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

Abstract

The invention provides a G protein-coupled bile acid receptor agonist and an application thereof, and in vitro biological experiments have found that it has G protein-coupled bile acid receptor agonist activity.

Description

G protein-coupled bile acid receptor agonists and their applications

technical field

The invention belongs to the field of small molecule medicines, in particular to a G protein-coupled bile acid receptor agonist and its application.

Background technique

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 (GPBAR1). TGR5 consists of a 330 amino acid peptide chain with 7 transmembrane domains. The intracellular part is bound to G protein, and has the typical structure and mode of action of G protein-coupled receptors. TGR5 is ubiquitously expressed in humans and mammals, its gene is located on chromosome 2q35 in humans, and its homology is highly conserved (>80%) in humans and mammals. TGR5 is highly expressed in placenta, spleen, lymph nodes, bone marrow, lung, brown fat, skeletal muscle, intestine, gallbladder, mononuclear-phagocytic cells, liver sinusoidal endothelial cells, Kupffer cells, bile duct epithelium and other tissue cells.

Bile acids are important components of bile and play an important role in fat metabolism. Bile acids mainly exist in the enterohepatic circulatory system and play a certain protective role through recycling, and only a small part of bile acids enter the peripheral circulation. Bile acids are natural ligands for TGR5. Studies have shown that bile acids play an important role in regulating glycolipid and energy metabolism, inflammatory response, atherosclerosis, metabolic syndrome and gynecological tumors by activating the TGR5 signaling pathway.

Inflammatory response: TGR5 is expressed in monocytes and various macrophages, and monocytes-phagocytic cells can secrete various inflammatory mediators and play an important role in regulating inflammatory responses. Studies have shown that activation of TGR5 can inhibit the expression of pro-inflammatory cytokines in monocytes-phagocytic cells through a variety of signaling pathways, resulting in an anti-inflammatory effect.

Atherosclerosis: Activation of TGR5 can alleviate AS lesions by inducing cAMP signaling in macrophages, attenuating the transcriptional activity of NF-κB, inhibiting the expression of pro-inflammatory cytokines, and reducing the uptake of OX-LDL by macrophages.

Metabolic syndrome (including glucose intolerance, insulin resistance, obesity, dyslipidemia, and hypertension): Monocyte-phagocytic TGR5 activation alleviates metabolic symptoms by suppressing inflammatory responses and reducing insulin resistance; TGR5 in pancreatic beta cells Activation can promote insulin secretion, indicating that TGR5-mediated signaling pathway can directly affect islet function and improve glucose metabolism; TGR5 agonists in thermogenic tissues can still promote energy metabolism by activating TGR5-cAMP-D2-T3-UCP signaling pathway, thereby Relieve obesity and insulin resistance.

Type II diabetes: GLP-1 release is induced by TGR5 signaling, and small molecules that increase GLP-1 secretion can be used to treat diabetes.

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

SUMMARY OF THE INVENTION

In view of this, the present invention aims to propose a G protein-coupled bile acid receptor agonist and its application.

To achieve the above object, the technical scheme of the present invention is achieved in this way:

The present invention provides a compound represented by structural formula (I) and a pharmaceutically acceptable salt thereof:

The common name of this compound is echinocystic acid, the English name is Echinocystic acid, the CAS number is 510-30-5, its molecular formula is C30H48O4, and its molecular weight is 472.700.

Use of the above compounds and their pharmaceutically acceptable salts in the preparation of medicaments for treating or preventing related diseases or disorders caused by low expression or low activity of G protein-coupled bile acid receptors.

Use of the above compounds and their pharmaceutically acceptable salts in the preparation of drugs for treating metabolic syndrome including glucose intolerance, insulin resistance, obesity, dyslipidemia and hypertension.

Use of the above compounds and their pharmaceutically acceptable salts in the preparation of medicaments for treating inflammatory diseases.

Use of the above-mentioned compounds and their pharmaceutically acceptable salts in the preparation of medicaments for treating atherosclerosis.

Use of the above compounds and their pharmaceutically acceptable salts in the preparation of a medicament for treating gallstone disease.

A pharmaceutical composition, comprising the above-mentioned compounds and their pharmaceutically acceptable salts and their pharmaceutically acceptable excipients.

The pharmaceutical composition also includes a pharmaceutically acceptable excipient, diluent or carrier. Specifically, such as syrup, gum arabic and starch. The pharmaceutical composition can be administered by intravenous, oral, sublingual, intramuscular or subcutaneous, mucocutaneous route.

The preparation form of the pharmaceutical composition is a liquid preparation or a solid preparation. Specifically, such as tablets, capsules and injections. Each dosage form can be prepared by conventional methods of pharmacy.

A compound medicine, comprising the above-mentioned compounds and their pharmaceutically acceptable salts and medicines that can be used in combination with them.

Compared with the prior art, the G protein-coupled bile acid receptor agonist of the present invention and its application have the following advantages:

The EC ₅₀ of the G protein-coupled bile acid receptor functional assay (hTGR5 cAMPAssay) of the compound 510-30-5 of the present invention was 2.626 μM.

Description of drawings

Figure 1 is the agonistic activity curve of compound 510-30-5 on G protein-coupled bile acid receptors in functional experiments;

Detailed ways

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

The present invention relates to compound 510-30-5 purchased from Chengdu Durst Biotechnology Co., Ltd.

The present invention will be described in detail below with reference to the embodiments.

Example

Experimental procedure

1. Preparation of cell suspension

a) All cells were cultured according to ATCC standards, and CHO-K1 was subjected to experiments in exponential growth phase.

b) Discard the medium.

c) Wash cells twice with PBS.

d) The cells were digested by adding trypsinization solution, and the digestion was terminated with complete medium.

e) Cells were collected and counted, and the experiment could be performed only when the cell viability was greater than 90%.

f) Seed ¹ *106 CHO-K1 cells into a 60mm cell culture dish.

g) The culture dish with the seeded cells was placed in a 37°C, 5% CO2 incubator overnight for cultivation.

2. Cell transfection

a) Equilibrate Lipo3000 transfection reagent at room temperature.

b) Add 19.8 μl Lipofectamine ^TM 3000 reagent to 250 μl Opti-MEM ^TM medium, be careful not to touch the tube wall, and mix by pipetting.

c) Add 6.6 μg DNA to 250 μl Opti-MEM ^TM medium, then add 13.2 μl P3000 ^TM reagent, and mix by pipetting gently.

d) Add the diluted DNA P3000 to the diluted Lipofectamine ^™ 3000 transfection reagent (1:1 ratio).

e) Gently blow and mix with a pipette, and equilibrate at room temperature for 10 minutes.

f) Add the mixed transfection reagent to a 60mm cell culture dish (see step 3.1).

g) Place the petri dish in a 37°C, 5% CO2 incubator for 5 hours.

3. Cell seed plate

a) Cells were seeded into 384 cell culture plates (6007680-50, PE), 8,000 cells per well, 25 μl of medium.

b) Cells were cultured overnight in a 37°C, 5% CO2 incubator.

4. Compound detection

a) Prepare experimental buffer: 1xHBSS, 20mM HEPES, 500μM IBMX and 0.1% BSA.

b) Prepare 5x compound: Dilute the compound diluted in DMSO (see step 2.3) 100-fold with buffer.

c) Discard the medium in the cell culture plate (see step 3.3.b) and add 16 μl of buffer to each well.

d) Add 4 μl of 5x diluted compound to each well (see step b).

e) Place in a 25°C incubator for 1 hour.

f) Preparation of cAMP-d2: 20-fold dilution with 1x lysis buffer.

g) Add 10 μl of cAMP-d2 (see step f) per well to the 384 cell culture plate (see step e).

h) Preparation of Anti cAMP-Eu3+-Cryptate: 20-fold dilution with 1x lysis buffer.

i) Add 10 μl of Anti cAMP-Eu3+-Cryptate (see step h) per well to the 384 cell culture plate (see step g).

j) Place in a 25°C incubator for 1 hour.

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

Through this example, a G protein-coupled bile acid receptor agonist was found, and its agonistic activity in vitro was 2.626 μM.

Claims (9)

1. A compound represented by structural formula (I) and a pharmaceutically acceptable salt thereof: 2. Use of the compound of claim 1 and a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prevention of related diseases or conditions caused by low expression or low activity of G protein-coupled bile acid receptors. 3. Use of the compound of claim 1 and a pharmaceutically acceptable salt thereof in the preparation of a drug for metabolic syndrome including glucose intolerance, insulin resistance, obesity, dyslipidemia and hypertension. 4. Use of the compound of claim 1 and a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of inflammatory diseases. 5. Use of the compound of claim 1 and a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating atherosclerosis. 6. Use of the compound of claim 1 and a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating gallstone disease. 7. A pharmaceutical composition, characterized in that it comprises the compound of claim 1 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient as active ingredients. 8. A compound medicine, characterized in that it comprises the G protein-coupled bile acid receptor agonist of claim 1 and a medicine that can be used in combination with it. 9. Use of the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating or preventing related diseases or conditions caused by low expression or low activity of G protein-coupled bile acid receptors.