CN110590681A - A 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 alkyl 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 ₄ haloalkyl groups. The novel quinazolinone compound of the present invention has a novel structure, has a good inhibitory effect on a variety of tumor cells, and has strong selectivity with BLM protein, strong binding ability, and can significantly induce DNA damage. Normal cells are less toxic, and have broad application space in the preparation of antitumor drugs.

Description

A novel quinazolinone compound and its preparation method and application

technical field

The present invention relates to the technical field of medicinal chemistry, and more specifically, relates to a novel quinazolinone compound and its preparation method and application.

Background technique

Malignant tumors are a large class of diseases that endanger human health. According to the World Health Organization (WHO), 8 million people die of cancer every year in the world, and nearly 2 million people die of cancer every year in China. Although the drug treatment of tumors has made great progress, and has become an indispensable main measure of current clinical treatment. However, problems such as high toxicity and side effects and drug resistance are still the main obstacles to clinical tumor drug treatment. The international medical community regards the development of innovative anti-tumor drugs with new targets and targeted therapy as a new hope for changing the status quo of cancer treatment, and it is also the leading direction of anti-tumor drug research in the new century.

DNA damage is an inevitable event in the life process of every cell, the source of gene mutation, and the source of cancer. In order to maintain the stability of the cell genome and the normal progress of life activities, cells have evolved a DNA damage repair system to deal with the damage caused by damage. Compared with normal cells, the damage repair system in cancer cells is generally defective, and the abnormal proliferation rate of cancer cells significantly increases the incidence of DNA damage, which makes the rapid proliferation of cancer cells particularly dependent on damage repair pathways. Therefore, regulating and interfering with DNA damage repair is considered to be an effective anti-tumor strategy. It is of great significance to develop anticancer drugs targeting key proteins in the DNA damage repair pathway.

Bloom's syndrome (BLM) protein is a kind of 3'-5' helicase, which can ensure the smooth progress of repair by unwinding the nucleic acid structure to form a single strand, and plays an important role in the damage repair process. Recent studies have found that reducing the expression of BLM in tumor cells through gene silencing technology can effectively inhibit tumor cell proliferation and increase the sensitivity of cancer cells to DNA damaging agents, which indicates that targeting BLM protein has potential anti-tumor effects. However, gene silencing technology has great limitations, and it is difficult to carry out druggable research. Therefore, the development of small molecule inhibitors directly targeting BLM helicase may be a new strategy for anticancer drug development.

The currently reported BLM inhibitors, on the one hand, are small in number; on the other hand, they have problems such as high intracellular onset concentration and unclear mode of action, so there is still a lot of room for research.

Contents of the invention

The object of the present invention is to provide a kind of novel quinazolinone compound. The compound of the present invention has a good inhibitory effect on a variety of tumor cells. Compared with ML216 (an existing BLM inhibitor), it can effectively inhibit the activity of BLM helicase at a lower effective concentration and induce DNA damage. And inhibit the proliferation of cancer cells, providing theoretical basis and experimental support for the strategy of developing new anticancer drugs based on the inhibition of BLM function.

Another object of the present invention is to provide a preparation method of the novel quinazolinone compounds.

Another object of the present invention is to provide applications of the novel quinazolinone compounds.

Above-mentioned purpose of the present invention is achieved by following scheme:

A novel quinazolinone compound, the structure of the compound is as 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 alkyl, amino or alkylamino;

R ₂ is hydrogen, C _1-4 alkyl or C _1-4 haloalkyl;

R ₃ is one or more of hydrogen, halogen, hydroxyl, nitro, C _1-4 alkyl or C _1-4 haloalkyl.

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

R is _hydrogen , methyl, ethyl, propyl, butyl, trifluoromethyl, trifluoroethyl or chloro-substituted ethyl;

_R is one of hydrogen, fluorine, chlorine, bromine, hydroxyl, nitro, methyl, ethyl, propyl, butyl, trifluoromethyl, trifluoroethyl, methoxy or ethoxy, or Various.

Preferably, the R ₁ is hydrogen, methyl, ethyl, amine or N,N-diethyl-methylamine;

R ₂ is hydrogen, methyl, ethyl, propyl, butyl or chlorine-substituted ethyl;

R ₃ is one or more of hydrogen, hydroxyl, nitro, methyl, ethyl, n-propyl, isopropyl or isobutyl.

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

The preparation method of described novel quinazolinone compounds is also within the protection scope of the present invention, comprising the following steps:

S1.2-Amino-4,5-difluorobenzoic acid reacts with acetic anhydride to obtain intermediate d1

S2. Reaction of intermediate d1 with compound NH ₂ -R ₂ to obtain intermediate d2

S3. Reaction of intermediate d2 with compound NH ₂ -CH ₂ -R ₁ to obtain intermediate d3

S4. Combining intermediate d3 with compound reaction, the target product as shown in formula I can be obtained;

Preferably, in step S1, the reaction temperature is 80-140°C.

Preferably, in step S2, the reaction molar ratio of the intermediate d1 to the compound NH ₂ -R ₂ is 1:5-10; the reaction temperature is 20-100° C.; the reaction time is 5 min-4 h.

Preferably, in step S3, the reaction molar ratio of the intermediate d2 to the compound NH ₂ -CH ₂ -R ₁ is 1:5-10; the reaction temperature is 80-120°C; the reaction time is 12h-48h.

Preferably, in the step S4, the intermediate d3 and the compound The molar ratio is 1:1.05~3.0, the reaction temperature is 50~120°C, and the reaction time is 12h~48h.

At the same time, the present invention also protects the application of the novel quinazolinone compound, its pharmaceutically acceptable salt, isomer or prodrug molecule in the preparation of antitumor drugs.

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

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

At the same time, the present invention also protects the application of the novel quinazolinone compound, its pharmaceutically acceptable salt, isomer or prodrug molecule in the preparation of BLM protein inhibitor drugs.

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

The novel quinazolinone compound of the present invention has a novel structure, has a good inhibitory effect on a variety of tumor cells, and has strong selectivity with BLM protein, strong binding ability, and can significantly induce DNA damage. Normal cells are less toxic, and have broad application space in the preparation of antitumor drugs.

Description of drawings

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

Fig. 2 is a graph showing the effect of novel quinazolinone derivatives provided by the present invention on the growth inhibition of transplanted tumors in mice.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, which are only used to explain the present invention, and are not intended to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

The preparation of embodiment 1 compound 1f

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

S1, synthesis of intermediate d1

Add 4.2g (24.3mmol) of 2-amino-4,5-difluorobenzoic acid into a 100mL reaction flask filled with 12mL of acetic anhydride, and react at 120°C for 1.5 hours. After cooling, a large amount of white solid precipitated out. The reaction liquid was rotary-evaporated under reduced pressure. After removing most of the acetic anhydride, a large amount of white solid appeared, which was filtered by suction and washed with ethanol to obtain the white solid d1, which was directly put into the next reaction.

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

S2, synthesis of intermediate d2

Take 1g of d1 (5.07mmol) and 9mL ammonia water in a 100mL reaction flask, condense and reflux at 70°C (put a balloon on the condenser tube to prevent ammonia gas from escaping) and react for 4 hours. Filtrate with suction, wash the solid with water to obtain a white solid d2, which is directly used for the next reaction.

S3, synthesis of intermediate d3

Take compound d2 (1g, 4.5mmol), N,N-diethylpropylenediamine (1.5ml, 10.7mmol), put it in a 15ml thick-walled pressure-resistant bottle, stir and react at 100°C for 12 hours, TLC spot plate monitoring found that After the reaction was complete, after extraction with dichloromethane and water, the organic layer was concentrated through a column (solvent ratio: DCM:MeOH=250:1) to obtain a yellow solid d3 with a yield of 83.7%.

S4, the synthesis of compound 1f

Take 1g of compound d3 (3.14mmol) and 0.52mL (3.45mmol) of 4-isopropylbenzaldehyde in a pressure tube, add 3mL of DMF and 26mL of TMSCl, and stir the reaction at 100°C for 12-24 hours. After the reaction was completed, the pH of the reaction system was adjusted to weak alkalinity in an ice bath. Precipitation was precipitated, filtered and dried with suction, and the filter residue was mixed and passed through a silica gel column to obtain a yellow product. Some reaction systems have no precipitation after adjusting the pH, then extract with DCM and water, spin the organic layer to dry and mix the sample, and then purify by silica gel column chromatography (the solvent ratio is DCM:MeOH=500:1→200:1). A light yellow solid was obtained, namely compound 1f, with a yield of 69.5%.

¹ H NMR (400MHz, CDCl ₃ ) 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, 5.8 Hz, 2H), 1.29(d, J=6.9Hz, 6H), 1.07(t, J=7.1Hz, 6H). ¹³ C NMR(101MHz, CDCl ₃ ) 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 C ₂₆ H ₃₃ N ₄ OF, 437.2711. Purity: 99.9% by HPLC.

Referring to the preparation process of the above-mentioned compound 1f, the ammonia water in step S2 and the N,N-diethylpropylenediamine in step S3 were respectively replaced according to Table 1 to prepare compound 2f and compound 9f.

Structures of compounds 2f to 9f in table 1

The structural identification data of compounds 2f to 9f are as follows:

Compound 2f:

¹ H NMR (400MHz, CDCl ₃ ) 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). ¹³ C NMR (101MHz, CDCl ₃ ) 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 C ₂₇ H ₃₅ N ₄ OF, 451.2868. Purity: 100.0% by HPLC.

Compound 3f:

¹ H NMR (400MHz, CDCl ₃ ) 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). ¹³ C NMR (101MHz, CDCl ₃ ) 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=7.9 Hz), 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 C ₂₇ H ₃₅ N ₄ OF, 451.2868. Purity: 100.0% by HPLC.

Compound 4f:

¹ H NMR (400MHz, CDCl ₃ ) 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=7.7Hz,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). ¹³ C NMR (101MHz, CDCl ₃ ) 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 C ₂₆ H ₃₃ N ₄ OF, 437.2711. Purity: 99.8% by HPLC.

Compound 5f:

¹ H NMR (400MHz, CDCl ₃ ) 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=8.0Hz,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). ¹³ CNMR (101MHz, CDCl ₃ ) 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=8.0 Hz), 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 C ₂₃ H ₃₃ N ₄ O ₂ F, 453.2661. Purity: 96.3% by HPLC.

Compound 6f:

¹ H NMR (400MHz, CDCl ₃ ) 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=7.7Hz,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). ¹³ C NMR (101MHz, CDCl ₃ )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 C ₂₈ H ₃₇ N ₄ OF, 465.3024. Purity: 96.6% by HPLC.

Compound 7f:

¹ H NMR (400MHz, CDCl ₃ ) 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=7.3Hz,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). ¹³ C NMR (101MHz, CDCl ₃ ) 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 C ₂₃ H ₂₆ N ₃ OF, 380.2133. Purity: 97.7% by HPLC .

Compound 8f:

¹ H NMR (400MHz, CDCl ₃ ) 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=7.7Hz,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). ¹³ C NMR(101MHz,CDCl ₃ )δ160.66(d,J=3.2 Hz), 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 C ₃₀ H ₄₁ N ₄ OF, 493.3337. Purity: 97.0% by HPLC.

Compound 9f:

¹ H NMR (400MHz, CDCl ₃ ) 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=6.9Hz,6H),1.07(t,J=7.0Hz,6H). ¹³ C NMR(101MHz,CDCl ₃ )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 experiment of helicase activity

The compound prepared in Example 1 was used as the test object to test its ability to inhibit the unwinding of BLM.

1. Anneal double-stranded Biotin forked-DNA with a final concentration of 10 nM at 95°C for 5 minutes and slowly cool down to room temperature to form a stable double helix structure;

2. Mix the purified BLM protein with different concentrations of compounds in the helicase working solution, and incubate at 37°C for 1 hour. The final concentration of the protein is 30nM, then mix the protein compound mixed solution with the DNA solution, and mix After homogenization, continue to incubate at 37°C for 1 hour;

3. After the incubation, add DNA Loading buffer to the sample to end the enzyme reaction. After mixing well, load the sample to 8% Native-PAGE with a buffer of 0.5×TB, 80V ice bath for about 3 hours, until bromine The phenol blue band is close to the edge of the electrophoresis tank;

4. After electrophoresis, transfer the DNA on Native-PAGE to a nitrocellulose membrane using a Bio-Red wet transfer apparatus, the buffer solution is 0.5×TB, and the transfer time is about 30 minutes in an 80V ice bath;

5. After transferring the membrane, place the nitrocellulose membrane under the UV crosslinker for about 180s, then block, label, wash and stain the crosslinked DNA according to the instructions of the chemiluminescence kit, and finally pass it through Tanon-4200SF A chemiluminescence instrument was used to take pictures, and the development results were quantified by ImageJ 2.0.

The measured results are shown in Figure 1, and it can be seen from Figure 1 that most of the quinazolinone compounds prepared in Example 1 have a strong ability to inhibit the unwinding of BLM, especially compounds 4f, 5f, 8f, 9f, the inhibition rate exceeds 90%.

Embodiment 3 MTT experiment

1. Inoculate the HCT116 cells in the logarithmic growth phase in a 96-well cell culture plate, the number of cells is 5000/well, and place them in a 5% CO2 incubator for 24 hours;

2. After the cells are completely adhered to the wall, discard the old medium, add medium containing different concentrations of compounds, and culture for different times according to the requirements of different experiments;

3. When testing, add 20 μL of MTT solution with a concentration of 2.5 mg/mL to each well of cells, and continue to incubate at 37 ° C for 4 h;

4. After MTT incubation, discard the old culture medium, add 100 μL of DMSO to each well, and the solution in the well is purple at this time. After oscillating evenly, use a multifunctional microplate reader to detect the absorbance of each well at a wavelength of 570nm, and calculate the half inhibitory concentration IC ₅₀ of the compound on cell proliferation according to the relationship between the cell survival rate and the dose.

The measured results are shown in Table 2.

Figure 2 Cytotoxicity test results of compounds

It can be seen from Table 2 that this kind of quinazolinone derivatives showed strong growth inhibitory activity on these three tumor cell lines, and compound 9f also showed strong inhibition on the proliferation of these three tumor cell lines. It can be seen that there is a certain correlation between the unwinding inhibition, blockade and cytotoxicity of the compound.

Example 4 Compound 9f inhibits the ability of tumor growth on the HCT116 xenograft nude mouse model

Firstly, a modeling was carried out, and HCT116 cells in the logarithmic growth phase with a density of 1×107 cells/100 μL were inoculated into the armpit of the forelimb of male BALB/C-nu/nu nude mice aged 3-4 weeks, and fed with After 4 weeks, the volume of the tumor increased to about 600mm ³ , and the tumor was removed for secondary modeling. The tumor was cut into a mass of about 5 mm ³ , and transplanted again into the armpit of the forelimb of a healthy male nude mouse weighing 14-17 g. After the tumor size grew to about 80 mm ³ , the nude mice were divided into groups for administration. A solvent group, a positive drug cisplatin 2.5 mg/kg group, and a 9f 5 mg/kg group were set up, and tumor-bearing nude mice were randomly assigned, 5 in each group, and administered intraperitoneally every other day for 28 days. The length and width data of the mouse tumors during the experiment were recorded before each administration, and the tumor size was calculated using the formula length × width 2/2.

The measured results are shown in Figure 2, the compound 9f administration group can inhibit the growth of tumor and has anti-tumor activity in vivo.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the scope of the present invention. For those of ordinary skill in the art, on the basis of the above descriptions and ideas, they can also make There is no need to and cannot exhaustively list all the implementation manners for other changes or changes in different forms. All modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the claims of the present invention.

Claims (10)

1. A novel quinazoline ketone compound is characterized in that the structure of the compound is shown as 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.

2. The novel quinazolinone compound of claim 1, wherein 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.

3. The novel quinazolinone compound of claim 2, wherein R is₁Is hydrogen, methyl, ethyl, amino or N, N-diethyl-methylamine;

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

R₃is one or more of hydrogen, hydroxyl, nitro, methyl, ethyl, n-propyl, isopropyl or isobutyl.

4. The novel quinazolinone compound of claim 3, wherein said compound has the structure of one of the following:

5. the process for preparing a novel quinazolinone compound according to any one of claims 1 to 4, comprising the steps of:

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 compoundReacting to obtain the compound shown in the formula IA target product;

6. the method for preparing a novel quinazolinone compound according to claim 5, 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; the reaction 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 compoundThe molar ratio of (1: 1.05) - (3.0), the reaction temperature of 50-120 ℃, and the reaction time of 12-48 h.

7. Use of the novel quinazolinone compound according to any one of claims 1 to 4, the pharmaceutically acceptable salts, isomers or prodrug molecules thereof for the preparation of antitumor drugs.

8. The use of claim 7, 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.

9. The use as claimed in claim 8, wherein the antitumor drug is in the form of injection, tablet, pill, capsule, suspension or emulsion.

10. Use of the novel quinazolinone compound according to any one of claims 1 to 4, the pharmaceutically acceptable salt, isomer or prodrug molecule thereof for the manufacture of a medicament for the treatment of BLM protein inhibitors.