CN110218205B

CN110218205B - 2, 4-diarylaminopyrimidine derivatives containing pyridine structure and application thereof

Info

Publication number: CN110218205B
Application number: CN201910601774.2A
Authority: CN
Inventors: 刘举; 陈烨; 丁实; 雷婷; 杨文婷; 贺一明; 陈阳; 史建涛; 宫益林
Original assignee: Liaoning University
Current assignee: Liaoning University
Priority date: 2019-07-05
Filing date: 2019-07-05
Publication date: 2022-04-05
Anticipated expiration: 2039-07-05
Also published as: CN110218205A

Abstract

The invention belongs to the field of medicines, and particularly relates to a pyridine structure-containing 2, 4-diarylaminopyrimidine derivative and pharmaceutically acceptable salts thereof. The 2, 4-diarylaminopyrimidine derivative containing a pyridine structure has a structure shown in a general formula (I). The invention also relates to a compound with a general formula (I) which has a strong effect of inhibiting c-Met kinase, and provides application of 2, 4-diarylaminopyrimidine derivatives containing a pyridine structure and pharmaceutically acceptable salts thereof in preparing medicines for treating and/or preventing diseases caused by abnormal high expression of c-Met kinase, in particular application in preparing medicines for treating and/or preventing cancers.

Description

2, 4-diarylaminopyrimidine derivatives containing pyridine structure and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to 2, 4-diarylaminopyrimidine derivatives containing a pyridine structure and pharmaceutically acceptable salts thereof, a preparation method thereof and a pharmaceutical composition containing the compounds. The invention also relates to application of the compounds and pharmaceutically acceptable salts thereof in preparing medicaments for treating diseases caused by abnormal high expression of c-Met kinase, in particular to application in preparing medicaments for treating and/or preventing cancers such as gastric cancer, colon cancer, lung glands, chronic myelogenous leukemia, gastrointestinal stromal tumors, imatinib-resistant gastrointestinal stromal tumors and the like.

Background

The national cancer center 2019 recent cancer report shows: cancer has become one of the major public health problems threatening the health of the Chinese population, and it has been shown according to the latest international data that the number of deaths due to cancer accounts for 23.91% of the total number of deaths, and the morbidity and mortality caused by malignant tumors have been rising since 2000.

Receptor Tyrosine Kinases (RTKs) have been found to play an important role in many intracellular signal transduction. Overexpression, dysfunction or inappropriate activation of RTKs can affect cell survival, proliferation and function, leading to cancer development and even resistance to cancer therapy. Therefore, inhibition of RTKs has become an attractive therapeutic intervention target. Among them, in many cancers, growth and metastasis of cancer cells are caused by abnormality of HGF/c-Met signaling pathway, and the search for highly efficient c-Met kinase inhibitors has become an effective means for treating cancer. c-Met was originally identified as an oncogenic fusion protein (Tpr/Met) which was discovered when cells were exposed to the carcinogen N-methyl-N' -nitro-N-nitrosoguanidine. c-Met was later shown to play an important role in embryo development, participating in the process of proliferation and differentiation of cells. c-Met and HGF are widely expressed in adult tissues but their biological activity is limited, and under stress or tissue injury under certain conditions, the expression of c-Met and HGF can regulate and participate in wound healing and tissue injury repair. In many tumor cells, c-Met genes have the defects of over-amplification, over-normal expression, mutation, splicing and the like, c-Met kinase is over-expressed, and the c-Met kinase is abnormally activated through ligand HGF mediated autocrine and paracrine pathways, such as thyroid cancer, colorectal cancer, ovarian cancer, pancreatic cancer, prostatic cancer, liver cancer, gastric cancer and the like. The abnormity of HGF/c-Met signal channel can reduce the adhesion between tumor cells; promoting extracellular matrix degradation and tumor cell proliferation; induce tumor angiogenesis, thereby enhancing the processes of tumor development, growth, invasion and metastasis. In addition, the high expression of c-Met kinase and HGF is also associated with the process of poor prognosis of tumors and the like. The c-Met kinase is taken as a target spot to block an HGF/c-Met signal channel which is abnormally activated in tumor cells, and the tumor cells can generate a series of changes such as morphological change, slow proliferation, reduced tumorigenesis, reduced invasive ability and the like. Thus, c-Met kinase has become an important target for anti-tumor targeted therapy.

Cabozantinib (Cabozantinib) is the first approved small molecule c-Met kinase inhibitor with 4-phenoxyquinoline mother nucleus for use in the postoperative advanced or metastatic medullary thyroid cancer, and the drug also has strong inhibitory effect on VEGFR-2, c-Kit, Flt-3 and other kinases. The other 4-phenoxyquinoline c-Met kinase inhibitor Foretinib is an analogue of cabozantinib, and clinical research shows that the forotinib has a remarkable proliferation inhibition effect on various human tumor cell strains and enters a phase III clinical research stage at present. On the other hand, pyridine derivatives are important heterocyclic compounds, and compounds containing pyridine structures often have wide biological activities such as anticancer and antibacterial activities. The compounds become a hotspot of research of researchers in the pharmaceutical field, and particularly are applied to antitumor drugs, such as: the marketed drugs Sorafenib, Rigefitinib, imatinib and the like all contain pyridine structural fragments. Scientific research finds that the substituted pyridine ring is used for replacing 6, 7-dimethoxyquinoline mother nucleus of cabozantinib, and the obtained Altiratinib and Golvatinib keep good c-Met kinase activity, have good anti-tumor research and development prospects, and are respectively in the stage I and stage II clinical research stages at present.

Disclosure of Invention

The invention aims to design and synthesize a series of novel 2, 4-diarylaminopyrimidine derivatives containing pyridine structures. In vitro activity screening shows that the compounds have antitumor activity and can be used for developing antitumor drugs.

The invention provides a pyridine structure-containing 2, 4-diaryl aminopyrimidine derivative shown as a general formula I and pharmaceutically acceptable salts thereof,

wherein:

R₁selected from hydrogen, C₁-C₁₀Alkyl radical, C₃-C₇Cycloalkyl, or halogenated C₁-C₁₀An alkyl group;

x is selected from 1 to 4 identical or different substituents: hydrogen, halogen, C₁-C₁₀Alkyl, or C₁-C₄An alkoxy group;

ar is selected from C₆-C₁₀Aryl or 5-10 membered heteroaryl; wherein the heteroaryl contains 1-3 heteroatoms selected from N, O or S, and Ar is substituted with 1-3R, which may be the same or different₂Substitution;

R₂selected from hydrogen, hydroxyl, halogen, nitro, ester group, amino, cyano, C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl, C₁-C₆Alkoxy radical, C₁-C₆Alkylthio, C substituted by hydroxy or by amino or by halogen₁-C₆Alkyl, C substituted by hydroxy or by amino or by halogen₁-C₆Alkoxy, mono-or di-C₁-C₆Alkyl-substituted amino, free or salified or esterified or amidated carboxyl, C₁-C₆Alkylsulfinyl radical, C₁-C₆Alkylsulfonyl, carbamoyl, C₁-C₆Alkyl amide amino, C₁-C₆Alkyl acyl and mono-or di-C₁-C₆Alkyl-substituted carbamoyl.

Further, the 2, 4-diarylaminopyrimidine derivatives containing pyridine structures and pharmaceutically acceptable salts thereof, wherein,

R₁is selected from C₁-C₁₀Alkyl, or C₃-C₇A cycloalkyl group;

x is selected from 1 to 4 identical or different substituents: hydrogen or halogen;

ar is selected from phenyl, naphthyl, or 5-10 membered heteroaryl; wherein the heteroaryl contains 1-3 heteroatoms selected from N, O or S, and Ar is substituted with 1-3R, which may be the same or different₂Substitution;

R₂selected from hydrogen, hydroxyl, halogen, nitro, ester group, amino, cyano, C₁-C₆Alkyl radical, C₁-C₆Alkoxy, halogenated C₁-C₆Alkyl, halogenated C₁-C₆Alkoxy, mono-or di-C₁-C₆Alkyl-substituted amino, C₁-C₆Alkylamido radical, C₁-C₆Alkylsulfonyl radical, C₁-C₆An alkanoyl group, or a carbamoyl group.

R₁selected from methyl, ethyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl;

x is selected from 1 to 2 identical or different substituents: hydrogen, fluorine or chlorine;

ar is selected from phenyl or 5-6 membered heteroaryl; wherein the heteroaryl contains 1-3 heteroatoms selected from N, O or S, and Ar is substituted with 1-3R, which may be the same or different₂Substitution;

R₂selected from hydrogen, hydroxy, halogen, nitro, amino, cyano, methyl, C₁-C₆Alkoxy, trifluoromethyl, trifluoromethoxy, difluoromethoxy, methylsulfonyl, mono-or di-C₁-C₆Alkyl-substituted amino or C₁-C₆An alkylamido group.

R₁selected from methyl, ethyl, or cyclopropyl;

x is selected from 1 to 2 identical or different substituents: hydrogen or fluorine;

ar is phenyl and Ar is substituted by 1 to 3 identical or different R₂Substitution;

R₂selected from hydrogen, halogen, methyl, methoxy or trifluoromethoxy.

Further, the above-mentioned 2, 4-diarylaminopyrimidine derivatives containing pyridine structure and their pharmaceutically acceptable salts have the following structural formula, but these compounds are not meant to limit the present invention in any way:

a pharmaceutical composition containing 2, 4-diarylaminopyrimidine derivatives containing pyridine structures and pharmaceutically acceptable salts thereof comprises the 2, 4-diarylaminopyrimidine derivatives containing pyridine structures and pharmaceutically acceptable salts thereof as active ingredients and pharmaceutically acceptable excipients.

Furthermore, the 2, 4-diarylaminopyrimidine derivatives containing pyridine structures, pharmaceutically acceptable salts thereof or the pharmaceutical composition can be applied to preparation of drugs for treating and/or preventing proliferative diseases.

Furthermore, the 2, 4-diarylaminopyrimidine derivatives containing pyridine structures and pharmaceutically acceptable salts thereof or the pharmaceutical composition containing pyridine structures are applied to the preparation of drugs for treating and/or preventing cancers.

Further, the 2, 4-diarylaminopyrimidine derivatives containing pyridine structures, pharmaceutically acceptable salts thereof or the pharmaceutical composition are applied to preparation of medicines for treating and/or preventing gastric cancer, colon cancer, pulmonary glands, chronic myelogenous leukemia, gastrointestinal stromal tumors and imatinib-resistant gastrointestinal stromal tumors.

Furthermore, the 2, 4-diarylaminopyrimidine derivatives having pyridine structure shown in formula I of the present invention can form pharmaceutically acceptable salts with acids according to some common methods in the field of the present invention. Pharmaceutically acceptable addition salts include inorganic and organic acid addition salts, with the following acids being particularly preferred: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, benzoic acid, and the like.

In the present invention, "halogen" means fluorine, chlorine, bromine or iodo; "alkyl" refers to straight or branched chain alkyl; "alkylene" refers to straight or branched chain alkylene; "cycloalkyl" refers to a substituted or unsubstituted cycloalkyl; "aryl" refers to phenyl with no substituents or with substituents; "heteroaryl" means a monocyclic or polycyclic ring system containing one or more heteroatoms selected from N, O, S, the ring system being aromatic, such as imidazolyl, pyridyl, pyrazolyl, (1,2,3) -and (1,2,4) -triazolyl, furyl, thienyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, naphthyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, and the like; "saturated or partially saturated heterocyclyl" refers to monocyclic or polycyclic ring systems containing one or more heteroatoms selected from N, O, S, such as pyrrolidinyl, morpholinyl, piperazinyl, piperidinyl, pyrazolidinyl, imidazolidinyl, thiazolinyl, and the like.

The invention has the beneficial effects that:

according to the invention, the c-Met enzyme activity test shows that the compound has a remarkable effect of inhibiting the c-Met kinase activity. Can be used for treating and/or preventing diseases caused by abnormal high expression of c-Met kinase, in particular to preparing medicaments for treating and/or preventing cancers.

According to the invention, through in vitro activity tests of inhibiting human gastric cancer cells MKN-45, human colon cancer cells HT-29, human lung adenocarcinoma A549, human chronic myelogenous leukemia cells K562, human gastrointestinal stromal tumor cells GIST882 and imatinib-resistant gastrointestinal stromal tumor cells GIST-1210, the compounds disclosed by the invention are proved to have a significant inhibiting effect on gastric cancer cells, colon cancer cells, lung adenocarcinoma, chronic myelogenous leukemia cells, gastrointestinal stromal tumor cells and imatinib-resistant gastrointestinal stromal tumor cells, and are particularly used for preparing medicines for treating and/or preventing gastric cancer, colon cancer, lung gland, chronic myelogenous leukemia, gastrointestinal stromal tumor and imatinib-resistant gastrointestinal stromal tumor.

Detailed Description

The examples and preparations provided below further illustrate and exemplify the compounds of the present invention and their methods of preparation. It should be understood that the scope of the following examples and preparations are not intended to limit the scope of the invention in any way. The following examples are intended to illustrate, but not limit, the scope of the invention. The nuclear magnetic resonance hydrogen spectrum of the compound is measured by Bruker ARX-600, and the mass spectrum is measured by Agilent6460 QQQ; all reagents used were analytically or chemically pure.

The following synthetic schemes describe the preparation of the derivatives of formula I of the present invention, all starting materials are prepared by the following synthetic schemes, by methods well known to those of ordinary skill in the art of organic chemistry or are commercially available. All final derivatives of the invention are prepared by the synthetic routes described below or by methods analogous thereto, which are well known to those of ordinary skill in the art of organic chemistry. All variables used in the synthetic routes described below are as defined below or in the claims.

The synthetic route is as follows:

general method for preparation

Step A N- (4-Chloropyridin-2-yl) Cyclopropylcarboxamide (a)

8.80g of 2-amino-4-chloropyridine and 20.80g of triethylamine were dissolved in 80mL of dichloromethane, and 30mL of a dichloromethane solution containing 9.30g of cyclopropylcarbonyl chloride was added dropwise to the solution under ice-bath conditions, and the temperature was raised to room temperature after completion of the addition. Stirring for 12h, and reacting, and mixing with 20% K₂CO₃Washing the solution and saturated saline solution for 3 times, separating organic phase, drying with anhydrous sodium sulfate, filtering, evaporating solvent to obtain crude product, and separating by column chromatography to obtain white solid as intermediate (a).

Step B N- (4- (2-fluoro-4-nitrophenoxy) pyridin-2-yl) cyclopropylcarboxamide (b)

Intermediate (a)8.00g and 2-fluoro-4-nitrophenol 15.98g were added to 100mL of chlorobenzene, 14Reacting at 0 ℃ for 40 h. Cooling to room temperature, concentrating under reduced pressure, dissolving the residue in an appropriate amount of dichloromethane, adding₂CO₃Washing the solution and saturated saline solution for 3 times, separating organic phase, drying with anhydrous sodium sulfate, filtering, evaporating solvent to obtain brown solid, and performing column chromatography to obtain light yellow solid product as intermediate (b).¹H NMR(600MHz,DMSO-d₆)δ11.00(s,1H),8.43(m,1H),8.30(d,J＝5.7Hz,1H),8.19(m,1H),7.76(d,J＝2.2Hz,1H),7.61(t,J＝8.5Hz,1H),6.86(m,1H),2.04–1.95(m,1H),0.78(t,J＝6.3Hz,4H)。

Step C N- (4- (4-amino-fluoro-2-fluorophenoxy) pyridin-2-yl) cyclopropylcarboxamide (c)

Adding 6.00g of intermediate (b), 5.28g of iron powder and 11.36g of acetic acid into 100mL of ethyl acetate, adding 20mL of water, refluxing for 2h at 80 ℃, filtering while hot after the reaction is finished, separating an organic phase, drying with anhydrous sodium sulfate, filtering, and evaporating the solvent under reduced pressure to obtain a white solid serving as intermediate (c).¹H NMR(600MHz,DMSO-d₆)δ10.79(s,1H),8.15(d,J＝5.7Hz,1H),7.59(s,1H),6.95(t,J＝9.0Hz,1H),6.67–6.61(m,1H),6.49(dd,J＝13.1,2.2Hz,1H),6.40(d,J＝8.7Hz,1H),5.44(s,2H),2.03–1.88(m,1H),0.76(br,4H)。

Step D N- (4- (4- ((2-Chloropyrimidin-4-yl) amino) -2-fluorophenoxy) pyridin-2-yl) cyclopropanecarboxamide (d)

Adding 3.00g of the intermediate (c), 1.87g of 2, 4-dichloropyrimidine and 1.35g of diisopropylethylamine into 50mL of isopropanol, refluxing for 25h at 83 ℃, distilling the solvent under reduced pressure, dissolving the residue in 80mL of dichloromethane, washing with a proper amount of water for 4 times, separating an organic phase, drying with anhydrous sodium sulfate, filtering, and evaporating the solvent under reduced pressure to obtain a crude product, and performing silica gel column chromatography on the crude product to obtain a light yellow solid product, namely the compound (d).¹H NMR(600MHz,DMSO-d₆)δ10.90(s,1H),10.34(s,1H),8.25-8.22(m,2H),7.88-7.60(m,2H),7.50–7.30(m,2H),6.85-6.71(m,2H),2.03–1.92(m,1H),0.84–0.72(m,4H)；MS(ESI)m/z(％):400.1[M+H]⁺,422.0[M+Na]⁺.

Step E general procedure for preparation of Compounds of examples 1-17:

adding 0.20g of compound (d) or analogue thereof and 1.3 equivalents of substituted arylamine into 5mL of isopropanol, adding 1.0 equivalent of p-toluenesulfonic acid, heating to 83 ℃, carrying out reflux reaction for 10-18 hours, cooling a reaction mixture to room temperature after the reaction is finished, precipitating a large amount of solid, filtering out the solid, washing a filter cake for three times by using isopropanol, and drying to obtain a white solid product.

The compounds of examples 1-17 were prepared according to the general procedure for the preparation, respectively, as shown in Table 1.

Table 1:

in vitro antitumor cell Activity

Partial compounds of the embodiment of the invention are subjected to in vitro inhibition of activity screening of human gastric cancer cells MKN-45, human colon cancer cells HT-29, human lung adenocarcinoma A549, human chronic myelogenous leukemia cells K562, human gastrointestinal stromal tumor cells GIST882, imatinib-resistant gastrointestinal stromal tumor cells GIST-1210 and c-met kinase activity screening.

In vitro cytotoxic Activity assay

(1) After the cells of human gastric cancer cells MKN-45, human colon cancer cells HT-29, human lung adenocarcinoma A549, human chronic myelogenous leukemia cells K562, human gastrointestinal stromal tumor cells GIST882 and imatinib-resistant gastrointestinal stromal tumor cells GIST-1210 are respectively recovered and passaged for 2-3 times of stabilization, the cells are digested from the bottom of the culture flask by using trypsin solution (0.25 percent). After pouring the cell digest into the centrifuge tube, the culture medium is added to stop the digestion. Centrifuging the centrifuge tube at 800r/min for 10min, discarding supernatant, adding 5mL culture solution, blowing and beating the mixed cells, sucking 10 μ L cell suspension, adding into cell counting plate, counting, and adjustingThe whole cell concentration was 10⁴Per well. 100. mu.L of the cell suspension was added to the 96-well plate except that the A1 well was a blank well and no cells were added. The 96-well plate was placed in an incubator for 24 h.

(2) The test sample was dissolved in 50. mu.L of dimethyl sulfoxide, and then an appropriate amount of culture solution was added to dissolve the sample to 2mg/mL of the liquid, and then the sample was diluted to 20,4,0.8,0.16, 0.032. mu.g/mL in a 24-well plate.

3 wells were added for each concentration, two columns of cells surrounding each, which were greatly affected by the environment, and only used as blank wells. The 96-well plate was placed in an incubator for 72 h.

(3) The drug-containing culture solution in the 96-well plate is discarded, the cells are washed twice by using Phosphate Buffer Solution (PBS), 100 mu L of MTT (tetrazole) (0.5mg/mL) is added into each well and put into an incubator for 4h, the MTT solution is discarded, and 100 mu L of dimethyl sulfoxide is added. And oscillating on a magnetic oscillator to fully dissolve the viable cells and the MTT reaction product formazan, and putting the formazan into an enzyme labeling instrument to measure the result. Determination of drug IC by Bliss method₅₀The value is obtained. The results of the activity of the compounds of the examples and the positive control drug, foretinib, on inhibiting human gastric cancer cells MKN-45, human colon cancer cells HT-29, human lung adenocarcinoma A549, human chronic myelogenous leukemia cells K562, human gastrointestinal stromal tumor cells GIST882, and imatinib-resistant gastrointestinal stromal tumor cells GIST-1210 are shown in Table 2.

c-Met enzyme Activity assay

The assay used to measure c-Met kinase activity is based on an enzyme-linked immunosorbent assay (ELISA). The specific operation is as follows:

the example compound, 50pM c-Met (His-tagged recombinant human Met (amino acid 974-terminus), expressed by baculovirus) and 5. mu.M ATP in assay buffer (25mM MOPS, pH 7.4, 5mM MgCl) were added to 0.25mg/mL PGT-coated plates at room temperature₂，0.5raM MnCl₂100 μ M sodium orthovanadate, 0.01% Triton X-100, 1mM DTT, and finally DMSO concentration 1% (v/v)) for 20 minutes. The reaction mixture was removed by washing and the phosphorylated polymer substrate was detected with 0.2. mu.g/mL of a phosphotyrosine-specific monoclonal antibody (PY20) conjugated with horseradish peroxidase (HRP). Adding 1M phosphoric acid to stop color development, and introducing at 450nmThe color of the developed substrate (TMB) was quantified spectrophotometrically. The data for the inhibition of c-Met kinase by the compounds of the examples and the positive control (foretinib) are shown in table 2.

The results of the compounds for inhibiting the activities of human gastric cancer cells MKN-45, human lung adenocarcinoma A549 and c-met kinase are shown in Table 2, and IC in the Table 2₅₀10.0 μ M ≦ IC expressed as A₅₀> 10.0 muM, denoted B, and ND denotes untested.

TABLE 2

As is clear from Table 2, the compounds of formula (I) to be protected by the present invention have good in vitro anti-tumor activity against both tumor cell lines and c-met kinase. The compounds have good development and application prospects of antitumor drugs.

While the invention has been described with reference to specific embodiments, modifications and equivalent arrangements will be apparent to those skilled in the art and are intended to be included within the scope of the invention.

Claims

1. 2, 4-diaryl aminopyrimidine derivatives containing pyridine structures and pharmaceutically acceptable salts thereof are characterized by having the following structural formula:

2. a pharmaceutical composition comprising a pyridine structure-containing 2, 4-diarylaminopyrimidine derivative and a pharmaceutically acceptable salt thereof, wherein the pyridine structure-containing 2, 4-diarylaminopyrimidine derivative and the pharmaceutically acceptable salt thereof according to claim 1 are contained as an active ingredient and a pharmaceutically acceptable excipient.

3. Use of the 2, 4-diarylaminopyrimidine derivatives containing pyridine structure and pharmaceutically acceptable salts thereof according to claim 1 or the pharmaceutical composition according to claim 2 in preparing drugs for treating and/or preventing proliferative diseases.

4. Use of the 2, 4-diarylaminopyrimidine derivatives containing pyridine structure and pharmaceutically acceptable salts thereof according to claim 1 or the pharmaceutical composition according to claim 2 in preparing medicine for treating and/or preventing cancer.

5. Use of the 2, 4-diarylaminopyrimidine derivatives containing pyridine structure and pharmaceutically acceptable salts thereof according to claim 1 or the pharmaceutical composition according to claim 2 for preparing drugs for treating and/or preventing gastric cancer, colon cancer, lung adenocarcinoma, chronic myelogenous leukemia and gastrointestinal stromal tumor, imatinib-resistant gastrointestinal stromal tumor.