WO2021036998A1 - 胆汁酸衍生物及其组合物和应用 - Google Patents

胆汁酸衍生物及其组合物和应用 Download PDF

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WO2021036998A1
WO2021036998A1 PCT/CN2020/110851 CN2020110851W WO2021036998A1 WO 2021036998 A1 WO2021036998 A1 WO 2021036998A1 CN 2020110851 W CN2020110851 W CN 2020110851W WO 2021036998 A1 WO2021036998 A1 WO 2021036998A1
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bile acid
acid derivative
carbon
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贾伟
谢国祥
翟宁
赵爱华
郑晓皎
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Shenzhen Yunhe Pharmaceutical Technology Partnership Ltd
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Priority to US17/637,450 priority patent/US12491190B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0066—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by a carbon atom forming part of an amide group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
    • C07J41/0061—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives one of the carbon atoms being part of an amide group
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • C07J9/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane containing a carboxylic function directly attached or attached by a chain containing only carbon atoms to the cyclopenta[a]hydrophenanthrene skeleton

Definitions

  • the invention belongs to the fields of medicine and biotechnology, and specifically relates to a new bile acid derivative, a preparation method thereof, a composition containing the derivative and application thereof.
  • FXR Farnesoid X Receptor
  • BM.Forman et al., Cell, 1995, 81 (5). ), 687-693 FXR is a member of the nuclear receptor family of ligand-activated transcription factors.
  • the nuclear receptor family includes receptors for steroids, retinoids, and thyroid hormones (DJ. Mangelsdorf et al., Cell, 1995, 83(6), 841 -850).
  • the relevant physiological ligands of FXR are bile acids (D. Parks et al., Science, 1999, 284(5418), 1362-1365).
  • chenodeoxycholic acid which regulates the expression of several genes involved in bile acid homeostasis.
  • FXR is expressed in the liver and is found throughout the gastrointestinal tract, including the esophagus, stomach, duodenum, small intestine, colon, ovary, adrenal gland, and kidney.
  • FXR also appears to be involved in paracrine and endocrine signal transduction by up-regulating the expression of cytokine fibroblast growth factor (J. Holt et al., Genes Dev., 2003, 17(13) ,1581-1591; T. Inagaki et al., Cell Metab., 2005, 2(4), 217-225).
  • the TGR5 receptor is a G protein-coupled receptor, which has been identified as a cell surface receptor that responds to bile acids (BA).
  • BA bile acids
  • the primary structure of TGR5 and its response to bile acids have been found to be highly conserved in TGR5 among humans, cattle, rabbits, rats and mice, which suggests that TGR5 has important physiological functions. It has been found that TGR5 is widely distributed not only in lymphoid tissues, but also in other tissues. High concentrations of TGR5 mRNA have been detected in placenta, spleen and monocytes/macrophages. Bile acids have been shown to induce the internalization of TGR5 fusion proteins from the cell membrane to the cytoplasm (Kawamata et al., J. Bio. Chem., 2003, 278, 9435). It has been found that TGR5 is the same as hGPCR19 reported by Takeda et al., FEBS Lett. 2002,520,97-101.
  • TGR5 is also associated with the intracellular accumulation of cAMP, which is widely expressed in various cell types.
  • cAMP a membrane receptor that is widely expressed in various cell types.
  • the activation of this membrane receptor in macrophages reduces the production of pro-inflammatory cytokines (Kawamata, Y. et al., J. Biol. Chem. 2003, 278, 9435-9440), while in adipocytes and monocytes BA stimulation of TGR5 enhances energy expenditure (Watanabe, M. et al. Nature. 2006, 439, 484-489).
  • D2 type 2 iodothyronine deiodinase
  • TGR5 knockout mice Consistent with the role of TGR5 in controlling energy metabolism, female TGR5 knockout mice showed significant fat accumulation with weight gain when challenged with a high-fat diet, indicating that TGR5 deficiency reduces energy expenditure and causes obesity (Maruyama, T., et al. , J. Endocrinol. 2006, 191, 197-205).
  • bile acid activation of membrane receptors has also been reported to promote the production of glucagon-like peptide 1 (GLP-1) in murine enteroendocrine cell lines (Katsuma, S., Biochem. Biophys. Res. Commun., 2005, 329, 386-390). Based on all the above observations, TGR5 is an attractive target for the treatment of diseases such as obesity, diabetes and metabolic syndrome.
  • TGR5 modulators can also be used to treat other diseases, such as central nervous system diseases and inflammatory diseases.
  • TGR5 modulators also provide methods to regulate the homeostasis of bile acids and cholesterol, fatty acid absorption, and protein and carbohydrate digestion.
  • fatty liver refers to a lesion with excessive accumulation of fat in liver cells due to various reasons. It is a common liver pathological change, rather than an independent disease. Fatty liver disease is seriously threatening the health of the Chinese people. It has become the second largest liver disease after viral hepatitis. The incidence rate is increasing, and the age of onset is getting younger.
  • Normal human liver tissue contains a small amount of fat, such as triglycerides, phospholipids, glycolipids, and cholesterol. Its weight is about 3% to 5% of the liver weight. If the accumulation of fat in the liver is too much, it will exceed 5% of the liver weight. Or when histologically more than 50% of liver cells have fatty degeneration, it can be called fatty liver.
  • the clinical manifestations are asymptomatic in mild cases and fierce in severe cases. Generally speaking, fatty liver is a reversible disease, and early diagnosis and timely treatment can often return to normal.
  • obeticholic acid can significantly reduce the degree of liver fibrosis in patients with non-alcoholic fatty liver disease, but the drug has an adverse effect on lipid metabolism, and the results of different clinical trials are not consistent.
  • the drug is currently approved in the United States for only indications It is primary biliary cirrhosis.
  • liver metabolism rate is high, and the benefit/risk ratio is a question worthy of discussion. Therefore, safe and effective drugs for the treatment of non-alcoholic fatty liver disease are a huge unmet need.
  • the present invention provides a group of bile acid derivatives and their compositions, which can be used to regulate or ameliorate diseases and symptoms mediated or caused by FXR or TGR5.
  • a bile acid derivative of the present invention is the structure of the following formula (I) or its stereoisomer, or its salt or ester,
  • R 2 is ⁇ -OH or H or CH 2 OH
  • R 3 is ⁇ -OH or H or ⁇ -OH or CH 3 ,
  • R 4 is H or CH 3 ,
  • R 5 is ⁇ -OH or H
  • the carbon to which the R 6 methyl group is connected can be in the S configuration or the R configuration; in the R 7 substituent, the carbon to which the Y group is connected can be in the S configuration or the R configuration.
  • the carbon to which the Y group is connected can be in the S configuration or the R configuration.
  • the bile acid derivative of the present invention is further preferably that R 1 is ⁇ -OH, R 2 is ⁇ -OH, R 3 is ⁇ -OH, R 4 is H, R 5 is H, R 6 is H, R 7 is OH .
  • the carbon to which the Y group is attached can be in the S configuration or the R configuration.
  • R 5 can be H or ⁇ -OH.
  • R 5 is H or ⁇ -OH.
  • the present invention further provides a composition comprising any one or more of the bile acid derivatives described above and a suitable carrier.
  • the present invention further provides a composition for treating and ameliorating diseases and symptoms mediated or caused by FXR or TGR5, which comprises an effective amount of any one or more bile acid derivatives as described above and a suitable carrier.
  • the effective amount refers to the daily dose of the composition containing any one or more bile acid derivatives as described above, 50-500 mg/kg of the patient's body weight.
  • the suitable carrier refers to a medically applicable excipient.
  • composition is an oral preparation, more preferably an ordinary tablet, chewable tablet, dispersible tablet, granule, solution, capsule or suspension, and further preferably an enteric-coated preparation or an enteric-coated sustained-release preparation.
  • the present invention further provides the use of the bile acid derivative as described above in the preparation of a composition for treating and ameliorating diseases and symptoms mediated or caused by FXR or TGR5.
  • the present invention further provides the use of any of the bile acid derivatives described above in the treatment and improvement of FXR or TGR5 mediated or caused diseases and symptoms related to liver damage.
  • the bile acid derivative when used in the above-mentioned use, can be optionally combined with a conventional hypoglycemic and lipid-lowering drug to be used for this purpose, and the conventional hypoglycemic and lipid-lowering drug is selected from liraglutide, Exenatide, abirutai.
  • the effective amount of bile acid derivatives refers to any one or more of the bile acid derivatives described above, 50-500 mg/kg of the patient's body weight.
  • the diseases and symptoms mediated or caused by FXR or TGR5 include the following diseases or symptoms: liver disease, hyperlipidemia, hypercholesterolemia, obesity, metabolic syndrome, cardiovascular disease, gastrointestinal disease, atherosclerosis -Like sclerosis and kidney disease.
  • the diseases and symptoms related to liver damage mediated or caused by FXR or TGR5 include the following diseases or symptoms: simple fatty liver, primary biliary cirrhosis, primary sclerosing cholangitis, liver fibrosis, liver Cirrhosis, non-alcoholic steatohepatitis and non-alcoholic fatty liver disease and their related liver damage, and further particularly refer to simple fatty liver, non-alcoholic steatohepatitis and their related liver damage.
  • the compounds described in this application include their pharmaceutically acceptable acid or base addition salts and esters.
  • the pharmaceutically acceptable salt may be formed of an inorganic acid, an organic acid, or an acidic amino acid.
  • the compound of the present invention has an acidic group, it can form a salt with metal, ammonia or organic amine or basic amino acid.
  • the compounds of the present invention can exhibit tautomerism, configurational isomerism, geometric isomerism and stereoisomerism. Although this application only presents limited isomeric forms, the compounds of the present invention shall cover any tautomerism, configurational isomerism, stereochemical or geometrical differences of one or more compounds having the utility described in this application. The configuration of the configuration, and the mixture of these different forms.
  • a group of 1 to 3 atoms refers to a group having 1, 2 or 3 atoms.
  • a group of 0-3 atoms is outside the above range, and there is a case in which it does not have the group.
  • the suitable carrier contained in the composition of the present invention refers to a pharmaceutically acceptable carrier or excipient or filler or diluent or other necessary auxiliary materials according to the knowledge of those skilled in the pharmaceutical field.
  • the composition contains a therapeutically effective amount of one or more of the bile acid derivatives of the present invention.
  • the composition can be used in a variety of ways, such as injection, oral, inhalation, implantation and the like.
  • bile acid derivatives of the present invention can be prepared according to the knowledge of those of ordinary skill in the art and under the guidance of the synthesis scheme in the examples.
  • Figure 1 shows the normal people, patients with non-alcoholic fatty liver disease, patients with non-alcoholic steatohepatitis, patients with non-alcoholic steatohepatitis-early liver fibrosis, patients with non-alcoholic steatohepatitis-late liver fibrosis, and Graph of average hygrocholic acid content in patients with non-alcoholic steatohepatitis-cirrhosis.
  • Figure 2 is a schematic diagram showing the significant reduction of liver triglycerides after 8 weeks of intervention with hyocholic acid, hyodeoxycholic acid, and 9 synthetic bile acid derivatives of the present invention.
  • Fig. 3 is a schematic diagram showing the significant reduction of serum triglycerides after 8 weeks of intervention with hyocholic acid, hyodeoxycholic acid, and 9 synthetic bile acid derivatives of the present invention.
  • Figure 4 shows the changes in blood glucose levels of mice after the intervention of hyocholic acid (HCA), hyodeoxycholic acid (HDCA) and 9 synthetic bile acid derivatives (50 mg/kg) of the present invention.
  • HCA hyocholic acid
  • HDCA hyodeoxycholic acid
  • 9 synthetic bile acid derivatives 50 mg/kg
  • NCI-H716 and STC-1 cells were treated with hyocholic acid, 6 kinds of bile acid derivatives and 19 kinds of other bile acid derivatives, treated at 50 ⁇ M for 48 hours, and it was found that by the action of TGR5 and FXR, hyocholic acid Its derivatives are more effective than other bile acids in up-regulating GLP-1 protein expression in enteroendocrine cell lines.
  • the preparation method includes the following steps:
  • the preparation method includes the following steps:
  • the preparation method includes the following steps:
  • the preparation method includes the following steps:
  • Hypocholic acid (1.632g, 4mmol) was dissolved in 30mL methanol, and 3 drops of concentrated sulfuric acid were added to catalyze the reaction overnight at room temperature. After the completion of the reaction was monitored by thin-layer chromatography, it was concentrated under reduced pressure to remove the solvent methanol. After the ethyl acetate was dissolved, it was washed with saturated sodium bicarbonate and brine successively. The organic phase was dried and concentrated to obtain 1.69 g of methyl hyocholic acid (H1).
  • Methyl hyocholic acid (1.69g, 4mmol) and 2,6-lutidine (4.29g, 40mmol) were dissolved in dichloromethane, protected by nitrogen, cooled to 0-5°C, and added dropwise to the reaction solution Tert-butyl dimethylsilyl trifluoromethanesulfonate (2.8 ml), after dripping, react at room temperature. After the completion of the reaction was monitored by thin-layer chromatography, the reaction solution was flash column chromatography to obtain 3.1 g of intermediate (H2).
  • the intermediate (H3) was dissolved in methanol (20ml), and 4 drops of concentrated hydrochloric acid were added to catalyze the removal of the TBS protecting group at room temperature. After the reaction was completed, the solvent methanol was removed by concentration under reduced pressure. The residue was dissolved in 10 ml of tetrahydrofuran/H 2 O (4:1), sodium hydroxide (0.34 g, 8.6 mmol) was added, and the reaction was carried out at room temperature.
  • Example 10 Hypocholic acid concentration is significantly reduced in fatty liver disease patients
  • Example 1 of the present invention a total of 200 subjects were enrolled, and 25 healthy people diagnosed by liver biopsy and 175 patients with fatty liver (including simple liver biopsy) confirmed by liver biopsy were detected using ultra-high performance liquid chromatography tandem mass spectrometry technology. Fatty amine, steatohepatitis, steatohepatitis with early liver fibrosis, steatohepatitis with advanced liver fibrosis, and steatohepatitis with liver cirrhosis) The content of metabolites such as bile acid, amino acids and fatty acids in serum samples, And the detection of corresponding clinical indicators. The test results found that hyurocholic acid was significantly reduced in patients with fatty liver disease (Figure 1).
  • Example 11 As shown in Figures 2 and 3, for hyocholic acid, hyodeoxycholic acid, and synthetic hyocholic acid derivatives (Table 1), it significantly improved the mouse serum hyperlipidemia caused by hyperlipidemia.
  • We used hyperlipidemia In the fat-induced obesity mouse model, the rats were given high-fat diet (HFD) at the same time as 50mg/Kg/day with hyotcholic acid and hyodeoxycholic acid respectively for 8 weeks.
  • HFD high-fat diet
  • mice in the hypocholic acid, hyodeoxycholic acid, and synthetic hypocholic acid derivatives groups were significantly lower than those in the simple high-fat diet group after 8 weeks, and the hypocholic acid and hyodeoxygen levels in the mice were The increase of bile acid can effectively improve the dyslipidemia of mice caused by high fat.
  • Example 12 The porcine bile acid series and the synthetic porcine bile acid derivatives (Table 1) (50 mg/kg/day) were orally administered to C57BL/6J mice. As shown in Figure 4, after one week of intervention, the results showed that the blood glucose of all intervention groups were significantly reduced.
  • Example 13 NCI-H716 cells were cultured, treated with 50 ⁇ M hyocholic acid and hyocholic acid derivatives, and a reported TGR5 agonist INT-777, the level of GLP-1 in the cell culture medium was measured, and all compounds were found Both can effectively promote the release of GLP-1 (Figure 5), and the release ability of compound ZN-1-102-1 for GLP-1 is better than hyotcholic acid and the existing TGR5 agonist INT-777.
  • Example 14 Treatment of NCI-H716 with 50 ⁇ M hyodeoxycholic acid, hyodeoxycholic acid, taurohyodeoxycholic acid, glycohyodeoxycholic acid, taurohyotocholic acid, glycohyotocholic acid and 19 other bile acids After 48 hours with STC-1 cells, it was found that through the action of TGR5 and FXR, hyocholic acid and its derivatives were more effective than other bile acids in up-regulating GLP-1 protein expression in enteroendocrine cell lines ( Figure 6). (a) Using real-time PCR to measure GLP-1 transcription, see Figure 6(a).

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Abstract

本发明提供了一种治疗脂肪肝病的新型胆汁酸衍生物其药物组合物和应用于制备治疗和改善FXR或TGR5介导或导致的疾病和症状的药物的用途。本发明胆汁酸衍生物在肠道抑制/或延缓细菌BSH/7a脱羟酶对胆酸的代谢，大大延长胆汁酸在肠道的有效生存时间，胆汁酸衍生物及其药物组合物可显著激动胆汁酸膜受体TGR5，促进肠内分泌细胞分泌胰高血糖素样肽1，改善肝脏脂肪蓄积，显著改善肝功能，提高糖耐量，具有较好的治疗脂肪肝病的效果。

Description

胆汁酸衍生物及其组合物和应用 技术领域
本发明属于医药、生物技术领域，具体涉及一种新的胆汁酸衍生物、其制备方法及包含有该衍生物的组合物及其应用。
背景技术
类法尼醇X受体(Farnesoid X Receptor,FXR)为最初从大鼠肝cDNA文库鉴定与昆虫蜕皮激素受体最紧密相关的孤儿核受体(BM.Forman等,Cell,1995,81(5),687-693)。FXR为配体活化转录因子的核受体家族成员，该核受体家族包括用于类固醇、类视色素、以及甲状腺激素的受体(DJ.Mangelsdorf等,Cell,1995,83(6),841-850)。FXR的相关生理学配体为胆汁酸类(D.Parks等,Science,1999,284(5418),1362-1365)。最有效的一个为鹅去氧胆酸(CDCA)，其调节参与胆汁酸动态平衡的数个基因的表达。FXR在肝脏中表达，遍布整个胃肠道，包括食道、胃、十二指肠、小肠、结肠、卵巢、肾上腺和肾。除了控制细胞内基因表达之外，FXR看起来还通过上调细胞因子成纤维细胞生长因子的表达而参与了旁分泌和内分泌信号转导(J.Holt等,Genes Dev.,2003,17(13),1581-1591；T.Inagaki等,Cell Metab.,2005,2(4),217-225)。
TGR5受体为G蛋白偶联受体，其已被鉴定为应答胆汁酸类(BA)的细胞表面受体。TGR5的一级结构及其应答胆汁酸类已被发现在人、牛、兔、大鼠和小鼠之间的TGR5中是高度保守的，由此提示TGR5具有重要的生理学功能。已发现TGR5不仅在淋巴组织、还在其他组织中广泛分布。已在胎盘、脾和单核细胞/巨噬细胞中检测到高浓度的TGR5mRNA。胆汁酸类已显示出诱导TGR5融合蛋白从细胞膜到细胞质的内化(Kawamata等,J.Bio.Chem.,2003,278,9435)。已发现TGR5与Takeda等,FEBS Lett.2002,520,97-101报道的hGPCR19相同。
TGR5还与cAMP的细胞内积聚相关，其在各种细胞类型中广泛表达。这种膜受体在巨噬细胞中的活化降低促炎细胞因子生成，(Kawamata,Y.等,J.Biol.Chem.2003,278,9435-9440)，而在脂肪细胞和单核细胞中BA对TGR5的刺激则增强能量消耗(Watanabe,M.等Nature.2006,439,484-489)。该后一作用涉及对2型碘甲状腺原氨酸脱碘酶(D2)的cAMP依赖性诱导，该酶通过局部地将T4转化为T3 而导致增加的甲状腺激素活性。与TGR5在控制能量代谢中的角色一致，雌性TGR5敲除小鼠当以高脂肪饮食挑战时显示了随体重增加显著的脂肪积累，表明TGR5缺乏降低能量消耗并引起肥胖(Maruyama,T.,等,J.Endocrinol.2006,191,197-205)。另外，并且与TGR5参与能量动态平衡一致，膜受体的胆汁酸活化也有报道促进鼠科肠内分泌细胞系中胰高血糖素样肽1(GLP-1)的产生(Katsuma,S.,Biochem.Biophys.Res.Commun.,2005,329,386-390)。基于全部上述观察结果，TGR5为治疗诸如肥胖、糖尿病和代谢综合征的疾病的引人注目的靶标。
除了使用TGR5激动剂治疗和防止代谢性疾病之外，调节TGR5调节剂的化合物也可用于治疗其他疾病，例如中枢神经疾病以及炎性疾病。TGR5调节剂也提供了调节胆汁酸和胆固醇动态平衡、脂肪酸吸收、以及蛋白质和碳水化合物消化的方法。
其中，脂肪肝(fatty liver)是指由于各种原因引起的肝细胞内脂肪堆积过多的病变，是一种常见的肝脏病理改变，而非一种独立的疾病。脂肪性肝病正严重威胁国人的健康，成为仅次于病毒性肝炎的第二大肝病，发病率在不断升高，且发病年龄日趋年轻化。正常人肝组织中含有少量的脂肪，如甘油三酯、磷脂、糖脂和胆固醇等，其重量约为肝重量的3％～5％，如果肝内脂肪蓄积太多，超过肝重量的5％或在组织学上肝细胞50％以上有脂肪变性时，就可称为脂肪肝。其临床表现轻者无症状，重者病情凶猛。一般而言，脂肪肝属可逆性疾病，早期诊断并及时治疗常可恢复正常。
目前针对该病的治疗手段也非常有限。临床试验表明奥贝胆酸可显著减轻非酒精性脂肪肝病患者肝纤维化程度，但是该药对脂代谢有不良影响，并且不同临床试验的结果并不一致，该药目前在美国获批适应症仅为原发性胆汁性肝硬化。指南推荐的保肝药物如水飞蓟素、双环醇、多烯磷酰胆碱、甘草酸制剂、还原性谷胱苷肽等，对于非酒精性脂肪肝病并没有非常确切的疗效证据。另外许多治疗肝病的药物一方面可以改善肝脏损伤，但是另一方面肝脏代谢率很高，获益/风险比是值得探讨的问题。因此，安全有效的治疗非酒精性脂肪肝病的药物是巨大的未满足的需求。
发明内容
本发明提供了一组胆汁酸衍生物及其组合物，可用于调节或改善FXR或TGR5介导或导致的疾病和症状。
本发明一种胆汁酸衍生物，为如下式(I)结构或其立体异构体、或其盐或酯，
Figure PCTCN2020110851-appb-000001
其中
R 1为α-OH或β-O(CH 2) aOH，其中a＝1-10，
R 2为α-OH或H或CH 2OH，
R 3为α-OH或H或β-OH或CH 3，
R 4为H或CH 3，
R 5为α-OH或H，
R 6为H或(CH 2) bCH 3，其中b＝0-3，
R 7为
Figure PCTCN2020110851-appb-000002
[X＝H或CH 3，Y＝CH 3或CH 2OH，Z＝COOH或SO 3H，n＝0-10；或R 7为OH或-O(CH 2) tCH 3，其中t＝0-3，
其中R 6甲基所连接的碳可以是S构型或R构型；R 7取代基中，Y基团所连接的碳可以是S构型或R构型。
本发明胆汁酸衍生物进一步优选为，R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4＝H，R 5为α-OH或-H，R 6为-H或(CH 2) bCH 3(b＝0-3)，R 7为
Figure PCTCN2020110851-appb-000003
[X＝H或CH 3，Y＝CH 3或CH 2OH，Z＝COOH或SO 3H，n＝0-10]或OH或-O(CH 2) tCH 3(t＝0-3)；其中R 6甲基所连接的碳可以是S 构型或R构型；R 7取代基中，Y基团所连接的碳可以是S构型或R构型。
本发明胆汁酸衍生物进一步优选为，R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为H，R 6为H或(CH 2) bCH 3(b＝0-3)，R 7为
Figure PCTCN2020110851-appb-000004
[X＝H或CH 3，Y＝CH 3或CH 2OH，Z＝COOH或SO 3H，n＝0-10]或R 7为OH或-O(CH 2) tCH 3(t＝0-3)，其中R 6甲基所连接的碳可以是S构型或R构型；R 7取代基中，Y基团所连接的碳可以是S构型或R构型。
本发明胆汁酸衍生物进一步优选为，R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为H，R 6为H，R 7为
Figure PCTCN2020110851-appb-000005
[X＝H或CH 3，Y＝CH 3或CH 2OH，Z＝COOH或SO 3H，n＝0-10]或R 7为OH或-O(CH 2) tCH 3(t＝0-3)，Y基团所连接的碳可以是S构型或R构型。
本发明胆汁酸衍生物进一步优选为，R 1为α-OH，R 2为α-OH，R 3为-α-OH，R 4为H，R 5为H，R 6为(CH 2) bCH 3(b＝0-3)，R 7为
Figure PCTCN2020110851-appb-000006
[X＝H或CH 3，Y＝CH 3或CH 2OH，Z＝COOH或SO 3H，n＝0-10]或R 7为OH或-O(CH 2) tCH 3(t＝0-3)；其中R 6甲基所连接的碳可以是S构型或R构型；R7取代基中，Y基团所连接的碳可以是S构型或R构型。
本发明胆汁酸衍生物进一步优选为，R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为H，R 6为H，R 7为OH或-O(CH 2) tCH 3(t＝0-3)。
本发明胆汁酸衍生物进一步优选为，R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为H，R 6为H，R 7为OH。
本发明胆汁酸衍生物进一步优选为，R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为H，R 6为H，R 7为-O(CH 2) tCH 3(t＝0-3)。
本发明胆汁酸衍生物进一步优选为，R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为H，R 6为H，R 7为
Figure PCTCN2020110851-appb-000007
[X＝H，Y＝CH 2OH，Z＝COOH，n＝0-10]，Y基团所连接的碳可以为S构型或R构型。
本发明胆汁酸衍生物进一步优选为，R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为H，R 6为H，R 7为
Figure PCTCN2020110851-appb-000008
[X＝H，Y＝CH 3，Z＝SO 3H，n＝0-10]，Y基团所连接的碳可以为S构型或R构型。
本发明胆汁酸衍生物进一步优选为，R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为H，R 6为H，R 7为
Figure PCTCN2020110851-appb-000009
[X＝H；Y＝CH 2OH，Z＝SO 3H，n＝0-10]，Y基团所连接的碳可以为S构型或R构型。
本发明胆汁酸衍生物进一步优选为，R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为H，R 6为H，R 7为
Figure PCTCN2020110851-appb-000010
[X＝H，Y＝CH 3，Z＝COOH，n＝0-10]，Y基团所连接的碳可以为S构型或R构型。
本发明胆汁酸衍生物进一步优选为，其中R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为α-OH或H，R 6为-(CH 2) bCH 3(b＝0-3)，R 7为
Figure PCTCN2020110851-appb-000011
[X＝H，Y＝CH 2OH，Z＝COOH，n＝0-10]；其中R 6甲基所连接的碳可以为S构型或R构型；R 7取代基中，Y基团所连接的碳可以为S构型或R构型。
本发明胆汁酸衍生物进一步优选为，其中R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为α-OH或H，R 6为-(CH 2) bCH 3(b＝0-3)，R 7为
Figure PCTCN2020110851-appb-000012
[X＝H，Y＝CH 3，Z＝SO 3H，n＝0-10]；其中R 6甲基所连接的碳可以为S构型或R构型；R 7取代基中，Y基团所连接的碳可以为S构型或R构型。
如本发明胆汁酸衍生物进一步优选为，其中R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为 H，R 5为α-OH或H，R 6为-(CH 2) bCH 3(b＝0-3)，R 7为
Figure PCTCN2020110851-appb-000013
[X＝H，Y＝CH 2OH，Z＝SO 3H，n＝0-10]；其中R 6甲基所连接的碳可以为S构型或R构型；R 7取代基中，Y基团所连接的碳可以为S构型或R构型。
本发明胆汁酸衍生物进一步优选为，其中R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H；R 5为α-OH或-H，R 6为-(CH 2) bCH 3(b＝0-3)，R 7为
Figure PCTCN2020110851-appb-000014
[X＝H，Y＝CH 3，Z＝COOH,n＝0-10]；其中R 6甲基所连接的碳或以为S构型或R构型；R 7取代基中，Y基团所连接的碳可以为S构型或R构型。
本发明胆汁酸衍生物进一步优选为，R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为H，R 6为H，R 7为
Figure PCTCN2020110851-appb-000015
[X＝CH 3，Y＝CH 2OH，Z＝COOH，n＝0-10]，Y基团所连接的碳可以为S构型或R构型。
本发明胆汁酸衍生物进一步优选为，R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为H，R 6为H，R 7为
Figure PCTCN2020110851-appb-000016
[X＝CH 3，Y＝CH 3，Z＝SO 3H，n＝0-10]，Y基团所连接的碳可以为S构型或R构型。
本发明胆汁酸衍生物进一步优选为，R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为H，R 6为H，R 7为
Figure PCTCN2020110851-appb-000017
[X＝CH 3，Y＝CH 2OH，Z＝SO 3H，n＝0-10]，Y基团所连接的碳可以为S构型或R构型。
本发明胆汁酸衍生物进一步优选为，R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为H，R 6为H，R 7为
Figure PCTCN2020110851-appb-000018
[X＝CH 3，Y＝CH 3，Z＝COOH，n＝0-10]；Y基团所连接的碳可以 为S构型或R构型。
本发明胆汁酸衍生物进一步优选为，其中R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为α-OH或H，R 6为-(CH 2) bCH 3(b＝0-3)，R 7为
Figure PCTCN2020110851-appb-000019
[X＝CH 3，Y＝CH 2OH，Z＝COOH，n＝0-10]；其中R 6甲基所连接的碳可以为S构型或R构型；R 7取代基中，Y基团所连接的碳可以为S构型或R构型。
本发明胆汁酸衍生物进一步优选为，其中R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为α-OH或H，R 6为-(CH 2) bCH 3(b＝0-3)，R 7为
Figure PCTCN2020110851-appb-000020
[X＝CH 3，Y＝CH 3，Z＝SO 3H，n＝0-10]；其中R 6甲基所连接的碳可以为S构型或R构型；R 7取代基中，Y基团所连接的碳可以为S构型或R构型。
本发明胆汁酸衍生物进一步优选为，其中R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为α-OH或H，R 6为-(CH 2) bCH 3(b＝0-3)，R 7为
Figure PCTCN2020110851-appb-000021
[X＝CH 3，Y＝CH 2OH，Z＝SO 3H，n＝0-10]；其中R 6甲基所连接的碳可以为S构型或R构型；R 7取代基中，Y基团所连接的碳可以为S构型或R构型。
本发明胆汁酸衍生物进一步优选为，其中R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为α-OH或H，R 6为-(CH2) bCH 3(b＝0-3)，R 7为
Figure PCTCN2020110851-appb-000022
[X＝CH 3，Y＝CH 3，Z＝COOH,n＝0-10]；其中R 6甲基所连接的碳可以为S构型或R构型；R 7取代基中，Y基团所连接的碳可以为S构型或R构型。
本发明胆汁酸衍生物的一种方案为，如前述式(I)基本结构，其中R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 7为
Figure PCTCN2020110851-appb-000023
[X＝H或CH 3，Y＝CH 3或CH 2OH，Z＝COOH或SO 3H，n＝0-10]或OH或-O(CH 2) tCH 3(t＝0-3)，Y基团所连接的碳可以为S构型或R构型。
作为方案之一，R 7为
Figure PCTCN2020110851-appb-000024
[X＝H或CH 3，Y＝CH 3或CH 2OH，Z＝COOH或SO 3H，n＝0-10],Y基团所连接的碳可以为S构型或R构型。在此结构基础上，R 6可以为-(CH 2) bCH 3(b＝0-3)或H。在前述结构基础上，R 5可以为H或α-OH。
作为方案之一，R 7为OH或-O(CH 2) tCH 3(t＝0-3)，在此结构基础上，R 6为-(CH 2) bCH 3(b＝0-3)或H。在前述结构基础上，R 5为H或α-OH。
本发明进一步提供包含如上所述任一种或多种胆汁酸衍生物和适宜载体的组合物。
本发明进一步提供用于治疗和改善FXR或TGR5介导或导致的疾病和症状的组合物，其包含有效量的如上所述任一种或多种胆汁酸衍生物和适宜的载体。
所述有效量指日剂量的组合物包含如上所述任一种或多种胆汁酸衍生物50-500mg/kg患者体重。
所述适宜的载体指医药上所适用的辅料。
所述组合物为口服制剂，进一步优选为普通片剂、咀嚼片、分散片、颗粒剂、溶液、胶囊剂或混悬剂，进一步优选为肠溶制剂或肠溶缓释制剂。
本发明进一步提供如上任一所述的胆汁酸衍生物在制备治疗和改善FXR或TGR5介导或导致的疾病和症状的组合物中的应用之用途。
本发明进一步提供如上任一所述的胆汁酸衍生物在治疗和改善FXR或TGR5介导或导致的与肝脏损伤有关的疾病和症状的治疗用途。
进一步优选地，当用于上述用途时，可以任选地将所述胆汁酸衍生物与常规降糖降脂肪药物联合应用于该用途，所述常规降糖降脂肪药选自利拉鲁肽，艾塞那肽，阿必鲁泰。
当用于上述用途时，有效量的胆汁酸衍生物指如上所述任一种或多种胆汁酸衍生物50-500mg/kg患者体重。
以下对本申请中所使用名词进行说明：
所述FXR或TGR5介导或导致的疾病和症状包括以下疾病或症状：肝病、高脂质血症、高胆固醇血症、肥胖症、代谢综合症、心血管疾病、胃肠道疾病、动脉粥样硬化和肾病。所述FXR或TGR5介导或导致的与肝脏损伤有关的疾病和症状包括以下疾病或症状：单纯性脂肪肝、原发性胆汁性肝硬化、原发性硬化性胆管炎、肝纤维化、肝硬 化、非酒精性脂肪性肝炎和非酒精性脂肪性肝病及它们相关的肝脏损伤，进一步特别指单纯性脂肪肝、非酒精性脂肪性肝炎以及与它们相关的肝脏损伤。
本申请中描述的化合物包括其在药学上可接受的酸或碱加成盐和酯。当本发明化合物具有碱性基团时，药学上可接受的盐可由无机酸、有机酸或酸性氨基酸形成。当本发明化合物具有酸性基团时，可与金属、氨或有机胺或碱性氨基酸形成盐。
本发明化合物可呈现互变异构、构型异构、几何异构和立体异构。虽然本申请书仅呈现有限的异构形式，但本发明化合物应涵盖具有本申请所描述的效用中的一种或多种化合物的任何互变异构、构型异构、立体化学或几何异构的构型，以及这些不同形式的混合物。
本文所列举的组合涵盖任何和所有可能的子范围和子范围组合。特别地，以举例目的，1-3个原子的基团指具有1、2或3个原子的基团。0-3个原子的基团表示在上述范围之外，进一步包含一种情形是不具有该基团。
本发明所述的组合物中包含的适宜的载体指根据制药领域一般技术人员的认知，在药学上可接受的载体或赋形剂或填充剂或稀释剂或其他必辅料。组合物包含治疗上有效量的本发明所述的胆汁酸衍生物的一种或几种。组合物可以多种方式使用，如注射、口服、吸入、植入等方式。
本发明胆汁酸衍生物可依据本领域一般技术人员所知，在实施例中合成方案的指引下制备得到。
附图说明
图1为本发明正常人、非酒精性脂肪性肝病病人、非酒精性脂肪性肝炎病人，非酒精性脂肪性肝炎-早期肝纤维化病人，非酒精性脂肪性肝炎-晚期肝纤维化病人以及非酒精性脂肪性肝炎-肝硬化病人的平均猪胆酸含量图。
图2为本发明猪胆酸，猪去氧胆酸，以及合成的9种胆汁酸衍生物干预8周后，肝脏甘油三酯显著降低的示意图。
图3为本发明猪胆酸，猪去氧胆酸，以及合成的9种胆汁酸衍生物干预8周后，血清甘油三脂显著降低的示意图。
图4为本发明猪胆酸(HCA)、猪去氧胆酸(HDCA)以及合成的9种胆汁酸衍生物(50mg/kg)干预一周后，小鼠血糖水平的变化。
图5. 50μM胆汁酸及其衍生物能有效地促进肠内分泌细胞系NCI-H716细胞中的GLP-1的分泌。
图6.用猪胆酸及6种胆汁酸衍生物和19种其他胆汁酸衍生物处理NCI-H716和STC-1细胞，在50μM下处理48小时，发现通过TGR5和FXR的作用，猪胆酸及其衍生物比其他胆汁酸更有效地上调肠内分泌细胞系中的GLP-1蛋白表达。
具体实施方式
为使本发明的目的、技术方案及效果更加清楚、明确，以下参照附图并举实施例对本发明进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
制备实施例中所有试剂和材料均购自商业供应商。
实施例中所使用的化合物的代号及其结构式如下表所示。
表1.合成的猪胆酸衍生物结构
Figure PCTCN2020110851-appb-000025
Figure PCTCN2020110851-appb-000026
实施例1:D-丙氨猪胆酸的合成
Figure PCTCN2020110851-appb-000027
制备方法包含如下步骤：
Figure PCTCN2020110851-appb-000028
将猪胆酸(0.204g，0.5mmol)，D-苯丙氨酸苄酯对甲苯磺酸盐(0.185g，0.52mmol)和N,N-二异丙基乙基胺(0.194mg，1.5mmol)溶于5ml二甲基甲酰胺中，搅拌均匀。于室温下，一次性加入四甲基脲六氟磷酸酯(0.209g，0.55mmol)至反应液中，并于该温度下反应1h。薄层色谱监控反应完全后，加入10ml水，并用乙酸乙酯提取2次，有机相依次用1N氢氧化钠，1N盐酸和饱和食盐水洗涤，之后将有机相干燥浓缩得到D-丙氨猪胆酸苄酯。
将上述得到中间体溶于10mL甲醇中，加入25mg10％钯碳，于室温下催化加氢。反应结束后，过滤除去Pd/C，滤液减压浓缩得到D-丙氨猪胆酸粗品，经柱层析纯化后得到D-丙氨猪胆酸0.216g，两步收率90％。ESI-MS(m/z)：959.6(2M+H) +。 1HNMR(300MHz，DMSO)：δ0.6(s,3H),0.83(s,3H),0.88(d,3H),1.23(d,3H),3.13(m,1H),3.59(m,2H),3.89(s,1H),4.14(t,1H),8.01(d,1H).
实施例2:L-丙氨猪胆酸的合成
Figure PCTCN2020110851-appb-000029
操作过程同实施例1，只是将D-丙氨酸苄酯对甲苯磺酸盐用L-丙氨酸苄酯盐酸盐代替，得到L-丙氨猪胆酸0.164g，收率86.2％。ESI-MS(m/z)：959.6(2M+H) +。 1HNMR(300MHz，DMSO)：δ0.6(s,3H),0.83(s,3H),0.88(d,3H),1.23(d,3H),3.13(m,1H),3.59(m,2H),3.89(s,1H),4.14(t,3H),4.19(m,1H),4.32(m,1H),8.06(d,1H).
实施例3:L-丝氨猪胆酸的合成
Figure PCTCN2020110851-appb-000030
操作过程同实施例1，只是将D-丙氨酸苄酯对甲苯磺酸盐用L－丝氨酸苄酯盐酸盐代替，得到L-丝氨猪胆酸0.352g，收率93.6％。ESI-MS(m/z)：991.6(2M+H) +。 1HNMR(300MHz，DMSO)：δ0.6(s,3H),0.83(s,3H),0.88(d,3H),3.13(m,2H),3.59(m,4H),3.89(s,1H),4.20-4.28(m,2H),4.19(m,1H),4.32(m,1H),8.06(d,1H).
实施例4:D-丝氨猪胆酸的合成
Figure PCTCN2020110851-appb-000031
操作过程同实施例1，只是将D-丙氨酸苄酯对甲苯磺酸盐用D－丝氨酸苄酯盐酸盐代替，得到D-丝氨猪胆酸0.334g，收率89.2％。ESI-MS(m/z)：991.6(2M+H) +。 1HNMR(300MHz，DMSO)：δ0.6(s,3H),0.83(s,3H),0.88(d,3H),3.13(m,2H),3.59(m,4H),3.89(s,1H),4.20-4.28(m,2H),4.19(m,1H),4.32(m,1H),8.06(d,1H).
实施例5:(2R)-2-((4R)-4-((3R,6R,7S,10R,13R,17R)-3,6,7-三羟基-10,13-二甲基十六氢-1H-环戊[a]菲蒽-17-基)戊酰胺基)丙烷-1-磺酸((2R)-2-((4R)-4- ((3R,6R,7S,10R,13R,17R)-3,6,7-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)propane-1-sulfonic acid)
Figure PCTCN2020110851-appb-000032
制备方法包含如下步骤：
Figure PCTCN2020110851-appb-000033
将猪胆酸(0.31g，0.76mmol)、(R)-2-氨基丙磺酸(0.1g，0.77mmol)和N,N-二异丙基乙基胺(0.29mg,2.28mmol)溶于5ml二甲基甲酰胺中，搅拌均匀。室温下，一次性加入四甲基脲六氟磷酸酯(0.32g，0.84mmol)至反应液中，并于该温度下反应1h。薄层色谱监控反应完全后，浓缩除去二甲基甲酰胺，之后加入10ml水，并用乙酸乙酯提取2次，水相用1N盐酸调节pH值至1-2，之后将水相浓缩至干，得到粗品。柱层析纯化后得到(2R)-2-((4R)-4-((3R,6R,7S,10R,13R,17R)-3,6,7-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)propane-1-sulfonic acid产品276mg，收率68.6％。ESI-MS(m/z)：1059.7(2M+H) +。 1HNMR(300MHz，CD 3OD)：δ0.69(s,3H),0.95(s,3H),0.99(d,3H),1.32(d,3H),2.71(s,1H),2.8-3.1(qd,2H),3.79(m,2H),4.34(m,1H).
实施例6:(2S)-2-((4R)-4-((3R,6R,7S,10R,13R,17R)-3,6,7-三羟基-10,13-二甲基十六氢-1H-环戊[a]菲蒽-17-基)戊酰胺基)丙烷-1-磺酸((2S)-2-((4R)-4-((3R,6R,7S,10R,13R,17R)-3,6,7-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)propane-1-sulfonic acid)
Figure PCTCN2020110851-appb-000034
操作过程同实施例5，只是将(R)-2-氨基丙磺酸用(S)-2-氨基丙磺酸代替，得到(2S)-2-((4R)-4-((3R,6R,7S,10R,13R,17R)-3,6,7-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)propane-1-sulfonic acid产品292mg，收率72.6％。ESI-MS(m/z)：1059.7(2M+H) +。 1HNMR(300MHz，CD 3OD)：δ0.69(s,3H),0.95(s,3H),0.99(d,3H),1.32(d,3H),2.8-3.1(qd,2H),3.79(m,2H),4.37(m,1H).
实施例7:N-甲基牛磺猪胆酸的合成
Figure PCTCN2020110851-appb-000035
操作过程同实施例5，只是将(R)-2-氨基丙磺酸用N-甲基牛磺酸代替，得到N-甲基牛磺猪胆酸164mg，收率40.8％。ESI-MS(m/z)：1059.7(2M+H) +。 1HNMR(300MHz，CD 3OD)：δ0.69(s,3H),0.93(s,3H),0.99(d,3H),1.3(s,3H),2.70(m,1H),2.93(m,1H),2.95-3.12(m,2H),3.13(m,1H),3.76(m,4H).
实施例8:N-甲基甘氨猪胆酸的合成
Figure PCTCN2020110851-appb-000036
制备方法包含如下步骤：
Figure PCTCN2020110851-appb-000037
将猪胆酸(0.31g，0.76mmol)，N-甲基甘氨酸乙酯盐酸盐(0.117g，0.8mmol)和N,N-二异丙基乙基胺(0.29g，2.28mmol)溶于5ml二甲基甲酰胺中，搅拌均匀。于室温下，一次性加入四甲基脲六氟磷酸酯(0.32g，0.836mmol)，并于该温度下反应1h。薄层色谱监控反应完全后，加入10ml水，并用乙酸乙酯提取2次，有机相依次用1N氢氧化钠，1N盐酸和饱和食盐水洗涤，之后将有机相干燥浓缩得到中间体N-甲基甘氨猪胆酸乙酯。
将上述中间体溶于10mL甲醇/水(4/1v/v)中，加入氢氧化钾(66mg)，于室温下水解。反应结束后，减压浓缩除去溶剂甲醇，残留物用5ml水稀释，并用1N盐酸调节pH至1-2，用乙酸乙酯提取2次，合并有机相，干燥浓缩得到N-甲基甘氨猪胆酸263mg，收率72.2％。ESI-MS(m/z)：959.6(2M+H) +。 1HNMR(300MHz，DMSO)：δ0.6(s,3H),0.83(s,3H),0.88(d,3H),1.31(s,3H),3.13(m,1H),3.59(m,2H),3.89(s,1H),4.19(m,1H),4.32(m,1H).
实施例9:(S)-23-甲基猪胆酸的合成
Figure PCTCN2020110851-appb-000038
制备方法包含如下步骤：
Figure PCTCN2020110851-appb-000039
将猪胆酸(1.632g，4mmol)溶于30mL甲醇中，加入3滴浓硫酸催化，室温下过夜反应。薄层色谱监控反应完全后，减压浓缩除去溶剂甲醇，乙酸乙酯溶解后，依次用饱和碳酸氢钠和食盐水洗涤。有机相干燥浓缩后得到猪胆酸甲酯(H1)1.69g。
将猪胆酸甲酯(1.69g，4mmol)和2,6-二甲基吡啶(4.29g，40mmol)溶于二氯甲烷中，氮气保护，降温至0-5℃，向反应液中滴加叔丁基二甲硅基三氟甲磺酸酯(2.8ml)，滴毕后，于室温下反应。薄层色谱监控反应完全后，将反应液快速柱层析得到中间体(H2)3.1g。
上述中间体H2和HMPA(4.35g,24mmol)加入无水四氢呋喃中，搅拌均匀，氮气保护下降温至－78℃。在该温度下反应30min后，向反应液中缓慢滴加碘甲烷(5.7g，40mmol)，滴毕后在该温度下继续反应1h，自然升温至室温过夜反应。薄层色谱反应完 全后，用饱和氯化铵溶液淬灭反应，乙酸乙酯萃取2次，合并有机相，并用饱和食盐水洗涤一次，有机相干燥浓缩得到残余物，用柱层析纯化得到中间体(H3)2.24g，三步收率71.8％。
将中间体(H3)溶于甲醇(20ml)中，加入4滴浓盐酸催化，室温下脱除TBS保护基。反应完全后减压浓缩除去溶剂甲醇。残余物用四氢呋喃/H 2O(4:1)10ml溶解，加入氢氧化钠(0.34g，8.6mmol)，室温下反应，水解完全后，乙酸乙酯提取2次，水相用1N盐酸调节pH值至1-2，乙酸乙酯萃取3次，合并有机相，干燥浓缩得到(S)-23-甲基猪胆酸和(R)-23-甲基猪胆酸的混合物，柱层析分离上述非对映异构体，得到(S)-23-甲基猪胆酸331mg，两步收率27.1％。ESI-MS(m/z)：959.6(2M+H) +. 1HNMR(300MHz,CD 3OD):0.68(S,3H),0.93(S,3H),0.98(d,3H),1.12(d,3H),2.57(m,1H),3.58(m,1H),3.78(m,2H).
实施例10:猪胆酸浓度在脂肪性肝病病人中显著降低
本发明中的试验样本得到本地伦理委员会的批准并获得所有受试者的知情同意书。本发明实施例1共入组200名受试者，使用超高效液相色谱串联质谱技术分别检测了经肝穿刺确诊的25例健康人和175例经肝穿刺活检证实的脂肪肝病人(包括单纯性脂肪胺，脂肪性肝炎，脂肪性肝炎伴随早期肝纤维化，脂肪性肝炎伴随晚期肝纤维化，以及脂肪性肝炎伴随肝硬化病人)血清标本中胆酸，氨基酸以及脂肪酸等代谢物的含量，以及相应临床指标的检测。检测结果发现猪胆酸在脂肪性肝病病人中显著降低(图1)。
实施例11：如图2和3所示，对于猪胆酸、猪去氧胆酸、以及合成的猪胆酸衍生物(表1)明显改善由高脂引起的小鼠血清高血脂，我们采用高脂诱导的肥胖小鼠模型，给予高脂饮食(HFD)的同时按50mg/Kg/day的剂量灌胃分别给予猪胆酸、猪去氧胆酸，持续8周时间。发现给猪胆酸、猪去氧胆酸以及合成猪胆酸衍生物组小鼠的甘油三酯水平在8周后均分别显著低于单纯的高脂饮食组小鼠，小鼠体内猪胆酸和猪去氧胆酸的升高能有效改善由高脂引起的小鼠血脂异常的作用。
实施例12：将猪胆汁酸系列以及合成的猪胆酸衍生物(表1)(50mg/kg/day)给C57BL/6J小鼠口服。如图4所示，干预一周后结果显示，所有干预组的血糖都显著降低。
实施例13:培养NCI-H716细胞，用50μM猪胆酸和猪胆酸衍生物，以及一个已经报道的TGR5的激动剂INT-777处理，测定细胞培养液中GLP-1的水平，发现所有化合物均能有效促进GLP-1的释放(图5)，并且化合物ZN-1-102-1对于GLP-1的释放能力优于猪胆酸和现有的TGR5激动剂INT-777。
实施例14:用50μM猪胆酸，猪去氧胆酸，牛磺猪去氧胆酸，甘氨猪去氧胆酸，牛磺猪胆酸，甘氨猪胆酸和19种其他胆汁酸处理NCI-H716和STC-1细胞48小时，发现通过TGR5和FXR的作用，猪胆酸及其衍生物比其他胆汁酸更有效地上调肠内分泌细胞系中的GLP-1蛋白表达(图6)。(a)使用实时PCR测量GLP-1转录，见图6(a)。(b)使用ELISA测量GLP-1分泌,见图6(b)。(c)NCI-H716和STC-1以及它们的TGR5敲低细胞用6种猪胆酸处理24小时，并使用蛋白质印迹测定细胞内GLP-1，p-CREB和总CREB。见图6(c)。(d)用50μM鹅去氧胆酸或5β-胆酸在有猪胆酸以及没有猪胆酸的情况下处理NCI-H716细胞24小时后的核和胞质级分中的FXR蛋白浓度。见图6(d)。*p<0.05，与对照相比。
可以理解的是，对本领域普通技人员来说，可以根据本发明的技术方案及其发明构思加以等同替换或改变，而所有这些改变或替换都应属于本发明所附的权利要求的保护范围。

Claims (37)

  1. 一种胆汁酸衍生物，为如下式(I)结构及其立体异构体、盐或酯，
    Figure PCTCN2020110851-appb-100001
    其中
    R 1为α-OH或β-O(CH 2) aOH，其中a＝1-10，
    R 2为α-OH或H或CH 2OH，
    R 3为α-OH或H或β-OH或CH 3，
    R 4为H或CH 3，
    R 5为α-OH或H，
    R 6为H或-(CH 2) bCH 3，其中b＝0-3，
    R 7为
    Figure PCTCN2020110851-appb-100002
    其中X＝H或CH 3，Y＝CH 3或CH 2OH，Z＝COOH或SO 3H，n＝0-10，或R 7为OH或-O(CH 2) tCH 3,其中t＝0-3，
    其中R 6甲基所连接的碳可以是S构型或R构型；R 7取代基中，Y基团所连接的碳可以是S构型或R构型。
  2. 如权利要求1所述胆汁酸衍生物，其特征在于，R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H。
  3. 如权利要求1所述胆汁酸衍生物，其特征在于，R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为H。
  4. 如权利要求3所述胆汁酸衍生物，其特征在于，R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为H，R 6为H。
  5. 如权利要求3所述胆汁酸衍生物，其特征在于，R 1为α-OH，R 2为α-OH，R 3为α-OH，R 4为H，R 5为H，R 6为-(CH 2) bCH 3，其中b＝0-3，其中R 6甲基所连接的碳可以是S构型或R构型。
  6. 如权利要求3所述胆汁酸衍生物，其特征在于，R 7为OH或-O(CH 2) tCH 3，其中t＝0-3。
  7. 如权利要求6所述胆汁酸衍生物，其特征在于，R 7为OH。
  8. 如权利要求6所述胆汁酸衍生物，其特征在于，R 7为-O(CH 2) tCH 3，其中t＝0-3。
  9. 如权利要去3所述胆汁酸衍生物，其特征在于，R 7为
    Figure PCTCN2020110851-appb-100003
    其中X＝H，Y＝CH 2OH，Z＝COOH，n＝0-10；Y基团所连接的碳可以为S构型或R构型。
  10. 如权利要求3所述胆汁酸衍生物，其特征在于，R 7为
    Figure PCTCN2020110851-appb-100004
    其中X＝H，Y＝CH 3，Z＝SO 3H，n＝0-10；Y基团所连接的碳可以为S构型或R构型。
  11. 如权利要求3所述胆汁酸衍生物，其特征在于，R 7为
    Figure PCTCN2020110851-appb-100005
    其中X＝H，Y＝CH 2OH，Z＝SO 3H，n＝0-10；Y基团所连接的碳可以为S构型或R构型。
  12. 如权利要求3所述胆汁酸衍生物，其特征在于，R 7为
    Figure PCTCN2020110851-appb-100006
    其中X＝H，Y＝CH 3，Z＝COOH,n＝0-10，Y基团所连接的碳可以为S构型或R构型。
  13. 如权利要求2所述胆汁酸衍生物，其特征在于，其中R 6为-(CH 2) bCH 3，其中b＝0-3，R 7为
    Figure PCTCN2020110851-appb-100007
    其中X＝H，Y＝CH 2OH，Z＝COOH，n＝0-10；其中R 6甲基所连接的碳可以为S构型或R构型；R 7取代基中，Y基团所连接的碳可以为S构型或R构型。
  14. 如权利要求2所述胆汁酸衍生物，其特征在于，其中R 6为-(CH 2) bCH 3，其中b＝0-3，R 7 为
    Figure PCTCN2020110851-appb-100008
    其中X＝H，Y＝CH 3，Z＝SO 3H，n＝0-10；其中R 6甲基所连接的碳可以为S构型或R构型；R 7取代基中，Y基团所连接的碳可以为S构型或R构型。
  15. 如权利要求2所述胆汁酸衍生物，其特征在于，其中R 6为-(CH 2) bCH 3，其中b＝0-3，R 7为
    Figure PCTCN2020110851-appb-100009
    其中，X＝H，Y＝CH 2OH，Z＝SO 3H，n＝0-10；其中R 6甲基所连接的碳可以为S构型或R构型；R 7取代基中，Y基团所连接的碳可以为S构型或R构型。
  16. 如权利要求2所述胆汁酸衍生物，其特征在于，其中R 6为-(CH 2) bCH 3，其中b＝0-3，R 7为
    Figure PCTCN2020110851-appb-100010
    其中，X＝H，Y＝CH 3，Z＝COOH,n＝0-10；其中R 6甲基所连接的碳可以为S构型或R构型；R 7取代基中，Y基团所连接的碳可以为S构型或R构型。
  17. 如权利要求3所述胆汁酸衍生物，其特征在于，R 7为
    Figure PCTCN2020110851-appb-100011
    其中X＝CH 3，Y＝CH 2OH，Z＝COOH，n＝0-10，Y基团所连接的碳可以为S构型或R构型。
  18. 如权利要求4所述胆汁酸衍生物，其特征在于，R 7为
    Figure PCTCN2020110851-appb-100012
    其中X＝CH 3，Y＝CH 3，Z＝SO 3H，n＝0-10，Y基团所连接的碳可以为S构型或R构型。
  19. 如权利要求3所述胆汁酸衍生物，其特征在于，R 7为
    Figure PCTCN2020110851-appb-100013
    其中，X＝CH 3，Y＝CH 2OH，Z＝SO 3H，n＝0-10，Y基团所连接的碳可以为S构型或R构型。
  20. 如权利要求3所述胆汁酸衍生物，其特征在于，R 7为
    Figure PCTCN2020110851-appb-100014
    其中，X＝CH 3，Y＝CH 3，Z＝COOH，n＝0-10，Y基团所连接的碳可以为S构型或R构型。
  21. 如权利要求3所述胆汁酸衍生物，其特征在于，R 6为-(CH 2) bCH 3，其中b＝0-3，R 7为
    Figure PCTCN2020110851-appb-100015
    其中，X＝CH 3，Y＝CH 2OH，Z＝COOH，n＝0-10；其中R 6甲基所连接的碳可以为S构型或R构型；R 7取代基中，Y基团所连接的碳可以为S构型或R构型。
  22. 如权利要求3所述胆汁酸衍生物，其特征在于，R 6为-(CH 2) bCH 3，其中b＝0-3，R 7为
    Figure PCTCN2020110851-appb-100016
    其中X＝CH 3，Y＝CH 3，Z＝SO 3H，n＝0-10；其中R 6甲基所连接的碳可以为S构型或R构型；R 7取代基中，Y基团所连接的碳可以为S构型或R构型。
  23. 如权利要求3所述胆汁酸衍生物，其特征在于，R 6为-(CH 2) bCH 3，其中b＝0-3，R 7为
    Figure PCTCN2020110851-appb-100017
    其中X＝CH 3，Y＝CH 2OH，Z＝SO 3H，n＝0-10；其中R 6甲基所连接的碳可以为S构型或R构型；R 7取代基中，Y基团所连接的碳可以为S构型或R构型。
  24. 如权利要求3所述胆汁酸衍生物，其特征在于，R 6为-(CH 2) bCH 3，其中b＝0-3，R 7为
    Figure PCTCN2020110851-appb-100018
    其中X＝CH 3，Y＝CH 3，Z＝COOH,n＝0-10；其中R 6甲基所连接的碳可以为S构型或R构型；R 7取代基中，Y基团所连接的碳可以为S构型或R构型。
  25. 如权利要求2所述胆汁酸衍生物，其特征在于，R 7为
    Figure PCTCN2020110851-appb-100019
    其中X＝H或CH 3，Y＝CH 3或CH 2OH，Z＝COOH或SO 3H，n＝0-10，或Y＝OH或-O(CH 2) tCH 3，其中t＝0-3；Y基团所连接的碳可以为S构型或R构型。
  26. 如权利要求25所述胆汁酸衍生物，其特征在于，R 7为
    Figure PCTCN2020110851-appb-100020
    其中X＝H或CH 3，Y＝CH 3或CH 2OH，Z＝COOH或SO 3H，n＝0-10，，Y基团所连接的碳可以为S构型或R构型。
  27. 如权利要求25所述胆汁酸衍生物，其特征在于，R 7为OH。
  28. 如权利要求25所述胆汁酸衍生物，其特征在于，R 7为-O(CH 2) tCH 3，其中t＝0-3。
  29. 如权利要求26至28任一所述胆汁酸衍生物，其特征在于，R 6为-(CH 2) bCH 3，其中b＝0-3，或R 6为H。
  30. 如权利要求26至28任一所述胆汁酸衍生物，其特征在于，R 5为α-OH或H。
  31. 一种用于治疗和改善FXR或TGR5介导或导致的疾病和症状的组合物，其包含有效量的权利要求1至30任一所述的胆汁酸衍生物和适宜的载体；其有效量指日剂量的组合物包含权利要求1至30任一所述的胆汁酸衍生物50-500mg/kg患者体重；所述适宜的载体指医药上所适用的辅料。
  32. 如权利要求30所述的用于治疗和改善FXR或TGR5介导或导致的疾病和症状的组合物，所述组合物为口服制剂。
  33. 如权利要求30所述的用于治疗和改善FXR或TGR5介导或导致的疾病和症状的组合物，所述FXR或TGR5介导或导致的疾病和症状包括以下疾病或症状：肝病、高脂质血症、高胆固醇血症、肥胖症、代谢综合症、心血管疾病、胃肠道疾病、动脉粥样硬化和肾病，其中所述肝病包括单纯性脂肪肝、原发性胆汁性肝硬化、原发性硬化性胆管炎、肝纤维化、肝硬化、非酒精性脂肪性肝炎和非酒精性脂肪性肝病及它们相关的肝脏损伤。
  34. 权利要求1至30任一所述的胆汁酸衍生物在制备治疗和改善FXR或TGR5介导或导致的疾病和症状的组合物中的应用。
  35. 权利要求1至30任一所述的胆汁酸衍生物用于治疗和改善FXR或TGR5介导或导致的疾病或症状的治疗方法，包括向具有所述疾病或症状的患者施用如权利要求1至19所述胆汁酸衍生物，施用剂量为日剂量50-500mg/kg患者体重。
  36. 如权利要求35所述的治疗方法，所述FXR或TGR5介导或导致的疾病和症状包括以下疾病或症状：肝病、高脂质血症、高胆固醇血症、肥胖症、代谢综合症、心血管疾病、胃肠道疾病、动脉粥样硬化和肾病，其中所述肝病包括单纯性脂肪肝、原发性胆汁性肝硬化、原发性硬化性胆管炎、肝纤维化、肝硬化、非酒精性脂肪性肝炎和非酒精性脂肪性肝病及它们相关的肝脏损伤。
  37. 如权利要求35所述的治疗方法，可以将所述胆汁酸衍生物与常规降糖降脂肪药物联合施用于患者，其中所述常规降糖降脂肪药选自利拉鲁肽、艾塞那肽、阿必鲁泰之一种或几种。
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