CN106632294A

CN106632294A - Spiro compound and medicinal use thereof

Info

Publication number: CN106632294A
Application number: CN201611159261.3A
Authority: CN
Inventors: 李小川
Original assignee: Ningbo Naxi Pharmaceutical Co Ltd
Current assignee: Ningbo Naxi Pharmaceutical Co Ltd
Priority date: 2016-12-15
Filing date: 2016-12-15
Publication date: 2017-05-10

Abstract

The invention relates to a spiro compound serving as a FXR receptor agonist, whose chemical structure is shown in type I, pharmaceutically acceptable salts of the spiro compound, or the enantiomers, diastereomers, tautomers, racemates, solvates, N-oxide or amino acid conjugates and the pharmaceutical compositions of the spiro compound, and the use in preparing drugs for treatment of diseases mediated by the FXR receptor.

Description

Spiro compound and pharmaceutical application thereof

Technical Field

The invention belongs to the technical field of organic chemistry, and particularly relates to a spiro compound and pharmaceutical application thereof.

Background

Recent studies have shown that Farnesoid X Receptor (FXR) agonists have anti-cholestatic, anti-fibrotic effects. FXR is a nuclear receptor, a sensor of bile acids, regulates the synthesis of bile acids and the flow of bile in the liver, and has effects on bile homeostasis, lipid metabolism, glycometabolism, and inflammatory/immune responses.

Research data show that 6-alpha-ethyl chenodeoxycholic acid (6-ECDCA, OCA) has 100 times of activation effect on FXR compared with chenodeoxycholic acid, and clinical studies show that OCA can be used for treating diseases related to Bile secretion (DrrugDiscovery today. volume 17, Numbers 17/18, 2012) such as Primary Biliary Cirrhosis (PBC), Portal hypertension (Portal hypertension), nonalcoholic steatohepatitis (NASH), Bile acid diarrhea (Bile acid diarrhea), alcoholic hepatitis, Primary Sclerosing Cholangitis (PSC).

However, the cholic acid compound OCA has been clinically manifested as side effects: one is that the side effects of pruritus were severe, with 33 (23%) of the 141 patients in OCA group experiencing pruritus, and 9 (6%) of the 142 patients in placebo group experiencing pruritus (P < 0.0001); secondly, the increase of low density lipoprotein cholesterol, which may cause some unacceptable side effects.

Therefore, development of a non-bile acid small-molecule FXR agonist is likely to avoid side effects such as pruritus and increase in low-density lipoprotein of OCA while maintaining drug efficacy.

A large body of literature has reported various FXR small molecule agonists: WO2000037077, WO2008025539, bioorg.med.chem.lett.19(2009) 2595-.

Although these FXR agonists have shown some agonist activity, each of these compounds has some drawbacks, such as the FXR agonist GW4064, which has low bioavailability and short half-life; the trans-stilbene in the structure is a potential toxic group (toxicoly, 1981, 22(2), 149-160; Toxicol appl. Pharmacol.2000, 167(1), 46-54); the olefinic bond in trans-stilbene is not stable to uv light and thus also presents potential toxicity problems. Therefore, there is a need to develop novel FXR agonists with improved drug properties. The drug property of the spiro compound provided by the invention is generally superior to that of GW4064, and the chemical structural formula of the spiro compound is shown as follows.

Disclosure of Invention

The invention aims to overcome the defect of the existing FXR agonist in the aspect of pharmacy, provide a novel compound capable of improving pharmacy properties, and is more suitable for treating, inhibiting or improving farnesoid X receptor-mediated diseases.

In order to solve the technical problems, the invention adopts the following technical scheme: a compound of formula (I), a pharmaceutically acceptable salt thereof, or an enantiomer, diastereomer, tautomer, racemate, solvate, N-oxide, or amino acid conjugate thereof:

wherein,

R₁selected from 5-10 membered aromatic ring group or 5-10 membered heteroaromatic ring group, wherein each group can be substituted with 1-3R₄Substitution;

R₂selected from hydrogen, C1-3 alkyl, haloC 1-3 alkyl or C3-6 cycloalkyl, wherein C3-6 cycloalkyl may be substituted by C1-3 alkyl, haloC 1-3 alkyl;

z is selected from the group consisting of 5-10 membered aromatic ring group or 5-10 membered heteroaromatic ring group, wherein each group may be substituted with 1-3R₄Substitution;

R₃is selected from-CO₂R₅、-CONR₅R₆、-CONR₅SO₂R₆、-CONR₅(CR₇)_1-4CO₂R₅、-SO₂R₅Or tetrazole;

R₄selected from halogen, C1-6 alkyl, haloC 1-6 alkyl, C1-6 alkoxy, haloC 1-6 alkoxy or C3-6 cycloalkyl;

R₅、R₆、R₇independently selected from hydrogen, C1-6 alkyl, haloC 1-6 alkyl, C3-6 cycloalkyl or haloC 3-6 cycloalkyl.

According to the invention, R₂Preferably cyclopropyl.

According to the invention, R₁Preferably by 1-3R₄Substituted phenyl; wherein R is₄Selected from halogen, C1-6 alkyl, haloC 1-6 alkyl, C1-6 alkoxy, haloC 1-6 alkoxy or C3-6 cycloalkyl.

According to the invention, Z is preferably phenyl, pyridyl, pyrazinyl, pyrimidinyl, furanyl, oxazolyl, thienyl, thiazolyl, pyrazole, benzothienyl,Benzofuranyl, benzimidazolyl, imid [1, 2-a ]]Pyridyl, pyrrole [1, 2-b ]]Pyrazinyl, indolyl, benzothiazolyl, benzisothiazolyl, benzoxazolyl, naphthyl, quinolinyl, isoquinolinyl, wherein each radical may be substituted by 1-3R₄Substitution; said R₄Selected from halogen, C1-6 alkyl, haloC 1-6 alkyl, C1-6 alkoxy, haloC 1-6 alkoxy or C3-6 cycloalkyl.

According to the invention, R₃preferably-CO₂R₅Wherein R is₅Selected from hydrogen or C1-6 alkyl, more preferably, R₅Independently selected from hydrogen.

Typical compounds according to the invention are exemplified by the following:

3- (5- ((5-cyclopropyl-3- (2, 6-dichlorophenyl) isoxazole-4-position) methoxy) hexahydrocyclopenta [ c ] pyrrole-2 (1H) -position) benzoic acid

4- (5- ((5-cyclopropyl-3- (2, 6-dichlorophenyl) isoxazole-4-position) methoxy) hexahydrocyclopenta [ c ] pyrrole-2 (1H) -position) benzoic acid

6- (5- ((5-cyclopropyl-3- (2, 6-dichlorophenyl) isoxazole-4-position) methoxy) hexahydrocyclopenta [ c ] pyrrole-2 (1H) -position) nicotinic acid

2- (5- ((5-cyclopropyl-3- (2, 6-dichlorophenyl) isoxazole-4-position) methoxy) hexahydrocyclopenta [ c ] pyrrole-2 (1H) -position) benzo [ d ] thiazole-6-carboxylic acid

5- (5- ((5-cyclopropyl-3- (2, 6-dichlorophenyl) isoxazol-4-yl) methoxy) hexahydrocyclopenta [ c ] pyrrole-2 (1H) -yl) -1-isopropyl-1H-pyrazole-3-carboxylic acid

5- (5- ((5-cyclopropyl-3- (2, 6-dichlorophenyl) isoxazol-4-position) methoxy) hexahydrocyclopenta [ c ] pyrrole-2 (1H) -position) -1-naphthoic acid

6- (5- ((5-cyclopropyl-3- (2, 6-dichlorophenyl) isoxazol-4-yl) methoxy) hexahydrocyclopenta [ c ] pyrrole-2 (1H) -yl) -1-methyl-1H-indole-2-carboxylic acid

And pharmaceutically acceptable salts thereof, or enantiomers, diastereomers, tautomers, racemates, solvates, N-oxides or amino acid conjugates thereof.

The invention further provides glycine, taurine or acylglucuronide conjugates of the compounds.

The invention further provides application of the compound, the pharmaceutically acceptable salt thereof, or one or more of enantiomers, diastereoisomers, tautomers, racemates and solvates of the compound and the pharmaceutically acceptable salt thereof in preparing a bile regulation secretion regulator or an FXR nuclear receptor activity regulator.

The invention further provides application of the compound, the pharmaceutically acceptable salt thereof, or one or more of enantiomers, diastereoisomers, tautomers, racemates and solvates of the compound and the pharmaceutically acceptable salt thereof in preparing a medicament for preventing and/or treating cholesteryl cholelithiasis, primary biliary cirrhosis, portal hypertension, nonalcoholic steatohepatitis, bile acid diarrhea, alcoholic hepatitis, primary sclerosing cholangitis or atherosclerosis, or diseases related to bile secretion.

The present invention further provides a pharmaceutical composition comprising a first therapeutic agent and a pharmaceutically acceptable matrix, wherein the first therapeutic agent is a compound selected from the compounds described herein, or a pharmaceutically acceptable salt thereof, or a combination of one or more of their enantiomers, diastereomers, tautomers, racemates and solvates.

Further, the pharmaceutical composition may further comprise a second therapeutic agent, which is a combination of one or more selected from the group consisting of drugs for treating cholestasis and the like.

The present invention also provides a method for preventing and/or treating cholesterolemia, primary biliary cirrhosis, portal hypertension, nonalcoholic steatohepatitis, bile acid diarrhea, alcoholic hepatitis, primary sclerosing cholangitis, or atherosclerosis, or a disease associated with bile secretion, using the composition.

In the present invention, terms used in the present application, including the specification and claims, are defined as follows, if not otherwise specified. It must be noted that, in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Conventional methods of mass spectrometry, nuclear magnetism, HPLC, protein chemistry, biochemistry, recombinant DNA technology and pharmacology are used, if not otherwise stated.

The term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon group containing 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms; C1-Cn alkyl represents a saturated aliphatic hydrocarbon group of 1-n carbon atoms, including straight chain and branched chain groups (e.g., "C1-C10 alkyl" means that the group is alkyl, and the number of carbon chain carbon atoms of the alkyl group is 1-10, i.e., 1, 2, or 3 carbon atoms, etc., up to and including 10 carbon atoms, and the limitation of 1-10 does not include the number of substituted carbon atoms on the alkyl group, such as "alkyl" in substituted alkylamino, and when the number of carbon atoms is not particularly limited, only the number of carbon atoms of the alkyl moiety indicated therein is 1-10, but does not include the number of carbon atoms of the substituent on the alkyl group and the number of carbon atoms of other substituents on the amino group.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "membered ring" includes any cyclic structure. The term "element" is intended to mean the number of backbone atoms constituting a ring. Thus, for example, cyclohexyl, pyridyl, pyranyl and thiopyranyl are six-membered rings; cyclopentyl, pyrrolyl, furanyl and thienyl are five-membered rings.

The term "optionally substituted" or "substituted" means that the reference group may be substituted with one or more additional groups individually and independently selected from one or more of the following groups: C1-C10 alkyl, C3-C20 cycloalkyl, C5-C10 aryl, C5-C10 heteroaryl, C2-C20 heteroalicyclic hydrocarbon, hydroxyl, C1-C5 alkoxy, alkylthio, arylthio, alkylsulfinyl, arylsulfonyl, alkylsulfonyl, arylsulfonyl, cyano, halo, carbonyl, thiocarbonyl, nitro, haloalkyl, fluoroalkyl or amino (including mono-substituted and di-substituted amino groups and protected derivatives thereof), and when the substituent groups are multiple, the substituent groups are the same or different. For example, the optional substitution may be halide, -CN, -NO2, or LsRs, wherein each Ls is independently selected from the following bonds: -O-, -C (═ O) O-, -S (═ O)2-, -NH-, -NHC (═ O) -, -C (═ O) NH-, S (═ O)2NH-, -NHs (═ O)2, -OC (═ O) NH-, -NHC (═ O) O-, or- (C1 to C10 alkyl); each Rs is selected from hydrogen, alkyl, fluoroalkyl, heteroalkyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl. Reference may be made to Greene and Wuts for protecting groups that may form protected derivatives of the above substituents. In one aspect, the optional substituents are selected from halogen, trifluoromethyl, hydroxy, cyano, nitro, -SO3H, -SO2NH2, -SO2Me, -NH2, -COOH, -CONH2, C1-C5 alkoxy, -N (CH3)2, and C1-C10 alkyl.

In certain embodiments, the compounds have one or more stereogenic centers, and each center independently exists in R or S form. The compounds mentioned herein include all diastereomers, enantiomers, epimers and mixtures thereof. Stereoisomers can be obtained by methods such as separation of stereoisomers by chiral chromatography columns.

The methods and formulae described herein include the use of N-oxides (if appropriate), crystalline forms (also known as polymorphs) or pharmaceutically acceptable salts of the compounds of formula I, and active metabolites of these compounds having the same activity. In some cases, the compounds may exist as tautomers. All tautomers are included within the scope of the compounds mentioned herein. In a particular embodiment, the compound is present in the form of a solvate, a pharmaceutically acceptable solvent such as water or ethanol, and the like. In other embodiments, the compounds are present in unsolvated forms.

The term "prodrug" refers to a biotransformed derivative of a drug molecule that undergoes enzymatic or chemical transformation in vivo to release the active parent drug, which then exerts the desired pharmacological effect. There are other descriptions of prodrugs, such as the Nat Rev Drug discovery, 2008 Mar; 7 (3): 255-70, by j.

The term "salt" as used herein refers to a cation and anion containing compound that can be produced by protonation of an acceptable proton site and/or deprotonation of an available proton site. Notably, protonation of the acceptable proton sites results in the formation of cationic species whose charge is balanced by the presence of physiological anions, while deprotonation of the available proton sites results in the formation of anionic species whose charge is balanced by the presence of physiological cations.

The term "pharmaceutically acceptable salt" means that the salt is pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to: (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as glycolic acid, pyruvic acid, lactic acid, malonic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1, 2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-p-toluenesulfonic acid, camphoric acid, dodecylsulfuric acid, gluconic acid, glutamic acid, salicylic acid, cis-hexadiene diacid, and the like; or (2) a base addition salt, and a conjugate base of any of the above inorganic acids, wherein the conjugate base comprises a base selected from the group consisting of Na +, K +, Mg²⁺、Ca²⁺NHgR 4-g +, wherein R' "is C1-3 alkyl and g is an integer selected from 0, 1, 2, 3, or 4. It is to be understood that all references to pharmaceutically acceptable salts include the solvent addition forms (solvates) or crystal forms (polymorphs) as defined herein of the same acid addition salt.

The terms "formulation" or "dosage form" refer specifically to solid and liquid formulations comprising the active compound and one skilled in the art will appreciate that the active ingredient may exist in different formulations depending on the desired dosage and pharmacokinetic parameters.

The term "acceptable", as used herein, means that a prescribed component or active ingredient does not unduly adversely affect the health of the general therapeutic target.

The term "subject" or "patient" includes mammals and non-mammals. Mammals include, but are not limited to, mammals: human, non-human primates such as orangutans, apes, and monkeys; agricultural animals such as cattle, horses, goats, sheep, pigs; domestic animals such as rabbits, dogs; the experimental animals include rodents, such as rats, mice, guinea pigs and the like. Non-mammalian animals include, but are not limited to, birds, fish, and the like. In a preferred embodiment, the mammal of choice is a human.

"Nuclear receptor" refers to a receptor that, typically in conjunction with other transcription factors, activates or inhibits transcription of one or more genes in the nucleus (but may also have a second messenger signaling effect). Nuclear receptors are activated by the natural cognate ligand for the receptor. Nuclear receptors are usually found in the cytoplasm or nucleus, rather than membrane-bound.

Farnesoid X Receptor (FXR), a member of the nuclear receptor superfamily, is mainly expressed in the intestinal system and is involved in important links such as bile acid metabolism and cholesterol metabolism. In natural environment, the ligand comprises primary bile acid chenodeoxycholic acid, secondary cholic acid lithocholic acid, deoxycholic acid and the like.

"therapeutically effective amount" refers to an amount of a compound that, when administered to a subject, is sufficient to effectively treat a disease or condition described herein. Although the amount of a compound that constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, and the age of the subject to be treated, it can be determined in a routine manner by one skilled in the art.

"modulating" refers to treating, preventing, inhibiting, enhancing or inducing a function, condition or disorder.

As used herein, "treating" encompasses treating a disease or disorder described herein in a subject (preferably a human) and includes:

i. inhibiting the disease or disorder, i.e., arresting its development; or

Alleviating the disease or condition, i.e., causing regression of the condition.

By "subject" is meant a warm-blooded animal, such as a mammal, preferably a human or a human child, that has or may have one or more of the diseases and disorders described herein.

"biliary cirrhosis" refers to cirrhosis caused by biliary obstruction and cholestasis, and is divided into Primary Biliary Cirrhosis (PBC) and secondary biliary cirrhosis. Primary biliary cirrhosis is generally considered to be an autoimmune disease.

"portal hypertension" refers to a group of symptoms caused by persistent increases in portal pressure. Most are caused by cirrhosis, and a few are secondary to obstruction of the portal trunk or hepatic veins and other factors of unknown cause. Portal vein pressure increases when portal vein blood fails to flow back through the liver into the inferior vena cava.

"non-alcoholic fatty liver disease" refers to a metabolic stress-induced liver injury closely associated with Insulin Resistance (IR) and genetic predisposition, with pathological changes similar to Alcoholic Liver Disease (ALD), but without a history of excessive alcohol consumption by patients, and a disease spectrum including non-alcoholic simple fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), and associated cirrhosis and hepatocellular carcinoma.

"bile acid diarrhea" refers to diarrhea caused by the arrival of large amounts of bile acids in the colon when the ileum has failed to absorb bile acids.

"Alcoholic hepatitis" refers to a liver disease caused by long-term excessive drinking. It is clinically characterized by nausea, vomiting, jaundice, hepatomegaly and tenderness, and may be complicated with hepatic failure and upper gastrointestinal bleeding.

"Primary sclerosing cholangitis" refers to a chronic cholestatic disease characterized by inflammation and fibrosis of the intrahepatic and extrahepatic bile ducts, which in turn leads to multifocal bile duct stenosis. Most patients eventually develop cirrhosis, portal hypertension, and decompensation of liver function.

The terms "comprising," "such as," "for example," and the like are intended to be exemplary embodiments and do not limit the scope of the invention.

As used herein, a compound or pharmaceutical composition, when administered, can ameliorate a disease, symptom, or condition, particularly severity, delay onset, slow progression, or reduce duration of a condition. Whether fixed or temporary, sustained or intermittent, may be due to or associated with administration.

All of the features described in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps involved in any method or process, may be present in any combination, except combinations where certain features or steps are mutually exclusive.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the FXR receptor agonist has high activity and stable metabolism, and is more suitable for treating, inhibiting or improving farnesoid X receptors.

Detailed Description

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended that the experiments below are all experiments that have been performed and that are only experiments that can be performed. The experimental method without specifying the specific conditions in the experimental examples is generally carried out under the conventional conditions or under the conditions recommended by the manufacturers, and all the chemical reagents used in the examples are analytical reagents and purchased from the national pharmaceutical group.

EXAMPLE 1 preparation of 3- (5- ((5-cyclopropyl-3- (2, 6-dichlorophenyl) isoxazole-4-position) methoxy) hexahydrocyclopenta [ c ] pyrrole-2 (1H) -position) benzoic acid (01)

Compound 01 was prepared according to the following scheme:

dissolving ketone (1.000g, 0.0044mol, 1eq) in 10mL methanol, adding sodium borohydride (0.503g, 0.0133mol, 3eq) in batches under ice bath, reacting for 30min under ice bath after adding, TLC shows that the raw materials are completely converted, concentrating and drying, dripping water and dilute hydrochloric acid for quenching, extracting for three times by ethyl acetate, combining organic layers, washing by saturated sodium bicarbonate solution, washing by brine, drying by anhydrous sodium sulfate, and concentrating and drying to obtain 0.990g of white-like solid.

Intermediates 1 to 3:

1-2(0.990g, 0.0044mol, 1.5eq) was added to 10mL of anhydrous tetrahydrofuran, 18-crown-6 (1.305g, 0.0049mol, 1.7eq) and potassium tert-butoxide (550mg, 0.0049mol, 1.7eq) were added under ice bath, and stirred for 30min under ice bath. This solution was added dropwise to a solution of compound 1-1(0.949g, 0.0029mol, 1.0eq) in 10mL of anhydrous THF, the temperature was maintained at 0 ℃ in an ice bath, and the mixture was allowed to spontaneously warm to room temperature and stirred overnight after the addition of the solution. TLC showed complete conversion of the starting material, and water was added to quench the ethyl acetate and the crude product was extracted by silica gel column chromatography (PE/EA: 4/1) to give 0.930g of a white solid.

Intermediates 1 to 4:

dissolving the intermediate 1(0.930g, 0.0019mol) in 5ml dichloromethane, adding 5ml trifluoroacetic acid at room temperature, stirring for 2 hours at room temperature, TLC shows that the raw material conversion is finished, concentrating under reduced pressure to dryness, adding saturated sodium carbonate to quench, extracting for three times by ethyl acetate, combining organic layers, washing with water and common salt water respectively, drying, concentrating to dryness to obtain 635mg of light yellow oily matter, and directly using in the next step.

Intermediates 1 to 6:

intermediates 1-4(150mg, 0.381mmol) and 5ml acetonitrile were charged to a stuffy pot and intermediates 1-5(200mg, 0.762mmol, 2eq) and triethylamine (77mg, 0.762mmol, 2eq) and copper acetate (76mg, 0.381mmol, 1eq) were added. The system was heated to 80 ℃ and stirred overnight. TLC showed complete conversion of intermediate 2, quenched with water, extracted three times with ethyl acetate, combined organic layers, washed successively with water and brine, dried and concentrated to dryness, and chromatographed on silica gel column (PE/EA ═ 10/1, 5/1) to give 105mg of a pale yellow oil.

Compound 01:

and (3) mixing the intermediate: 1-6(105mg, 0.199mmol, 1.0eq), dissolved in 5ml THF and 5ml MeOH, added potassium hydroxide (0.034mg, 0.597mmol, 3.0eq), and 1ml water, heated to 90 deg.C and stirred for 4 hours, TLC indicated complete conversion of the starting material. The methanol and THF were concentrated, the residue was taken up in water, the aqueous layer was washed twice with MTBE and adjusted to PH 2 with 1N hydrochloric acid, EA extracted (20ml × 2), dried and concentrated to dryness to give 48mg of a brown solid.

¹H NMR(400MHz，CD₃Cl)：7.44-7.42(m，1H)，7.39-7.20(m，5H)，6.78(d，J＝7.2，1H)，4.21(s，2H)，3.91-3.88(m，1H)，3.44-3.40(m，2H)，3.14-3.11(m，2H)，2.66-2.60(m，2H)，2.10-2.03(m，3H)，1.40-1.20(m，4H)，1.08-1.05(m，2H).ESI-MS m/z：511.3(M-H)^-。

Preparation of intermediate 1-1:

intermediates 1 to 8:

3mol/L sodium hydroxide solution (21ml, 0.063mol, 1.1eq) was added dropwise to a suspended 3.3ml aqueous hydroxylamine hydrochloride (4.37g, 0.063mol, 1.1eq) solution at 0 ℃. Then, the mixed solution was dropped into a mixed solution of 50ml of ethanol and 2, 6-dichlorobenzaldehyde (10g, 0.057mol, 1.0eq), and after dropping, the mixture was heated to 90 ℃ overnight. After the reaction was completed, the reaction solution was concentrated to dryness, 29.3ml (H2O: EtOH 10: 1) of a solution was added to the concentrated dry solid, followed by slurry crystallization, filtration, cake draining, and vacuum drying at 45 ℃ to obtain intermediate 1-8, white solid 10.48g, yield 96.5%.

Intermediates 1 to 9

2, 6-Dichlorobenzohydroxylamine (10.48g, 0.055mol, 1.0eq) was dissolved in 63ml of DMF and N-chlorosuccinimide (7.36g, 0.28mol, 1.0eq) was added in portions. After the addition was complete, the mixture was stirred at 40 ℃ for 1 h. After the reaction, the reaction mixture was cooled to room temperature, poured into 200ml of ice water at 0 ℃ and extracted once with 200ml of methyl t-butyl ether, and the aqueous layer was discarded. The organic layer was washed with brine, dried over anhydrous sodium sulfate, evaporated to dryness at 30 ℃ to give a solid oil, triturated with 6ml of n-hexane to give a solid, filtered, and the filter cake was dried under vacuum to give 8-1-2 g of an intermediate as a yellow solid in a yield of 97.7%.

Intermediates 1 to 10

Triethylamine (10.91g, 0.11mol, 2.0eq) was added to a mixture of methyl 3-cyclopropyl-3-oxopropanoate (7.66g, 0.054mol, 1.0eq), stirred at room temperature for 30min and cooled to 10 ℃. The intermediate 1 to 9(12.10g, 0.054mol, 1.0eq) was dissolved in 24.2mL of ethanol and slowly added to the reaction mixture at an internal temperature of not more than 24 ℃. Further, the reaction was stirred at room temperature overnight.

After the reaction, the reaction solution was diluted with 45ml of ea, washed with 15ml of water, separated, the aqueous layer was extracted once with ethyl acetate, the organic layers were combined, washed with brine, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated to 10% of the total amount to form a precipitate, triturated with ether, slurried, filtered, and the filter cake was vacuum-dried to give an intermediate 1-10, 8.48g of a white solid, with a yield of 54%.

Intermediates 1 to 11:

1.5mol/L diisobutylaluminum hydride (38ml, 0.057mol, 2.1eq) in toluene was added dropwise to a mixed solution of 1-10(8.48g, 0.027mol, 1.0eq) of intermediate and 54ml of THF, and the temperature was controlled at about 0 ℃. After the addition, the mixture was stirred at room temperature for 2 hours.

After the reaction, 1.8g of methanol was added, and the mixture was stirred for 10min, and then 27ml of water and 54ml of EA were added dropwise. The precipitate formed was filtered through celite and the mother liquor was vacuum dried. Adding n-hexane into the solid, mashing, filtering, and drying the filter cake under vacuum to obtain 1-11 intermediate and 8.46g white solid.

Intermediate 1-1:

8.46g of intermediate 1-11(8.46g, 0.030mol, 1.0eq) and 60ml of DCM were dissolved, triphenylphosphine (11.71g, 0.045mol, 1.5eq) was added, the mixture was cooled to 0-10 ℃ and carbon tetrabromide (14.82g, 0.045mol, 1.5eq) was added in portions. The reaction was stirred at room temperature for 2 h. After the reaction, the reaction solution was mixed and purified by column chromatography with PE: EA of 10: 1 to obtain intermediate 1-1, white crystals 7.35g, and the yield was 71.2%.

Example 2.

Compounds 2-7 were prepared according to the procedure described in example 1, with only corresponding replacement of the starting materials to give the target compounds. Compound number, compound structure and HNMR/MS details are listed in table 1:

TABLE 1 Compound number, Compound Structure and HNMR/MS results

Example 3 Farnesoid X Receptor (FXR) activation assay

The FXR reporter gene method was used to test the activating activity of the compounds of the invention as follows:

I. cell culture

a. Trypsinized and cells seeded at the appropriate density in 10ml of complete matrix.

b. Cells were cultured at 37 ℃ under 5% CO2 for 24 hours.

Cell inoculation and transfection

FuGENE HD was used as transfection reagent.

a. Transfection mixtures were prepared as follows.

pBIND-FXR(ng/well)	25
		pG5Luc(ng/well)	25
FuGENE HD(ul/well)	0.15
		No FBS media(ul/well)	1.85
Total mix(ul/well)	2.5

b. Mix well by vigorously beating the tube and incubate for 15 minutes at room temperature.

c. Trypsinized and cell density determined.

d. The cell fluid was diluted to the desired volume at a density of 600,000 cells/ml.

e. The desired volume of transfection mixture (prepared in two aliquots) was added and 100 ul/well of cell fluid was suspended on the assay plate.

f. The assay plates were incubated at 37 ℃ with 5% CO2 for 24 hours.

Treating the compound

a. Compound stocks were prepared to 10mM working concentration for FXR, and then diluted sequentially 3-fold with 100% DMSO.

b. 10ul of compound was added to 90ul of complete matrix.

c. Add 5ul of compound solution to each well.

d. The assay plates were incubated at 37 ℃ with 5% CO2 for 18 hours.

Dual luciferase reporter method

Firefly and marine coelenterazine luciferase signals were analyzed by the dual-luciferase reporter detection system of Promega. An EnVision multi-label microplate detector was used as the chemiluminescence meter.

Calculation of results

a. Normalization of the values was achieved by splitting the firefly fluorescence signal into renilla signals. "F/R" means "Firefly/Renilla". Normalization eliminated differences in the number of cells in each well and transfection efficiency.

b. Calculation of% Activation value [% ]

The% activation value is calculated by the following equation,

x is the "F/R" value for each concentration point. Min is the "F/R" average for the drug-free control group. Max is the "F/R" average of the reference control.

c. EC50 was calculated using GraphPrism 5.0.

Results are shown in table 2.

TABLE 2 results of the experiment

Compound (I)	EC₅₀，(nM)	Compound (I)	EC₅₀，(nM)
				01	1983	05	890
02	988	06	563
				03	5000	07	479
04	321	GW4064	308

In conclusion, the compounds of the present invention showed very good FXR receptor activating activity, and even some of the compound activating activity was comparable to that of the positive control compound GW 4064.

Pharmacokinetic Properties of the Compound of example 4

The pharmacokinetic properties of the compounds of formula I can be demonstrated by hepatic microsomal metabolic stability assays:

1. preparing a buffer solution: and (3) buffer solution A: 1.0L of 0.1M potassium dihydrogen phosphate buffer (containing 1.0mM EDTA); and (3) buffer solution B: 1.0L of 0.1M dipotassium hydrogen phosphate buffer (containing 1.0mM EDTA); and (3) buffer C: 0.1M potassium phosphate buffer (containing 1.0mM EDTA), pH 7.4, buffer A was added to 700mL buffer B, and the reaction was stopped when the pH reached 7.4.

2. Compound dosing solution: 500 μ M solution: add 10. mu.L of 10mM DMSO stock to 190. mu.L of ACN; 1.5 μ M dosing solution (dissolved in liver microsomes, final concentration of liver microsomes 0.75 mg/mL): mu.L of 500. mu.M solution and 18.75. mu.L of 20mg/mL liver microsomes were added to 479.75uL of buffer C.

3. NADPH solution (6mM in buffer C).

4. 30L of 1.5. mu.M dosing solution was added to 96-well plates at positions set at different time points. Preheat at 37 ℃ for 10 minutes.

5. 15L of NADPH solution (6mM) was added to the position set as the 45 minute time point, and the timer was started.

6. At 30min, 15 min, 5 min, 15LNADPH solution (6mM) was added to the corresponding time point locations.

7. At the end of the incubation (0 min), 135L ACN (containing internal standard) was added to the positions set for all time points. Then 15L of NADPH solution (6mM) was added to the position set at 0 min.

8. Centrifuging: 3220 g was centrifuged for 10 min.

9. 50 μ L of the supernatant was removed, mixed with 50 μ L of ultrapure water (Millipore), and subjected to LC/MS analysis.

The results of the experiments with the compounds of the examples are given in table 3.

TABLE 3 Experimental results for Compounds

Compound (I)	t_1/2(minutes)
		01	55
03	73
		04	105
GW4064	30

The result shows that the invention provides a compound which has novel structure and is more stable than the positive control compound GW4064 in metabolism.

It should be understood that various changes and modifications can be made by those skilled in the art after reading the above disclosure, and equivalents also fall within the scope of the invention as defined by the appended claims.

Claims

1. A compound of formula (I), a pharmaceutically acceptable salt thereof, or an enantiomer, diastereomer, tautomer, racemate, solvate, N-oxide, or amino acid conjugate thereof:

wherein,

R₁selected from 5-10 membered aromatic ring group or 5-10 membered heteroaromatic ring group, wherein each group may beBy 1-3R₄Substitution;

R₃is selected from-CO₂R₅、-CONR₅R₆、-CONR₅SO₂R₆、-CONR₅(CR7)_1-4CO₂R₅、-SO₂R₅Or tetrazole;

2. The compound of claim 1, wherein R is₂Selected from cyclopropyl.

3. The compound of claims 1-2, wherein R is₁Selected from the group consisting of 1-3R₄Substituted phenyl; wherein R is₄Selected from halogen, C1-6 alkyl, haloC 1-6 alkyl, C1-6 alkoxy, haloC 1-6 alkoxy or C3-6 cycloalkyl.

4. A compound of claims 1-3 wherein Z is selected from the group consisting of phenyl, pyridyl, pyrazinyl, pyrimidinyl, furanyl, oxazolyl, thienyl, thiazolyl, pyrazole, benzothienyl, benzofuranyl, benzimidazolyl, imidazo [1, 2-a ]]Pyridyl, pyrrole [1, 2-b ]]Pyrazinyl, indolyl, benzothiazolyl, benzisothiazolyl, benzoxazolyl, naphthyl, quinolinyl, isoquinolinyl, wherein each radical may be substituted by 1-3R₄Substitution; said R₄Selected from halogen, C1-6 alkyl, halo C1-6 alkyl, C1-6 alkoxy, halo C1-6 alkoxy or C3-6 cycloalkyl.

5. A compound according to claims 1-4, wherein R is₃Is selected from-CO₂R₅Wherein R is₅Selected from hydrogen or C1-6 alkyl.

6. The compound of claims 1-5, wherein the compound is selected from the group consisting of:

7. A compound according to claims 1 to 6, wherein the compound is a glycine, taurine or acylglucuronide conjugate thereof.

8. Use of a compound according to any one of claims 1 to 7, a pharmaceutically acceptable salt thereof, or one or more of enantiomers, diastereomers, tautomers, racemates and solvates thereof in the preparation of a bile secretion-regulating modulator or a modulator of FXR nuclear receptor activity.

9. Use of a compound according to any one of claims 1 to 7, a pharmaceutically acceptable salt thereof, or one or more of an enantiomer, a diastereomer, a tautomer, a racemate, and a solvate thereof, in the manufacture of a medicament for the prevention and/or treatment of cholesterol gallstones, primary biliary cirrhosis, portal hypertension, non-alcoholic steatohepatitis, bile acid diarrhea, alcoholic hepatitis, primary sclerosing cholangitis, or atherosclerosis, or a disease associated with bile secretion.

10. A pharmaceutical composition comprising an effective amount of a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, or one or more of its enantiomers, diastereomers, tautomers, racemates and solvates, and at least one pharmaceutically acceptable carrier or excipient.

11. A pharmaceutical composition according to claim 10, wherein: the pharmaceutical composition may further comprise a second therapeutic agent which is a combination of one or more selected from the group consisting of agents for treating cholestasis and the like.

12. A pharmaceutical composition according to claims 10-11, wherein: the pharmaceutical composition is used for preparing medicines for preventing and/or treating cholesterolemia, primary biliary cirrhosis, portal hypertension, nonalcoholic steatohepatitis, bile acid diarrhea, alcoholic hepatitis, primary sclerosing cholangitis or atherosclerosis, or diseases related to bile secretion.