Small-molecule inhibitor for treating bile duct cancer
By developing the GPRC5A small molecule inhibitor 4-((3-(diphenylamino)-3-oxopropyl)amino)-4-oxobutyric acid, the problem of difficulty in inhibiting GPRC5A activity in the prior art has been solved, effective treatment for cholangiocarcinoma has been achieved, and a direct small molecule targeted treatment plan has been provided.
Patent Information
- Application Number
- CN202510455426.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-11
- Publication Date
- 2025-07-11
AI Technical Summary
The prior art is difficult to effectively inhibit the activity of GPRC5A, resulting in limited complexity of gene editing technology in the treatment of cholangiocarcinoma and lack of direct small molecule inhibitors.
It provides a GPRC5A small molecule inhibitor 4-((3-(diphenylamino)-3-oxopropyl)amino)-4-oxobutyric acid and its pharmaceutically acceptable salt. By directly acting on the GPRC5A protein, it inhibits its signaling and is prepared into tablets, capsules, granules or lyophilized powder injections.
This compound can effectively inhibit the signaling of GPRC5A, delay the progression of cholangiocarcinoma, and provide new small molecule targeted therapy solutions, overcoming the technical limitations of gene editing therapy.
Smart Images
Figure CN120289322A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a small molecule inhibitor for treating cholangiocarcinoma. Background Art
[0002] Biliary tract cancer (BTC) is the second most common malignant tumor in the hepatobiliary system after hepatocellular carcinoma, mainly including gallbladder cancer and cholangiocarcinoma (CCA). The early clinical features of cholangiocarcinoma are often not obvious, early diagnosis is difficult, and the prognosis is poor after diagnosis.
[0003] G protein-coupled receptors (GPCRs) are an important family of signal transduction proteins that participate in various biological reactions of cells. GPRC5A, as a major member of the GPRC5A family, participates in cellular responses to DNA damage, repair, and many other biological processes. GPRC5A plays an important role in the occurrence and development of tumors. Especially in cholangiocarcinoma, colorectal cancer (CRC), gastric cancer, and pancreatic ductal adenocarcinoma, its overexpression is closely related to poor prognosis. Among them, in colorectal cancer, liquid chromatography analysis shows that the expression of GPRC5A in polyps is lower than that in metastatic and non-metastatic CRC samples, suggesting that GPRC5A may also be used as a biomarker to distinguish CRC from normal tissues. Therefore, GPRC5A has become an important target in cancer treatment. In previous studies, it was found that inhibiting GPRC5A can effectively slow down the proliferation of cholangiocarcinoma tumors. Previous studies have shown that knocking down the GPRC5A gene can inhibit the growth, proliferation, colony formation, and migration ability of cholangiocarcinoma cell lines. Existing studies have not reported compounds that can effectively inhibit GPRC5A signal transduction, and only stay at inhibiting its function by knocking down the GPRC5A gene expression. However, these research methods usually face technical limitations.
[0004] Therefore, there is an urgent need for a new GPRC5A inhibitor that can not only effectively inhibit the activity of GPRC5A but also has stronger operability and potential clinical application value. Small molecule inhibitors can directly act on the GPRC5A protein, quickly and effectively inhibit its activity, and at the same time do not rely on the complexity of gene editing technology, having better prospects for drug development. Summary of the Invention
[0005] In view of the deficiencies of the prior art, the present invention provides a small molecule inhibitor for treating cholangiocarcinoma. The technical solution provided by the present invention is as follows:
[0006] As the first aspect of the present invention, it lies in providing a small molecule inhibitor for treating cholangiocarcinoma, and the small molecule inhibitor is a GPRC5A small molecule inhibitor.
[0007] Furthermore, the small molecule inhibitor of GPRC5A is 4-((3-(diphenylamino)-3-oxopropyl)amino)-4-oxobutyric acid and its pharmaceutically acceptable salts, and its SMILES code is:
[0008] OC(=O)CCC(=O)NCCC(=O)N(c1ccccc1)c2ccccc2, and the structural formula is as follows:
[0009]
[0010] In view of the complexity of gene editing technology in the treatment of cholangiocarcinoma, the present invention provides a small molecule inhibitor of GPRC5A. Through BRET experiments, it is verified that this compound has an affinity for GPRC5A, has a good inhibitory effect on GPRC5A, and thus has an inhibitory effect on tumors.
[0011] As the second aspect of the present invention, it lies in providing the application of the small molecule inhibitor of GPRC5A in the preparation of anti-cancer drugs. The small molecule inhibitor of GPRC5A is 4-((3-(diphenylamino)-3-oxopropyl)amino)-4-oxobutyric acid and its pharmaceutically acceptable salts, and its SMILES code is:
[0012] OC(=O)CCC(=O)NCCC(=O)N(c1ccccc1)c2ccccc2, and the structural formula is as follows:
[0013]
[0014] This compound has an affinity for GPRC5A, has a good inhibitory effect on GPRC5A, and thus has an inhibitory effect on tumors.
[0015] As the third aspect of the present invention, it lies in providing an anti-cholangiocarcinoma drug, which contains 4-((3-(diphenylamino)-3-oxopropyl)amino)-4-oxobutyric acid and its pharmaceutically acceptable salts.
[0016] Furthermore, the anti-cholangiocarcinoma drug also includes pharmaceutically acceptable carriers and excipients.
[0017] The small molecule inhibitor of GPRC5A provided by the present invention can be formulated and administered alone or in combination with one or more carriers. It can be administered in oral dosage forms, such as tablets, capsules, granules, etc.; or in injection dosage forms, such as freeze-dried powder injections. The dosage forms of the drugs of the present invention can be prepared according to methods well-known in the pharmaceutical field.
[0018] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:
[0019] (1) The present invention provides a small molecule inhibitor for treating cholangiocarcinoma. This compound can effectively inhibit the signal transduction and activity of GPRC5A, thereby inhibiting various tumor growth and metastasis, providing a new small molecule targeted therapy for tumor treatment, and overcoming the defect of the technical limitation of gene editing therapy in the field of cholangiocarcinoma treatment.
[0020] (2) The present invention provides a compound of a GPRC5A inhibitor. This compound can effectively inhibit the function of GPRC5A. The study on the formation of tumors in the liver of cholangiocarcinoma has proved that this compound can significantly delay the progression of cholangiocarcinoma and can effectively treat cholangiocarcinoma. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.
[0022] Figure 1 It is a result diagram of the Gq signaling pathway in the BRET experiment in Example 1.
[0023] Figure 2 It is a result diagram of the Gi signaling pathway in the BRET experiment in Example 1.
[0024] Figure 3 It is a diagram of the formation of orthotopic tumors after injection of liver cholangiocarcinoma in Example 2; among them, (a) is the normal saline group, and (b) is the inhibitor group.
[0025] Figure 4 It is an index of liver weight after orthotopic injection of liver cholangiocarcinoma in Example 2; among them, (a) is the normal saline group, and (b) is the inhibitor group.
[0026] Figure 5 It is the inhibitory effect on Transwell invasion at different concentrations in Example 3; among them, (a) is the diagram of the transwell migration experiment, and (b) is the statistical chart of the migrated cells in the transwell migration experiment. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0027] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments and the accompanying drawings. It should be noted that the specific embodiments below are only for illustration and do not limit the present invention.
[0028] 4-((3-(Diphenylamino)-3-oxopropyl)amino)-4-oxobutyric acid was purchased from TargetMol.
[0029] 4-((3-(Diphenylamino)-3-oxopropyl)amino)-4-oxobutyric acid is an aromatic compound. It is a brown powder at room temperature, with a molecular weight of 327.36 g / mol, showing weak acidity. Its molecular structure has two benzene rings connected by a nitrogen atom, and a carboxyl group is connected at the other end of the compound.
[0030] In the experimental design, the inhibitory effect of 4-((3-(diphenylamino)-3-oxopropyl)amino)-4-oxobutyric acid on GPRC5A was evaluated by BRET assay.
[0031] Example 1, 4-((3-(diphenylamino)-3-oxopropyl)amino)-4-oxobutyric acid can effectively inhibit the downstream signaling pathway of GPRC5A (BRET assay)
[0032] Verification of the compound by BRET assay
[0033] The method was carried out according to the reference "Structural and signaling mechanisms of TAAR1 enabled preferential agonist design".
[0034] Plasmids encoding the intracellular biosensors (i.e., RLucI-Gαq and GFP10-Gβγ), or the plasmid of GPRC5AWT, were co-transfected with the cDNA plasmid encoding GPRC5A. To calculate the netBRET ratio of the activation of the intracellular biosensor, the pIRES(puro)-RLucII vector was used to subtract the background signal. At the time of transfection, linear PEI (1 mg / mL) was used as the transfection reagent, and the ratio of DNA to PEI was 3:1. Up to 2 μg of plasmid DNA was prepared using Opti-MEM and transfected into 3.5×10 5 cells, and 3.5×10 5 cells were seeded in a white opaque 96-well plate with a volume of 100 μL. 48 hours after transfection, the medium was removed and replaced with HBSS containing 20 mM HEPES, and incubated at 37 °C for 1 hour. Coelenterazine 400A (5 μM) substrate was added 5 minutes before reading the BRET signal, and different concentrations of -methoxy-2-[(4-propan-2-ylphenyl)methoxy]phenyl]methanamine (10 -9 M, 10 -6 M, 10 -3 M) were added 10 minutes before reading the BRET signal. The RLucII and GFP10 signals were collected using a microplate reader, with RLucII emission at 410 nm and GFP10 emission at 515 nm. The BRET ratio was determined by calculating the ratio of the receptor signal (GFP10) to the donor signal (RLucII).
[0035] Composed of Figure 1 and Figure 2 It can be seen that under the stimulation of 4-((3-(diphenylamino)-3-oxopropyl)amino)-4-oxobutyric acid, the ratio of RlucII / GFP10 increases with the increase of the inhibitor concentration, indicating that the transfer of Rluc fluorescence signal to GFP signal decreases, and the dissociation degree of G protein downstream of GPRC5A decreases with the increase of the inhibitor concentration, which can prove that the compound has an inhibitory effect on the G protein pathway of GPRC5A.
[0036] Example 2. In order to verify that 4-((3-(diphenylamino)-3-oxopropyl)amino)-4-oxobutyric acid can effectively inhibit tumor growth, the present invention established an orthotopic model in wild-type mice by intrahepatic injection of mouse iCCA cell line LD-1.
[0037] Construction of mouse orthotopic injection model:
[0038] Treatment of mouse cholangiocarcinoma cell line LD-1: Place the LD-1 cell line in a 10 cm dish for culture. When the cell density reaches 70-80%, aspirate the culture medium, add 3 ml of PBS along the side wall to wash the cells and then aspirate, add 1 ml of trypsin, and place the cells in an incubator at 37 °C for digestion for 3 min. Add 3 ml of 1640 medium containing 10% FBS to terminate digestion. Gently pipette the cells down, transfer them to a 15 ml tube, centrifuge at 1000 rpm for 3 min, aspirate the supernatant, add 1 ml of PBS to resuspend, and repeat the washing three times. Add 100 ul of PBS to resuspend to make the density about 10 8 / ml.
[0039] After anesthetizing C57 female mice (purchased from Jicui Yakang) by inhaling isoflurane, remove the hair on the abdomen of the mice using a hair clipper. Use sterilized scissors and forceps to cut open the abdominal skin and peritoneum of the mice along the midline of the abdomen to expose the liver. Aspirate 20 ul of cell suspension with an insulin syringe and inject it into the right lobe of the liver. It can be seen that the liver turns significantly white. Compress the injection site with a sterile cotton swab for 30 s, and suture the peritoneum and the surface skin layer by layer. The mice can be sacrificed one week later, and hard masses the size of mung beans can be seen in the livers of the mice, indicating successful model establishment.
[0040] One week after orthotopic injection, the inhibitor group was intraperitoneally injected with 100 μl of 4-((3-(diphenylamino)-3-oxopropyl)amino)-4-oxobutyric acid (10 μM), and the normal saline group was intraperitoneally injected with 100 μl of normal saline. Inject once every 2 days for one week to treat the mice. Weigh the livers of the mice and keep the liver photos.
[0041] The results are as Figure 3 and Figure 4As shown, it can be seen that the size of the tumor on the liver surface in the inhibitor group is significantly smaller than that in the normal saline group, and the liver mass of mice of the same age in the inhibitor group is significantly smaller than that in the normal saline group.
[0042] Example 3, Transwell invasion assay
[0043] Steps of the Transwell experiment: 1. Observe the growth state of HCCC-9810 cells. Remove the culture medium from the cells with good growth state, and add serum-free medium to starve for 24 h; 2. Prepare the samples with serum-free medium, set the concentration of the inhibitor solution to 2 times the final concentration, and prepare the concentrations to be 2×10 -10 , 2×10 -10 , 2×10 -10 , 2×10 -10 , 2×10 -6 ; 3. Add 500 μl of complete medium containing 10% FBS to the lower chamber of a 24-well plate, and place the Transwell chamber into the 24-well plate with forceps; 4. Digest the starved cells, resuspend them with serum-free medium to make the cell density 2×10 5 ; 5. Add to the chamber according to the ratio of inhibitor solution: cell suspension = 1:1. First add 100 uL of the sample working solution, and then add 100 uL of the cell suspension. At this time, the sample concentration is diluted 2 times to be the final concentration; 6. Incubate the 24-well plate at 37 °C, 5% CO2, and 90% humidity for 24 hours; 7. Take out the Transwell chamber, remove the culture solution, and gently wipe the Matrigel and cells in the chamber with a cotton swab or cotton moistened with PBS; 8. Add 600 μl of 4% paraformaldehyde fixative to a clean well of the 24-well plate, and place the chamber in it for fixation for 30 minutes; 9. Discard the fixative, and wash the inside and outside of the chamber with PBS once; 10. Add 600 uL of crystal violet staining solution to a clean well of the 24-well plate, and place the chamber in it for staining for 10 minutes; 11. Take out the chamber, and wash the inside and outside of the chamber with PBS three times; 12. After proper air drying, observe qualitatively under a microscope; Take pictures of 5 fields of view and use ImageJ to count and take the average value for quantitative research.
[0044] The results are as Figure 5 shown. The IC50 of the GPRC5A small molecule inhibitor provided by the present invention is 12.6 nM, and this compound has a good therapeutic effect on cholangiocarcinoma.
[0045] Example 4, A drug for treating cholangiocarcinoma
[0046] It contains 4-((3-(diphenylamino)-3-oxopropyl)amino)-4-oxobutyric acid and its pharmaceutically acceptable salts.
[0047] Furthermore, the drug for treating cholangiocarcinoma further includes pharmaceutically acceptable carriers and excipients.
[0048] The GPRC5A small molecule inhibitor provided by the present invention can be formulated and administered alone or in combination with one or more carriers. It can be administered in oral dosage forms such as tablets, capsules, granules, etc.; it can also be administered in injection dosage forms such as lyophilized powder for injection. The dosage forms of the drugs of the present invention can be prepared according to methods well known in the pharmaceutical field.
[0049] When preparing an oral preparation, suitable fillers, binders, disintegrants, lubricants, etc. can be added. Commonly used fillers include starch, dextrin, microcrystalline cellulose, lactose, pregelatinized starch, mannitol, etc.; commonly used binders include sodium carboxymethyl cellulose, PVP-K30, hydroxypropyl cellulose, starch paste, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, gelatinized starch, etc.; commonly used disintegrants include dry starch, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, etc.; commonly used lubricants include magnesium stearate, talc, sodium lauryl sulfate, colloidal silica, etc.
[0050] It may also include glidants, which include one or more of colloidal silica, colloidal silicon dioxide, and silicon dioxide; it may also include flavoring agents, which include one or more of steviol glycosides, aspartame, and sodium cyclamate.
[0051] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.
Claims
1. A small molecule inhibitor for treating cholangiocarcinoma, characterized in that, The small molecule inhibitor is a small molecule inhibitor of GPRC5A.
2. The small molecule inhibitor for treating cholangiocarcinoma according to claim 1, wherein The small molecule inhibitor of GPRC5A is 4-((3-(diphenylamino)-3-oxopropyl)amino)-4-oxobutyric acid and its pharmaceutically acceptable salts, and the structural formula is as follows:
3. Application of the small molecule inhibitor of GPRC5A in the preparation of drugs for treating cancer.
4. Use of the GPRC5A small molecule inhibitor according to claim 3 in the preparation of a medicament for treating cancer, characterized in that, The small molecule inhibitor of GPRC5A is 4-((3-(diphenylamino)-3-oxopropyl)amino)-4-oxobutyric acid and its pharmaceutically acceptable salts.
5. A drug for treating cholangiocarcinoma, characterized in that, It contains 4-((3-(diphenylamino)-3-oxopropyl)amino)-4-oxobutyric acid and its pharmaceutically acceptable salts.
6. The cholangiocarcinoma treatment drug according to claim 5, wherein The drug for treating cholangiocarcinoma further comprises a pharmaceutically acceptable carrier and excipients.
7. The drug for treating cholangiocarcinoma according to claim 5, characterized in that, The dosage form of the drug for treating cholangiocarcinoma is selected from tablets, capsules, granules or injections.