CN110563792A

CN110563792A - G protein coupled bile acid receptor agonist and application thereof

Info

Publication number: CN110563792A
Application number: CN201910846807.XA
Authority: CN
Inventors: 林建平; 梁璐; 王目阔
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2019-09-09
Filing date: 2019-09-09
Publication date: 2019-12-13

Abstract

The invention provides 1G protein-coupled bile acid receptor agonist and application thereof, and in-vitro biological experiments show that the G protein-coupled bile acid receptor agonist has G protein-coupled bile acid receptor agonistic activity.

Description

G protein coupled bile acid receptor agonist and application thereof

Technical Field

The invention belongs to the field of small molecule drugs, and particularly relates to 1G protein-coupled bile acid receptor agonist and application thereof.

Background

There are two bile acid receptors discovered so far: one is the bile acid nuclear receptor FXR and the other is the bile acid membrane receptor TGR5(GPBAR 1). TGR5 consists of 330 amino acids in a peptide chain containing 7 transmembrane domains, and the intracellular part is combined with G protein, and has the typical structure and action mode of a G protein coupled receptor. TGR5 is ubiquitously expressed in humans and mammals, where its gene is located on chromosome 2q35, and homology is highly conserved (> 80%) in humans and mammals. TGR5 is highly expressed in tissue cells such as placenta, spleen, lymph node, bone marrow, lung, brown fat, skeletal muscle, intestine, gallbladder, mononuclear phagocyte, liver sinus endothelial cell, Kupffer cell, bile duct epithelium, etc.

Bile acid is an important component of bile and plays an important role in fat metabolism. Bile acids are mainly present in the enterohepatic circulatory system and play a certain protective role by recirculation, only a small part of bile acids enter peripheral circulation. Bile acids are natural ligands of TGR 5. The research shows that bile acid plays an important role in regulating glycolipid and energy metabolism, inflammatory reaction, atherosclerosis, metabolic syndrome and gynecological tumor by activating TGR5 signal path.

Inflammatory reaction: TGR5 is expressed in monocytes and various macrophages, and the monocyte-phagocytes can secrete various inflammatory mediators and play an important role in regulating inflammatory response. Through research, after TGR5 is activated, proinflammatory cytokine expression of mononuclear-phagocyte can be inhibited through a plurality of signal paths, and an anti-inflammatory effect is generated.

Atherosclerosis: activation of TGR5 can reduce the AS pathology by inducing cAMP signaling in macrophages, decreasing the transcriptional activity of NF- κ B, inhibiting the expression of pro-inflammatory cytokines, and decreasing macrophage uptake of OX-LDL.

Metabolic syndrome (including glucose intolerance, insulin resistance, obesity, dyslipidemia, and hypertension): monocyte-phagocyte TGR5 activation can relieve metabolic symptoms by inhibiting inflammatory responses, relieving insulin resistance; activation of TGR5 in islet beta cells promotes insulin secretion, suggesting that TGR 5-mediated signaling pathways may directly affect islet function and improve glucose metabolism; TGR5 agonist in thermogenic tissue can promote energy metabolism by activating TGR5-cAMP-D2-T3-UCP signaling pathway, thereby relieving obesity and insulin resistance.

Type II diabetes: small molecules that increase GLP-1 secretion by inducing GLP-1 release through TGR5 signaling may be useful for treating diabetes.

Obesity: PYY is secreted from intestinal L-cells with GLP-1 with meals, and the PYY system can be used as a target for treating obesity, so activation of TGR5 is beneficial for treating obesity.

Disclosure of Invention

in view of the above, the present invention aims to provide 1G protein-coupled bile acid receptor agonist and its application.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

The present invention provides a compound represented by structural formula (I):

The compound is commonly named Echinocystic acid, the English name is Echinocystic acid, the CAS number is 510-30-5, the molecular formula is C30H48O4, and the molecular weight is 472.700.

The application of the compound and the pharmaceutically acceptable salt thereof in preparing medicaments for treating or preventing related diseases or symptoms caused by low expression or low activity of a G protein-coupled bile acid receptor.

The use of the above compounds and their pharmaceutically acceptable salts for the manufacture of a medicament for the treatment of metabolic syndrome including glucose intolerance, insulin resistance, obesity, dyslipidemia and hypertension.

The application of the compound and the pharmaceutically acceptable salt thereof in preparing the medicines for treating inflammatory diseases.

The application of the compound and the pharmaceutically acceptable salt thereof in preparing the medicines for treating atherosclerosis.

The application of the compound and the pharmaceutically acceptable salt thereof in preparing the medicine for treating the gall-stone disease.

a pharmaceutical composition comprises the above compound and its pharmaceutically acceptable salt and pharmaceutically acceptable excipient.

The pharmaceutical composition further comprises a pharmaceutically acceptable excipient, diluent or carrier. Specifically, syrup, gum arabic, starch, etc. can be used. The pharmaceutical composition can be administered by intravenous, oral, sublingual, intramuscular or subcutaneous routes, or by the skin mucosa route.

The pharmaceutical composition is prepared in a liquid preparation or a solid preparation. Such as tablet, capsule and injection. The preparation can be prepared by a conventional pharmaceutical method.

A compound medicine comprises the compound and pharmaceutically acceptable salts thereof and medicines capable of being combined with the compound and the pharmaceutically acceptable salts thereof.

Compared with the prior art, the G protein coupled bile acid receptor stimulant and the application thereof have the following advantages:

EC for G protein-coupled bile acid receptor function Assay (hTGR5 cAMP Assay) of Compound 510-30-5 of the present invention₅₀2.626 μ M.

Drawings

FIG. 1 is a graph of the agonistic activity of compounds 510-30-5 on G protein-coupled bile acid receptors in a functional assay;

Detailed Description

Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.

the present invention relates to compound 510-30-5, which is available from Dorset Biotechnology, Inc.

The present invention will be described in detail with reference to examples.

Examples

Experimental procedure

1. Cell suspension preparation

a) all cells were cultured according to ATCC standard procedures, and CHO-K1 was tested in exponential growth phase.

b) The medium was discarded.

c) Cells were washed 2 times with PBS.

d) The cells were digested by adding pancreatin digest and the digestion was stopped with complete medium.

e) Cells were collected and counted and experiments were only performed if the cell viability rate was greater than 90%.

f) Variety 1x 10⁶CHO-K1 cells were plated into 60mm cell culture dishes.

g) The well-seeded cell culture dish was placed in a 5% CO2 incubator at 37 ℃ overnight for culture.

2. Cell transfection

a) The Lipo3000 transfection reagent was allowed to equilibrate to room temperature.

b) Adding 19.8 μ l Lipofectamine^TM3000 reagents to 250. mu.l Opti-MEM^TMThe culture medium is sucked and blown by a pipette gun to be mixed evenly without touching the tube wall.

c) Add 6.6. mu.g of DNA to 250. mu.l of Opti-MEM^TMTo the medium, 13.2. mu. l P3000 was added^TMAnd (5) slightly blowing and sucking the reagent by using a pipette gun, and uniformly mixing.

d) The diluted DNA P3000 was added to the diluted Lipofectamine^TM3000 transfection reagents (1: 1 ratio).

e) Gently blow and suck the mixture by a pipette gun, and balance the mixture at room temperature for 10 min.

f) The mixed transfection reagent was added to a 60mm cell culture dish (see step 3.1).

g) The dishes were incubated for 5h at 37 ℃ in a 5% CO2 incubator.

3. Cell plate

a) Cells were plated in 384 cell culture plates (6007680-50, PE) at 8,000 cells per well in 25. mu.l of medium.

b) Cells were cultured overnight at 37 ℃ in a 5% CO2 incubator.

4. Compound detection

a) Preparation of assay buffer: 1xHBSS, 20mM HEPES, 500. mu.M IBMX and 0.1% BSA.

b) Preparation of 5 × compound: the DMSO diluted compound (see step 2.3) was diluted 100-fold with buffer.

c) The medium in the cell culture plate was discarded (see step 3.3.b) and 16. mu.l buffer was added per well.

d) Add 4. mu.l of 5 Xdiluted compound to each well (see step b).

e) The mixture was incubated at 25 ℃ for 1 hour.

f) Preparation of cAMP-d 2: diluted 20-fold with 1x lysis buffer.

g) Add 10. mu.l cAMP-d2 per well (see step f) to 384 cell culture plates (see step e).

h) Preparing Anti cAMP-Eu3+ -Cryptate: diluted 20-fold with 1x lysis buffer.

i) Mu.l of Anti cAMP-Eu3+ -Cryptate (see step h) was added to 384 cell culture plates (see step g) per well.

j) The mixture was incubated at 25 ℃ for 1 hour.

Envision 2104 measures the ratio of 665nm and 615nm wavelengths.

By this example, a G protein-coupled bile acid receptor agonist was found, the agonistic activity of which in vitro was 2.626 μ M.

Claims

1. A compound represented by structural formula (I):

2. Use of a compound of claim 1 and pharmaceutically acceptable salts thereof in the manufacture of a medicament for the treatment or prevention of diseases or conditions associated with under-expression or under-activity of G-protein coupled bile acid receptors.

3. The use of a compound of claim 1 and pharmaceutically acceptable salts thereof in the manufacture of a medicament for the treatment of metabolic syndrome including glucose intolerance, insulin resistance, obesity, dyslipidemia and hypertension.

4. Use of a compound of claim 1 and pharmaceutically acceptable salts thereof for the manufacture of a medicament for the treatment of inflammatory diseases.

5. The use of a compound of claim 1, and pharmaceutically acceptable salts thereof, for the manufacture of a medicament for the treatment of atherosclerosis.

6. The use of a compound of claim 1 and pharmaceutically acceptable salts thereof in the manufacture of a medicament for the treatment of cholecystolithiasis.

7. A pharmaceutical composition characterized by: comprising as an active ingredient a compound according to claim 1 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient.

8. A compound medicine is characterized in that: comprising the G protein-coupled bile acid receptor agonist of claim 1 in combination with a drug.

9. Use of the pharmaceutical composition according to claim 7 for the preparation of a medicament for the treatment or prevention of a disease or disorder associated with under-expression or under-activity of a G protein-coupled bile acid receptor.