CN114437161B - Zidovudine spliced 4-aniline quinazoline compound and preparation method and application thereof - Google Patents

Zidovudine spliced 4-aniline quinazoline compound and preparation method and application thereof Download PDF

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CN114437161B
CN114437161B CN202210103168.XA CN202210103168A CN114437161B CN 114437161 B CN114437161 B CN 114437161B CN 202210103168 A CN202210103168 A CN 202210103168A CN 114437161 B CN114437161 B CN 114437161B
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梁光平
杨俊�
朱绪秀
文露滴
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ZUNYI MEDICAL AND PHARMACEUTICAL COLLEGE
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Abstract

The invention discloses a zidovudine spliced 4-aniline quinazoline compound and a preparation method and application thereof. The compound contains zidovudine and 4-aniline quinazoline skeleton structure, has potential biological activity, provides a compound source for biological activity screening, has important application value in medicine screening and pharmaceutical industry, and finds that the compound has obvious inhibition effect on a human non-small cell lung cancer cell line (A549), a human breast cancer cell line (MCF-7), a human cervical cancer cell line (Hela), a human liver cancer cell line (HepG 2) and a human lung adenocarcinoma cisplatin-resistant strain (A549/DDP) through in vitro antitumor activity screening, the antitumor effect intensity of part of the compound is more than 10 times that of positive control erlotinib, and the compound can be found to cooperatively play the antitumor effect from a plurality of signal paths through inhibiting EGFR enzyme activity, so that the compound has great potential to be developed into a novel antitumor drug.

Description

Zidovudine spliced 4-aniline quinazoline compound and preparation method and application thereof
Technical Field
The invention relates to the technical field of chemistry, in particular to a zidovudine spliced 4-aniline quinazoline compound and application thereof in anti-tumor related medicines.
Background
Zidovudine (Zidovudine), also known as azidothymidine, is commonly used alone or in combination with other drugs primarily for the treatment of Human Immunodeficiency Virus (HIV) infections. Recent studies have shown that zidovudine can exert antitumor and radiosensitization effects by inhibiting telomerase activity, and its antitumor-related studies have been directed to the treatment of various tumors such as breast cancer, cervical cancer, ovarian cancer, vaginal cancer, T-lymphoma, burkitt's lymphoma, glioma, liver cancer, stomach cancer, bladder cancer, parathyroid cancer, colon cancer, lung cancer, prostate cancer, kaposi's sarcoma, head and neck cancer, squamous cell carcinoma of throat, oral tumor, leukemia, etc., and the study of the efficacy of malignant tumors such as parathyroid cancer, colon cancer, gastrointestinal cancer, etc., has entered the stage of clinical study.
The 4-aniline quinazoline compound is a small molecular tyrosine kinase inhibitor acting on an intracellular tyrosine kinase active region of an epidermal growth factor (Epidermal Growth Factor R eceptor, EGFR), gefitinib, erlotinib, lapatinib, afatinib and the like are marketed, and a plurality of other candidate medicines of the compound are in clinical research stages, so that the 4-aniline quinazoline compound is one of hot spot fields for researching anti-tumor medicines in recent years. However, these 4-anilinoquinazoline compounds only have an inhibitory effect on tumors, cannot effectively kill cancer cells, have about 60% of patients with non-small cell lung cancer, and have T790M action site drug resistance mutation, and the subsequent drug treatment has very limited selection, and other inhibitors almost have similar drug resistance problems. Improving and overcoming the defects of 4-aniline quinazoline EGFR inhibitors has become a difficulty in researching the inhibitors.
Because EGFR is over-expressed in 40-80% of patients with non-small cell cancers (such as breast cancer, head and neck cancer, bladder cancer, colon cancer, ovarian cancer and prostate cancer), the EGFR is a good anti-tumor action target, and azidothymidine has sensitization effect on tumor treatment. According to the principle of drug molecule splicing, zidovudine and a 4-arylamine quinazoline EGFR inhibitor are spliced, and the novel molecule obtained by splicing is not predicted to have the effect of multi-target synergistic effect on tumors.
Based on the facts, the azide structure in the zidovudine structure is utilized to splice and synthesize with 4-aniline quinazoline compounds with terminal alkyne substitution through classical Click reaction, and the splice products are found to have good anti-tumor activity through tests, so that the splice products can be further developed and researched, and meanwhile, compound sources can be provided for biological activity screening, so that the splice products have important application value to the screening of medicines and the pharmaceutical industry.
Disclosure of Invention
The purpose of the invention is that: provides a zidovudine spliced 4-aniline quinazoline compound, a preparation method and application thereof, which are important medicine molecular analogues and medicine intermediate analogues, provide compound sources for biological activity screening, have important application value for medicine screening and pharmaceutical industry, and have economic and simple synthesis method.
The invention also discovers the application of the compounds in preparing medicines for preventing and treating tumor diseases.
The invention is realized in the following way: zidovudine spliced 4-aniline quinazoline compound has a structure shown in a general formula (I) or a general formula (II):
wherein: r is a hydrogen atom or a 2-propynyloxy group;
R 1 is a hydrogen atom, hydroxyl group, acetyl group, methoxy group, ethoxy group, methyl group, fluorine atom, chlorine atom, bromine atom, iodine atom, 2-methoxyethoxy group, N-methyleneacrylamido group, O-2-tetrahydrofuranyl group, O- (1-methylpiperidin-4-yl) group, 1-methylpiperidin-4-yl-methoxy group, phenyl group, O-benzyl group, thiazol-2-ylmethoxy group, thiazolyl group, furyl group, pyrrolyl group, oxazolyl group, thienyl group, pyrazolyl group, pyrimidinyl group, sulfonamide group, 3-methoxypyrazin-2-yl-sulfonamide group, 5-methyl-3-isoxazolyl-sulfonamide group or acetylsulfonamide group substituted on the benzene ring;
R 2 1 to 5 hydrogen atoms, fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, hydroxyl groups, methoxy groups, acetyl groups, N-dimethyl groups, alkynyl groups, phenyl groups, O- (1-methylpiperidin-4-yl) groups, 1-methylpiperidin-4-yl-methoxy groups, benzyl groups, O-benzyl groups, thiofuran groups, substituted on the benzene ringOxazol-2-ylmethoxy, thiazolyl, furanyl, pyrrolyl, oxazolyl, thiophenyl, pyrazolyl, pyrimidinyl, sulfonamide, 3-methoxypyrazin-2-yl-sulfonamide, 5-methyl-3-isoxazolyl-sulfonamide, or acetyl sulfonamide.
The preparation method of the zidovudine spliced 4-aniline quinazoline compound comprises the following steps of:
dissolving a 6-hydroxy-4-aniline quinazoline compound (1) in an organic solvent, reacting to generate an intermediate 2 under the action of propargyl bromide and an acid binding agent, and then reacting with zidovudine (3) under the action of a catalyst to obtain a compound 4 shown in a formula (I), wherein the reaction route is as follows:
wherein the preparation method of the general formula (II) is as follows: dissolving a 7-hydroxy-4-aniline quinazoline compound (5) in an organic solvent, reacting to generate an intermediate 6 under the action of propargyl bromide and an acid binding agent, and then reacting with zidovudine (3) under the action of a catalyst to obtain a compound 7 shown in a formula (II), wherein the reaction route is as follows:
wherein: r is a hydrogen atom or a 2-propynyloxy group;
R 1 is a hydrogen atom, hydroxyl group, acetyl group, methoxy group, ethoxy group, methyl group, fluorine atom, chlorine atom, bromine atom, iodine atom, 2-methoxyethoxy group, N-methyleneacrylamido group, O-2-tetrahydrofuranyl group, O- (1-methylpiperidin-4-yl) group, 1-methylpiperidin-4-yl-methoxy group, phenyl group, O-benzyl group, thiazol-2-ylmethoxy group, thiazolyl group, furyl group, pyrrolyl group, oxazolyl group, thienyl group, pyrazolyl group, pyrimidinyl group, sulfonamide group, 3-methoxypyrazin-2-yl-sulfonamide group, 5-methyl-3-isoxazolyl-sulfonamide group or acetylsulfonamide group substituted on the benzene ring;
R 2 is a benzene ring1 to 5 hydrogen atoms, fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, hydroxyl groups, methoxy groups, acetyl groups, N-dimethyl groups, alkynyl groups, phenyl groups, O- (1-methylpiperidin-4-yl), 1-methylpiperidin-4-yl-methoxy groups, benzyl groups, O-benzyl groups, thiazol-2-ylmethoxy groups, thiazolyl groups, furyl groups, pyrrolyl groups, oxazolyl groups, thienyl groups, pyrazolyl groups, pyrimidinyl groups, sulfonamide groups, 3-methoxypyrazine-2-yl-sulfonamide groups, 5-methyl-3-isoxazolyl-sulfonamide groups or acetyl sulfonamide groups.
The organic solvent is methanol, ethanol, N '-dimethylformamide, N' -dimethylacetamide, 1, 4-dioxane, tetrahydrofuran, dichloromethane, chloroform or toluene.
The catalyst is one or a combination of more of sodium L-ascorbate, copper sulfate or cuprous iodide;
examples of the catalyst are as follows (but it is emphasized that the condensing agent of the present invention is not limited to the following ones):
the acid binding agent is one or a combination of several of triethylamine, N '-diisopropylethylamine, 1, 8-diazo hetero-double spiro [5.4.0] undec-7-ene, potassium carbonate, potassium hydroxide, sodium bicarbonate or N, N' -diisopropylamine.
Examples of the acid-binding agent are as follows (but it is emphasized that the acid-binding agent of the present invention is not limited to the following expression):
the reaction temperature is between-20 ℃ and 200 ℃ and the reaction time is between 0 and 72 hours.
The application of the zidovudine spliced 4-aniline quinazoline compound in preparing antitumor drugs.
By adopting the technical scheme, the 4-aniline quinazoline compound and propargyl bromide are dissolved in an organic solvent according to the mol ratio of 1:1.0-1:5.0, an intermediate is obtained under the action of an acid binding agent, and then the azidothymidine spliced 4-aniline quinazoline compound with the general formula (I) or the general formula (II) is prepared under the condition of a catalyst. The compound contains azidothymidine and 4-aniline quinazoline skeleton structure, has potential bioactivity, provides a compound source for bioactivity screening, has important application value to medicine screening and pharmaceutical industry, and has good inhibition effect on human non-small cell lung cancer cell line (A549), human breast cancer cell line (MCF-7), human cervical cancer cell line (Hela), human liver cancer cell line (HepG 2) and human lung adenocarcinoma cisplatin-resistant strain (A549/DDP) through in vitro antitumor activity screening, thus being likely to be further developed into a novel medicine for preventing and treating tumors. The operation method of the invention is very economical and simple, the product yield is higher, the dissolubility is better, the raw materials are cheap and easy to obtain, and the anti-tumor activity of the invention can be further researched and developed.
Drawings
FIG. 1 is a schematic representation of the design of zidovudine-spliced 4-anilinoquinazoline compounds.
Figure 2 shows high resolution mass spectrometry data for compound IVb of example 1.
FIG. 3 is a graph of nuclear magnetic resonance spectrum of compound IVb of example 1.
Detailed Description
[ example 1 ]
The chemical synthesis of the zidovudine spliced 4-aniline quinazoline compound with the general formula (I) is shown in table 1, the preparation methods of the compounds IVa to IVd are completely the same, but it is emphasized that the compound of the invention is not limited to the contents shown in table 1.
TABLE 1 chemical Synthesis of zidovudine spliced 4-anilinoquinazoline Compounds of formula (I)
Quinazoline intermediates (Ia, 0.10g,0.32 mmol) were placed in a 25mL reaction tube, 10mL of N, N-dimethylformamide was added, stirred to dissolve them completely, anhydrous potassium carbonate (0.13 g,0.95mmol,3.0 eq) was added again, stirred for 10min, propargyl bromide (45.0 mg,0.38mmol,1.2 eq) was slowly added, stirred at room temperature for reaction, monitored by Thin Layer Chromatography (TLC) for about 15 hours, the reaction mixture was diluted with 50mL of ethyl acetate, then 50mL of water was added, the aqueous layer was extracted once with 40mL of ethyl acetate, the organic layers were combined, washed with water (100 mL. Times.2) and concentrated under reduced pressure to give intermediates which were used directly in the next reaction without purification.
The above concentrated solution was taken to dry oily compound IIa (70 mg,0.20 mmol), zidovudine (53 mg,0.20mmol,1.0 eq.) in a 25mL eggplant-shaped bottle, 5mL of N, N-dimethylformamide and 0.6mL of water were added, stirred to dissolve completely, sodium ascorbate (47 mg,0.24mmol,1.2 eq.) and cuprous iodide (38 mg,0.20mmol,1.0 eq.) were added sequentially under dark conditions, N-diisopropylethylamine (8 mg,0.060mmol,0.3 eq.) were added, the reaction was continued with stirring at room temperature, monitored by Thin Layer Chromatography (TLC), the reaction was completed for about 12 hours, the reaction solution was diluted with 40mL of ethyl acetate, then extracted with 30mL of ethyl acetate once, the organic layers were combined, washed with water (80 mL. Times 2), the organic layers were concentrated under reduced pressure to obtain a crude product, and the aqueous layer was separated and purified by a silica gel column (eluent: methanol: 9: 1: a) to obtain the compound a). Brown powder, yield 17.9%; melting point, high resolution mass spectrum and nmr data are as follows: m.p.182.5 ℃ -184.1 ℃; HRMS, m/z calcd.for C 27 H 25 O 5 N 8 BrNa([M+Na]+):643.1024,found:643.1006. 1 H NMR(600MHz,DMSO-d 6 )δ:11.37(s,1H),9.82(s,1H),8.59(s,1H),8.54(s,1H),8.25(s,1H),8.22–8.18(m,1H),7.99–7.95(t,J＝8.0Hz,1H),7.84(s,1H),7.80–7.77(m,1H),7.60–7.57(m,1H),7.38(t,J＝8.1Hz,1H),7.32–7.30(m,1H),7.24–7.15(m,1H),6.46(t,J＝6.7Hz,1H),5.47–5.43(m,1H),5.39(s,2H),4.30–4.20(m,1H),3.75–3.71(m,1H),3.68–3.63(m,1H),2.81–2.74(m,1H),2.71–2.65(m,1H),1.82(s,3H). 13 C NMR(151MHz,DMSO-d 6 )δ:164.2,157.2,156.7,152.8,151.7,150.9,145.8,143.0,141.6,136.7,130.9,126.4,124.9,124.6,121.7,121.2,116.2,110.1,105.5,104.1,85.0,84.4,62.6,61.3,59.9,37.7,12.7.
Compound IVb: white powder, collectingThe rate is 15.5%; m.p.182.9 ℃ -184.7 ℃; HRMS, m/z calcd.for C 27 H 24 O 5 N 8 BrFNa([M+Na]+):661.0929,found:661.0909. 1 H NMR(600MHz,DMSO-d 6 )δ:11.38(s,1H),9.75(s,1H),8.52(s,1H),8.48(s,1H),8.06(d,J＝2.3Hz,1H),7.86(dd,J＝6.7,2.3Hz,1H),7.84(s,1H),7.79(d,J＝9.1Hz,1H),7.59(dd,J＝9.1,2.5Hz,1H),7.53–7.48(m,1H),7.36(t,J＝9.5Hz,1H),6.46(t,J＝6.6Hz,1H),5.44(dt,J＝8.8,5.3Hz,1H),5.35(s,2H),5.32(t,J＝5.2Hz,1H),4.27(dd,J＝8.7,3.6Hz,1H),3.76–3.71(m,1H),3.69–3.64(m,1H),2.81–2.75(m,1H),2.72–2.65(m,1H),1.82(s,3H). 13 C NMR(151MHz,DMSO-d 6 )δ:164.2,158.0,157.2,156.6,155.5,153.1,150.9,145.8,143.0,136.7,130.8,130.0,129.9,128.7(d,J＝13.5Hz),125.3,124.8,118.5(d,J＝21.8Hz),115.8(d,J＝25.7Hz),110.1,103.7,84.8,84.4,62.4,61.3,59.9,37.7,12.7.
Compound IVc: red powder, yield 67.4%; m.p.196.1-198.2 deg.c; HRMS, m/z calcd.for C 27 H 24 O 5 N 8 ClFNa([M+Na]+):617.1434,found:617.1412. 1 H NMR(600MHz,DMSO-d 6 )δ:11.37(s,1H),9.80(s,1H),8.55(s,1H),8.18(s,1H),8.11(s,1H),7.96–7.67(m,3H),7.60(s,1H),7.53–7.28(m,2H),6.46(s,1H),5.45(s,1H),5.33(s,3H),4.28(s,1H),3.73(s,1H),3.67(s,1H),2.77(s,1H),2.71(d,J＝16.1Hz,1H),1.80(d,J＝23.4Hz,3H). 13 C NMR(151MHz,DMSO-d 6 )δ:164.2,156.9,156.8,154.8,154.7,153.2,150.9,143.1,137.1,137.0,136.7,125.1,124.6,124.3,124.0,123.1(d,J＝6.4Hz),119.4(d,J＝18.2Hz),117.1(d,J＝20.7Hz),110.1,104.2,84.9,84.4,62.4,61.3,60.0,37.7,12.7.
Compound IVd: white powder, yield 22.1%; m.p.201.6-203.5 ℃; HRMS, m/z calcd.for C 28 H 27 O 6 N 8 ClF([M+H]+):625.1721,found:625.1706. 1 H NMR(600MHz,DMSO-d 6 )δ:11.38(s,1H),9.72(s,1H),8.52(d,J＝17.2Hz,2H),8.19(dd,J＝6.9,2.6Hz,1H),8.13(s,1H),7.88–7.85(m,1H),7.84(d,J＝1.1Hz,1H),7.45(t,J＝9.1Hz,1H),7.24(s,1H),6.46(q,J＝6.3Hz,1H),5.46(dt,J＝8.7,5.2Hz,1H),5.35(s,2H),4.29–4.25(m,1H),3.93(s,3H),3.73(dt,J＝16.2,8.1Hz,1H),3.69–3.63(m,1H),2.81–2.75(m,1H),2.71–2.65(m,1H),1.82(s,3H). 13 C NMR(151MHz,DMSO-d 6 )δ:164.2,156.6,154.9,154.4,153.3,152.8,150.9,148.1,147.7,142.7,137.3,136.7,125.2,123.8,122.6(d,J＝7.0Hz),119.2(d,J＝17.9Hz),117.0(d,J＝22.0Hz),110.1,107.9,104.0,85.0,84.4,62.9,61.3,59.9,56.3,37.7,12.7.
[ example 2 ]
The chemical synthesis of the zidovudine spliced 4-aniline quinazoline compound with the general formula (II) is shown in table 2, the preparation methods of the compounds VIIa-VIIb are completely the same, but it is emphasized that the compound of the invention is not limited to the contents shown in table 2.
TABLE 2 chemical Synthesis of zidovudine spliced 4-anilinoquinazoline Compounds of formula (II)
Quinazoline intermediates (Va, 0.10g,0.31 mmol) were placed in a 25mL reaction tube, 10mL of N, N-dimethylformamide was added, stirred to dissolve them completely, anhydrous potassium carbonate (0.13 g,0.95mmol,3.0 eq) was added again, stirred for 10min, propargyl bromide (45.0 mg,0.38mmol,1.2 eq) was slowly added, stirred at room temperature for reaction, monitored by Thin Layer Chromatography (TLC), reacted for about 15 hours to completion, the reaction solution was diluted with 50mL of ethyl acetate, then 50mL of water was added, the aqueous layer was extracted once with 40mL of ethyl acetate, the organic layers were combined, washed with water (100 mL. Times.2), the product was concentrated under reduced pressure to give intermediate IIa, and the product was used directly for the next reaction without purification.
The above concentrated to dryness was taken out as an oily compound IIa (85 mg,0.24 mmol), zidovudine (64 mg,0.24mmol,1.0 eq.) in a 25mL eggplant-shaped bottle, 5mL of N, N-dimethylformamide and 0.6mL of water were added and stirred to dissolve completely, sodium ascorbate (56 mg,0.29mmol,1.2 eq.) and cuprous iodide (45 mg,0.24mmol,1.0 eq.) were added sequentially, N-diisopropylethylamine (10 mg,0.071mmol,0.3 eq.) were added under dark condition, the reaction was continued with stirring at room temperature, monitored by Thin Layer Chromatography (TLC) and 40mL of acetic acid was added to the reaction solution for about 12 hoursDiluting with ethyl ester, adding 40mL of water for extraction, extracting the water layer once again with 30mL of ethyl acetate, combining the organic layers, adding water (80 mL multiplied by 2), washing the organic layers, concentrating the solvent under reduced pressure to obtain a crude product, and separating and purifying the crude product by using a silica gel column (eluent: dichloromethane: methanol=9:1) to obtain the compound VIIa. Brown yellow powder, yield 40.3%; melting point, high resolution mass spectrum and nmr data are as follows: m.p.199.8 ℃ -202.7 ℃; HRMS, m/z calcd.for C 28 H 26 O 6 N 8 ClFNa([M+Na]+):647.1540,found:647.1522. 1 H NMR(600MHz,DMSO-d 6 )δ:11.37(s,1H),9.65(s,1H),8.53(d,J＝10.2Hz,1H),8.51(s,1H),8.15(dd,J＝6.8,2.6Hz,1H),7.88(s,1H),7.84(d,J＝1.0Hz,1H),7.82(dd,J＝5.9,3.2Hz,1H),7.49(s,1H),7.44(d,J＝9.1Hz,1H),6.49–6.42(m,1H),5.44(dt,J＝8.7,5.4Hz,1H),5.35(s,2H),4.26(dt,J＝7.3,3.6Hz,1H),3.95(s,3H),3.82(dd,J＝8.7,4.1Hz,1H),3.73(dd,J＝12.0,3.4Hz,1H),3.67–3.63(m,1H),2.81–2.74(m,1H),2.68(dt,J＝18.9,6.6Hz,1H),1.82(d,J＝0.9Hz,3H). 13 C NMR(151MHz,DMSO-d 6 )δ:164.4,156.5,154.4,153.5,152.8,150.9,149.5,147.4,142.6,137.3,136.7,125.3,123.9,122.8(d,J＝7.0Hz),119.2(d,J＝18.5Hz),117.0(d,J＝21.9Hz),110.1,108.9,102.5,84.9,84.4,62.2,61.2,59.8,56.7,37.7,12.7.
Compound VIIb: brown yellow powder, yield 6.8%; m.p.230.7-232.9 ℃; HRMS, m/z calcd.for C 28 H 26 O 6 N 8 IClNa([M+Na]+):755.0601,found:755.0582. 1 H NMR(600MHz,DMSO-d 6 )δ:11.37(s,1H),9.57(s,1H),8.51(s,1H),8.49–8.30(m,1H),7.96(t,J＝2.9Hz,1H),7.87(s,1H),7.83(d,J＝1.0Hz,1H),7.77(dd,J＝8.3,1.9Hz,1H),7.52(d,J＝5.8Hz,1H),7.36(d,J＝8.3Hz,1H),6.45(t,J＝6.6Hz,1H),5.46–5.41(m,1H),5.35(s,2H),5.30(t,J＝5.2Hz,1H),4.26(dd,J＝8.8,3.6Hz,1H),3.92(s,3H),3.75–3.69(m,1H),3.68–3.62(m,1H),2.80–2.74(m,1H),2.71–2.65(m,1H),1.82(s,3H). 13 C NMR(151MHz,DMSO-d 6 )δ:164.2,153.4,150.9,149.4,144.6,142.6,142.5,140.3,137.9,137.0,136.8,136.7,132.5,131.8,125.3,115.1,110.1,102.6,94.6,91.7,84.9,84.4,62.2,61.2,59.8,56.5,37.7,12.7.
[ example 3 ]
The in vitro antitumor activity of the compounds IVa-IVd and VIIa-VIIb on a human non-small cell lung cancer cell line (A549), a human breast cancer cell line (MCF-7), a human cervical cancer cell line (Hela), a human liver cancer cell line (HepG 2) and a human lung adenocarcinoma cisplatin-resistant strain (A549/DDP) is tested by adopting an MTT method, and the test method is described by using A549 cells. However, it should be emphasized that the compounds of the present invention are not limited to cytotoxicity expressed by human non-small cell lung cancer cell line (A549), human breast cancer cell line (MCF-7), human cervical cancer cell line (Hela), human liver cancer cell line (HepG 2), and human lung adenocarcinoma cisplatin-resistant strain (A549/DDP).
(a) Cell resuscitation: taking out A549 cells from liquid nitrogen, rapidly placing the cells into a water bath kettle at 37 ℃, and slightly shaking a freezing storage tube to dissolve the freezing storage liquid; transferring the cells into a centrifuge tube containing 5ml of culture medium after dissolution, centrifuging to collect the cells, centrifuging at room temperature of 1000rmp for 5min, and discarding the supernatant; suspending cells in complete culture medium containing 10% fetal bovine serum, inoculating into culture dish, gently stirring, and culturing at 37deg.C under 5% CO2 saturated humidity.
(b) When the density of cells reached 80%, cells were passaged: discarding the culture medium, and washing with PBS; adding 1-2ml of 0.25% trypsin to digest the cells, observing under a microscope, and digesting for 1-2min to ensure that the cells are separated from each other and rounded, namely the digestion is completed; rapidly discarding pancreatin, adding complete culture medium, blowing cells, making into single cell suspension, passaging at 37deg.C and 5% CO at a ratio of 1:3 2 And (5) performing expansion culture under saturated humidity conditions.
(c) Cell treatment: taking cells in logarithmic growth phase and good growth state at 3×10 3 The cells were plated in 96-well plates with blank groups and incubated overnight at 37 ℃ (100 μl sterile PBS was added to the wells surrounding the cell wells); drug concentration was set at 0.01. Mu.M, 0.1. Mu.M, 1. Mu.M, 10. Mu.M, 100. Mu.M; after 48 hours of action, 10. Mu.L MTT was added to each well and incubated at 37℃for 4 hours; the culture supernatant was carefully aspirated, 150. Mu.L of DMSO was added and shaken for 10min to dissolve the reduced MTT crystalline formazan (formazan), and the absorbance OD for each well was measured at 568nm by a microplate reader. Cell proliferation inhibition = 1- (experimental OD-blank OD)/(control OD-blank OD), a549 cell half-populationConcentration-inhibiting IC 50 Analysis was performed by spss software.
The methods for detecting HepG2, hela, MCF-7 and A549/DDP tumor cells are the same as those of A549 cells, and the test results are shown in Table 3.
TABLE 3 inhibition of 5 tumor cells by Compounds IVa-IVd, VIIa-VIIb
As can be seen from Table 3, compound VIIa was shown to be IC against HepG2, A549, hela, MCF-7, A549/DDP tumor cells 50 The inhibition effect of 1.27 mu mol/L, 2.18 mu mol/L, 1.95 mu mol/L, 6.54 mu mol/L and 5.03 mu mol/L on the 5 cells is better than that of the positive control zidovudine, and particularly, the inhibition effect of the compound VIIa on the Hela cells is most prominent and 36 times that of the zidovudine. Compound IVb also inhibited MCF-7 cells 27.6 times more than zidovudine. The inhibition of HepG2 cells by compounds VIIa, VIIb is better than erlotinib and zidovudine, respectively. Wherein, the inhibition effect of the compound VIIb on HepG2 cells is 11.2 times and 7.7 times of that of erlotinib and zidovudine respectively. The inhibition of HepG2 cells by compound VIIa was also 7.0 times and 4.8 times that of erlotinib and zidovudine, respectively.
[ example 4 ]
The 4-aniline quinazoline skeleton compound has obvious inhibition effect on Epidermal Growth Factor (EGFR). In order to further examine the inhibition effect of azidothymidine spliced 4-aniline quinazoline compounds shown in the formula (I) or the formula (II) on EGFR, after the effect A549 cells of the compounds IVa to IVd and VIIa to VIIb are detected by adopting an enzyme-linked immunosorbent assay (ELISA), the concentration of EGFR in supernatant fluid is as follows:
pretreatment: the MTT assay method of example 3 was performed by subjecting compounds IVa to IVd and VIIa to VIIb to respective actions of A549 cells at 0.01. Mu.M, 0.1. Mu.M, 1. Mu.M, 10. Mu.M and 100. Mu.M, and centrifuging at 3000rpm for 10min to obtain supernatants.
After pretreatment, blank holes, standard holes and sample holes to be tested are respectively arranged. Blank Kong Jiabiao standard and sample dilutions 100 μl, yu Kong are added with 100 μl of standard or sample to be tested, respectively. The ELISA plate was covered and incubated at 37℃for 90 minutes. The liquid in the wells was discarded, dried without washing the plate, 100. Mu.L of biotinylated antibody working solution (prepared within 20 minutes before use) was added to each well, and the ELISA plate was covered with the coating film and incubated at 37℃for 1 hour. The liquid was discarded, the plate was washed 3 times, each time soaked for 30s, approximately 350 μl/well, spun dry and the liquid in the well was tapped dry on absorbent paper. 100 μl of enzyme conjugate working solution (prepared within 20 minutes before use) was added to each well, and the mixture was incubated at 37deg.C for 30 minutes. The liquid in the holes is discarded, the plate is dried by spin-drying and washed 5 times. The color reagent (TMB) was added to each well at 90. Mu.L, and the ELISA plate was incubated at 37℃for 15 minutes in the absence of light. The reaction was stopped by adding 50. Mu.L of stop solution to each well, and the blue color turned yellow immediately. The optical density (OD value) of each well was measured immediately with an enzyme-labeled instrument at a wavelength of 450nm
The EGFR concentrations in the supernatants after the compounds IVa-IVd, VIIa-VIIb had been applied to A549 cells at concentrations of 0.01. Mu.M, 0.1. Mu.M, 1. Mu.M, 10. Mu.M, 100. Mu.M, respectively, are shown in Table 4.
TABLE 4 inhibition of EGFR by Compounds IVa-IVd, VIIa-VIIb
As can be seen from the results in Table 4, after the compounds IVa to IVd, VIIa to VIIb act on A549 cells at the concentrations of 0.01. Mu.M, 0.1. Mu.M, 1. Mu.M, 10. Mu.M and 100. Mu.M, respectively, the EGFR concentration in the cell supernatant was lower than that of the normal A549 cells in the group without administration, indicating that the compounds IVa to IVd, VIIa to VIIb had a certain inhibitory effect on EGFR, and the inhibitory effect was enhanced with the increase of the administration concentration. IC for EGFR inhibition by Compounds VIc, VIIa 50 IC with strength also superior to zidovudine, but with EGFR inhibition by all compounds 50 The intensity is far lower than that of erlotinib, which indirectly suggests that the compounds IVa-IVd, VIIa-VIIb are inThe anti-tumor effect is not only achieved by inhibiting EGFR enzyme when the anti-tumor effect is well inhibited on cells, but also can be achieved by other signal paths.
Conclusion of study: the zidovudine spliced 4-aniline quinazoline compound shown in the formula (I) or the formula (II) has obvious inhibition effect on human non-small cell lung cancer cells (A549), human breast cancer cells (MCF-7), human cervical cancer cells (Hela), human liver cancer cells (HepG 2) and human lung adenocarcinoma cisplatin-resistant cells (A549/DDP), and the inhibition effect of partial spliced compounds on tumor cells is obviously improved. The inhibition effect of the compound VIIb on HepG2 cells is 11.2 times and 7.7 times of that of erlotinib and zidovudine, and the inhibition effect of the compounds IVb and VIIa on MCF-7 and Hela cells is 27.6 times and 36 times of that of zidovudine. Through a series of experimental researches on late antitumor mechanisms and the like, the compounds are likely to be developed into antitumor drugs for clinical use. Therefore, the compound has potential of being developed into an anti-tumor drug, has certain economic and clinical values, and deserves to be continuously and intensively studied.

Claims (7)

1. A zidovudine spliced 4-aniline quinazoline compound is characterized in that: the compound has a structure shown as a general formula (I) or a general formula (II):
；
wherein: r is a hydrogen atom;
R 1 is a hydrogen atom or methoxy group substituted on the benzene ring;
R 2 1 to 5 fluorine atoms, chlorine atoms, bromine atoms or iodine atoms substituted on the benzene ring.
2. A method for preparing the zidovudine spliced 4-aniline quinazoline compound as claimed in claim 1, wherein the preparation method of the general formula (I) is as follows:
dissolving a 6-hydroxy-4-aniline quinazoline compound (1) in an organic solvent, reacting to generate an intermediate 2 under the action of propargyl bromide and an acid binding agent, and then reacting with zidovudine (3) under the action of a catalyst to obtain a compound 4 shown in a formula (I), wherein the reaction route is as follows:
；
wherein the preparation method of the general formula (II) is as follows: dissolving a 7-hydroxy-4-aniline quinazoline compound (5) in an organic solvent, reacting to generate an intermediate 6 under the action of propargyl bromide and an acid binding agent, and then reacting with zidovudine (3) under the action of a catalyst to obtain a compound 7 shown in a formula (II), wherein the reaction route is as follows:
；
wherein: r is a hydrogen atom;
R 1 is a hydrogen atom or methoxy group substituted on the benzene ring;
R 2 1 to 5 fluorine atoms, chlorine atoms, bromine atoms or iodine atoms substituted on the benzene ring.
3. The preparation method according to claim 2, characterized in that: the organic solvent is methanol, ethanol, N '-dimethylformamide, N' -dimethylacetamide, 1, 4-dioxane, tetrahydrofuran, dichloromethane, chloroform or toluene.
4. The preparation method according to claim 2, characterized in that: the catalyst is L-sodium ascorbate, copper sulfate and cuprous iodide; the acid binding agent is one or a combination of several of triethylamine, N '-diisopropylethylamine, 1, 8-diazo hetero-double spiro [5.4.0] undec-7-ene, potassium carbonate, potassium hydroxide, sodium bicarbonate or N, N' -diisopropylamine.
5. The preparation method according to claim 2, characterized in that: the reaction temperature is-20-200 ℃ and the reaction time is 0-72 hours.
6. Use of the zidovudine spliced 4-aniline quinazoline compound as claimed in claim 1 in the preparation of anti-tumor drugs.
7. The application of the zidovudine spliced 4-aniline quinazoline compound in preparing antitumor drugs according to claim 6, which is characterized in that: the anti-tumor is non-small cell lung cancer cells, human breast cancer cells, human cervical cancer cells, human liver cancer cells or human lung adenocarcinoma cisplatin-resistant cells.
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Citations (1)

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WO2008002039A1 (en) * 2006-06-28 2008-01-03 Hanmi Pharm. Co., Ltd. Quinazoline derivatives for inhibiting the growth of cancer cell

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Title
梁光平等.新型齐多夫定与4-苯胺喹唑啉骨架拼接产物的合成及体外抗肿瘤活性.《有机化学》.2022,第42卷2793-2805. *

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