CN104910239A - Pentacyclic triterpene compound and its preparation method, its pharmaceutical composition and purpose thereof - Google Patents

Abstract

The present invention relates to a pentacyclic triterpenoid compound represented by general formula (I) and a preparation method thereof, a pharmaceutical composition containing the compound, the application of the compound in the preparation of a drug for treating type 2 diabetes, and the The application of said compound in the treatment of type 2 diabetes.

Description

Pentacyclic triterpenoids, preparation method, pharmaceutical composition and use thereof

technical field

The present invention relates to a class of TGR5 (G protein-coupled bile acid membrane receptor) agonists, in particular to a class of pentacyclic triterpenoids, a preparation method thereof, and a pharmaceutical composition comprising the compound, and Said compounds can act as TGR5 agonists.

Background technique

Diabetes is caused by different etiologies (such as genetic factors, immune dysfunction, microbial infection and its toxins, free radicals, mental factors, etc.) that lead to hypofunction of pancreatic β cells and/or resistance of body cells to insulin. A characteristic metabolic disorder syndrome. According to the statistics of the International Diabetes Federation (IDF), in 2010, there were more than 43 million people with diabetes among the people aged 20 to 79 in China, and the prevalence rate reached 4.5%. Diabetes has become a disease that endangers human safety and health.

At present, the drugs for the treatment of diabetes mainly include insulin secretion promoters (sulfonylureas, repaglinide), insulin sensitizers (biguanides, thiazolidinediones) and α-glucosidase inhibitors (acarbose). Sugar), but they often have side effects of varying degrees, such as hypoglycemia, weight gain, and cardiovascular side effects. It is urgent to develop new antidiabetic drugs that act on new targets and avoid the side effects of traditional antidiabetic drugs.

TGR5 is a G protein-coupled receptor (GPCR) expressed in brown adipose tissue and muscle. Before TGR5 was discovered in 2002 as a specific receptor for endogenous metabolites of bile acids, it had long been recognized as a detergent capable of dissolving fatty acids, fat-soluble vitamins, and cholesterol, thereby facilitating their digestive transport . It has therefore only been endowed with limited therapeutic applications. Before the discovery of TGR5, the orphan farnesoid X receptor (FXR) was the only receptor known to be activated by bile acid analogs. Through activation of TGR5, cholic acid stimulates the activation of type 2 deiodinase, which leads to increased mitochondrial function and energy expenditure. It has also been reported that activation of TGR5 by cholic acid can lead to the secretion of glucagon-like peptide 1 (GLP1) in mouse enteroendocrine cell lines. These data suggest that TGR5 is an important target for the treatment of diabetes and related metabolic disorders.

Currently known TGR5 agonists mainly include two categories. One is the chemical synthesis of small molecules. Although most of these compounds have very strong agonistic activity, and some even have an EC ₅₀ value lower than 10nM, due to the strong activation of TGR5 receptors on the gallbladder caused by these compounds, the relaxation of smooth muscle and the promotion of gallbladder filling are serious. Side effects of increased gallbladder volume. Another class of natural product-like molecules, including steroids and other types of natural products, is less active than synthetic molecules, but due to their structural advantages, this class of compounds can overcome the gallbladder toxicity of chemically synthesized molecules. The most notable among them is INT-777 (Pellicciari, R. et al. J. Med. Chem. 2009, 52, 7958-7961), which is a derivative of cholic acid, and its agonistic activity on TGR5 reaches EC50 of 0.82 nM, has entered the clinical stage. However, compound INT777 has the disadvantage of being difficult to prepare. Starting from cholic acid, the synthetic route has as many as twelve steps, and extremely harsh reaction conditions are used many times. The inventors have been working on the structural modification of the compound INT777 for a long time, hoping to find new diabetes drugs with better activity and lower toxicity. The invention discloses a class of pentacyclic triterpenoid compounds, the main feature of which is the inversion of the 3-position hydroxyl group. After the β-type hydroxyl group of natural products such as betulinic acid is reversed to the α-type hydroxyl group, its TGR5 agonistic activity is significantly increased. And with the transformation of other sites of this series of compounds, the degree of activity improvement after hydroxyl inversion is also different. The present invention discloses the influence of this structure on the activity, and obtains a series of compounds with excellent properties. Compared with INT777, it is not only easy to synthesize, but also its TGR5 agonistic activity is significantly better than INT777, and it is expected to become a new drug for this target. New drugs for the treatment of type 2 diabetes.

Contents of the invention

The object of the present invention is to design and synthesize pentacyclic triterpenoids with the structure described in general formula (I).

Another object of the present invention is to provide a preparation method of the compound.

Another object of the present invention is to provide a pharmaceutical composition containing the compound as an active ingredient.

Another object of the present invention is to provide the application of the compound described in the present invention in the preparation of medicaments for treating type 2 diabetes.

Another object of the present invention is to provide the application of the compound described in the present invention in the treatment of diabetes.

The present invention provides a kind of pentacyclic triterpenoid compound, it has the structure shown in following general formula (I):

in:

R ₁ is hydrogen, hydroxyl, halogen or C ₁ -C ₆ alkyl;

R ₂ is hydrogen, hydroxyl, halogen, oxo group (=O), =N-OH, C ₁ -C ₆ alkylcarbonyloxy group, 3 to 8-membered cycloalkylcarbonyloxy group, C ₁ - C ₆ alkyl, 3 to 8 membered cycloalkyl or Wherein, Rc is C ₁ -C ₆ alkyl or hydrogen;

R ₃ and R ₈ are each independently hydrogen; hydroxyl; halogen; amino C ₁ -C ₆ alkyl substituted by C ₁ -C ₆ alkyl; unsubstituted or substituted C ₁ -C ₆ alkyl, wherein, substituted The substituents in the C ₁ -C ₆ alkyl are selected from hydroxyl, halogen, oxo group (=O), =N-OH, epoxypropylene, amino and hydroxyl C ₁ -C ₆ alkylamino More than one substituent; unsubstituted or substituted C ₁ -C ₆ alkenyl, wherein the substituted C ₁ -C ₆ alkenyl includes groups selected from hydroxyl, halogen, oxo (=O), =N- One or more substituents of OH, amino and hydroxyl C ₁ -C ₆ alkylamino;

Wherein, R ₉ is H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, hydroxyl, hydroxyl C ₁ -C ₆ alkyl, -CH ₂ OC(O)R ₁₀ , -CH ₂ OC( O)OR ₁₁ , -CH ₂ OC(O)CH ₂ OR ₁₂ ;

R ₁₀ , R ₁₁ and R ₁₂ are each independently substituted or unsubstituted 5 to 8 membered aryl; substituted or unsubstituted 3 to 8 membered cycloalkyl; 5 to 8 membered arylamino; containing N, S or 5- to 8-membered heteroaryl with at least one heteroatom in O; C ₁ -C ₆ alkyl, halogenated C ₁ -C ₆ alkyl; hydroxy C ₁ -C ₆ alkyl; BocNH(CH ₂ ) _m O(CH ₂ ) _n -, wherein each m and n are the same or different, and are each independently an integer from 1 to 6; wherein, substituted 5 to 8-membered aryl or substituted 3 to 6 The substituents in the 8-membered cycloalkyl group are selected from one or more substituents in hydroxyl, halogen, C ₁ -C ₆ alkyl, and C ₁ -C ₆ alkoxy;

R ₄ is hydrogen, hydroxyl, halogen or C ₁ -C ₆ alkyl;

R ₅ is independently selected from the group consisting of _hydrogen ; _hydroxy ; hydroxy C _{1 -C 6} alkyl; halogen; C ₁ -C ₆ alkyl; _o CH ₂ CH ₂ R ₁₄ ; -C(O)NH(CH ₂ CH ₂ O) _p CH ₂ CH ₂ R ₁₅ ; or -C(O)NH(CH ₂ ) _q C(O)OH; wherein, o and p are independently 0, 1, 2 or 3; and q is an integer from 3 to 8;

R ₁₃ is hydrogen; hydroxycarbonyl C ₁ -C ₈ alkylamino; hydroxy; unsubstituted or C ₁ -C ₆ alkyl substituted piperazinyl; benzyloxy group; C ₁ -C ₆ alkoxy ; tert-butoxycarbonyl C ₁ -C ₆ alkyloxy; or hydroxycarbonyl C ₁ -C ₆ alkylamino;

R ₁₄ and R ₁₅ are independently hydrogen; amino; 5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanylamino; C ₁ -C ₆ amido ; tert-butoxycarboxamido; or C ₁ -C ₆ alkoxy;

R ₆ and R ₇ are each independently hydrogen, hydroxyl, halogen, hydroxycarbonyl or C ₁ -C ₆ alkoxycarbonyl;

R _a and R _b are each independently hydrogen, hydroxyl, halogen, C ₁ -C ₆ alkyl or hydroxy C ₁ -C ₆ alkyl;

Z is a methylene group or a direct bond;

Indicates a single or double bond.

Further preferably, _R3 and _R8 are each independently hydrogen, hydroxyl, methyl, ethyl,

In the present invention unless otherwise indicated, Indicates the junction site;

Preferably, R is methyl, formaldehyde, _-COOH , methoxycarbonyl,

In a preferred embodiment of the present invention, further preferably, the general formula (I) described in the present invention has the structure shown in the following general formula (II):

The definition of each substituent in the general formula (II) is the same as that in the general formula (I).

Further preferably, the general formula (I) described in the present invention has the structure shown in the following general formula (III) or (IV):

Wherein, in the general formula (III) or (IV), the definition of each substituent is the same as that in the general formula (I).

In the specification, the term "C ₁ -C ₆ alkyl" may be straight chain or branched C ₁ -C ₆ alkyl, especially, may be methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, isobutyl, pentyl, neopentyl or hexyl; preferably linear or branched C ₁ -C ₃ alkyl.

In the description, the term "5-8 membered aryl" is an aromatic group with 5-8 membered rings, preferably phenyl;

In the description, the term "5 to 8 membered cycloalkyl" refers to a cycloalkyl group having a 5 to 8 membered ring, specifically, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl;

The pentacyclic triterpenoid compound of general formula (I) structure of the present invention is specifically:

According to another aspect, the present invention provides a method for preparing the compound represented by general formula (I).

The preparation method includes the following preparation routes:

Route 1:

Route two:

Route three:

Specifically, the Dess-Martin oxidation of compound 2 in dichloromethane gave compound 16, which was then reacted with borane in dry THF under the catalysis of (S)-CBS to give the 3-OH inverted Compound 17, finally compound 17 is debenzylated by catalytic hydrogenation in methanol to obtain compound 1', compound 1' is reacted with alcohol or amine to obtain compound 2', compound 2' is reacted with acid or acid chloride to obtain compound 3';

Route 4:

Wherein, R ₁₆ and R ₁₇ are respectively hydrogen, C ₁ -C ₆ alkyl or hydroxy C ₁ -C ₆ alkyl;

R ₁₈ is a C ₁ -C ₆ alkyl group; the rest of the substituents are as defined in the general formula (I).

The present invention also provides a pharmaceutical composition for treating type 2 diabetes, which contains one or more pentacyclic triterpenoids represented by general formula (I) of the present invention as active ingredients. The composition may further include pharmaceutically conventional adjuvants, such as dispersants, excipients, disintegrants, antioxidants, sweeteners, coating agents and the like.

According to another aspect of the present invention, the use of the compound represented by the general formula (I) in the preparation of a drug for treating type 2 diabetes is provided.

According to yet another aspect of the present invention, the use of the compound represented by the general formula (I) as a TGR5 agonist is provided.

According to another aspect of the present invention, the application of the compound represented by the general formula (I) in the treatment of diabetes is provided.

Beneficial effect

The present invention designs and synthesizes a new class of pentacyclic triterpenoids, which can effectively stimulate TGR5, and is useful for making medicines for treating type 2 diabetes, and overcomes the gallbladder problems existing in the existing chemically synthesized small-molecule TGR5 agonists. Compared with the positive control INT777, it has a simpler synthesis method and milder reaction conditions. The raw materials of the pentacyclic triterpenes in the present invention are abundant in nature and have the structural advantages of natural products.

Detailed ways

The present invention will be further described below in conjunction with specific examples, but the present invention is not limited to these examples.

Compound preparation example

In the following preparation examples, NMR is measured with a Mercury-Vx 300M instrument produced by Varian, NMR calibration: δH 7.26ppm (CDCl ₃ ), 2.50ppm (DMSO-d ₆ ); Determination by coupled instrument or SHIMADZU GCMS-QP5050A; reagents are mainly provided by Shanghai Chemical Reagent Company; TLC thin-layer chromatography silica gel plate is produced by Shandong Yantai Huiyou Silica Gel Development Co., Ltd., model HSGF 254; normal phase column chromatography silica gel used for compound purification It is produced by Shandong Qingdao Ocean Chemical Factory, model zcx-11, 200-300 mesh.

Preparation Example 1 (Compound No.: C33)

(1) Benzyl betulinate

Dissolve the raw material betulinic acid (4g, 8.76mmol) (purchased from Xi'an Haoxuan Biotechnology Co., Ltd.) in DMF (50mL) at room temperature, add anhydrous potassium carbonate (2.4g, 17.37mmol), and slowly drop it under stirring After adding benzyl chloride (1.2 mL, 10.52 mmol) dropwise, the reaction solution was moved to 50° C. and stirred overnight. The next day, the mixture was cooled to room temperature, diluted with 100 mL of deionized water, extracted with ethyl acetate (2×100 mL), and the combined organic layers were washed with deionized water and saturated brine, dried over sodium sulfate and reduced Pressure distillation gave the desired white solid compound benzyl betulinate (4.62 g), molar yield: 97%. ¹ H NMR (300MHz, CDCl ₃ )δ7.34(m,5H),5.09(d,1H,J=11.7Hz),5.17(d,1H,J=11.7Hz),4.75(s,1H),4.62 (s,1H),3.21-3.15(m,1H),2.92-2.80(m,1H),2.10-1.90(m,2H),1.87-1.69(m,2H),1.64(s,3H),1.64 -0.96(m, other alicyclic protons), 1.04(s,3H), 1.00(s,3H), 0.94(s,3H), 0.91(s,3H), 0.87(s,3H), 0.84(s, 3H); ESI-MS (m/z): 569.4 (M+Na) ⁺ (calc. for C ₃₇ H ₅₄ O ₃ : 546.41).

(2) Benzyl 3-carbonyl betulinate

The product from the previous step (4.62g, 8.64mmol) was dissolved in dichloromethane (100mL) in an ice-water bath, and Dess-Martin oxidant (4.3g, 10.15mmol) was added in batches, slowly raised to room temperature and stirred overnight. The next day, the reaction mixture was filtered and spin-dried, and separated by column chromatography with petroleum ether/ethyl acetate as an eluent system of 20:1 to obtain the compound 3-carbonyl betulinic acid benzyl ester (4.39g) as a white Solid, molar yield: 95%. ¹ H NMR (300MHz, CDCl ₃ )δ7.34(m,5H),5.09(d,1H,J=11.7Hz),5.17(d,1H,J=11.7Hz),4.75(s,1H),4.62 (s,1H),2.92-2.80(m,1H),2.49-2.39(m,2H),2.10-2.04(m,2H),1.92-1.80(m,2H),1.78-1.68(m,2H) ,1.65(s,3H),1.50-1.16(m,other alicyclic protons),1.04(s,3H),1.00(s,3H),0.94(s,3H),0.91(s,3H),0.87( s, 3H), 0.84 (s, 3H); ESI-MS (m/z): 567.3 (M+Na) ⁺ (calc. for C ₃₇ H ₅₂ O ₃ : 544.39).

(3) Benzyl 3-α-hydroxybetulinate

Add the previous product (1.58g, 2.90mmol) and S-(-)-2-methyloxazoboridine (80mg, 0.29mmol) to a 100mL oven-dried round-bottomed flask, and add fresh sodium wire-treated THF (50 mL). Slowly add 10M borane-tetrahydrofuran solution (0.32mL) dropwise at room temperature, control the rate of addition, and complete the addition within 10 minutes. Stir at room temperature for 10 minutes. TLC monitoring shows that the reaction has been completed. Move the reaction bottle to an ice-water bath, slowly add methanol dropwise to quench the reaction, spin the solvent until no more bubbles are generated, and use petroleum ether/ethyl acetate as an eluent system of 20:1 for column chromatography separation. The compound 3-α-hydroxybetulinate benzyl ester (790 mg) was obtained as a white solid, molar yield: 50%. ¹ H NMR (300MHz, CDCl ₃ )δ7.34(m,5H),5.09(d,1H,J=11.7Hz),5.17(d,1H,J=11.7Hz),4.73(s,1H),4.60 (s,1H),3.39(s,1H),3.02-2.96(m,1H),2.28-2.16(m,2H),1.98-1.95(m,2H),1.68(s,3H),1.64-0.96 (m, other alicyclic protons),1.04(s,3H),1.00(s,3H),0.94(s,3H),0.91(s,3H),0.87(s,3H),0.84(s,3H) ; ESI-MS (m/z): 569.4 (M+Na) ⁺ (theoretical for C ₃₇ H ₅₄ O ₃ : 546.41).

(4) 3-α-Hydroxybetulinic acid

The product from the previous step (100mg, 0.18mmol) was dissolved in methanol (8mL) and a small amount of ethyl acetate. After changing the nitrogen, quickly add 10% Pd/C, then change the nitrogen and hydrogen, and stir at room temperature. One hour later, TLC detected that the reaction was complete. Pd/C was filtered off after changing the nitrogen gas, and the reaction solution was spin-dried and separated by column chromatography with petroleum ether/ethyl acetate as an eluent system of 10:1 to obtain compound C33 (78 mg) as a white solid with a molar yield of : 94%. ¹ H NMR (300MHz, CDCl ₃ )δ4.73(s,1H),4.60(s,1H),3.39(s,1H),3.02-2.96(m,1H),2.28-2.16(m,2H), 1.98-1.95(m,2H),1.68(s,3H),1.64-0.96(m,other alicyclic protons),1.04(s,3H),1.00(s,3H),0.94(s,3H),0.91 (s,3H), 0.87(s,3H), 0.84(s,3H); ESI-MS (m/z): 479.3 ( _M +Na) ⁺ (theoretical for _C30H48O3 : _456.36 ).

Preparation example two (compound number: C34)

Betulinic acid (1.2g, 2.63mmol) was dissolved in methanol/ethyl acetate (40mL/10mL). After changing the nitrogen, add a catalytic amount of Pd/C, and then change the nitrogen and hydrogen. Stir at room temperature for 2 days, and detect the reaction by TLC completely. After changing the nitrogen gas, the reaction solution was filtered, spin-dried, and separated by column chromatography with petroleum ether:ethyl acetate at a polarity of 10:1. The product C34 was obtained as a white solid (1.04 g, 2.27 mmol) with a molar yield of 86%. ¹ H NMR (300MHz, CDCl ₃ ) δ3.13 (t, 1H, J=9.0, 6.9Hz), 2.28-2.16 (m, 2H), 1.98-1.78 (m, 4H), 1.64-0.96 (m, others alicyclic proton),0.96(s,3H),0.93(s,3H),0.92(s,3H),0.90(s,3H),0.89(s,3H),0.87(s,3H),0.78(s ,3H); ESI-MS (m/z): 481.3 (M+Na) ⁺ (theoretical for C ₃₀ H ₅₀ O ₃ : 458.38).

Using the same method, using different natural products or compounds as raw materials (raw material oleanolic acid: C2, raw material ursolic acid: C7, raw material glycyrrhetinic acid: C107, raw material betulin: C29, raw material: C34), According to the same method of Preparation Example 1, the following compounds or intermediates were synthesized:

Preparation Example 3 3-α-Acetoxy Betulinic Acid (Compound No.: C97)

(1) Benzyl 3-α-acetoxybetulinate

3-α-Hydroxybetulinate benzyl ester (107mg, 0.20mmol) and a catalytic amount of DMAP (10mg, 0.08mmol) were dissolved in dichloromethane (10mL), triethylamine (82mL, 0.60mmol) was added, and ice-water bath Acetic anhydride (42 mL, 0.60 mmol) was added dropwise and reacted at room temperature for 12 hours. TLC showed that the reaction was complete. After concentrating to remove the solvent, diluting with ethyl acetate, washing with water and saturated aqueous sodium chloride solution respectively, drying and concentrating the organic phase, and purifying the residue by column chromatography with petroleum ether/ethyl acetate as the eluent of 20:1 The compound 3-α-acetoxy betulinate benzyl ester was obtained as white solid (77 mg, 0.13 mmol), molar yield: 65%. ¹ H NMR (300MHz, CDCl ₃ )δ7.34(m,5H),5.09(d,1H,J=11.7Hz),5.17(d,1H,J=11.7Hz),4.62(s,1H),2.84 -2.20(m,4H),2.08(s,3H),1.98-1.15(m,other alicyclic protons),1.01(s,3H),0.94(s,3H),0.92(s,3H),0.85( s,3H), 0.83(s,3H), 0.76(s,3H), 0.74(s,3H); ESI-MS(m/z): 611.4(M+Na) ⁺ (C ₃₉ H ₅₆ O _4theoretical Value: 588.42).

(2) 3-α-Acetoxy betulinic acid

The product from the previous step (77mg, 0.13mmol) was dissolved in methanol (8mL) and a small amount of ethyl acetate. After changing the nitrogen, quickly add 10% Pd/C, and then change the nitrogen and hydrogen, and stir at room temperature. One hour later, TLC detected that the reaction was complete. Pd/C was filtered off after changing the nitrogen, and the reaction solution was spin-dried and separated by column chromatography with petroleum ether/ethyl acetate as an eluent system of 10:1 to obtain the compound 3-α-acetoxy betulinic acid (60mg ,0.12mmol), as a white solid, molar yield: 92%. ¹ H NMR (300MHz, CDCl ₃ ) δ4.62(s, 1H), 2.84-2.20(m, 4H), 2.08(s, 3H), 1.98-1.15(m, other alicyclic protons), 1.01(s, 3H),0.94(s,3H),0.92(s,3H),0.85(s,3H),0.83(s,3H),0.76(s,3H),0.74(s,3H); ESI-MS(m /z): 521.3 (M ₊ Na) ⁺ (calc. for _C32H50O4 : _498.37 ).

Synthesize following compound with the same method of embodiment three and different acid anhydrides:

Preparation Example Four (Compound No. C46)

(1) Benzyl 3-α-hydroxy-20,21-epoxy betulinate

The intermediate 3-α-hydroxybetulinic acid benzyl ester (90mg, 0.17mmol) was dissolved in carbon dichloride (10mL), m-chloroperoxybenzoic acid (71mg, 0.34mmol) was slowly added in an ice-water bath, and stirred at room temperature After 3 hours, TLC detected that the reaction was complete, adding a saturated solution of sodium sulfite to quench the reaction, washing the organic phase with water, drying and concentrating the resulting residue, and performing column chromatography separation with an eluent system of petroleum ether/ethyl acetate of 6:1 to obtain compound 3 -74 mg of benzyl α-hydroxy-20,21-epoxy betulinate, a white solid, molar yield: 80.4%. ¹ H NMR (300MHz, CDCl ₃ )δ7.34(m,5H),5.09(d,1H,J=11.7Hz),5.17(d,1H,J=11.7Hz),3.40(s,1H),2.64 (t,2H),2.25(m,2H),2.14(m,2H),1.97(m,2H),1.78(m,2H),1.76-1.32(m, other alicyclic protons),1.28(s, 3H),0.98(s,3H),0.93(s,3H),0.92(s,3H),0.90(s,3H),0.86(s,3H),0.82(s,3H); ESI-MS(m /z): 585.4 (M ₊ Na) ⁺ (calc. for _C37H54O4 : _562.40 ).

(2) 3-α-Hydroxy-20,21-epoxy betulinic acid

The product from the previous step (74mg, 0.14mmol) was dissolved in methanol (8mL) and a small amount of ethyl acetate. After changing the nitrogen, quickly add 10% Pd/C, and then change the nitrogen and hydrogen, and stir at room temperature. One hour later, TLC detected that the reaction was complete. Pd/C was filtered off after changing the nitrogen, and the reaction solution was spin-dried and separated by column chromatography with petroleum ether/ethyl acetate as the eluent system of 6:1 to obtain the compound 3-α-hydroxyl-20,21-epoxy Betulinic acid (38 mg, 0.08 mmol), a white solid, molar yield: 58%. ¹ H NMR (300MHz, CDCl ₃ )δ3.40(s,1H),2.64(t,2H),2.25(m,2H),2.14(m,2H),1.97(m,2H),1.78(m, 2H),1.76-1.32(m, other alicyclic protons),1.28(s,3H),0.98(s,3H),0.93(s,3H),0.92(s,3H),0.90(s,3H), 0.86 (s, 3H), 0.82 (s, 3H); ESI-MS (m/z): 495.3 (M+Na) ⁺ (theoretical for C ₃₀ H ₄₈ O ₄ : 472.36).

Synthesize C36 with the same method of embodiment four:

Preparation Example five (compound number: C48)

(1) Benzyl 3-α-hydroxy-20-formyl betulinate

The intermediate 3-α-hydroxy-20,21-epoxy betulinic acid benzyl ester (68mg, 0.12mmol) was dissolved in carbon trichloride (10mL), two drops of concentrated hydrochloric acid were added dropwise, refluxed for 1h, and the reaction was complete by TLC detection , direct drying and concentrating the obtained residue with petroleum ether/ethyl acetate as the eluent system of 4:1 to carry out column chromatography separation to obtain compound 3-α-hydroxy-20-formyl betulinic acid benzyl ester 50 mg, which is white Solid, molar yield: 73.6%. ¹ H NMR (300MHz, CDCl ₃ ) δ9.84(s, 1H), 7.34(m, 5H), 5.09(d, 1H, J=11.7Hz), 5.17(d, 1H, J=11.7Hz), 3.39 (s,1H),3.33(d,1H,J=4.2Hz),2.40(td,1H,J=11.7,2.7Hz),2.28(td,1H,J=11.7,2.7Hz),2.30-2.18( m,2H),1.98-1.84(m,2H),1.69-0.96(m,other alicyclic protons),1.11(s,3H),0.93(s,3H),0.90(s,3H),0.89(s ,3H), 0.87(s,3H), 0.78(s,3H); ESI-MS (m/z): 585.4 (M+Na) ⁺ (theoretical for C ₃₇ H ₅₄ O ₄ : 562.40).

(2) 3-α-Hydroxy-20-formyl betulinic acid

The product from the previous step (50mg, 0.088mmol) was dissolved in methanol (5mL) and a small amount of ethyl acetate. After changing the nitrogen, quickly add 10% Pd/C, then change the nitrogen and hydrogen, and stir at room temperature. One hour later, TLC detected that the reaction was complete. Pd/C was filtered off after changing the nitrogen gas, and the reaction solution was spin-dried and separated by column chromatography with petroleum ether/ethyl acetate as the eluent system of 2:1 to obtain the compound 3-α-hydroxy-20-formyl betulin Acid (36.7mg, 0.078mmol), white solid, molar yield: 87.3%. ¹ H NMR (300MHz, CDCl ₃ )δ9.84(s,1H),3.39(s,1H),3.33(d,1H,J=4.2Hz),2.58(m,1H),2.40(m,1H) ,2.23(m,2H),1.98-1.84(m,2H),1.69-0.96(m,other alicyclic protons),1.12(d,3H,J＝9.0Hz),0.96(s,3H),0.93( s,3H), 0.87(s,3H), 0.84(s,3H), 0.81(s,3H); ESI-MS(m/z): 495.2(M+Na) ⁺ (C ₃₀ H ₄₈ O ₄ Theoretical Value: 472.36).

Preparation example six (compound number: C53)

(1) Benzyl 3-α-hydroxy-20-(2'-hydroxyethylamino) betulinate

The intermediate 3-α-hydroxy-20-formyl betulinic acid benzyl ester (20mg, 0.036mmol) and sodium cyanoborohydride (11mg, 0.018mmol) were stirred at room temperature for 2h, then ethanolamine (11μL, 0.18mmol) was added dropwise ) Stir at room temperature overnight, TLC detects that the reaction is complete, the saturated sodium bicarbonate solution quenches the reaction, dilutes with water and extracts twice with ethyl acetate, after washing with saturated brine, the organic phase is dried and concentrated, and the obtained residue is 20: The eluent system of 1 was separated by column chromatography to obtain 12 mg of compound 3-α-hydroxy-20-(2'-hydroxyethylamino) betulinate benzyl ester as a white solid, molar yield: 60%. ¹ H NMR (300MHz, CDCl ₃ +CD ₃ OD) δ7.34(m, 5H), 5.09(d, 1H, J=11.7Hz), 5.17(d, 1H, J=11.7Hz), 3.49(t, 2H, J＝4.5Hz), 3.00(s, 1H), 2.75(t, 2H, J＝4.5Hz), 2.57-2.46(m, 2H), 2.10-1.85(m, 4H), 1.55-0.90(m , other alicyclic protons), 0.93(s,3H),0.66(s,3H),0.62(d,3H,J＝6.0Hz),0.58(s,3H),0.52(s,3H),0.48(s ,3H); ESI-MS (m/z): 608.5 (M+H) ⁺ (theoretical for C ₃₈ H ₅₉ NO ₄ : 607.91).

(2) 3-α-Hydroxy-20-(2'-hydroxyethylamino) betulinic acid

The product from the previous step (12mg, 0.020mmol) was dissolved in methanol (2mL) and a small amount of ethyl acetate. After changing the nitrogen, quickly add 10% Pd/C, then change the nitrogen and hydrogen, and stir at room temperature. One hour later, TLC detected that the reaction was complete. Pd/C was filtered off after changing the nitrogen gas, and the reaction solution was spin-dried and separated by column chromatography with an eluent system of petroleum ether/ethyl acetate of 4:1 to obtain the compound 3-α-hydroxyl-20-(2'- Hydroxyethylamino) betulinic acid (9.3 mg, 0.018 mmol), a white solid, molar yield: 91%. ¹ H NMR (300MHz, CDCl ₃ +CD ₃ OD) δ3.49(t, 2H, J=4.5Hz), 3.00(s, 1H), 2.75(t, 2H, J=4.5Hz), 2.57-2.46( m, 2H), 2.10-1.85(m, 4H), 1.55-0.90(m, other alicyclic protons), 0.93(s, 3H), 0.66(s, 3H), 0.62(d, 3H, J＝6.0Hz ), 0.58(s,3H), 0.52(s,3H), 0.48(s,3H); ESI-MS(m/z): 518.4(M+H) ⁺ (C ₃₂ H ₅₅ NO ₄ Theoretical value: 517.41 ).

Preparation Example 7 (Compound No.: C47)

(1) Benzyl 3-α-hydroxy-22-hydroxybetulinate

Selenium dioxide (22 mg, 0.22 mmol) and tert-butyl hydroperoxide (165 μL, 5.5 M solution in tetrahydrofuran) were dissolved in dry dichloromethane (20 mL), and a catalytic amount of acetic acid (6 μL, 0.1 eq.), after stirring at this temperature for ten minutes, slowly add a dichloromethane solution (5mL) of intermediate 3-α-hydroxy betulinate benzyl ester (242mg, 0.44mmol), slowly warming up to room temperature and stirring Overnight, TLC detected that the reaction was complete, adding a saturated solution of sodium sulfite to quench the reaction, extracting with dichloromethane and washing the organic phase with water, drying and concentrating the obtained residue was directly used in the next step. The crude product was dissolved in methanol (20 mL), and sodium borohydride (25 mg, 0.66 mmol) was added in batches under an ice-water bath. After one hour of reaction, TLC detected that the reaction was complete, and the reaction was quenched by adding a saturated solution of ammonium chloride dropwise, extracted with ethyl acetate and used After washing with saturated brine, the organic phase was dried and concentrated, and then separated by column chromatography with petroleum ether/ethyl acetate as the eluent system of 2:1 to obtain 197 mg of the compound 3-α-hydroxy-22-hydroxybetulinic acid benzyl ester, It is a white solid, molar yield: 79.2%. ¹ H NMR (300MHz, CDCl ₃ )δ7.34(m,5H),5.09(d,1H,J=11.7Hz),5.17(d,1H,J=11.7Hz),4.97(s,1H),4.92 (s,1H),4.12(m,2H),3.39(s,1H),2.88(td,1H,J=12.0,3.0Hz),2.32-2.26(m,2H),2.21-2.10(m,2H ),1.98-1.77(m,2H),1.64-0.96(m,other alicyclic protons),0.99(s,3H),0.94(s,3H),0.91(s,3H),0.87(s,3H) , 0.84 (s, 3H); ESI-MS (m/z): 585.4 (M+Na) ⁺ (theoretical for C ₃₇ H ₅₄ O ₄ : 562.40).

(2) 3-α-hydroxy-22-hydroxy betulinic acid

The product from the previous step (197mg, 0.35mmol) was dissolved in methanol (10mL) and a small amount of ethyl acetate. After changing the nitrogen, quickly add 10% Pd/C, and then change the nitrogen and hydrogen, and stir at room temperature. One hour later, TLC detected that the reaction was complete. Pd/C was filtered off after changing the nitrogen gas, and the reaction solution was spin-dried and separated by column chromatography with petroleum ether/ethyl acetate as the eluent system of 2:1 to obtain the compound 3-α-hydroxy-22-hydroxy betulinic acid (140mg, 0.30mmol), as a white solid, molar yield: 85%. ¹ H NMR (300MHz, CDCl ₃ ) δ4.97(s, 1H), 4.92(s, 1H), 4.12(m, 2H), 3.39(s, 1H), 2.88(td, 1H, J=12.0, 3.0 Hz), 2.32-2.26(m, 2H), 2.21-2.10(m, 2H), 1.98-1.77(m, 2H), 1.64-0.96(m, other alicyclic protons), 0.99(s, 3H), 0.94 (s,3H),0.91(s,3H),0.87(s,3H),0.84(s,3H); ESI-MS(m/z):495.3(M+Na) ⁺ (C ₃₀ H ₄₈ O ₄ Theoretical value: 472.36).

Preparation Example Eight (Compound No. C111)

Betulin (250mg, 0.56mmol) was dissolved in dichloromethane (20mL), and Dess-Martin oxidant (718mg, 4.5mmol) was slowly added at room temperature, and the reaction was refluxed for 4h, and the reaction was complete by TLC. The reaction mixture was filtered and spin-dried, and separated by column chromatography with petroleum ether/ethyl acetate as an eluent system of 10:1 to obtain compound 3-carbonyl-betulin (145mg) as a white solid, molar yield : 58.7%. ¹ H NMR (300MHz, CDCl ₃ )δ9.66(s,1H),4.75(s,1H),4.62(s,1H),2.92-2.80(m,1H),2.49-2.39(m,2H), 2.10-2.04(m,2H),1.92-1.80(m,2H),1.78-1.68(m,2H),1.65(s,3H),1.50-1.16(m,other alicyclic protons),1.04(s, 3H),1.00(s,3H),0.94(s,3H),0.91(s,3H),0.87(s,3H),0.84(s,3H); ESI-MS(m/z):456.3(M +Na) ⁺ (C ₃₀ H ₄₆ O ₂ calc.: 438.35).

Preparation Example Nine (Compound No. C37)

Betulinic acid (41 mg, 0.09 mmol) was dissolved in dry THF (5 mL), and BH ₃ -Me ₂ S in THF (2 M) was added dropwise in an ice-water bath, stirred for one hour, and then brought to room temperature. The next day, the reaction solution was moved to an ice-water bath, and ethanol (280 μL), saturated sodium acetate solution (200 μL), and 30% hydrogen peroxide solution (140 μL) were added sequentially. Stir overnight at room temperature, and TLC detects that the reaction is complete. Dilute with water, extract with ethyl acetate and wash with saturated brine. After the organic phase was dried and concentrated, it was separated by column chromatography with chloroform/methanol as the eluent system of 50:1 to obtain 19 mg of compound 20(29)-reduced-29-hydroxybetulinic acid as a white solid, molar yield: 45.3 %. ¹ H NMR (300MHz, CD ₃ OD) δ3.73 (dd, 1H, J = 9.0, 3.0Hz), 3.34 (s, 1H), 3.13 (dd, 1H, J = 9.0, 6.0Hz), 2.36-2.12 (m,4H),1.83-1.08(m,other alicyclic protons),0.99(s,3H),0.96(s,3H),0.95(s,3H),0.94(s,3H),0.87(s, 3H), 0.75 (s, 3H); ESI-MS (m/z): 497.3 (M+Na) ⁺ (calc. for C ₃₀ H ₅₀ O ₄ : 474.37).

Preparation Example 10 (Compound No. C115)

20(29)-Reduced betulinic acid (37mg, 0.08mmol) and EDCI (21mg, 0.12mmol) in DMF (2mL), added triethylamine (15μL), HOBt (15mg, 0.12mmol) and N-Boc- 2-(2-Aminoethoxy)ethylamine (15mg, 0.08mmol), stirred overnight at 50°C. The next day, TLC detected that the reaction was complete, and the reaction solution was diluted with water, extracted with ethyl acetate and washed with saturated brine. The organic phase was dried and concentrated, and then separated by column chromatography with chloroform/methanol as an eluent system of 80:1 to obtain 45 mg of the target compound as a white solid with a molar yield of 86.5%. ¹ H NMR (300MHz, CDCl ₃ ) δ5.93 (t, 1H, J = 5.1Hz), 4.84 (brs, 1H), 3.48 (m, 4H), 3.13 (dd, 1H, J = 9.0, 6.0Hz) ,3.30(m,4H),2.40(td,1H,J=12.0,3.0Hz),2.23(td,1H,J=12.0,3.0Hz),1.44(s,9H),1.96-1.09(m,other alicyclic proton),0.96(s,3H),0.93(s,3H),0.92(s,3H),0.90(s,3H),0.89(s,3H),0.87(s,3H),0.78(s ,3H); ESI-MS (m/z): 667.5 (M+Na) ⁺ (theoretical for C ₃₉ H ₆₈ N ₂ O ₅ : 644.51).

Synthesize following compound with the same method of embodiment ten and different amines:

Preparation Example 11 (Compound No. C39)

Intermediate 3-carbonyl-20(29)-reduced betulinic acid (82mg, 0.18mmol), hydroxylamine hydrochloride (25mg, 0.37mmol) and pyridine (37μL, 0.46mmol) in absolute ethanol (5mL), stirred overnight at room temperature , and the next day TLC detected that the reaction was complete. Directly concentrated and separated by column chromatography with petroleum ether/ethyl acetate as an eluent system of 10:1, 77 mg of the target compound was obtained as a white solid with a molar yield of 91.2%. ¹ H NMR (300MHz, CDCl ₃ )ClR (300MHz,: remove the benzyl group and reduce the double bond at the same time to obtain 0.92(s,3H), 0.90(s,3 other alicyclic protons), 0.94(s,3H), 0.91(s,3H),0.90(s,3H),0.88(s,3H),0.85(s,3H),0.84(s,3H),0.76(s,3H); ESI-MS(m/z) : 494.3 (M+Na) ⁺ (calc. for C ₃₀ H ₄₉ NO ₃ : 471.37).

Preparation Example 12 (Compound No. C119)

Molecular sieves and dry dichloromethane (10 mL) were placed in a dry 50 mL round bottom flask, and redistilled tetraisopropyl titanium oxide (1 μL, 0.0037 mmol) and D-(-)- DIPT (1 μL, 0.0056 eq). After stirring for 15 minutes, 5.5 M TBHP in THF (10 μL, 0.056 mmol) was added and stirred for 30 minutes. Add the dichloromethane solution (2 mL) of the benzyl ester (21 mg, 0.037 mmol) of the product obtained in Example 7 dropwise, and place it in a -20°C refrigerator overnight. The next day, the temperature of the reaction solution was raised to zero, and 5N sodium hydroxide saturated saline solution (50 μL) was added. After stirring for one hour, the molecular sieves were removed by filtration. The system was separated by column chromatography to obtain 16 mg of the target compound as a white solid, with a molar yield of 76.2%. NMR proves that C-17 is a product mainly in S configuration. ¹ H NMR (300MHz, CDCl ₃ )δ7.34(m,5H),5.09(s,2H),3.80-3.64(m,2H),3.40(s,1H),2.92(d,1H,J＝4.5 Hz), 2.63(d, 1H, J=4.5Hz), 2.35-2.24(m, 2H), 2.01-1.92(m, 4H), 1.86-1.75(m, 4H), 1.57-1.28(m, other fat ring proton), 0.97(s,3H), 0.93(s,3H), 0.92(s,3H), 0.86(s,3H), 0.82(s,3H). The product was directly debenzylated under the catalysis of palladium carbon according to the previous steps to obtain 12 mg of the target product as a white solid with a molar yield of 89.2%. ¹ H NMR (300MHz, CDCl ₃ ) δ3.76 (d, 1H, J = 12.0Hz), 3.67 (d, 1H, J = 12.0Hz), 3.40 (s, 1H), 2.91 (d, 1H, J = 6.0Hz), 2.62(d, 1H, J=6.0Hz), 2.35-2.24(m, 2H), 2.01-1.92(m, 4H), 1.86-1.75(m, 4H), 1.57-1.28(m, others alicyclic proton), 0.97(s,3H), 0.93(s,3H), 0.92(s,3H), 0.86(s,3H), 0.82(s,3H); ESI-MS(m/z):511.3 (M+Na) ⁺ ₍ calc. for _C30H48O5 : _488.35 ).

Synthesize the following compounds in the same manner as in Example 12:

Preparation Example 13 (compound number C75)

The benzyl ester (16mg, 0.028mmol) and DMAP (1mg, 0.1eq) of the product obtained in Preparation Example 12 were added dropwise to dichloromethane (5ml), and TEA (6μL, 0.04mmol) and acetyl chloride (3μL , 0.04mmol), stirred overnight at room temperature. The next day, TLC detected that the reaction was complete. Directly concentrated and separated by column chromatography with petroleum ether/ethyl acetate 2:1 eluent system, 14 mg of the target compound was obtained as a white solid with a molar yield of 82.3%. The product was directly debenzylated under the catalysis of palladium carbon according to the previous steps to obtain 6 mg of the target product as a white solid, molar yield: 50%. ¹ H NMR (300MHz, CDCl ₃ ) δ4.35 (d, 1H, J = 12.0Hz), 4.04 (d, 1H, J = 12.0Hz), 3.40 (s, 1H), 2.76 (d, 1H, J = 6.0Hz), 2.66(d, 1H, J=6.0Hz), 2.35-2.24(m, 2H), 2.09(s, 3H), 2.01-1.92(m, 4H), 1.86-1.75(m, 4H), 1.57-1.28(m, other alicyclic protons), 0.97(s,3H), 0.93(s,3H), 0.92(s,3H), 0.86(s,3H), 0.82(s,3H); ESI-MS (m/z): _553.3 (M+Na) ⁺ (calc. for _C32H50O6 : _530.36 ).

Synthesize the following compounds with the same method of Example 13 and different acid chlorides:

Preparation Example 14: Synthesis of Compound C94

Compound A2 (2mL, 19.9mmol) and triethylamine (2.8mL, 19.9mmol) in dichloromethane (40mL), slowly added benzyl alcohol chloroformate (2.72mL, 19.9mmol) dropwise under ice-water bath, at room temperature The reaction was stirred overnight. The next day, TLC detected that the reaction was complete, the reaction system was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (chloroform/methanol=40/1, V/V) to obtain intermediate S29 (4.45 g, yield 93.1%) , a colorless oil. ¹ H NMR (300MHz, CDCl ₃ ) δ7.36-7.26 (m, 5H), 5.29 (brs, 1H), 5.10 (s, 2H), 3.71 (t, 2H, J=4.8Hz), 3.54 (m, 4H), 3.40 (t, 2H, J = 5.1 Hz), 2.31 (brs, 1H).

The obtained intermediate S29 (1.49g, 6.23mmol) was dissolved in acetonitrile/water (10mL/5mL) mixed solvent, and diacetoxyiodobenzene (6g, 18.68mmol) and tetramethylpiperidine (TEMPO , 292mg, 1.87mmol), the reaction was stirred overnight at room temperature. The next day, TLC detected that the reaction was complete, adding saturated sodium sulfite solution and stirring for 10 min, adjusting the pH to 5 with 1N hydrochloric acid solution, extracting with dichloromethane, combining the organic phases and washing with saturated brine, drying the organic phases, and concentrating under reduced pressure to obtain a residue After purification by silica gel column chromatography (chloroform/methanol=20/1, V/V), the intermediate S30 (1.23 g, yield 77.8%) was obtained as a yellow oil. ¹ H NMR (300MHz, CDCl ₃ ) δ7.36-7.26 (m, 5H), 5.42 (brs, 1H), 5.10 (s, 2H), 4.12 (s, 2H), 3.64 (t, 2H, J = 4.8 Hz), 3.43 (t, 2H, J = 5.1 Hz).

Intermediate S14 (31mg, 0.054mmol), S30 (27mg, 0.107mmol), EDCI (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) (20mg, 0.107mmol) and DMAP (4-dimethylaminopyridine) (7mg, 0.054mmol) in DMF (N,N-dimethylformamide) (5mL) were reacted overnight at room temperature, the next day the reaction solution was diluted with water, extracted with ethyl acetate, The organic phase was washed twice with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated and the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether=1/2) to obtain intermediate S31 (33 mg, Yield 76.7%), white solid. ¹ H NMR (300MHz, CDCl ₃ )δ7.36-7.26 (m, 5H), 5.45 (brs, 1H), 5.10 (m, 2H), 4.43 (d, 1H, J=12.0Hz), 4.20 (s, 1H), 4.13(s, 2H), 3.61(t, 2H, J=5.1Hz), 3.41(t, 2H, J=5.1Hz), 3.39(s, 1H), 2.63(dd, 2H, J=8.7 ,4.8Hz),2.30(m,2H),2.10(m,2H),1.90(m,4H),1.82(m,4H),1.76-1.32(m, other alicyclic hydrocarbon protons),0.90(s, 3H), 0.87(s,3H), 0.84(s,3H), 0.81(s,3H), 0.73(s,3H).

The obtained intermediate S31 was dissolved in methanol, and catalytic hydrogenolysis was carried out in the presence of Pd-C under normal pressure, and the benzyloxycarbonyl group and the benzyl group of C-20 were removed at the same time to obtain intermediate S32, which was directly carried out in the next step without purification. . Intermediate S32 (30mg, 0.05mmol), active ester Biotin-OSu (17mg, 0.05mmol) and triethylamine (200μL) were dissolved in dry DMF (2mL) solvent, heated to 50°C and stirred overnight. After cooling to room temperature, the reaction system was diluted with water (20 mL), extracted with ethyl acetate, the combined organic phases were washed with saturated brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated, and the resulting crude product was purified by silica gel column chromatography to obtain compound C82 (Yield 60%), white solid. ¹ H NMR (300MHz, CDCl ₃ )δ6.75(brs,1H),5.33(brs,1H),5.23(brs,1H),4.63(d,1H,J=12.0Hz),4.50(t,1H, J＝6.0Hz), 4.31(t, 1H, J＝6.0Hz), 4.10(m, 2H+2H), 3.94(d, 1H, J＝12.0Hz), 3.65(m, 2H), 3.48(m, 2H), 3.39(s, 1H), 3.19(m, 1H), 2.90(m, 1H), 2.72(d, 1H, J=6.0Hz), 2.67(d, 1H, J=6.0Hz), 2.25( m, 4H), 1.95 (m, 4H), 1.78-1.09 (m, other alicyclic hydrocarbon protons), 0.95 (s, 3H), 0.93 (s, 3H), 0.87 (s, 3H), 0.83 (s, 3H), 0.81(s, 3H).

Test Example 1

TGR5 agonist test example

TGR5 agonism test Example 1 Compound mediates the accumulation of intracellular cyclic adenosine monophosphate 3'-5'-cyclic adenosine monophosphate (cAMP) by activating TGR5

1. Purpose of the test

HEK293 cells transiently transfected with TGR5 were stimulated with compounds, and then detected by Homogeneous Time-Resolved Fluorescence (HTRF), in order to detect whether these compounds increase intracellular cAMP accumulation through TGR5-mediated.

2. Test principle

G _αs protein-coupled bile acid receptor TGR5 will undergo structural changes after binding to agonists, thereby activating adenylate cyclase (AC), further catalyzing ATP to generate cAMP, and cAMP is activated in phosphodiesterase (PDE ) is further degraded into AMP, and IBMX can inhibit the activity of PDE, thereby inhibiting the degradation of cAMP into AMP. Therefore, the amount of accumulated cAMP can be detected by adding IBMX in the experiment, and the amount of cAMP can directly reflect whether the compound activates or inhibits GPCR-mediated AC. The cAMP produced by the HEK293 cells transiently transfected with TGR5 and the d2-labeled cAMP provided by the kit compete for the antigen-binding site of the anti-cAMP antibody. When monoclonal antibodies labeled with europium or terbium are combined with d2-labeled cAMP, there will be a relatively large signal, and as the cAMP produced in the cells increases, the signal will gradually decrease, resulting in a decrease in fluorescence readings. The effect of the compound on the accumulation of intracellular cAMP can therefore be reflected by the fluorescence readout.

3. Experimental samples

Dissolve the compound in DMSO before the test, prepare the mother solution, and dilute it with the culture medium to the required concentration when using it, and set INT-777 and Lithocholic Acid as the positive control of the test to detect whether the reaction of each test is normal or not .

4. Experimental method

4.1. Prepare the compound to be tested with 1xPBS to double the final concentration. The final concentration is 100 μM, 10 μM, 1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM, DMSO (each well contains 1% DMSO).

4.2. Cell treatment:

4.2.1. Digest the cells with trypsin, and then suspend them with serum-free medium.

4.2.2. Determine the cell density. At the same time, add IBMX (final concentration: 500 μM) to the serum-free medium, and the cell number is 2000/5 μl/well.

4.2.3. Add 5 μl of the compound to be tested & 5 μl of IBMX-containing cell suspension to mix, seal the 384-well plate with foil paper, and react at room temperature in the dark for no more than 30 minutes.

4.3. Detection substrate configuration

4.3.1. Dilute 1μl cAMP-d2 to 20μl with cAMP&cGMP conjugates&lysis buffer

4.3.2. Dilute 1μl anti-cAMP-Cryptate to 20μl with cAMP&cGMP conjugates&lysis buffer

4.3.3 After 30 minutes, add 5 μl (1.3.1) + 5 μl (1.3.2), seal the 384-well plate with tin foil, and react at room temperature for 1 minute in the dark.

4.4 After 60 minutes, the Envision2101 multifunctional microplate microplate reader (PerkinElmer) reads.

5. Experimental results: (Take fourteen compounds such as C29, C33, C40, and C98 as examples, but not limited to these compounds)

Table 1 Compound TGR5 agonistic activity test test

Note: EC ₅₀ is the evaluation of the agonistic activity of the sample drug on TGR5, half of the 50% effective concentration. NR indicates no activity at a concentration of 100 μM.

6. Results and discussion:

These compounds can increase intracellular cAMP accumulation in TGR5-expressing HEK293 cells, and their activities are dose-dependent, and the EC ₅₀ values are shown in Table 1. However, in HEK293 cells that do not express TGR5, these compounds failed to cause cAMP accumulation. The results further demonstrate that these compounds are agonists of the TGR5 receptor. And it is shown in the table that the agonistic activity of TGR5 in the 3-α configuration is significantly higher than that in the 3-β configuration.

Test embodiment two:

Compounds fail to activate reporter gene expression mediated by the nuclear receptor FXR

1. Purpose of the test

HEK293 cells using the transient expression plasmid pBind-FXR and the reporter gene plasmid pGL4.31 were stimulated with compounds, and then Steady- Stable luciferase detection system is used to detect whether these compounds increase the expression level of luciferase in cells through FXR.

2. Test principle

Mammalian one-hybrid, also known as GAL4 chimera receptor assay, was developed in recent years and is mainly used in nuclear receptor (Nuclearreceptor, NR) function A new technology for screening and evaluating the physiological activity of its ligands. The mechanism of this technology is to utilize two similar main domains in the molecular structure of yeast cell transcription factor GAL4 and mammalian cell nuclear receptor: ligand-binding domain (LBD) and DNA-binding domain (DBD). The ligand-binding domain (LBD) of the body was fused with the DNA-binding domain (DBD) of the yeast cell transcription factor GAL4 to form a chimeric protein expression plasmid, and then co-transfected with the reporter plasmid containing GAL4-specific response elements to animal cells. Expression levels of reporter genes to assess agonistic or antagonistic activity of nuclear receptor ligands.

3. Experimental samples

Before the test, dissolve the compound in DMSO, prepare the mother solution, and dilute it with the culture medium to the required concentration when using it, and set GW4604 as the positive control of the test to detect whether the reaction of each test is normal or not.

4. Experimental method

4.1. Transient transfer: Transiently transfer the expression plasmid pBind-FXR and the reporter gene plasmid pGL4.31 to HEK293 cells, and then inoculate 10,000 cells/well into 384-well plates in 10% FBS high-glucose DMEM at 37°C. Incubate for 12 hours under 5% CO2 condition.

4.2 Add compound: in the agonist test, dilute the agonist positive control and the compound to a final concentration of 10× and directly add to the cell culture plate with a volume of 5 μl, and continue to culture at 37°C and 5% CO2 for 24 hours.

4.3. Detection: 24 hours after dosing, use Steady- Stable luciferase detection system for detection. Aspirate 25 μL of medium from each well, add 25 μL of luciferase activity detection reagent, and shake for 5 minutes. Finally, the chemiluminescence count value was detected in Envision2101 multifunctional microplate microplate reader.

5. Experimental results: (take fourteen compounds such as C29, C33, C40, C98 as examples, but not limited to these compounds)

Table 2 Compound FXR agonistic activity test test

Note: EC ₅₀ is the evaluation of the agonistic activity of the sample drug on TGR5, half of the 50% effective concentration. NR indicates no activity at a concentration of 100 μM.

6. Results and discussion

In the transient nuclear receptor FXR, GW4604 can activate FXR and increase the expression of luciferase in cells by activating FXR, and its activity is dose-dependent. However, these compounds cannot increase the expression of luciferase in cells by activating FXR. The results indicated that these compounds could not act through the nuclear receptor FXR, which further indicated that these compounds had a certain selection specificity.

Claims (10)

1. A compound having a structure shown in general formula (I), Wherein, R ₁ is hydrogen, hydroxyl, halogen or C ₁ -C ₆ alkyl; R ₂ is hydrogen; hydroxyl; halogen; oxo group (=O); =N-OH; C ₁ -C ₆ alkylcarbonyloxy group _; C ₆ alkyl; 3 to 8 membered cycloalkyl; or Wherein, R _c is C ₁ -C ₆ alkyl or hydrogen; R ₃ and R ₈ are each independently: hydrogen; hydroxyl; halogen; amino C ₁ -C ₆ alkyl substituted by C ₁ -C ₆ alkyl; unsubstituted or substituted C ₁ -C ₆ alkyl, wherein, The substituent in the substituted C ₁ -C ₆ alkyl is selected from hydroxyl, halogen, oxo group (=O), =N-OH, epoxypropylene, amino or hydroxyl C ₁ -C ₆ alkylamino; Unsubstituted or substituted C ₁ -C ₆ alkenyl, wherein the substituents in the substituted C ₁ -C ₆ alkenyl are selected from hydroxyl, halogen, oxo (=O), =N-OH, amino or hydroxy C ₁ -C ₆ alkylamino; Wherein, R ₉ is H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, hydroxyl, hydroxyl C ₁ -C ₆ alkyl, -CH ₂ OC(O)R ₁₀ , -CH ₂ OC( O)OR ₁₁ , -CH ₂ OC(O)CH ₂ OR ₁₂ ; Wherein, R ₁₀ , R ₁₁ and R ₁₂ are each independently substituted or unsubstituted 5-8 membered aryl; substituted or unsubstituted 3-8 membered cycloalkyl; 5-8 membered arylamino; containing N, 5- to 8-membered heteroaryl with at least one heteroatom in S or O; C ₁ -C ₆ alkyl; halogenated C ₁ -C ₆ alkyl; hydroxy C ₁ -C ₆ alkyl; BocNH(CH ₂ ) _m O(CH ₂ ) _n -, wherein each m and n are the same or different, and are each independently an integer from 1 to 6; wherein, substituted 5 to 8-membered aryl or substituted 3 to 6 The substituents in the 8-membered cycloalkyl group are selected from hydroxyl, halogen, C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy; R ₄ is hydrogen, hydroxyl, halogen or C ₁ -C ₆ alkyl; R ₅ is independently selected from the group consisting of _hydrogen ; _hydroxy ; hydroxy C _{1 -C 6} alkyl; halogen; C ₁ -C ₆ alkyl; _o CH ₂ CH ₂ R ₁₄ ; -C(O)NH(CH ₂ CH ₂ O) _p CH ₂ CH ₂ R ₁₅ ; wherein, o and p are independently 0, 1, 2 or 3; or -C( O)NH(CH ₂ ) _q C(O)OH, q is an integer from 3 to 8; R ₁₃ is hydrogen; hydroxycarbonyl C ₁ -C ₈ alkylamino; hydroxy; unsubstituted or C ₁ -C ₆ alkyl substituted piperazinyl; benzyloxy group; C ₁ -C ₆ alkoxy ; tert-butoxycarbonyl C ₁ -C ₆ alkyloxy; or hydroxycarbonyl C ₁ -C ₆ alkylamino; R ₁₄ and R ₁₅ are independently hydrogen; amino; 5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanylamino; C ₁ -C ₆ amido ; tert-butoxycarboxamido; or C ₁ -C ₆ alkoxy; R ₆ and R ₇ are each independently hydrogen, hydroxyl, halogen, hydroxycarbonyl or C ₁ -C ₆ alkoxycarbonyl; R _a and R _b are each independently hydrogen, hydroxyl, halogen, C ₁ -C ₆ alkyl or hydroxy C ₁ -C ₆ alkyl; Z is a methylene group or a direct bond; Indicates a single or double bond. 2. The compound according to claim ₁ , wherein _R and R are each independently hydrogen, hydroxyl, methyl, ethyl, 3. The compound according to claim ₁ , wherein R is methyl, formaldehyde, -COOH, methoxycarbonyl, 4. The compound according to claim 1, wherein the compound has a structure shown in the following general formula (II): Wherein, the definition of each substituent is the same as defined in claim 1. 5. The compound according to claim 1, wherein the compound has the structure of the following general formula (III) or (IV): Wherein, the definition of each substituent is the same as that defined in claim 1. 6. The compound according to claim 1, wherein the compound has the following structure: 7. a preparation method of the compound described in claim 1, it comprises following preparation route: Wherein, R ₁₆ and R ₁₇ are respectively hydrogen, C ₁ -C ₆ alkyl or hydroxy C ₁ -C ₆ alkyl; R ₁₈ is a C ₁ -C ₆ alkyl group; the rest of the substituents are the same as defined in claim 1. 8. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6 as an active ingredient. 9. Use of the compound according to any one of claims 1 to 6 in the preparation of a medicament for treating type 2 diabetes. 10. Use of a compound according to any one of claims 1 to 6 as a TGR5 agonist.