CN112007032B

CN112007032B - Application of compound in preparation of small molecule inhibitor or cancer treatment drug, small molecule inhibitor and cancer treatment drug

Info

Publication number: CN112007032B
Application number: CN202010971677.5A
Authority: CN
Inventors: 刘�文; 冉挺; 彭冰灵; 李文娟
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2020-09-16
Filing date: 2020-09-16
Publication date: 2021-10-22
Anticipated expiration: 2040-09-16
Also published as: CN112007032A

Abstract

The invention belongs to the field of anti-cancer drugs, and particularly relates to application of a compound (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidine-2-yl ] piperazine-1-yl } ethyl) dimethylamine in preparation of a small molecule inhibitor or a drug for treating cancer, and the small molecule inhibitor and the drug for treating cancer. The invention provides a compound (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidine-2-yl ] piperazine-1-yl } ethyl) dimethylamine and application of medicinal salts thereof in preparation of a CARM1 small molecule inhibitor, wherein the structural formula of the compound is shown as a formula I. The compound can effectively inhibit the enzymatic activity of CARM1, has the half inhibitory concentration of 77.15 mu M of the enzymatic activity, and can play a technical effect of inhibiting the proliferation of cancer cells.

Description

Application of compound in preparation of small molecule inhibitor or cancer treatment drug, small molecule inhibitor and cancer treatment drug

Technical Field

The invention belongs to the field of anti-cancer drugs, and particularly relates to application of a compound (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidine-2-yl ] piperazine-1-yl } ethyl) dimethylamine in preparation of a small molecule inhibitor or a drug for treating cancer, and the small molecule inhibitor and the drug for treating cancer.

Background

Cancer has become one of the most deaths, the incidence of cancer has also increased year by year, and the age of onset of cancer has also become younger. Therefore, the regulation factor which plays a key role in the development of cancer is searched, and the effective and specific small molecule inhibitor is designed by taking the regulation factor as a drug target, so that the method has important academic value and social benefit.

Post-translational modification of histones is one of the important regulatory mechanisms in epigenetics. Such modifications include histone phosphorylation, acetylation, methylation, ubiquitination, SUMO, ADP-ribosylation, etc., and typically occur at the amino terminus of histones, affecting processes such as gene transcription and DNA damage repair. Histone methylation is one of the most common modifications found to date, occurring at the arginine or lysine residue at the amino terminus of histone proteins. The histone methyltransferase, the histone demethylase and the methylation recognition protein respectively play roles in writing, erasing and reading histone methylation, and jointly regulate the methylation state of histone. Arginine methylation is an important post-translational modification that occurs widely in cells, mediated by the protein arginine methyltransferase (PRMTs) to transfer the methyl group of the methylated cofactor SAM to the nitrogen atom of the arginine side chain. PRMTs can either methylate histones or methylate non-histones. Arginine methyltransferases are largely classified into three classes, depending on the methylation pattern: type I is responsible for catalyzing monomethylation and asymmetric bimethylation, including PRMT1, PRMT2, PRMT3, PRMT4(CARM1), PRMT6, and PRMT 8; type II is responsible for monomethylation modifications and symmetric double methylation, including PRMT5 and PRMT 9; type III catalyzes only the monomethylation of arginine, and is PRMT 7. The research finds that the arginine methylation is involved in a series of biological processes such as gene expression regulation, cell signal transduction regulation, protein localization, DNA damage repair, RNA processing and the like. Abnormal expression and dysfunction of PRMTs are associated with various diseases and play an important role in the development, invasion and metastasis of malignant tumors.

Arginine methyltransferase 4 (PRMT 4)/coactivator-associated arginine methyltransferase 1 (CARM1) is one of the earliest discovered PRMTs family members, both in the nucleus and cytoplasm, and its methylation substrates include histone and non-histone proteins. Although CARM1 can exert biological functions in a non-methylation dependent manner, such as self-methylation and protein-protein interactions, its methylase activity has a critical influence on biological functions. In one aspect, methylation of histones generated by CARM1 (H3R17me2a and H3R26me2a) activates nuclear receptor or transcription factor mediated gene transcription. On the other hand, CARM1 can also regulate gene transcription by methylated non-histone substrates, and affect post-transcriptional regulation and intranuclear retention of mRNA, glutamine metabolism, and radiation-induced DNA damage repair, etc. At the cellular level, the methylation activity of CARM1 can regulate cell cycle, cell proliferation, cell differentiation, autophagy, stem cell pluripotency, and the like. The expression and function abnormality of CARM1 are closely related to the occurrence and development of various cancers (breast cancer, colorectal cancer, lung cancer, liver cancer, ovarian cancer, prostatic cancer, oral cancer, osteosarcoma, acute myelocytic leukemia and the like). In the case of breast cancer, CARM1 is highly expressed in breast cancer including estrogen positive, HER-2 positive, and triple negative. Moreover, the expression of CARM1 positively correlated with the malignancy of breast cancer. CARM1 is used as an estrogen coactivator transcription factor, is enriched in a promoter region of an estrogen receptor alpha (ER alpha) target gene, and can up-regulate the expression of the estrogen target gene by combining with a transcription coactivator p 160. Recent studies have shown that CARM1 also up-regulates the expression of E2F1 and CCND1 by activating the transcription factor AIB1 in estrogen-stimulated breast cancer cells, promoting tumor cell growth. CARM1 methylates the transcriptional coactivator p/CIP, increasing the activity and stability of p/CIP. In the estrogen-induced MCF7 cells, p/CIP interacts with activated ER alpha, and JAK/STAT signal channels downstream of p/CIP are activated, so that the proliferation capacity of tumor cells is enhanced. In addition, CARM1 can interact with oncotropin PELP1 to activate ER alpha, activate PELP-mediated gene expression, and promote invasion and migration capability of breast cancer cells MCF 7. In breast cancer cells, estrogen receptor independent signaling pathway regulated by CARM1 was also found. CARM1 methylates BAF155, improves expression of chromatin remodeling factor SWI/SNF mediated oncogene c-Myc, and promotes proliferation of triple negative breast cancer cell MDA-MB-231. Meanwhile, the CARM1 methylated MED12 in the cell can inhibit the transcription expression of a cell cycle inhibitor p21 and reduce the drug resistance of breast cancer to chemotherapeutic drugs. CARM1 overexpression was also found in the HER-2 positive breast cancer cytoplasm. From the above, CARM1 shows a promoting effect on breast cancer (especially ER positive breast cancer), and particularly shows potential carcinogenicity in breast cancer which is ineffective in traditional drug therapy, so that it has a new hope as a tumor drug target for solving the dilemma faced by current breast cancer therapy. In addition, the research finds that CARM1 has a promoting effect on the occurrence and development of various cancers such as breast cancer, colorectal cancer, lung cancer, liver cancer, ovarian cancer, prostate cancer, oral cancer, osteosarcoma, acute myelocytic leukemia and the like.

In conclusion, CARM1 has multiple biological functions as an epigenetic regulator, and the function research of arginine methylation in cancer shows that the epigenetic regulator is a potential antitumor drug target, but no inhibitor aiming at CARM1 enters clinical research at present.

Disclosure of Invention

In order to solve the problems, the invention provides application of a compound (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidine-2-yl ] piperazine-1-yl } ethyl) dimethylamine in preparation of a small molecule inhibitor or a medicament for treating cancer, and the small molecule inhibitor and the medicament for treating the cancer. The (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidin-2-yl ] piperazin-1-yl } ethyl) dimethylamine provided by the invention has obvious CARM1 methylation inhibition activity, and in the application of the (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidin-2-yl ] piperazin-1-yl } ethyl) dimethylamine provided by the invention, the small molecule inhibitor can effectively inhibit the activity of cancer cells, so that the technical effect of treating cancer is achieved.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides application of (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidine-2-yl ] piperazine-1-yl } ethyl) dimethylamine and pharmaceutically acceptable salts thereof in preparation of a CARM1 small molecule inhibitor.

The invention also provides a CARM1 small molecule inhibitor, wherein the CARM1 small molecule inhibitor comprises (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidin-2-yl ] piperazin-1-yl } ethyl) dimethylamine, pharmaceutically acceptable salts thereof and pharmaceutically acceptable auxiliary materials.

Preferably, the pharmaceutically acceptable salt of (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidin-2-yl ] piperazin-1-yl } ethyl) dimethylamine comprises a salt of (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidin-2-yl ] piperazin-1-yl } ethyl) dimethylamine with an acid and/or a salt with an acid salt of an inorganic base as defined in claim 1.

Preferably, the acid comprises hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, benzenesulphonic acid, p-benzenesulphonic acid, naphthalenesulphonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

Preferably, the acid salt of the inorganic base includes a salt containing a basic metal cation, a salt containing an alkaline earth metal cation, or a salt containing an ammonium ion.

The invention also provides application of (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidine-2-yl ] piperazine-1-yl } ethyl) dimethylamine and pharmaceutically acceptable salts thereof or the small molecule inhibitor in preparing a medicament for treating cancer.

The invention provides a medicine for treating cancer, which comprises (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidine-2-yl ] piperazine-1-yl } ethyl) dimethylamine and medicinal salts thereof or the micromolecule inhibitor and pharmaceutically acceptable auxiliary materials.

Preferably, the cancer comprises breast cancer, colorectal cancer, lung cancer, liver cancer, ovarian cancer, prostate cancer, oral cancer, osteosarcoma and acute myeloid leukemia.

The present invention provides (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidin-2-yl)]Piperazine-1-yl } ethyl) dimethylamine and the application of the medicinal salt thereof in the preparation of CARM1 small molecule inhibitors. In the present invention, (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidin-2-yl]Piperazin-1-yl } ethyl) dimethylamine, numbered WL 25. WL25 is capable of effectively inhibiting the enzyme activity of CARM1, and the application example shows that IC of WL25₅₀77.15 μ M, and has strong ability of inhibiting CARM 1. WL25 is useful in medicaments for the prevention or treatment of diseases associated with CARM1 inhibitors, especially in medicaments for the treatment of cancer. Under the same concentration, the inhibition rate of WL25 is higher than that of the current CARM1 enzyme activity inhibitor EZM 2302.

Detailed Description

The invention provides application of (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidine-2-yl ] piperazine-1-yl } ethyl) dimethylamine and pharmaceutically acceptable salts thereof in preparation of a CARM1 small molecule inhibitor. The (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidine-2-yl ] piperazine-1-yl } ethyl) dimethylamine provided by the invention has obvious CARM1 methylation inhibition activity. The structural formula of the (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidine-2-yl ] piperazine-1-yl } ethyl) dimethylamine provided by the invention is shown in a formula I:

in the present invention, the (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidin-2-yl group]Piperazin-1-yl } ethyl) dimethylamine of formula C₂₃H₂₉N₅O, purchased from a chembridge molecular library, linked as: https:// www.hit2lead.com/result. aspsearch ═ 68273648 and chembridge compound No. 98992594.

The invention also provides a CARM1 small molecule inhibitor, wherein the CARM1 small molecule inhibitor comprises the (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidin-2-yl ] piperazin-1-yl } ethyl) dimethylamine, a pharmaceutically acceptable salt thereof and pharmaceutically acceptable auxiliary materials. In the present invention, the adjuvant preferably includes a pharmaceutically acceptable carrier or excipient; the pharmaceutically acceptable salt of (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidin-2-yl ] piperazin-1-yl } ethyl) dimethylamine preferably comprises a salt of the above-mentioned (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidin-2-yl ] piperazin-1-yl } ethyl) dimethylamine with an acid and/or with an acid salt of an inorganic base; the acid preferably comprises hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, benzenesulphonic acid, p-benzenesulphonic acid, naphthalenesulphonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid; the acid salt of the inorganic base preferably includes a salt containing a basic metal cation, a salt containing an alkaline earth metal cation, or a salt containing an ammonium ion. The (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidine-2-yl ] piperazine-1-yl } ethyl) dimethylamine related by the invention can be used for preparing a CARM1 inhibitor and influencing the growth and proliferation of cancer cells.

The invention also provides application of (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidine-2-yl ] piperazine-1-yl } ethyl) dimethylamine and pharmaceutically acceptable salts thereof or the small molecule inhibitor in preparing a medicament for treating cancer. In the present invention, the cancer preferably includes breast cancer, colorectal cancer, lung cancer, liver cancer, ovarian cancer, prostate cancer, oral cancer, osteosarcoma and acute myeloid leukemia. The medicine provided by the invention has a remarkable technical effect of inhibiting cancer cell proliferation, and can effectively improve the efficiency of inhibiting cancer cell proliferation compared with the prior art, thereby playing a role in treating cancer. In a specific application example, the medicine provided by the invention has a technical effect of inhibiting breast cancer cells.

The invention provides a medicine for treating cancer, which comprises (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidine-2-yl ] piperazine-1-yl } ethyl) dimethylamine and medicinal salts thereof or the micromolecule inhibitor and pharmaceutically acceptable auxiliary materials. In the present invention, the adjuvant preferably includes a pharmaceutically acceptable carrier or excipient; the cancer preferably comprises breast cancer, colorectal cancer, lung cancer, liver cancer, ovarian cancer, prostate cancer, oral cancer, osteosarcoma and acute myeloid leukemia.

To further illustrate the present invention, the following description will be made in detail with reference to the application of the compound (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidin-2-yl ] piperazin-1-yl } ethyl) dimethylamine provided by the present invention in the preparation of small molecule inhibitors or drugs for treating cancer, and small molecule inhibitors and drugs for treating cancer, but they should not be construed as limiting the scope of the present invention.

Application example 1

The detection was carried out using MTase-Glo Methyltransferase Assay kit manufactured by Promega. The first step is as follows: the methylation reaction system comprises: 4 × reaction buffer (80mM Tris-HCl 8.0, 200mM NaCl, 4mM EDTA, 12mM MgCl2, 0.4mg/ml BSA), 20uM SAM, 25uM histone substrate, 0.5ng CARM1 and a DMSO Solution of WL25 with a concentration gradient, methylation reaction at 37 ℃ for 1 hour after adding MTase-Glo Reagent, reaction at 37 ℃ for 30 minutes after completing the methylation reaction by adding MTase-Glo Detection Solution, and then transferring to a 96-well plate to measure fluorescence intensity with GlMax Discover System. In this test, WL25 was not added to the system of the negative control group, methyltransferase and WL25 were not added to the system of the blank control group, and the concentrations of WL25 of the experimental group were 0.01. mu.M, 0.05. mu.M, 0.1. mu.M, 0.5. mu.M, 1. mu.M, 6.25. mu.M, 12.5. mu.M, 25. mu.M, 50. mu.M, 75. mu.M, 100. mu.M, 200. mu.M, 300. mu.M, 400. mu.M, 600. mu.M, 800. mu.M, 1000. mu.M and 2000. mu.M, respectively. The test is also provided with another pairComparative experiments, all reaction conditions were in accordance with those described above, substituting WL25 for EZM2302 (available from ProbeChem, cat # PC-61030). The fluorescence intensity values of the experimental group and the control group under the concentration gradient were analyzed by using GraphPad Prism6 software, and the enzyme inhibitory activity was calculated (half inhibitory concentration IC was used in this test)₅₀Representation). Wherein the data for enzyme inhibitory activity detected in each experiment are shown in tables 1 and 2.

TABLE 1 enzyme inhibitory Activity of WL25

Compound (I)	IC₅₀(micromolar, μ M)
		WL25	77.15

TABLE 2 enzyme inhibitory Activity of WL25 and EZM2302

As can be seen from tables 1 and 2, WL25 has a significant inhibitory activity against CARM1 methylation. Meanwhile, the inhibition rate of WL25 is obviously higher than that of EZM2302, the half inhibition concentration is lower, and compared with the prior art, WL25 has higher capability of inhibiting CARM 1. Since CARM1 has a key role in the growth and proliferation of cancer cells, WL25 can be used in drugs for preventing or treating diseases related to CARM1 inhibitors, especially related drugs for treating cancers.

Application example 2

Different breast cancer cell lines (MCF7, T47D, BT474, MDA-MB-231, MDA-MB-468, HCC1806 and HCC1937) are plated in a 96-well plate, and the plating density is 20-30 percent. After 24 hours, DMSO solutions containing different concentrations of WL25 were added. The positive control group was supplemented with EZM2302 DMSO solution, and the blank was supplemented with DMSO only solution. The test drugs (WL25 and EZM2302) established 12 drug concentration gradients: 400. mu.M, 200. mu.M, 100. mu.M, 80. mu.M, 60. mu.M, 40. mu.M, 20. mu.M, 10. mu.M, 5. mu.M, 2.5. mu.M, 1. mu.M and 0.5. mu.M. Each concentration gradient consists of 3 replicates. Cell exchange solution before dosing. After 48 hours of dosing, use (CellTiter)

AQueous single solution cell proliferation assay kit) was used to detect cytotoxicity colorimetrically. CellTiter

The AQueous single solution reagent comprises a 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazole [3- (4, 5-dimethylthiozol-2-yl) -5- (3-carboxymethy loxyphenyl) -2- (4-sulfopheny) -2H-tetrazolium, inner salt; MTS]And an electron coupling reagent (ethiophenazine, PES). Add 20. mu.l CellTiter per 100. mu.l medium

AQueous solution reagent, 5% CO at 37 ℃₂The culture was carried out in an incubator for 1 hour. The reaction was stopped by adding 25. mu.l of 10% SDS. Absorbance data at 490nm was recorded using a Thermo Multiskan MK3 microplate reader. Data were compiled and IC's of drugs were analyzed using GraphPad Prism6 software₅₀The value is obtained. The results are shown in Table 3.

TABLE 3 inhibition of cell proliferation data

IC50	WL25(μM)	EZM2302(μM)	Blank group
				MCF7	7.69	44.76	—
T47D	20.3	61.14	—
				BT474	9.13	53.54	—
MDA-MB-231	16.07	58.88	—
				MDA-MB-468	8.65	21.76	—
HCC1806	12.11	54.64	—
				HCC1937	8.47	80.64	—

As shown in table 3, WL25 has a significant technical effect of inhibiting breast cancer cell proliferation, and compared with EZM2302, it is effective in improving the efficiency of inhibiting breast cancer cell proliferation. WL25 is useful in medicaments for the prevention or treatment of diseases associated with CARM1 inhibitors, especially in medicaments for the treatment of cancer.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

Use of (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidin-2-yl ] piperazin-1-yl } ethyl) dimethylamine, and pharmaceutically acceptable salts thereof, in the manufacture of a medicament for the treatment of cancer; the cancer is breast cancer.
2. The use of claim 1, wherein the drug is a CARM1 small molecule inhibitor.
3. Use according to claim 1 or 2, wherein the pharmaceutically acceptable salt of (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidin-2-yl ] piperazin-1-yl } ethyl) dimethylamine comprises a salt of (2- {4- [5- (3-furyl) -4- (4-methylphenyl) pyrimidin-2-yl ] piperazin-1-yl } ethyl) dimethylamine with an acid.
4. Use according to claim 3, wherein the acid comprises hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, benzenesulphonic acid, naphthalenesulphonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.