CN110590681B - A kind of novel quinazolinone compound and its preparation method and application - Google Patents

Abstract

The invention discloses a novel quinazolinone compound and its preparation method and application. The structure of the compound is shown in formula I; wherein, R ₁ is hydrogen, halogen, C _1-4 alkyl, C _1-4 alkoxy, C _1-4 haloalkyl, C _1-6 amino-substituted alkane R ₂ is hydrogen, C _1-4 alkyl or C _1-4 haloalkyl; R ₃ is hydrogen, halogen, hydroxyl, nitro, C _1-4 alkyl or C _{1- 4} One or more of haloalkyl. The novel quinazolinone compound of the present invention has a novel structure, has a good inhibitory effect on various tumor cells, has strong selectivity with BLM protein, has strong binding ability, can significantly induce DNA damage, and has a strong inhibitory effect on various tumor cells. Normal cells are less toxic and have broad application space in the preparation of antitumor drugs.

Description

Novel quinazoline ketone compound and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicinal chemistry, and particularly relates to a novel quinazolinone compound and a preparation method and application thereof.

Background

Malignant tumors are a large group of diseases that endanger human health, and according to the World Health Organization (WHO), 800 million people die from cancer every year worldwide, while nearly 200 million people die from cancer every year in china. Despite the great progress made in the medical treatment of tumors, it is becoming an indispensable main measure for current clinical treatment. However, the problems of high toxic and side effects, drug resistance and the like are still the main obstacles in clinical tumor drug treatment. The international medical community considers the research and development of new antitumor drugs with new action targets and targeted therapy as a new hope for changing the current situation of tumor therapy and is also the leading direction of the research of the antitumor drugs in the new century.

DNA damage is an inevitable event in the life of every cell, and is the source of genetic mutations and also the source of cancer. In order to maintain the genome of a cell stable and to perform its life activities normally, the cell has evolved a DNA damage repair system to cope with damage caused by damage. Compared with normal cells, the damage repair system in cancer cells has defects generally, and the abnormal proliferation speed of the cancer cells obviously improves the incidence rate of DNA damage, so that the rapid proliferation of the cancer cells is additionally dependent on damage repair channels. Therefore, regulation and intervention in DNA damage repair is considered to be an effective anti-tumor strategy. The key protein in the targeted DNA damage repair pathway is of great significance in developing anticancer drugs.

Bloom's syndrome (BLM) protein is a type of 3' -5' helicase, can ensure smooth repair by a mode of forming a single chain by unwinding a nucleic acid structure, and plays an important role in the process of repairing damage. Recent research shows that the expression of BLM in tumor cells is reduced through a gene silencing technology, the proliferation of the tumor cells can be effectively inhibited, and the sensitivity of cancer cells to DNA damaging agents is improved, which indicates that targeted BLM protein has potential anti-tumor effect. However, the gene silencing technology has great limitation and is difficult to develop patent medicine research. Therefore, the development of small molecule inhibitors that directly target BLM helicase may be a new strategy for the development of anticancer drugs.

The BLM inhibitors reported in the past, on the one hand, are in relatively small amounts; on the other hand, the method has problems of high concentration of intracellular effect, unclear mode of action and the like, and thus has a large research space.

Disclosure of Invention

The invention aims to provide a novel quinazoline ketone compound. The compound disclosed by the invention has a good inhibition effect on various tumor cells, can effectively inhibit the activity of BLM helicase at a lower onset concentration compared with ML216 (the existing BLM inhibitor), induces DNA damage, inhibits cancer cell proliferation, and provides theoretical basis and experimental support for developing a novel anti-cancer drug strategy based on BLM function inhibition.

Another object of the present invention is to provide a method for preparing the novel quinazolinone compound.

Still another object of the present invention is to provide the use of the novel quinazolinone compounds.

The above object of the present invention is achieved by the following scheme:

a novel quinazoline ketone compound has a structure shown in a formula I:

wherein R is₁Is hydrogen, halogen, C_1～4Alkyl radical, C_1～4Alkoxy radical, C_1～4Haloalkyl, C_1～6Amino-substituted alkyl, amino or alkylamino;

R₂is hydrogen, C_1～4Alkyl or C_1～4A haloalkyl group;

R₃is hydrogen, halogen, hydroxy, nitro, C_1～4Alkyl or C_1～4One or more of haloalkyl groups.

Preferably, said R is₁Is hydrogen, fluorine, chlorine, bromine, methyl, ethyl, N-propyl, isopropyl, trifluoromethyl, trifluoroethyl, methoxy, ethoxy, amino or N, N-diethyl-methylamine;

R₂hydrogen, methyl, ethyl, propyl, butyl, trifluoromethyl, trifluoroethyl or chloro-substituted ethyl;

R₃is one or more of hydrogen, fluorine, chlorine, bromine, hydroxyl, nitro, methyl, ethyl, propyl, butyl, trifluoromethyl, trifluoroethyl, methoxy or ethoxy.

Preferably, said R is₁Is hydrogen, methyl, ethyl, amino or N, N-diethyl-methylamine;

R₂hydrogen, methyl, ethyl, propyl, butyl or chlorine substituted ethyl;

R₃is hydrogen, hydroxy, nitro, methyl, ethyl, n-propyl, isopropyl or isobutylOne or more of (a).

Preferably, the structure of the compound is one of the following structures:

the preparation method of the novel quinazoline ketone compound is also within the protection scope of the invention, and comprises the following steps:

s1, 2-amino-4, 5-difluorobenzoic acid is reacted with acetic anhydride to obtain an intermediate d1

S2, intermediate d1 and compound NH₂-R₂Reaction to give intermediate d2

S3, mixing the intermediate d2 with a compound NH₂-CH₂-R₁Reaction to give intermediate d3

S4, mixing the intermediate d3 with a compound

Reacting to obtain a target product shown as a formula I;

preferably, in step S1, the reaction temperature is 80-140 ℃.

Preferably, in step S2, intermediate d1 is reacted with compound NH₂-R₂The reaction molar ratio of (A) to (B) is 1: 5-10; the reaction temperature is 20-100 ℃; the reaction time is 5 min-4 h.

Preferably, in step S3, intermediate d2 is reacted with compound NH₂-CH₂-R₁The reaction molar ratio of (A) to (B) is 1: 5-10; the reaction temperature is 80-120 ℃; the reaction time is 12-48 h.

Preferably, in the step S4, the intermediate d3 is reacted with a compound

The molar ratio of (1: 1.05) - (3.0), the reaction temperature of 50-120 ℃, and the reaction time of 12-48 h.

The invention also protects the application of the novel quinazoline ketone compound, the pharmaceutically acceptable salt, the isomer or the prodrug molecule thereof in preparing the antitumor drugs.

Preferably, the anti-tumor drug is one or more drugs against colon cancer, liver cancer, leukemia, small cell lung cancer, skin cancer, epithelial cell cancer, prostate cancer, non-small cell lung cancer, nasopharyngeal carcinoma, glioblastoma, lymphoma or melanoma.

Preferably, the dosage form of the anti-tumor drug is injection, tablet, pill, capsule, suspension or emulsion.

The invention also protects the application of the novel quinazolinone compound, the pharmaceutically acceptable salt, the isomer or the prodrug molecule thereof in preparing BLM protein inhibitor drugs.

Compared with the prior art, the invention has the following beneficial effects:

the novel quinazolinone compound has a novel structure, has a good inhibition effect on various tumor cells, has strong selectivity with BLM protein and strong binding capacity, can remarkably induce DNA damage, has low toxicity to normal cells, and has a wide application space in preparing antitumor drugs.

Drawings

Fig. 1 is a graph showing the effect of novel quinazolinone derivatives on the inhibition of BLM protein activity according to the present invention.

FIG. 2 is a graph showing the effect of novel quinazolinone derivatives on the growth inhibition of mouse transplantable tumors.

Detailed Description

The present invention is further described in detail below with reference to specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

EXAMPLE 1 preparation of Compound 1f

The preparation process of compound 1f is specifically as follows:

synthesis of S1 and intermediate d1

4.2g (24.3mmol) of 2-amino-4, 5-difluorobenzoic acid was charged into a 100mL reaction flask containing 12mL of acetic anhydride and reacted at 120 ℃ for 1.5 hours. After cooling, a large amount of white solid precipitated. And (3) carrying out reduced pressure rotary evaporation on the reaction liquid, removing most acetic anhydride to obtain a large amount of white solid, carrying out suction filtration, washing with ethanol to obtain a white solid d1, and directly carrying out the next step of reaction.

¹H NMR(400MHz,DMSO)δ8.13(dd,J＝9.9,8.5Hz,1H),7.74(dd,J＝11.1,7.2 Hz,1H),2.40(s,3H).

Synthesis of S2 and intermediate d2

1g of d1(5.07mmol) and 9mL of aqueous ammonia were placed in a 100mL reaction flask and condensed under reflux (a balloon was placed on the condenser tube to prevent escape of ammonia) at 70 ℃ for 4 hours. Suction filtration and washing of the solid with water gave a white solid d2 which was directly taken to the next reaction step.

Synthesis of S3 and intermediate d3

Compound d2(1g, 4.5mmol), N-diethylpropanediamine (1.5ml, 10.7mmol) were taken and reacted in a 15ml thick-walled pressure-resistant flask with stirring at 100 ℃ for 12 hours, after completion of the reaction as monitored by TLC plates, the organic layer was concentrated on a column (solvent ratio DCM: MeOH: 250:1) to give d3 as a yellow solid in 83.7% yield.

S4 Synthesis of Compound 1f

1g of compound d3(3.14mmol) and 0.52mL (3.45mmol) of 4-isopropylbenzaldehyde were put in a pressure-resistant tube, 3mL of DMF and 26mL of TMSCl were added, and the mixture was stirred at 100 ℃ for reaction for 12 to 24 hours. After the reaction is finished, the pH value of the reaction system is adjusted to be alkalescent under ice bath, precipitates are separated out, the reaction system is subjected to suction filtration and drying, and filter residues are mixed with a sample and are passed through a silica gel column to obtain a yellow product. Some reaction systems were adjusted to pH and no precipitate precipitated, and were extracted with DCM and water, and the organic layer was spin-dried and sample-stirred and purified by silica gel column chromatography (solvent ratio DCM: MeOH ═ 500:1 → 200: 1). This gave a pale yellow solid, compound 1f, in 69.5% yield.

¹H NMR(400MHz,CDCl₃)δ11.43(s,1H),7.86(d,J＝16.4Hz,1H),7.78(d,J＝ 11.5Hz,1H),7.58(d,J＝8.1Hz,2H),7.31(d,J＝8.1Hz,2H),6.91(s,1H),6.87(s,1H), 6.78(d,J＝7.7Hz,1H),3.36(dd,J＝10.3,5.6Hz,2H),2.97(dq,J＝13.8,7.0Hz,1H), 2.66–2.60(m,2H),2.56(q,J＝7.1Hz,4H),1.87(dt,J＝11.5,5.8Hz,2H),1.29(d,J＝ 6.9Hz,6H),1.07(t,J＝7.1Hz,6H).¹³C NMR(101MHz,CDCl₃)δ163.26,151.20, 150.81,149.67,148.86,143.99(d,J＝15.2Hz),138.15,133.09,127.83(2C),127.10 (2C),120.71,109.59(d,J＝20.7Hz),108.94(d,J＝12.1Hz),105.67(d,J＝2.7Hz), 52.95,46.90(2C),43.83,34.10,25.03,23.86(2C),11.76(2C).ESI-HRMS[M+H]⁺ m/z＝437.2707,calcd for C₂₆H₃₃N₄OF,437.2711.Purity:99.9％by HPLC.

Referring to the above preparation process of compound 1f, ammonia water in step S2 and N, N-diethylpropanediamine in step S3 were replaced as in table 1, respectively, to prepare compound 2f and compound 9 f.

TABLE 1 structures of Compounds 2f to 9f

The structural identification data for compounds 2f to 9f are shown below:

compound 2 f:

¹H NMR(400MHz,CDCl₃)δ12.27(s,1H),7.98(d,J＝16.5Hz,1H),7.79(d,J＝ 11.5Hz,1H),7.58(d,J＝8.0Hz,2H),7.22(d,J＝8.0Hz,2H),6.94(s,1H),6.90(s,1H), 6.78(d,J＝7.7Hz,1H),3.35(dd,J＝10.3,5.5Hz,2H),2.65–2.59(m,2H),2.59–2.49 (m,6H),1.98–1.82(m,3H),1.07(t,J＝7.1Hz,6H),0.94(d,J＝6.6Hz,6H).¹³C NMR (101MHz,CDCl₃)δ163.35(d,J＝3.5Hz),151.26,150.88(d,J＝244.5Hz),148.90, 143.98(d,J＝14.0Hz),143.72,138.22,133.01,129.74(2C),127.59(2C),120.68, 109.57(d,J＝20.7Hz),108.93(d,J＝7.8Hz),105.69(d,J＝4.1Hz),52.96,46.91(2C), 45.37,43.83,30.23,25.05,22.43(2C),11.77(2C).ESI-HRMS[M+H]⁺m/z＝451.2869, calcd for C₂₇H₃₅N₄OF,451.2868.Purity:100.0％by HPLC.

compound 3 f:

¹H NMR(400MHz,CDCl₃)δ7.81(d,J＝15.4Hz,1H),7.67(d,J＝11.7Hz,1H), 7.47(d,J＝8.1Hz,2H),7.19(s,2H),6.98(d,J＝15.4Hz,1H),6.67(d,J＝7.7Hz,1H), 6.61(s,1H),3.64(s,3H),3.27(q,J＝4.5Hz,2H),2.94–2.79(m,2H),2.54(t,J＝4.9Hz, 2H),2.49(d,J＝6.1Hz,4H),1.80(t,J＝4.4Hz,2H),1.21(d,J＝6.9Hz,6H),1.00(t,J＝ 6.8Hz,6H).¹³C NMR(101MHz,CDCl₃)δ161.59(d,J＝3.5Hz),152.43–151.67(m), 150.85,149.70,146.57,143.48(d,J＝14.1Hz),140.28,133.18,127.84(2C),127.01 (2C),118.49,110.12(d,J＝20.8Hz),108.95(d,J＝7.9Hz),105.35(d,J＝4.2Hz),52.87, 46.88(2C),43.77,34.06,30.50,25.15,23.84(2C),11.74(2C).ESI-HRMS[M+H]⁺ m/z＝451.2862,calcd for C₂₇H₃₅N₄OF,451.2868.Purity:100.0％by HPLC.

compound 4 f:

¹H NMR(400MHz,CDCl₃)δ7.88(d,J＝15.4Hz,1H),7.76(d,J＝11.6Hz,1H), 7.54(d,J＝8.0Hz,2H),7.27(d,J＝5.8Hz,2H),7.04(d,J＝15.4Hz,1H),6.78(d,J＝7.7 Hz,1H),5.35(s,1H),3.70(s,3H),3.28(dd,J＝5.1Hz,2H),3.00–2.88(m,1H),2.79(t,J＝5.7Hz,2H),2.61(dd,J＝14.0,6.9Hz,4H),1.28(d,J＝6.9Hz,6H),1.06(t,J＝7.1Hz, 6H).¹³C NMR(101MHz,CDCl₃)δ161.55(d,J＝3.4Hz),152.14(d,J＝26.2Hz),150.93, 149.59,146.47,143.02(d,J＝13.8Hz),140.41,133.14,127.86(2C),127.02(2C), 118.39,110.37(d,J＝20.9Hz),109.50(d,J＝7.9Hz),106.09(d,J＝3.8Hz),51.00, 46.77(2C),40.19,34.07,30.53,23.83(2C),11.72(2C).ESI-HRMS[M+H]⁺ m/z＝437.2703,calcd for C₂₆H₃₃N₄OF,437.2711.Purity:99.8％by HPLC.

compound 5 f:

¹H NMR(400MHz,CDCl₃)δ8.21(d,J＝15.6Hz,1H),7.75(d,J＝11.6Hz,1H), 7.38(d,J＝8.0Hz,1H),7.09(d,J＝15.6Hz,1H),6.80(d,J＝7.6Hz,1H),6.72(d,J＝8.0 Hz,1H),6.67(s,1H),5.28(s,1H),3.65(s,3H),3.17(dd,J＝4.9Hz,2H),2.82–2.73(m, 1H),2.70(t,J＝5.8Hz,2H),2.56(q,J＝7.0Hz,4H),1.14(d,J＝6.9Hz,6H),1.02(t,J＝ 7.1Hz,6H).¹³C NMR(101MHz,CDCl₃)δ161.57(d,J＝3.0Hz),156.13,153.88, 152.58,150.68(d,J＝244.3Hz),146.12,143.13(d,J＝13.6Hz),137.28,128.42, 120.69,118.52,117.61,114.82,110.36(d,J＝21.0Hz),109.16(d,J＝8.0Hz),105.37(d, J＝3.4Hz),50.90,46.66(2C),40.12,33.91,30.78,23.64(2C),11.63(2C).ESI-HRMS [M+H]⁺m/z＝453.2661,calcd for C₂₃H₃₃N₄O₂F,453.2661.Purity:96.3％by HPLC.

compound 6 f:

¹H NMR(400MHz,CDCl₃)δ7.83(d,J＝15.3Hz,1H),7.65(d,J＝11.7Hz,1H), 7.45(d,J＝8.1Hz,2H),7.19(d,J＝8.1Hz,2H),6.96(d,J＝15.3Hz,1H),6.66(d,J＝7.7 Hz,1H),6.56(s,1H),4.20(q,J＝7.1Hz,2H),3.26(dd,J＝10.4,5.6Hz,2H),2.94–2.80 (m,1H),2.53(t,J＝5.9Hz,2H),2.47(q,J＝7.1Hz,4H),1.83–1.74(m,2H),1.32(t,J＝ 7.1Hz,3H),1.20(d,J＝6.9Hz,6H),0.98(t,J＝7.1Hz,6H).¹³C NMR(101MHz,CDCl₃) δ160.08(d,J＝3.0Hz),150.64,149.84(d,J＝244.2Hz),149.69,145.55,142.37(d,J＝ 14.0Hz),139.38,132.20,126.74(2C),125.94(2C),117.18,108.97(d,J＝20.7Hz), 108.10(d,J＝8.0Hz),104.31(d,J＝4.0Hz),51.67,45.81(2C),42.59,37.35,32.98, 24.05,22.78(2C),13.43,10.60(2C).ESI-HRMS[M+H]⁺m/z＝465.3024,calcd for C₂₈H₃₇N₄OF,465.3024.Purity:96.6％by HPLC.

compound 7 f:

¹H NMR(400MHz,CDCl₃)δ7.93(d,J＝15.1Hz,1H),7.76(d,J＝11.7Hz,1H), 7.54(d,J＝8.0Hz,2H),7.28(d,J＝8.1Hz,2H),7.04(d,J＝15.3Hz,1H),6.83(d,J＝7.3 Hz,1H),4.43(q,J＝2.7Hz,1H),4.29(q,J＝7.1Hz,2H),3.37–3.27(m,2H),2.95(m, 1H),1.41(t,J＝7.1Hz,3H),1.36(t,J＝7.2Hz,3H),1.28(d,J＝6.9Hz,6H).¹³C NMR (101MHz,CDCl₃)δ161.10(d,J＝3.2Hz),151.83(d,J＝17.3Hz),150.88,149.32, 146.42,142.79(d,J＝13.5Hz),140.73,133.21,127.83,127.02,118.08,110.29(d,J＝ 21.0Hz),109.73(d,J＝8.0Hz),105.84(d,J＝3.4Hz),38.51,37.80,34.06,23.83(2C), 14.42(d,J＝5.7Hz).ESI-HRMS[M+H]⁺m/z＝380.2118,calcd for C₂₃H₂₆N₃OF,380.2133. Purity:97.7％by HPLC.

compound 8 f:

¹H NMR(400MHz,CDCl₃)δ7.90(d,J＝15.3Hz,1H),7.73(d,J＝11.7Hz,1H), 7.51(d,J＝8.1Hz,2H),7.27(d,J＝8.5Hz,2H),7.04(d,J＝15.3Hz,1H),6.75(d,J＝7.7 Hz,1H),6.60(s,1H),4.10(d,J＝6.7Hz,2H),3.36(q,J＝4.0Hz,2H),2.95(q,J＝13.9, 6.9Hz,1H),2.61(m,6H),2.23–2.07(m,1H),1.89(s,2H),1.28(d,J＝6.9Hz,6H),1.09 (t,6H),1.00(d,J＝6.7Hz,6H).¹³C NMR(101MHz,CDCl₃)δ160.66(d,J＝3.2Hz), 151.02(d,J＝13.3Hz),149.71,148.65,145.47,142.36(d,J＝14.0Hz),139.07,132.31, 126.70(2C),126.01(2C),117.72,109.31(d,J＝20.7Hz),108.12(d,J＝7.8Hz),104.30 (d,J＝3.9Hz),51.49,48.50,45.82(2C),42.41,33.02,28.03,23.98,22.80(2C),19.08 (2C),10.43(2C).ESI-HRMS[M+H]⁺m/z＝493.3337,calcd for C₃₀H₄₁N₄OF,493.3337. Purity:97.0％by HPLC.

compound 9 f:

¹H NMR(400MHz,CDCl₃)δ7.86(d,J＝15.3Hz,1H),7.71(d,J＝11.6Hz,1H), 7.53(d,J＝8.0Hz,2H),7.27(d,J＝8.5Hz,2H),7.16(d,J＝15.3Hz,1H),6.82(s,1H), 6.75(d,J＝7.7Hz,1H),4.52(t,J＝6.6Hz,2H),3.88(t,J＝6.5Hz,2H),3.35(dd,J＝10.5, 5.3Hz,2H),3.01–2.86(m,1H),2.67–2.49(m,6H),1.91–1.81(m,2H),1.28(d,J＝6.9 Hz,6H),1.07(t,J＝7.0Hz,6H).¹³C NMR(101MHz,CDCl₃)δ161.25(d,J＝3.5Hz), 152.69–151.61(m),150.94,149.75,146.61,143.76(d,J＝14.4Hz),140.85,133.11, 127.85(2C),127.04(2C),118.30,110.04(d,J＝20.8Hz),108.70(d,J＝8.0Hz),105.49 (d,J＝4.2Hz),52.80(d,J＝1.2Hz),46.87(2C),45.02,43.70(d,J＝1.2Hz),41.24, 34.07,24.98,23.83(2C),11.62(2C).ESI-HRMS[M+H]⁺m/z＝499.2640,calcd for C₂₈H₃₆N₄OFCl,499.2634.Purity:97.0％by HPLC.

example 2 EMSA verification of helicase Activity experiment

The compound prepared in example 1 was used as a test subject and tested for its ability to inhibit unwinding of BLM.

1. Annealing the double-stranded Biotin formed-DNA with the final concentration of 10nM for 5min at 95 ℃ and slowly cooling to room temperature to form a stable double-helix structure;

2. uniformly mixing the purified BLM protein and compounds with different concentrations in helicase working solution, incubating for 1h at 37 ℃, wherein the final concentration of the protein is 30nM, then mixing the protein compound mixed solution with the DNA solution, and continuously incubating for 1h at 37 ℃ after uniformly mixing;

3. after incubation is finished, adding DNA Loading buffer into the sample to finish enzyme reaction, fully and uniformly mixing, Loading the sample to 8% Native-PAGE, wherein the buffer solution is 0.5 XTB, and carrying out ice bath at 80V for about 3 hours until a bromophenol blue strip approaches the edge of an electrophoresis tank;

4. after electrophoresis, DNA on Native-PAGE is transferred to a nitrocellulose membrane by adopting a Bio-Red wet transfer membrane instrument, the buffer solution is 0.5 XTB, and the 80V ice-bath transfer membrane time is about 30 min;

5. after membrane transfer, the nitrocellulose membrane was crosslinked for about 180s under an ultraviolet crosslinking instrument, followed by blocking, labeling, washing, and staining of the crosslinked DNA as specified in the chemiluminescence kit, and finally photographed by a Tanon-4200SF chemiluminescence instrument and quantified by ImageJ 2.0.

As shown in fig. 1, it can be seen from fig. 1 that most of the quinazolinone compounds prepared in example 1 showed strong BLM unwinding inhibition, especially compounds 4f, 5f, 8f and 9f, with an inhibition rate of over 90%.

Example 3 MTT assay

1. Inoculating HCT116 cells in a logarithmic growth phase to a 96-well cell culture plate, wherein the number of the cells is 5000 per well, and culturing the cells in an incubator containing 5% CO2 for 24 hours;

2. after the cells are completely attached to the wall, discarding the old culture medium, adding culture media containing compounds with different concentrations, and respectively culturing for different times according to the requirements of different experiments;

3. during detection, 20 mu L of MTT solution with the concentration of 2.5mg/mL is added into each hole of cells, and the cells are continuously cultured for 4 hours at 37 ℃;

after MTT incubation, old medium was discarded and 100. mu.L of DMSO was added to each well, at which time the solution in the well was purple. After uniform oscillation, the absorption value of each hole is detected at the wavelength of 570nm by using a multifunctional microplate reader, and the half inhibition concentration IC of the compound on cell proliferation is obtained according to the relationship between the cell survival rate and the dosage₅₀。

The results are shown in Table 2.

Table 2 results of cytotoxicity experiments with Compounds

As can be seen from table 2, the quinazolinone derivatives exhibit strong proliferation inhibitory activity on all three tumor cells, wherein the compound 9f also exhibits strong inhibitory activity on the proliferation of all three tumor cells, and thus a certain correlation exists between the unwinding inhibition, blocking and cytotoxicity of the compound.

EXAMPLE 4 ability of Compound 9f to inhibit tumor growth in nude mouse model of HCT116 transplantable tumors

First, a model was made by inoculating HCT116 cells at a density of 1X 107 cells/100. mu.L in logarithmic growth phase to the axilla of the forelimb of male BALB/C-nu/nu nude mice aged 3-4 weeks, and after feeding for 4 weeks, the tumor volume was increased to about 600mm³And taking out the tumor to prepare for secondary modeling. Cutting the tumor into 5mm³The left and right masses were transplanted again to the axilla of the forelimb of healthy male nude mice weighing 14-17 g. When the size of the tumor grows to 80mm³Left and right, the nude mice were started to be administered in groups. A solvent group, a positive medicine cisplatin 2.5mg/kg group and a 9f 5mg/kg group are arranged, tumor-bearing nude mice are randomly distributed, 5 mice in each group are injected in the abdominal cavity every other day for administration, and the administration lasts for 28 days. The length and width of the mouse tumor during the experiment were recorded before each dose and the tumor size was calculated using the formula length x width 2/2.

As shown in FIG. 2, the compound 9f administered group inhibited tumor growth and exhibited antitumor activity in vivo.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims (7)

1. A quinazolinone compound, characterized in that the structure of the compound is one of the following structures:

2. the method for preparing quinazolinone compounds according to claim 1, comprising the following steps:

s1, 2-amino-4, 5-difluorobenzoic acid is reacted with acetic anhydride to obtain an intermediate d1

S2, intermediate d1 and compound NH₂-R₂Reaction to give intermediate d2

S3, mixing the intermediate d2 with a compound NH₂-CH₂-R₁Reaction to give intermediate d3

S4, mixing the intermediate d3 with a compound

Reacting to obtain a target product shown as a formula I;

3. the method for preparing quinazolinone compounds according to claim 2, wherein the reaction temperature in step S1 is 80-140 ℃;

in step S2, intermediate d1 reacts with compound NH₂-R₂The reaction molar ratio of (A) to (B) is 1: 5-10; reaction temperatureThe temperature is 20-100 ℃; the reaction time is 5 min-4 h;

in step S3, intermediate d2 reacts with compound NH₂-CH₂-R₁The reaction molar ratio of (A) to (B) is 1: 5-10; the reaction temperature is 80-120 ℃; the reaction time is 12-48 h;

in the step S4, the intermediate d3 and the compound

The molar ratio of (1: 1.05) - (3.0), the reaction temperature of 50-120 ℃, and the reaction time of 12-48 h.

4. The quinazolinone compound according to claim 1, and pharmaceutically acceptable salts thereof for use in preparing antitumor drugs.

5. The use of claim 4, wherein the anti-neoplastic agent is an agent against one or more of ovarian cancer, cervical cancer, breast cancer, lung adenocarcinoma, colon cancer, liver cancer, leukemia, small cell lung cancer, skin cancer, epithelial cell cancer, prostate cancer, non-small cell lung cancer, nasopharyngeal cancer, glioblastoma, lymphoma or melanoma.

6. The use as claimed in claim 5, wherein the antitumor drug is in the form of injection, tablet, pill, capsule, suspension or emulsion.

7. The use of a quinazolinone compound according to claim 1, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of BLM protein inhibitors.

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