invention are described in detail below with reference to the embodiments, but the protection scope of the present invention is not limited thereto.

[0035] Example 1: Synthesis and structure confirmation of target compound 1

[0036] The synthetic route is as follows:

[0037]

[0038] The specific steps are:

[0039] 0.1 mol of CDCA was dissolved in 200 mL of methanol with 1 mL of sulfuric acid added, stirred overnight, and the solvent removed under vacuum. The residue was dissolved in ethyl acetate, washed with water and brine, and dried over sodium sulfate. The solvent was removed under vacuum to yield a pale yellow oil. The product was dissolved in 200 mL of toluene, and freshly prepared AgCO (22 mM) adsorbed on silicate was gradually added to oxidize the hydroxyl group at the -3 position of the methylated 5β-reduced CDCAC. Water was removed and the filtrate was concentrated under vacuum to yield a yellow solid. This solid was added to 100 mL of anhydrous chloroform containing 24 mM methylal. Approximately 3 g of phosphorus pentoxide was added portionwise and stirred vigorously for 0.5 hour, followed by an additional 4 hours at room temperature. The reaction mixture was then poured into 400 mL of ice water. The organic layer was separated, washed with 1 M hydrochloric acid, water, and brine, dried over anhydrous sodium sulfate, and the solvent removed under vacuum to yield a yellow oil. It was dissolved in 100 mL of dimethyl sulfoxide, and 4.4 mM 2-iodobenzoic acid was added. A catalytic amount of trifluoroacetic acid was added and the reaction was carried out at 40°C for 3 days. The suspension was filtered through celite and the filtrate was dissolved in 200 mL of ethyl acetate, washed and dried over anhydrous sodium sulfate to obtain a yellow oil. It was added to 30 mL of liquid ammonia in 10 mL of tetrahydrofuran at -78°C. During the stirring process, small pieces of lithium (3 mg, 0.4 mM) were gradually added, followed by tert-butanol. The mixture was stirred at -78°C for 5 minutes and then quenched with 1 mL of saturated ammonium chloride. The mixture was extracted with ethyl acetate (3×20 mL). The organic phase was washed with brine and dried over anhydrous sodium sulfate to obtain a colorless oil. Under argon protection, it was stirred in 30 mL of dry tetrahydrofuran at -78°C while slowly adding potassium tri-sec-butylborohydride (1.2 mL, 1 M in tetrahydrofuran, 1.2 mM). The reaction mixture was stirred at -78 ° C for 2 hours and then quenched by adding 1 mL of saturated aqueous ammonium chloride solution. Tetrahydrofuran was removed under vacuum, and the mixture was extracted with ethyl acetate (2×20 mL), and the organic phase was washed with water (1×10 mL) and brine (1×10 mL). The organic layer was dried over anhydrous sodium sulfate. The product was obtained as a white solid (300 mg). It was stirred at room temperature in 30 mL of methanol while slowly adding 10 ml of 1 M hydrochloric acid solution. The reaction mixture was refluxed for 2 hours, and then after the reaction system was cooled to room temperature, 10 ml of 2 M potassium hydroxide aqueous solution was added. The reaction was then carried out under reflux overnight (8 hours) and then cooled to room temperature. The organic solvent was removed under vacuum, and the aqueous solution was acidified with 1 M hydrochloric acid. A white solid precipitated out and filtered. Alternatively, for extremely fine precipitates, the suspension was centrifuged at 10,000×g. The solid was washed with cold water, then collected and dried in a vacuum. The product was obtained as a white solid. This synthesis method was based on a previous report by Li et al. (Tetrahedron Letters, 2011, 52, 4137-4139).To a solution of 5 mg / mL of the above product and 100 mM EEDQ (2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline) in acetonitrile:tert-butanol (3:1, v / v) was added an equal volume of 125 mM taurine and 100 mM sodium hydroxide in water, and the mixture was shaken at 80°C for 2 hours. After the reaction, the pH of the resulting solution was adjusted to 7 with 100 mM hydrochloric acid, resulting in the precipitation of a white precipitate. The solvent was removed under vacuum to obtain the product as a white solid with a purity of 99.7%. The HPLC-ELSD pattern is shown below. Figure 1 shown.

[0040] The mass spectrum of target compound 1 is as follows Figure 2 As shown. MS m / z:544.2672([C24H45NO6S+Na] + );545.2723([C24H45NO6S+1+Na] + );546.2709([C24H45NO6S+2+Na] + ).

[0041] The H NMR spectrum data of target compound 1 are as follows: Figure 3 shown. 1 H NMR(CD3OD)δ:3.98(1H),3.78(1H),3.62(2H,-CH2-SO3H),3.00(2H,-CH2-NH),2.1 4-1.15(m,-CH2,-CH),0.98(3H,d,21-Me),0.82(3H,s,19-Me),0.70(3H,s,18-Me).

[0042] The NMR carbon spectrum data of target compound 1 are as follows: Figure 4 shown. 13 C NMR(CD3OD)δ:67.36(C-3),65.82(C-7),55.86(C-17),50.35(C-2'),50.05(C-14),45.64 (C-13),42.32(C-5),39.54(C-12),39.51(C-8),36.34(C-4),35.79(C-1),35.54(C-1'),3 5.26(C-6),35.09(C-20),32.67(C-10),31.90(C-23),31.75(C-9),31.19(C-22),28.12( C-2),27.80(C-16),23.12(C-15),20.31(C-19),17.52(C-11),10.89(C-21),9.25(C-18).

[0043] The target compound 1 was analyzed by LC-MS, and its retention time was compared with the isomers with the same secondary fragment ion spectrum in the BA-Finder 2.0 bile acid database (Analytical Chemistry, 2022, 94, 16, 6242-6250; Analyst, 2023, 148 (21): 5380-5389; J Chromatogr A. 2024, 1715: 464602; https: / / bafinder.github.io). It was found that its retention time was 10.37 minutes, which was different from the isomers TCDCA (retention time of 10.51 minutes) and TDCA (retention time of 10.89 minutes). Figure 5 Based on the above information, it can be determined that the target compound 1 is the C5 chiral isomer of TCDCA, named TalloCDCA, and its chemical structure is as follows:

[0044]

[0045] Example 2: Activity test (inhibitory effect on FXR reporter gene)

[0046] 1. Experimental Materials

[0047] 1. Instruments

[0048] EnVision multifunctional microplate reader (PerkinElmer, USA), STERI-CYCLE CO2 cell culture incubator (Thermo Fisher, USA).

[0049] 2. Reagents and Materials

[0050] High-glucose DMEM (Gibco, 11965092), fetal bovine serum (Gibco, 10099 / 142), Dual Luciferase Reporter Assay kit (Vazyme, DL101-01), FXR Agonist Assay Kit (BPSBioscience, 78131), FXR Binding Buffer (BPSBioscience, 210726), Lipofectamine TM3000 transfection reagent (Thermo Fisher, L3000001), pRL Renilla Luciferase Control Reporter Vectors (Promega, E2231), GW4064 (Selleck Chemicals, S2782), CDCA (Shanghai Yuanye Biotechnology Co., Ltd., B20347), TCA (Shanghai Yuanye Biotechnology Co., Ltd., B26949).

[0051] 2. Experimental Methods

[0052] 1. Plasmid construction

[0053] The CDS sequences of the NR1H4 gene encoding human FXR protein, the RXRA gene encoding human RXRα protein, and the SLC10A2 gene encoding ABST protein were searched on the NCBI website, and transcription factor overexpression plasmids were constructed using the pcDNA3.1 vector. The promoter region sequence (-2000 bp to +1 bp) of the FGF19 gene encoding human FGF19 protein was searched, and the firefly luciferase main reporter gene was constructed using the pGL3 vector. The pRL-TK Renilla luciferase reporter gene plasmid was selected as an internal reference and purchased from Promega Corporation, USA.

[0054] 2. Cell plating, transfection, and drug administration

[0055] HEK-293T cells in the logarithmic growth phase were taken, the culture medium was discarded, and the cells were washed twice with PBS. The PBS was discarded and the cells were digested with trypsin at room temperature for 30 seconds. High-glucose DMEM complete medium twice the volume of trypsin was added to gently blow down the cells and collect the cell suspension. The cells were centrifuged at 1000 r / min. -1 Centrifuge for 5 minutes; discard the supernatant, resuspend in high-glucose DMEM complete medium (containing 10% fetal bovine serum but without double antibody), mix gently by blowing 10 times, and count the cells using a hemocytometer; resuspend in high-glucose DMEM complete medium (containing 10% fetal bovine serum but without double antibody), further dilute the cell suspension, and inoculate into a 96-well plate to make approximately 40,000 cells per well; place the 96-well plate in a 37°C, 5% CO2 incubator for overnight culture.

[0056] Microscopic examination, when the cell confluence reaches 60% to 80%, transfection is performed. TM The operating instructions of the 3000 transfection kit were used to dissolve the plasmid (add 2 μL of L3000 reagent per μg of plasmid) and Lipofectamine TM3000 reagent, mix by blowing gently 3-5 times, let it stand for 10 minutes, and add 10 μL of the mixture to each well of the 96-well plate; each well contains 12 ng of pGL3-FGF19 gene promoter plasmid, 4 ng of pCDNA 3.1-NR1H4 plasmid, 4 ng of pcDNA 3.1-RXRA plasmid, 12 ng of pRL-TK internal reference reporter gene and 4 ng of pCDNA 3.1-SLC10A2 plasmid, as well as 0.3 μL of Lipofectamine TM 3000 reagent. Place the 96-well plate in a 37°C, 5% CO2 incubator for 6 hours and then replace with drug-containing culture medium.

[0057] The groups were set as follows: DMSO control group: 0.1 mL of high glucose incomplete medium containing 0.2% DMSO was added to each well. TCA agonist group: 0.1 mL of 100 μmol·L -1 TCA high glucose incomplete medium. TalloCDCA treatment group: add 0.1mL containing 100μmol·L -1 TCA and 100 μmol·L -1 TalloCDCA high-glucose incomplete medium. TCDCA treatment group: add 0.1mL containing 100μmol·L -1 TCA and 100 μmol·L -1 TCDCA high-glucose incomplete medium was used for further culture at 37°C and 5% CO2 for 24 h.

[0058] 3. Dual luciferase reporter gene assay

[0059] After incubation, a dual-luciferase reporter gene assay was performed according to the manufacturer's instructions. The Luminescence module was selected on the microplate reader. The primary reporter reading was corrected using the internal reference reading and further corrected using the control group correction value to obtain the RLU values ​​for all wells. The inhibition rate was calculated using the control group mean as 0% and the agonist group mean as 100%. Multiple data sets were statistically analyzed using one-way ANOVA, and multiple comparisons were performed using the Tukey test.

[0060] 3. Experimental Results

[0061] The results are as follows Figure 6 As shown, at 100 μmol·L -1 In the presence of TCA (an FXR agonist), 100 μmol·L -1TCDCA and TalloCDCA (target compound 1) showed significant FXR antagonistic effects (p<0.01), among which: the inhibition rate of TCDCA was 20.2%, and the inhibition rate of TalloCDCA was 60.2%. The antagonistic effect of the latter on FXR was 3 times that of the former.

[0062] Example 3: Activity Test (Promoting Cholesterol Metabolism, Lowering Cholesterol)

[0063] 1. Experimental Materials

[0064] Cholesterol (HY-N0322, MedChemExpress, USA); total cholesterol assay kit (A111-1-1, Nanjing Jiancheng Bioengineering Institute); HepG2 human hepatoma cell line.

[0065] 2. Experimental Methods

[0066] The methods for HepG2 cell plating, drug administration, and total cholesterol determination are as follows:

[0067] Take HepG2 cells grown to the logarithmic growth phase, discard the culture medium, add PBS to wash twice, discard the PBS, add trypsin to digest at 37℃ for 3min, add high-glucose DMEM complete medium twice the volume of trypsin, blow the cells gently, collect the cell suspension, and spin at 1000r·min -1 Centrifuge for 5 minutes; discard the supernatant, resuspend in high-glucose DMEM complete medium (containing 10% fetal bovine serum but without double antibody), blow gently 10 times to mix, and count the cells using a hemocytometer; resuspend in high-glucose DMEM complete medium (containing 10% fetal bovine serum but without double antibody) and further dilute the cell suspension, inoculate in 6-well plates, and inoculate 5×10 cells per well. 5 The cells were cultured for 12 h at 37°C and 5% CO2. The culture medium was discarded and replaced with high-glucose DMEM incomplete medium, and serum starvation was continued for 12 h.

[0068] The culture medium was discarded, the cells were washed twice with PBS, and then replaced with drug-containing culture medium. The drug-treated group was set up as follows: in the control group, 2 mL of cholesterol (10 μg mL) prepared in high-glucose DMEM incomplete culture medium was added to each well. -1 ) and CDCA (100 μmol·L -1 ) mixed solution; TalloCDCA group: 2 mL of high-glucose DMEM incomplete medium prepared with cholesterol (10 μg mL -1 )、CDCA(100μmol·L -1 ) and TalloCDCA (100 μmol·L -1 ) mixed solution; TCDCA group: 2 mL of high-glucose DMEM incomplete medium prepared with cholesterol (10 μg mL -1)、CDCA(100μmol·L -1 ) and TCDCA (100 μmol·L -1 After replacement, continue culturing at 37°C, 5% CO2 for 24 hours.

[0069] After the culture is completed, the culture medium is discarded, the cells are washed twice with PBS, 0.2 mL of anhydrous ethanol is added to each well, the cells are scraped with a cell scraper, and the total cholesterol content of the cells is determined according to the operating instructions.

[0070] 3. Experimental Results

[0071] In the above cell model, the FXR activator CDCA inhibits the cell's ability to metabolize cholesterol by activating FXR. By comparing the total cholesterol (TC) levels in the cells after adding the test compound, the ability of the test compound to antagonize FXR and promote cell cholesterol metabolism can be evaluated. Figure 7 As shown, compared with the control group (100%), the relative content of TC in HepG2 cells in the TalloCDCA-treated group (39.7%) was significantly decreased and significantly lower than that in the TCDCA-treated group (84.4%). The decrease in the former was 3.9 times that of the latter. This result shows that TalloCDCA (i.e., the target compound 1 provided by the present invention) has the effect of antagonizing FXR to promote cellular cholesterol metabolism and has the potential to be developed into a cholesterol-lowering drug for the treatment of hypercholesterolemia / hyperlipidemia. At the same time, based on the role of high cholesterol in obesity and non-alcoholic fatty liver disease, TalloCDCA also has the potential to be developed into a drug for the treatment of obesity and non-alcoholic fatty liver disease.

[0072] Those skilled in the art will appreciate that the sodium salt of the bile acid derivative of the present invention can also be obtained according to Example 1, which is a chiral isomer of sodium taurochenodeoxycholate. According to general principles in the art, the sodium salt has approximately 3-4 times the activity of sodium taurochenodeoxycholate in antagonizing the farnesoid X receptor and antagonizing FXR to promote cellular cholesterol metabolism.

[0073] In summary, the present invention has discovered a novel bile acid derivative, which is a chiral isomer of taurochenodeoxycholic acid (salt). Those skilled in the art are aware that chiral isomers generally have similar biological activities. However, the bile acid derivative provided by the present invention exhibits 3-4 times greater farnesoid X receptor antagonist activity than its chiral isomer, a surprising technical effect. Therefore, the compounds provided by the present invention can be used to prepare farnesoid X receptor antagonists and have the potential to be developed into drugs that prevent, treat, or alleviate diseases by antagonizing farnesoid X receptors.

[0074] The purpose of the above embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. A bile acid derivative having the following chemical structure, or a pharmaceutically acceptable salt thereof:

2. Use of the bile acid derivative according to claim 1 for preparing a farnesoid X receptor antagonist.

3. Use of the bile acid derivative according to claim 1 for preparing a medicament for preventing, treating or alleviating a disease by antagonizing farnesoid X receptor.

4. The use according to claim 3, characterized in that: These diseases include cholestatic liver injury, hypercholesterolemia / hyperlipidemia, diabetes, obesity, non-alcoholic fatty liver disease, esophageal cancer, and pancreatic cancer.

5. Use of the pharmaceutically acceptable salt according to claim 1 for preparing a farnesoid X receptor antagonist.

6. Use of the pharmaceutically acceptable salt according to claim 1 for preparing a medicament for preventing, treating or alleviating a disease by antagonizing farnesoid X receptor.

7. The use according to claim 6, characterized in that: These diseases include cholestatic liver injury, hypercholesterolemia / hyperlipidemia, diabetes, obesity, non-alcoholic fatty liver disease, esophageal cancer, and pancreatic cancer.

8. A sodium salt of the bile acid derivative according to claim 1.

9. Use of the sodium salt according to claim 8 for preparing a farnesoid X receptor antagonist.

10. Use of the sodium salt according to claim 8 for preparing cholesterol-lowering drugs.

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