CN114315815A - Compounds for modulating FXR activity and uses thereof - Google Patents

Abstract

The present invention provides a compound having the structure of formula (I), a pharmaceutically acceptable salt, ester or stereoisomer thereof, wherein M, Q, T, W, Y and Z are independently selected from carbon and nitrogen, and at least one of M, Q, T, W, Y and Z is nitrogen, for use in modulating FXR activity.

Description

Compounds for modulating FXR activity and uses thereof

Technical Field

The present invention relates to the field of pharmaceuticals associated with FXR mediated diseases. In particular, the invention relates to a compound for regulating FXR activity, a preparation method and a pharmaceutical application thereof.

Background

Farnesoid X Receptor (FXR) is a member of the nuclear hormone receptor superfamily and is expressed primarily in the liver, kidney and intestine. It functions as a heterodimer with the Retinoid X Receptor (RXR) and binds to response elements in the promoter of target genes to regulate gene transcription. The FXR-RXR heterodimer binds with highest affinity to the inverted repeat-1 (IR-1) response element, where the hexamers that bind to the consensus receptor (consensus receptor) are separated by one nucleotide. FXR is part of a interrelated process in which FXR is activated by bile acids (the end products of cholesterol metabolism) which are used to inhibit cholesterol catabolism.

FXR is a key regulator of cholesterol homeostasis, triglyceride synthesis and adipogenesis (Crawley, Expert Opinion. patents (2010),20(8): 1047-. FXR-related indications include, in addition to treatment of dyslipidemia, obesity, vitamin D-related diseases, intestinal diseases, drug-induced side effects, and hepatitis, hepatobiliary disease, chronic hepatitis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cholestasis, liver fibrosis, cirrhosis, hepatitis b, metabolic diseases, lipid metabolism diseases, carbohydrate metabolism diseases, cardiovascular metabolism diseases, atherosclerosis, type II diabetes, and diabetic complications.

A variety of compounds capable of acting as FXR modulators (or referred to as FXR-agonists) have been developed, such as those small molecule FXR modulators disclosed in WO200037077, WO2003/015771, WO2004/048349, WO2007/076260, WO2007/092751, WO2007/140174, WO2007/140183, WO2008/051942, WO2008/157270, WO2009/005998, WO2009/012125, WO2009/149795, WO2008/025539, WO2008/025540, WO2012/087520, WO2012/087521, WO2012/087519, WO 2013/and WO 2015/036442.

Despite advances in the development of novel FXR agonists, there is still great room for improvement.

Disclosure of Invention

It is therefore an object of the present invention to provide novel compounds for modulating FXR activity, as well as methods of preparation and pharmaceutical use thereof, which exhibit physicochemical, in vitro and/or in vivo ADME (absorption, distribution, metabolism and excretion) properties and/or superior pharmacokinetics in vivo over known FXR agonists.

Definition of

The following definitions are made for the purpose of explaining the present specification, and where appropriate, singular terms also include the plural and vice versa.

As used herein, the term "C_1-6Alkyl "denotes an alkyl group having 1 to 6, in particular up to 4, carbon atoms, which is straight-chain or branched with single or multiple branches, for example a butyl group such as n-butyl, sec-butyl, isobutyl, tert-butyl; propyl, such as n-propyl or isopropyl; ethyl or methyl; more particularly, methyl, isopropyl or tert-butyl.

As used herein, "C" is_1-6Alkoxy "means" C_1-6alkyl-O- ", and in particular methoxy, ethoxy, isopropoxy or tert-butoxy.

As used herein, the term "C_3-6Cycloalkyl "means a cycloalkyl group having 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. C_3-6Cycloalkyl groups may optionally be substituted by C_1-6Alkyl and/or halogenAnd (4) substitution.

As used herein, the term "C_4-7Alkylcycloalkyl "refers to a combination of alkyl and cycloalkyl groups such that the total number of carbon atoms is from 4 to 7. E.g. C₄Alkylcycloalkyl includes methylenecyclopropyl.

The term "5-10 membered aryl" as used herein refers to a 5-10 membered monocyclic, bicyclic or tricyclic aromatic ring system. Typically, the aryl group is a5 or 6 membered ring system.

As used herein, the term "5-10 membered heteroaryl" refers to a 5-10 membered monocyclic, bicyclic, or tricyclic aromatic ring system having 1-4 heteroatoms. Typically, the heteroaryl group is a5 or 6 membered ring system. Furthermore, the term "heteroaryl" as used herein may include monovalent or divalent heteroaryl groups.

As used herein, the term "halogen" or "halo" refers to one or more of fluorine, chlorine, bromine and iodine, especially fluorine or chlorine.

As used herein, the term "C_1-6Haloalkyl "means an alkyl group substituted with one or more halogen atoms, especially C_1-6Fluoroalkyl or C_1-6Chloroalkyl groups such as trifluoromethyl and 2,2, 2-trifluoroethyl.

As used herein, the term "pharmaceutically acceptable excipients" may include solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial and antifungal agents), isotonic agents, absorption delaying agents, salts, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, pigments, and the like, and combinations thereof, known to those skilled in the art. Any conventional carrier is contemplated for use in the therapeutic or pharmaceutical compositions unless incompatible with the active ingredient.

As used herein, the term "therapeutically effective amount" refers to an amount of active substance sufficient to achieve the therapeutic effect. Thus, a therapeutically effective amount of an active substance for the treatment of a condition mediated by FXR will be sufficient for the treatment of a condition mediated by FXR.

In one aspect, the present invention provides a compound having the structure of formula (I), a pharmaceutically acceptable salt, ester or stereoisomer thereof, for use in modulating FXR activity:

wherein M, Q, T, W, Y and Z are each independently selected from C and N, and at least one of M, Q, T, W, Y and Z is N;

R¹、R²and R³Each independently selected from H, halogen, unsubstituted or halogen substituted C_1-6Alkyl and unsubstituted or halogen-substituted C_1-6Alkoxy, provided that R is¹、R²And R³Is not hydrogen;

R⁰selected from unsubstituted or halogen-substituted C_1-6Alkyl radical, C_3-6Cycloalkyl and C_4-7An alkyl cycloalkyl group;

X¹and X²Independently selected from H and halogen;

wherein L is selected from a 5-10 membered aryl or a 5-10 membered heteroaryl containing one or more heteroatoms selected from N, O and S, wherein the 5-10 membered aryl or 5-10 membered heteroaryl is substituted with R⁴Substituted and optionally further substituted by R⁵Substitution;

wherein R is⁴Selected from-COOH, -CH₂COOH、-NHSO₂CF₃、-SO₂NH-C_1-6Alkyl, -SO₃H、-CONHSO₂-C_1-6Alkyl, -CONHSO₂-C_3-6Cycloalkyl, -CONHSO₂-5-10 membered aryl and aryl substituted by C_1-6Alkyl substituted-CONHSO₂-a 5-10 membered aryl group; and wherein R⁵Selected from H, C_1-6Alkyl, halogen, C_1-6Haloalkyl, -O- (C)_1-6Alkyl) and-NH- (C)_1-6Alkyl groups).

In a preferred embodiment of the invention, R¹、R²And R³Each independently selected from H, halogen and C_1-3Perfluoroalkoxy, e.g. H, Cl, F and-O-CF₃. Specifically, in one embodiment, R¹And R²Are all Cl, and R³Is H; in another embodiment, R¹And R²Are all Cl, and R³Is F; in yet another embodiment, R¹And R²Are all Cl, R³is-O-CH₃(ii) a In yet another embodiment, R¹is-O-CF₃And R is²And R³Are all H.

In a preferred embodiment of the invention, R⁰Is isopropyl or cyclopropyl.

In one embodiment of the invention, L is phenyl, which is substituted with R⁴Substituted and optionally substituted by R⁵And (4) substitution. In another embodiment, L is a 5-10 membered heteroaryl having one or more heteroatoms selected from N, O and S. In a preferred embodiment, L is a 5-6 membered heteroaryl having one or more heteroatoms selected from N, O and S. In another preferred embodiment, L is substituted with R⁴And optionally R⁵A substituted pyridyl group.

In a preferred embodiment of the invention, R⁴Selected from-COOH, -CH₂COOH、-CONHSO₂-C_1-6Alkyl and-CONHSO₂-C_3-6One of cycloalkyl groups. In a more preferred embodiment of the invention, R⁴is-COOH or-CH₂COOH。

Preferably, R⁵Selected from H, C_1-3One of an alkyl group and a halogen.

For example, in a preferred embodiment of the invention, L is pyridyl; r⁴is-COOH; r⁵Is H or halogen.

Preferably, the halogen in the above substituents is fluorine or chlorine.

In a most preferred embodiment of the invention, the compound of formula (I) has one of the following structures:

in another aspect, the present invention provides a process for the preparation of a compound of formula (I), a pharmaceutically acceptable salt, ester or stereoisomer thereof, which process comprises employing one of the following synthetic routes:

route a:

route B:

the two general routes are specifically described below.

Route a:

(a) the halogenated compound of formula (a1) is reacted with a hydroxyl group-containing compound (a2) to give an ether of formula (A3). The reaction is carried out with a base in a polar solvent, preferably cesium carbonate or potassium carbonate or a similar base in DMF or acetonitrile or the like;

(b) converting compound (A3) to the boronic acid ester (a4), preferably under Pd-catalyzed conditions;

(c) with oxidizing agents, e.g. NaClO₂Or H₂O₂Oxidizing the compound (a4) to a hydroxy compound (a 5);

(d) reacting the resulting hydroxy compound of formula (A5) with a halogenated compound X-L to give a compound of formula (I),

according to the preparation method provided by the invention, X is preferably bromine or chlorine, and more preferably chlorine; x₄Preferably bromine or iodine, more preferably bromine.

Route B:

(a) reacting the hydroxy compound (B1) with a halo compound X-L to give an ether of formula (B2), wherein the reaction is with a base in a polar solvent, preferably cesium carbonate or potassium carbonate or similar base in DMF or acetonitrile or the like;

(b) converting compound (B2) to the boronic acid ester (B3), preferably under Pd-catalyzed conditions;

(c) with oxidizing agents, e.g. NaClO₂Or H₂O₂The compound(B3) Oxidation to a hydroxy compound (B4);

(d) compound (B4) is converted to compound (I) using the conditions described in scheme a.

In another aspect, the present invention also provides a pharmaceutical composition comprising at least one compound of formula (I) or a prodrug compound thereof, or a pharmaceutically acceptable salt, ester, or stereoisomer thereof, as an active ingredient, and a pharmaceutically acceptable excipient.

The pharmaceutical compositions of the present invention may additionally comprise as active ingredient one or more other compounds, such as prodrug compounds or other nuclear receptor modulators.

The pharmaceutical compositions are suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular and intravenous), ocular, pulmonary (nasal or buccal inhalation) or nasal administration, although the most suitable method of administration in a particular case will depend on the nature and severity of the condition being treated and on the nature of the active ingredient. The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.

The compounds of formula (I) and their pharmaceutically acceptable salts exhibit valuable pharmacological activities when tested in vitro binding assays and cellular assays and are therefore useful as pharmaceuticals. In particular, the compounds of the invention are FXR agonists and are useful as medicaments for the treatment of FXR mediated diseases such as liver damage caused by non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), Primary Biliary Cirrhosis (PBC), cholestatic liver disease, chronic liver disease, hepatitis c, alcoholic liver disease, liver fibrosis, Primary Sclerosing Cholangitis (PSC), gallstones, biliary atresia, lower urinary tract symptoms and Benign Prostatic Hyperplasia (BPH), ureteral stones, obesity, type 2 diabetes, atherosclerosis, hypercholesterolemia and hyperlipidemia. The compounds of the invention are also useful for lowering total cholesterol, lowering LDL cholesterol, lowering VLDL cholesterol, raising HDL levels, and/or lowering triglyceride levels.

The invention also provides the use of a pharmaceutically acceptable salt, ester or stereoisomer of a compound of formula (I) in the manufacture of a medicament for the treatment of FXR mediated diseases, optionally in combination with a second therapeutic agent. The second therapeutic agent is useful for treating liver damage caused by, for example, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), Primary Biliary Cirrhosis (PBC), cholestatic liver disease, chronic liver disease, hepatitis c infection, alcoholic liver disease, liver fibrosis, Primary Sclerosing Cholangitis (PSC), gallstones, biliary atresia, lower urinary tract symptoms and Benign Prostatic Hyperplasia (BPH), ureteral stones, obesity, type 2 diabetes, atherosclerosis, hypercholesterolemia, or hyperlipidemia.

Unless otherwise indicated, the term "compound of the invention" refers to a compound of formula (I), a prodrug thereof, a salt of the compound and/or a salt of the prodrug of the compound, a hydrate or solvate of the compound, and stereoisomers (including diastereomers and enantiomers), tautomers, isotopically labeled compounds (including deuterium substitutions) and polymorphs of the compound.

Salts of the compounds of the present invention may be prepared by methods known to those skilled in the art. For example, treatment of a compound of the invention with a suitable base or acid in a suitable solvent gives the corresponding salt.

Salts included in the term "pharmaceutically acceptable salts" refer to non-toxic salts of the compounds of the present invention. Preferred are alkali metal salts of carboxylic acids, such as sodium, potassium, lithium, calcium, magnesium, aluminum, zinc, N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine salts. Other pharmaceutically unacceptable salts may also be useful in the preparation of the compounds of the present invention and should be considered to constitute a further aspect of the invention.

All starting materials, reagents, acids, bases, solvents and catalysts for the synthesis of the compounds of the invention are commercially available or can be produced by organic synthesis methods known to the person skilled in the art. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., such as) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed.

Drawings

In order to more clearly explain technical solutions in embodiments of the present invention, drawings for describing embodiments of the present invention will be briefly described below. It is to be understood that the drawings in the following description are of some embodiments of the invention only.

FIG. 1 shows the binding activity of compounds of the invention to FXR;

FIG. 2 shows the FXR activating activity of the compounds of the present invention.

Detailed Description

The invention is further illustrated with reference to the following examples. It should be noted that the following examples are only for illustration and are not intended to limit the present invention. Various modifications of the invention in light of the teachings herein will be suggested to one skilled in the art and are to be included within the scope of the appended claims.

Example 1

Preparation of 6- ((3- ((5-cyclopropyl-3- (2, 6-dichlorophenyl) isoxazol-4-yl) methoxy) quinolin-7-yl) oxy) nicotinic acid (Compound 1)

(a) Compound 1A-2(1.0g, 4.46mmol, 1.0 equiv.), Compound 1A-1(1.55g, 4.46mmol, 1.0 equiv.) and cesium carbonate (2.90g, 8.92mmol, 2.0 equiv.) were dissolved in DMF (10ml) and stirred at 65 ℃ for 2 hours. After cooling to room temperature, 10ml of water and 10ml of EA (ethyl acetate) were added and the organic phase was concentrated to dryness to give compound 1A-3(1.61g, yield: 73.5%, which was used without further purification in the next step), i.e.: 4- (((6-bromoquinolin-2-yl) oxy) methyl) -5-cyclopropyl-3- (2, 6-dichlorophenyl) isoxazole, lcms (esi): calculated as C₂₂H₁₅BrCl₂N₂O₂；[M+H]⁺: 489.0, found: 489.0.

(b) compound 1A-3(200mg, 0.408mmol, 1.0 equiv.) is dissolved in anhydrous THF (2ml) then N₂KOAc (80mg, 0.816mmol, 2.0 equiv.), Pd (dppf) was added thereto₂Cl₂(30mg, 0.408mmol, 0.1 equiv.) and bis (pinacol) diboron (104mg, 0.408mmol, 1.0 equiv.) and the reaction mixture was heated to reflux for 2 h. After cooling to room temperature, 10ml of water and 10ml of EA were added and the organic phase was concentrated to dryness. Purification by silica gel column chromatography (PE (petroleum ether)/EA ═ 3/1) gave compound 1A-4(134mg, yield: 61.2%), i.e.: 5-cyclopropyl-3- (2, 6-dichlorophenyl) -4- (((((6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) quinolin-2-yl) oxy) methyl) isoxazole, LCMS (ESI) calculated as C₂₈H₂₇BCl₂N₂O₄；[M+H]⁺: 537.1, found: 537.1.

(c) compound 1A-4(100mg) was added slowly to EtOH (2ml) followed by 30% H₂O₂Aqueous solution (1 ml). The reaction mixture was stirred at room temperature for 1h, over saturated Na₂SO₃The aqueous solution was quenched and extracted with EA. The organic phase was concentrated and purified by silica gel column chromatography (PE/EA ═ 3/1) to give compound 1A-5(57mg, yield: 72.1%). Lcms (esi): calculated as C₂₂H₁₆Cl₂N₂O₃；[M+H]⁺: 427.1, found: 427.1.

(d) compound 1A-5(50mg, 0.117mmol, 1.0 equiv.), 1A-6(25.3mg, 0.117mmol, 1.0 equiv.) and cesium carbonate (76.0mg, 0.234mmol, 2.0 HemslAmount) was stirred in DMF (1ml) and reacted at 65 ℃ for 2 hours. After cooling to room temperature, 5ml of water and 5ml of EA were added to conduct extraction, and the organic phase was concentrated to dryness to obtain Compound 1A-7(41mg, yield: 62.3%). Lcms (esi): calculated as C₂₉H₂₁Cl₂N₃O₅；[M+H]⁺: 562.1, found: 562.1.

(e) compounds 1A-7(30mg) were dissolved in MeOH (1ml) and then incubated under N₂Next, 10% aqueous NaOH (0.5ml) was added, and the reaction mixture was heated to reflux for 1 hour. The pH of the reaction solution was adjusted to 3 to 4 by adding 1N HCl solution, and then extracted by adding 5ml EA. The organic phase was concentrated and purified on a column (PE/EA/AcOH 1/1/0.01 elution) to give the title compound 1(21mg, yield: 71.7%).

¹H NMR(400MHz,DMSO-d₆) δ 8.63(d, J ═ 2.3Hz,1H),8.37(d, J ═ 2.9Hz,1H),8.30(dd, J ═ 8.6,2.4Hz,1H),7.86(d, J ═ 8.9Hz,1H),7.80(d, J ═ 2.9Hz,1H),7.66(d, J ═ 2.4Hz,1H),7.60(d, J ═ 8.0Hz,2H),7.51(dd, J ═ 9.0,7.1Hz,1H),7.42(dd, J ═ 8.9,2.4Hz,1H),7.18(d, J ═ 8.6Hz,1H),5.07(s,2H), 1.26-1.10 (m,5H), 5.c, (C) calculated as (lcm, J: (C)₂₈H₁₉Cl₂N₃O₅；[M+H]⁺548.1, found 548.1.

Example 2

Preparation of 6- (((7- ((5-cyclopropyl-3- (2, 6-dichlorophenyl) isoxazol-4-yl) methoxy) quinolin-3-yl) oxy) nicotinic acid (Compound 2)

(a) Compound 2B-1(1.0g, 4.46mmol, 1.0 equiv.), Compound 1A-6(0.96g, 4.46mmol, 1.0 equiv.) and cesium carbonate (2.90g, 8.92mmol, 2.0 equiv.) were dissolved in DMF (10ml) and stirred at 65 ℃ for 2 hours. After cooling to room temperature10ml of water and 10ml of EA (ethyl acetate) were added, and the organic phase was concentrated to dryness to obtain compound 2B-2(1.1g, yield: 68.7%) as follows: methyl 6- ((7-bromoquinolin-3-yl) oxy) nicotinate, lcms (esi): calculated as C16H11BrN2O 3; [ M + H ]]⁺: 359.0, found: 359.0.

(b) compound 2B-2(200mg, 0.56mmol, 1.0 equiv.) is dissolved in anhydrous THF (2ml) then N₂KOAc (110mg, 1.12mmol, 2.0 equiv.), Pd (dppf) was added thereto₂Cl₂(41mg, 0.056mmol, 0.1 equiv.) and bis (pinacolato) diboron (142mg, 0.56mmol, 1.0 equiv.) the reaction mixture was heated to reflux for 2 h. After cooling to room temperature, 10ml of water and 10ml of EtOAc were added and extracted and the organic phase was concentrated to dryness. The residue was purified by silica gel column chromatography (PE: EA ═ 3:1) to obtain compound 2B-3(146mg, yield: 64.6%), which was: methyl 6- ((7- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) quinolin-3-yl) oxy) nicotinate. Lcms (esi): calculated as C₂₂H₂₃BN₂O₅；[M+H]⁺: 407.2, found: 407.2.

(c) to a solution of Compound 2B-3(100mg) in EtOH (2ml) was added slowly 30% H₂O₂Aqueous solution (1 ml). The reaction mixture was stirred at room temperature for 1h, over saturated Na₂SO₃The aqueous solution was quenched and extracted with EA. The organic phase was concentrated and purified by silica gel column chromatography (PE/EA ═ 3/1) to give compound 2B-4(44mg, yield: 61.0%). Lcms (esi): calculated as C₁₆H₁₂N₂O₄；[M+H]⁺: 297.1, found: 297.1.

(d) compound 2B-4(0.2g, 0.67mmol, 1.0 equiv.), 2A-1(0.2g, 0.67mmol, 1.0 equiv.) and cesium carbonate (0.435g, 0.94mmol, 2.0 equiv.) were stirred in DMF (10ml) and stirred at 65 ℃ for 2 hours. After cooling to room temperature, 10ml of water and 10ml of EtOAc were added, and the organic phase was concentrated to dryness to obtain compound 2B-5(0.26g, yield: 68.4%). Lcms (esi): calculated as C₂₉H₂₁Cl₂N₃O₅；[M+H]⁺: 562.1, found: 562.1.

(e) compound 2B-5(100mg) was dissolved in anhydrous THF (2ml) under nitrogen, then N₂Next, 10% aqueous NaOH (1ml) was added and the reaction mixture was heated to reflux for 1 hour. The pH of the reaction solution was adjusted to 3 to 4 by adding 1N HCl solution and the aqueous phase was extracted with 10ml EA. The organic phase was concentrated and purified by silica gel column chromatography (PE/EA/AcOH 1/1/0.01 elution) to give the title compound 2(46mg, yield: 47.2%).

¹H NMR(400MHz,DMSO-d₆) δ 8.73(d, J ═ 2.7Hz,1H),8.62(s,1H),8.32(d, J ═ 8.6Hz,1H),8.13(d, J ═ 2.8Hz,1H),7.80(d, J ═ 9.0Hz,1H),7.59(d, J ═ 8.0Hz,2H),7.51(d, J ═ 7.7Hz,1H),7.44(d, J ═ 2.5Hz,1H),7.25(d, J ═ 8.6Hz,1H), 7.11-6.96 (m,1H),5.07(s,2H), 1.27-1.04 (m,5H), esi (C) calculated as C₂₈H₁₉Cl₂N₃O₅；[M+H]⁺548.1, found 548.1.

Biological examples

Example A: FXR agonist binding Capacity

In this example, the analysis of the binding activity of compounds 1 and 2 prepared in examples 1 and 2 to FXR was performed using a recruitment experiment of time-resolved fluorescence resonance energy transfer co-activating peptides. The experiment used a LanthaScreen-labeled anti-GST antibody, fluorescein-labeled SRC2-2 coactivator peptide, and GST-tagged FXR-LBD. The LanthaScreen-labeled anti-GST antibody indirectly labels FXR-LBD by binding to a GST tag. FXR-LBD can form heterodimer FXR-LBD/RXR alpha with RXR alpha, and the compound can bind to the FXR-LBD/RXR alpha and cause the change of FXR-LBD conformation, so that the recruitment capacity of the heterodimer on SRC2-2 coactivator peptide is increased. At the same time, FXR-LBD (with GST tag) was caused to bind more tightly to SRC2-2 coactivator peptide, and then anti-GST antibody (with LanthaScreen tag) was added, and LanthaScreen reacted with fluorescein to generate fluorescence. Therefore, the stronger the binding activity of the compound to FXR, the higher the intensity of the excited fluorescence. The results are shown in FIG. 1.

Experimental Material

1. Protein: glutathione-S-transferase (GST) -tagged human FXR protein (Invitrogen)

2. Co-activator: fluorescein-labeled steroid receptor co-activator (SRC2-2) (Invitrogen)

3. Detection reagent: LanthaScreen time resolved fluorescence assay kit (Invitrogen)

Experimental methods

1. Compounds were made as 10mM DMSO stock solutions and stored in a freezer at-20 ℃ for long periods of time.

2. Compounds were diluted to 1mM before the experiment and then 3-fold to 10 concentration points with DMSO. Then, these 10 concentration points were diluted 50-fold using transcription helper regulatory factor buffer G (Invitrogen, PV4553) to be working solutions. To each well of the 384-well plate, 10. mu.l of each working solution was added.

3. A solution of human FXR protein (final concentration 20nM) was prepared in cooled buffer G and 5 μ l of human FXR protein solution was added to each well of the 384-well plate.

4. A mixture of buffer G containing 2. mu.M fluorescein-labeled steroid receptor co-activator and 20nM GST antibody was prepared.

5. Mu.l of the mixture prepared in step 4 was added to a 384 well plate.

6. Centrifuge 384 well plates at 1000g for 1 min.

7. Incubate for 1 hour at room temperature in the dark.

8. Read 384 well plates at 520 and 495nm on an Envision 2104 reader.

9. EC50 values for compound activation were calculated.

The experimental results show that compound 1 and compound 2 bind to FXR-bound EC₅₀110nM and 129nM, respectively.

Example B: FXR agonist activating ability

This example is intended to evaluate the agonistic effect of compounds 1 and 2 prepared in examples 1 and 2 on FXR. The FXR-LBD and GAL4 chimeric expression genes were inserted into the pBIND plasmid (Promega, E1581). The expression vector and luciferase reporter vector (pGL4.35 vector carrying luciferase reporter gene, the expression of which is driven by stably integrated GAL4 promoter) were co-expressed in HEK293T cells. When the compound binds to the FXR moiety in the chimeric protein, activated FXR will initiate downstream gene expression, i.e., binding to the GAL4 promoter binding site on the reporter vector via the GAL4 moiety in the chimeric protein, and stimulate expression of the reporter luciferase. After adding a substrate of luciferase, judging the agonistic activity of the compound on FXR according to the strength of a fluorescent luminescence signal. The results are shown in FIG. 2.

Experimental Material

1. Cell line: HEK293T (Invitrogen)

2. Expression plasmid: pBIND-hFXR-LBD-GAL4(Promega), pGL4.35-luciferase (Promega)

3. Cell culture medium: DMEM medium containing 10% serum and penicillin/streptomycin double antibody

4. Detection reagent: Steady-Glo fluorescence detection System (Promega).

5. Transfection reagent: TransIT-293 transfection reagent (MIRUS BIO)

Experimental methods

1. Compounds were made as 10mM DMSO stock solutions and stored in a freezer at-20 ℃ for long periods of time.

2. The HEK293T cells were revived to 5.5X 10⁶In a 10cm dish and at 5% CO₂Incubate at 37 ℃ for 16 hours in an incubator.

3. The transfection reagents were returned to room temperature prior to transfection. The Trans-IT solution was added dropwise to Opti-MEM and mixed by inversion for 5 minutes. The expression plasmid was added, mixed by inversion, and incubated at room temperature for 20 minutes.

4. The transfection mixture of step 3 was added to the 10cm petri dish prepared in step 2 and incubated at 5% CO₂Incubate in the incubator for 5-6 hours.

5. Compounds were diluted 3-fold to 10 concentration points using DMSO; add 25 μ Ι of compound per well in 384-well plates using an Echo 550 sonic pipette; HEK293T cells were added to 384-well plates at a concentration of 15,000 cells/well; at 37 ℃ in 5% CO₂Incubate in incubator for 16-20 hours.

6. Add 25. mu.l Steady-Glo fluorescent reagent to each well and read the fluorescence data on an Envision 2104 plate reader.

7. Calculation of activated EC of Compounds₅₀The value is obtained.

The experimental results show that the compound 1 and the compound 2 activate the EC of FXR₅₀1.63. mu.M and 2.92. mu.M, respectively.

Claims (9)

1. A compound, pharmaceutically acceptable salt, ester or stereoisomer thereof, having the structure of formula (I) for modulating FXR activity:

wherein: m, Q, T, W, Y and Z are independently selected from carbon and nitrogen, and at least one of M, Q, T, W, Y and Z is nitrogen.

R¹、R²And R³Each independently selected from H, halogen, unsubstituted or halogen substituted C_1-6Alkyl and unsubstituted or halogen-substituted C_1-6Alkoxy, provided that R is¹、R²And R³At least one of which is not hydrogen, R⁰Selected from unsubstituted or halogen-substituted C_1-6Alkyl radical, C_3-6Cycloalkyl and C_4-7An alkyl cycloalkyl group;

X¹and X²Independently selected from H and halogen;

wherein L is selected from a 5-10 membered aryl or a 5-10 membered heteroaryl containing one or more heteroatoms selected from N, O and S, wherein the 5-10 membered aryl or 5-10 membered heteroaryl is substituted with R⁴Substituted and optionally further substituted by R⁵Substitution;

wherein R is⁴Selected from-COOH, -CH₂COOH、-NHSO₂CF₃、-SO₂NH-C_1-6Alkyl, -SO₃H、-CONHSO₂-C_1-6Alkyl, -CONHSO₂-C_3-6Cycloalkyl, -CONHSO₂-5-10 membered aryl and aryl substituted by C_1-6Alkyl substituted-CONHSO₂-a 5-10 membered aryl group; and wherein R⁵Selected from H, C_1-6Alkyl, halogen, C_1-6Haloalkyl, -O- (C)_1-6Alkyl) and-NH- (C)_1-6Alkyl groups).

2. The compound of claim 1, wherein R¹、R²And R³Each independently selected from H, halogen and C_1-3Perfluoroalkoxy, and R⁰Is isopropyl or cyclopropyl, preferably, R¹、R²And R³Each independently selected from H, Cl, F and-O-CF₃(ii) a Further preferably, R¹And R²Are all Cl, and R³Is H, F or-O-CF₃Or R is¹is-O-CF₃And R is²And R³Are all H.

3. The compound of claim 1, wherein L is phenyl, which is substituted with R⁴Substituted and optionally substituted by R⁵Substitution; or L is a 5-10 membered heteroaryl group with one or more heteroatoms selected from N, O and S, substituted with R⁴Substituted and optionally substituted by R⁵Substitution;

preferably, L is a 5-6 membered heteroaryl group with one or more heteroatoms selected from N, O and S, substituted with R⁴Substituted and optionally substituted by R⁵Substitution; further preferably, L is substituted with R⁴And optionally R⁵A substituted pyridyl group;

preferably, R⁴Selected from-COOH, -CH₂COOH、-CONHSO₂-C_1-6Alkyl and-CONHSO₂-C_3-6One of cycloalkyl, further preferably R⁴is-COOH or-CH₂COOH；R⁵Selected from H, C_1-3One of alkyl and halogen;

preferably, L is pyridyl; r⁴is-COOH; and R is⁵Is H or halogen.

4. A compound according to any one of claims 1 to 3, wherein the halogen is fluorine or chlorine.

5. The compound of claim 1, wherein the compound has one of the following structures:

6. a pharmaceutical composition comprising a therapeutically effective amount of a compound of any one of claims 1 to 5, or a pharmaceutically acceptable salt, ester, or stereoisomer thereof, and a pharmaceutically acceptable adjuvant.

7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition further comprises a second therapeutic agent for treating liver damage caused by non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, primary biliary cirrhosis, cholestatic liver disease, chronic liver disease, hepatitis C infection, alcoholic liver disease, liver fibrosis, primary sclerosing cholangitis, gallstones, biliary atresia, lower urinary tract symptoms and benign prostatic hyperplasia, ureteral stones, obesity, type 2 diabetes, atherosclerosis, hypercholesterolemia, or hyperlipidemia.

8. Use of a compound of any one of claims 1 to 5, or a pharmaceutically acceptable salt, ester or stereoisomer thereof, or a pharmaceutical composition of claim 6 or 7, for the manufacture of a medicament for the treatment of a disease mediated by FXR.

9. The use according to claim 8, wherein the disease is liver damage caused by non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, primary biliary cirrhosis, cholestatic liver disease, chronic liver disease, hepatitis c infection, alcoholic liver disease, liver fibrosis, primary sclerosing cholangitis, gallstones, biliary atresia, lower urinary tract symptoms and benign prostatic hyperplasia, ureteral stones, obesity, type 2 diabetes, atherosclerosis, hypercholesterolemia, or hyperlipidemia.

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