1h-benzo[e]indol-2(3h)-one spiro derivative, synthetic method and application thereof

Abstract

The present application belongs to the field of pharmaceutical chemistry, and particularly relates to a 1H-benzo[e]indol-2(3H)-ketone spiro derivative, a synthetic method and application thereof. The present application constructs a class of spiro derivatives with a novel spiro skeleton based on 1H-benzo[e]indol-2(3H)-ketone, and provides a green synthesis method for the spiro derivatives. And through in vitro and in vivo anti-tumor activity research, a plurality of compounds with anti-tumor activity are successfully screened, wherein the best compound can inhibit the colony formation, cell migration and invasion of ovarian cancer cells in a concentration-dependent manner, and it is further found that in the ovarian cancer cells, the best compound can induce cell apoptosis and mediate the expression of cell apoptosis-related proteins, and can also significantly induce cell pyroptosis and up-regulate the expression of cell pyroptosis-related proteins GSDME-N and Caspase-3, and is a promising pyroptosis inducer with anti-tumor activity.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to a 1H-benzo[e]indole-2(3H)-one spirocyclic derivative, its synthesis method, and its application. Background Technology

[0002] Cancer remains a serious threat to public health, and drug therapy is one of the most important tools in oncology. Due to the significant heterogeneity and complex pathogenesis of tumors, finding new methods to induce tumor cell death and developing new treatments has always been a top priority. Pyroptosis, also known as inflammatory cell necrosis, is a novel type of programmed cell death mediated by the Gasdermin family. Initial research on pyroptosis primarily focused on inducing inflammation-related processes. However, recent studies have found that inducing tumor cell pyroptosis holds promise as a new approach to cancer treatment. Nevertheless, research and clinical translation of small molecule drugs for inducing pyroptosis are still in their early stages, thus there is an urgent need to discover highly effective, low-toxicity small molecule drugs with the potential to induce tumor pyroptosis.

[0003] Hydroxyindole is an important drug skeleton, widely found in natural products and synthetic drugs. Over the past few decades, hydroxyindole has received considerable attention as the active pharmacophore of antitumor drugs. Although hydroxyindole-based drugs such as sunitinib have achieved some clinical success, modification of the hydroxyindole active skeleton remains a promising research direction for developing novel, highly effective, and low-toxicity antitumor drugs. Among these, 1H-Benzo[e]indole-2(3H)-one (a structural skeleton with one more benzene ring than hydroxyindole) has been discovered to possess antibacterial and antiviral activities, and in some aspects, even better activity than hydroxyindole. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the existing technology and to provide a 1H-benzo[e]indole-2(3H)-one spirocyclic derivative, its synthesis method and its application.

[0005] In a first aspect, the present invention provides a 1H-benzo[e]indole-2(3H)-ketone spirocyclic derivative, the chemical formula of which is shown in general formula (I):

[0006]

[0007] R1 is H, or a meta-substituted halogen or alkyl group.

[0008] Preferably, the chemical formula of the 1H-benzo[e]indole-2(3H)-ketone spirocyclic derivative is any one of the following general formulas (II)-(VII):

[0009]

[0010] Preferably, the chemical formula of the 1H-benzo[e]indole-2(3H)-one spirocyclic derivative is shown in general formula (V) or (VII).

[0011] Preferably, R1 is a meta-substituted halogen.

[0012] Preferably, the chemical formula of the 1H-benzo[e]indole-2(3H)-one spirocyclic derivative is shown in general formula (VI).

[0013] A second aspect of the present invention provides a method for synthesizing the 1H-benzo[e]indole-2(3H)-ketone spirocyclic derivative as described above, the synthetic reaction formula of which is as follows:

[0014]

[0015] Preferably, the solvent used in the reaction is water with added surfactant.

[0016] Preferably, the solvent used in the reaction is 10wt% SDS / H2O, and the reaction temperature is 80℃.

[0017] A third aspect of the present invention provides the use of 1H-benzo[e]indole-2(3H)-one spirocyclic derivatives or pharmaceutical salts thereof represented by the above general formula (V) or (VII) for the preparation of antitumor drugs.

[0018] In a fourth aspect, the present invention provides an antitumor drug comprising a 1H-benzo[e]indole-2(3H)-one spirocyclic derivative or a pharmaceutical salt thereof as shown in the above general formula (V) or (VII).

[0019] This invention constructs a class of spirocyclic derivatives with novel spirocyclic skeletons based on 1H-benzo[e]indole-2(3H)-one, and provides a green synthetic method for these spirocyclic derivatives. Furthermore, through in vitro and in vivo antitumor activity studies, a variety of compounds with antitumor activity were successfully screened. Among them, the optimal compound inhibited colony formation, cell migration, and invasion of ovarian cancer cells in a concentration-dependent manner. Further, it was found that in ovarian cancer cells, this optimal compound, in addition to inducing apoptosis and mediating the expression of apoptosis-related proteins, also significantly induced pyroptosis and upregulated the expression of pyroptosis-related proteins GSDME-N and Caspase-3, making it a promising pyroptosis inducer with antitumor activity. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0021] Figure 1 The single-crystal X-ray structure of compound A1 is shown (CCDC 2266098).

[0022] Figure 2 To demonstrate the growth inhibition of CP70 cells (A) and AGS cells (B) by the compound (10 μM), the positive control was used with 5-FU and oxaliplatin. The drugs were incubated for 72 hours, and the final assay was performed using the MTT assay.

[0023] Figure 3 To obtain the results of quantitative structure-activity relationship analysis, the activity data of the compound in CP70 cells (A) and AGS cells (B) were obtained using the random forest method;

[0024] Figure 4 Results of experiments on the inhibition of CP70 cell growth, migration, and invasion by the active compound A9: (AB) CP70 cells were incubated with A9 and oxaliplatin for 24 hours, then digested and diffused into Transwell chambers. After 72 hours, the cells were stained with crystal violet and photographed using an inverted fluorescence microscope. CP70 cells were incubated with different concentrations of A9. When visible colonies formed in the negative control wells, crystal violet staining and photographing were performed. (CD) The effect of A9 on CP70 cell migration was shown by photographing cells incubated with A9 and oxaliplatin at 0 hours and 72 hours using an inverted fluorescence microscope, along with the analysis results. (EF) The effect of A9 on CP70 cell invasion. *P<0.05, **P<0.01, and ****P<0.0001.

[0025] Figure 5 The effect of compound A9 on apoptosis in CP70 cells: (A) The effect of the drug on apoptosis was detected by flow cytometry after 30 hours of incubation; (BD) The expression of apoptosis-related proteins was detected by Western blotting after 72 hours of incubation with A9. Among them, the expression of Cleaved-PARP and BAX increased, while the expression level of Bcl-2 protein decreased. *P<0.05,***P<0.001, and****P<0.0001;

[0026] Figure 6Results of experiments on A9-induced pyroptosis in CP70 and A2780 cells: (A) Morphological changes of CP70 cells observed under an inverted microscope after 24 hours of incubation with different concentrations of A9 and oxaliplatin; (B, E) Expression of pyroptosis-related proteins after 72 hours of incubation with A9 via Western blotting, showing increased expression of GSDME-N and decreased expression of total GSDME; (C) Morphological changes of A2780 cells observed under an inverted microscope after 46 hours of incubation with different concentrations of A9 and oxaliplatin; (D, F) Expression of pyroptosis-related proteins after 48 hours of incubation with A9 via Western blotting, showing increased expression of GSDME-N and decreased expression of total GSDME. *P<0.05, and **P<0.01;

[0027] Figure 7 The in vivo anti-ovarian cancer activity of compound A9: (A, B) refers to the reaction of A2780 cells (10 7 (a 100 μL injection) was administered subcutaneously to the right axilla of nude mice. After tumor formation, the mice were treated for 18 days. During this period, the tumors of the nude mice were photographed and their volume was measured every other day. The tumor volume of the nude mice in the A9 (5 mg / kg / d) group was significantly smaller than that in the solvent group. (C) The weight of the tumors removed on the day of sacrifice. (D) The weight changes of the solvent group and the A9 (5 mg / kg / d) group over 18 days. No significant changes in weight were observed. (E) The expression of pyroptosis-related proteins in A9 nude mice after intraperitoneal injection for 18 days was detected by Western blotting. Among them, the expression of GSDME-N increased and the expression level of total GSDME protein decreased. (F) The quantitative analysis of the expression of total GSDME protein, GSDME-N and Caspase3 protein. *P<0.05. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0029] Example 1 Synthesis of 1H-benzo[e]indole-2(3H)-ketospirocyclic derivative:

[0030] (1) Add 1 mmol of indigo, 1 mmol of 2-naphthylamine, and 1,3-dicarbonyl compound (1 mmol) to 2 mL of solvent, and react according to the conditions in Table 1. Monitor the reaction by TLC. After the reaction is complete, cool the reaction mixture to room temperature, filter the mixture, and wash the precipitate with hot water. Recrystallize from ethanol and dry to obtain the target product. The yields are shown in Table 1.

[0031] Table 1. Optimization of reaction solvents, temperatures, and other conditions for the synthesis of compound A1.

[0032]

[0033]

[0034] The study began with a catalyst-free reaction of indigo, 2-naphthylamine, and 2-thiobarbituric acid. Table 1 shows that the desired product A1 was only 20% yielded in water. The use of the cationic surfactant sodium dodecyl sulfate (SDS) in the reaction improved the yield of A1. Other surfactants were then applied, and moderate yields were observed when using 10 wt.% Triton X-100 / H2O, 10 wt.% TBAB / H2O, 10 wt.% TEBAC / H2O, and 10 wt.% CTMAB / H2O as solvents. The optimal choice was to react at 80°C with 10 wt% SDS / H2O as the solvent. To confirm the structure of the obtained compound, the single-crystal X-ray structure of compound A1 was also obtained. Figure 1 )

[0035] (2) 1 mmol of indigo, 1 mmol of 2-naphthylamine, and 1,3-dicarbonyl compound (1 mmol) were added to a 10 wt.% SDS / H2O (2 mL) solution, and the reaction was carried out at 80 °C. The reaction was monitored by TLC. After the reaction was complete, the reaction mixture was cooled to room temperature, filtered, and the precipitate was washed with hot water. The target product was obtained by recrystallization from ethanol and drying. The structural formulas of all synthesized compounds A1-A21 are shown in Table 2.

[0036]

[0037] Table 2. Structures of 1H-benzo[e]indole-2(3H)-ketone spirocyclic derivatives A1-A21

[0038]

[0039]

[0040] The chemical data for all the 1H-Benzo[e]indole-2(3H)-one spirocyclic derivatives synthesized above are as follows: 2'-thioxo-2',3'-dihydro-1'H-spiro[benzo[e]indole-1,5'-pyrimido[4,5-b]quinoline]-2,4'(3H,10'H)-dione(Al)

[0041] White solid,mp>300℃,yield 91%,362.253mg. 1H NMR(400MHz,

[0042] DMSO-d6)δ=11.96-12.04(m,2H),10.69(s,1H),9.12(s,1H),7.84(d,J=8Hz,1H),7.38-7.39(m,1H),7.27-7.29(m,2H),7.21-7.22(m,1H),7.08-7.14(m,2H),6.78(s,2H),6.58-6.60(m,1H)ppm. 13 C NMR(100MHz,DMSO-d6)δ=180.8,173.8,159.0,145.6,139.6,134.2,129.9,129.6,129.2,128.6,128.5,128.4,127.1,126.5,123.9,122.9,121.4,120.6,116.9,111.7,88.6,51.0 ppm.HRMS(ESI)[M+H]-Calcd for C 22 H 13 N4O2S397.0765,found 397.0769.

[0043] 1'H-spiro[benzo[e]indole-1,5'-pyrimido[4,5-b]quinoline]-2,2',4'(3H,3'H,10'H)-trione(A2)

[0044] White solid,m.p.>300℃,yield 90%,343.899 mg. 1 H NMR(400 MHz,

[0045] DMSO-d6)δ=10.55-10.61(m,3H),9.32(s,1H),7.82(s,2H),7.40(s,1H),7.10-7.27(m,5H),6.75(s,1H),6.59(s,1H)ppm. 13 C NMR(100 MHz,DMSO-d6)δ=181.4,161.8,150.0,146.0,139.4,134.9,129.9,129.5,129.4,128.9,128.5,128.3,126.9,126.5,123.5,122.8,121.7,120.6,116.5,111.7,84.3,51.2 ppm.HRMS(ESI)[M+H] - Calcd forC22 H 13 N4O3 381.0993,found 381.0991.

[0046] 1',3'-dimethyl-1'H-spiro[benzo[e]indole-1,5'-pyrimido[4,5-b]quinoline]-2,2',4'(3H,3'H,10'H)-trione(A3)

[0047] White solid,m.p.>300℃,yield 80%,328.112 mg. 1 H NMR(400 MHz,

[0048] DMSO-d6)δ=10.63(s,1H),9.53(s,1H),7.81(s,2H),6.99-7.55(m,6H),6.77(s,1H),6.60(s,1 H),3.62(s,3H),2.50(s,3H)ppm. 13 C NMR(100 MHz,DMSO-d6)δ=181.5,159.6,150.5,146.0,139.4,135.3,129.9,129.6,129.4,128.9,128.4,128.2,126.9,126.2,123.9,122.7,121.5,120.9,117.1,111.7,84.9,52.1,30.3,27.4 ppm.HRMS(ESI)[M+H] - Calcd for C 24 H 17 N4O3 409.1306,found 409.1302.

[0049] 3,4-dihydro-2H-spiro[acridine-9,1'-benzo[e]indole]-1,2'(3'H,10H)-dione(A4)

[0050] White solid,m.p.>300℃,yield 84%,307.558 mg.1H NMR(400 MHz,DMSO-d6)δ=10.66(s,1H),9.83(s,1H),7.74(s,3H),6.99-7.34(m,4H),6.75-6.85(m,3H),2.56-2.80(m,2H),2.00-2.27(m,2H),1.63-1.95(m,2H)ppm.13 C NMR(100 MHz,DMSO-d6)δ=192.3,180.9,152.4,142.2,137.1,134.5,131.9,130.8,129.8,129.1,127.5,126.7,123.9,123.4,122.6,121.6,117.6,114.4,108.6,108.2,52.2,37.7,27.6,20.6 ppm.HRMS(ESI)[M+H] - Calcd for C 24 H 17 N2O2 365.1296,found 365.1297.

[0051] 3,3-dimethyl-3,4-dihydro-2H-spiro[acridine-9,1'-benzo[e]indole]-1,2'(3'H,10H)-dione(A5)

[0052] White solid,m.p.>300℃,yield 83%,327.161 mg. 1 H NMR(400 MHz,

[0053] DMSO-d6)δ=10.55-10.72(m,1H),9.84-9.95(m,1H),7.83(s,3H),6.65-7.52(m,7H),2.58(s,2 H),1.99-2.18(m,2H),1.03-1.12(m,6H)ppm. 13 C NMR(100 MHz,DMSO-d6)δ=192.0,180.9,150.6,142.1,136.9,134.6,132.0,130.8,129.9,129.1,127.6,126.7,123.8,123.5,122.7,121.6,117.6,114.4,108.7,106.8,52.2,51.2,40.8,31.8,28.5,26.6ppm.HRMS(ESI)[M+H] + Calcd for C 26 H 22 N2O2 395.1754,found395.1752.

[0054] 7'-methyl-2'-thioxo-2',3'-dihydro-1'H-spiro[benzo[e]indole-1,5'-pyrimido[4,5-b]quinoline]-2,4'(3H,10'H)-dione(A6)

[0055] White solid,m.p.>300℃,yield 88%,362.648 mg. 1 H NMR(400 MHz,

[0056] DMSO-d6)δ=11.96(s,2H),10.65(s,1H),9.06(s,1H),7.83(d,J=8 Hz,1H),7.19-7.35(m,4 H),6.95-7.00(m,2H),6.36(s,1H),1.97(s,3H)ppm. 13 C NMR(100MHz,DMSO-d6)δ=180.9,173.7,159.0,145.6,139.6,139.6,132.9,131.9,129.9,129.6,129.3,129.2,128.6,128.4,127.1,126.4,122.9,121.2,116.9,111.7,88.4,51.0,20.3 ppm.HRMS(ESI)[M+H] - Calcd for C 23 H 15 N4O3 411.0921,found 411.0922.

[0057] 7'-methyl-1'H-spiro[benzo[e]indole-1,5'-pyrimido[4,5-b]quinoline]-2,2',4'(3H,3'H,10'H)-trione(A7)

[0058] White solid,m.p.>300℃,yield 90%,356.508 mg. 1 H NMR(400MHz,

[0059] DMSO-d6)δ=10.59(s,1 H),10.48-10.52(m,2H),9.24(s,1H),7.80-7.83(m,2H),7.37-7.39(m,1H),7.18-7.30(m,3H),7.00(d,J=8 Hz,1H),6.93(d,J=8 Hz,1H),6.38(s,1H),1.97(s,3H)ppm. 13 C NMR(100 MHz,DMSO-d6)δ=181.4,161.7,150.0,146.0,139.4,132.6,132.3,129.9,129.5,129.2,129.0,128.8,128.4,126.8,126.3,122.7,121.5,120.7,116.5,111.7,84.1,51.1,20.3 ppm.HRMS(ESI)[M+H] - Calcd for C 23 H 15 N4O3395.1150,found 395.1158.

[0060] 7'-chloro-1'H-spiro[benzo[e]indole-1,5'-pyrimido[4,5-b]quinoline]-2,2',4'(3H,3'H,10'H)-trione(A8)

[0061] White solid,m.p.>300℃,yield 88%,366.142 mg. 1 H NMR(400 MHz,

[0062] DMSO-d6)δ=10.68-10.72(m,2H),10.58(s,1H),9.51(s,1H),7.84-7.86(m,2H),7.18-7.37(m,6H),6.46(s,1H)ppm. 13 C NMR(100 MHz,DMSO-d6)δ=180.9,161.6,149.9,145.8,139.3,134.2,129.9,129.6,129.3,128.8,128.4,128.3,127.1,126.7,125.5,123.5,122.9,120.3,118.5,111.7,84.0,51.1 ppm.HRMS(ESI)[M+H] - Calcd forC 22 H12 CIN4O3 415.0603,found 415.0601.

[0063] 9'-chlorospiro[benzo[e]indole-1,7'-chromeno[4,3-b]quinoline]-2,6'(3H,12'H)-dione(A9)

[0064] White solid,m.p.>300℃,yield 82%,369.066 mg. 1 H NMR(400 MHz,

[0065] DMSO-d6)δ=10.87(s,1H),10.43(s,1H),8.35-8.49(m,1H),7.86-7.88(m,2H),7.61-7.74(m,1 H),7.43-7.58(m,2H),7.09-7.35(m,6H),6.57(s,1H)ppm. 13 CNMR(100 MHz,DMSO-d6)δ=181.0,158.9,152.2,144.7,139.4,134.2,132.8,130.0,129.8,129.0,128.8,128.3,127.6,127.5,125.6,124.2,123.4,123.2,123.1,120.2,119.0,117.0,112.5,111.9,94.7,52.1 ppm.HRMS(ESI)[M+H]-Calcd forC 27 H 14 CIN2O3 449.0698,found449.0697.

[0066] 7'-fluoro-1'H-spiro[benzo[e]indole-1,5'-pyrimido[4,5-b]quinoline]-2,2',4'(3H,3'H,10'H)-trione(A10)

[0067] White solid,m.p.>300℃,yield 87%,348.087 mg. 1 H NMR(400 MHz,

[0068] DMSO-d6)δ=10.69-10.72(m,2H),10.59(s,1H),9.45(s,1H),7.83-7.85(m,2H),7.26-7.33(m,2H),7.17-7.24(m,2H),6.99-7.04(m,1H),6.27-6.30(m,1H)ppm. 13 CNMR(100MHz,DMSO-d6)δ=181.0,161.8,159.3,156.9,150.1,146.1,139.4,131.8,130.0,129.6,129.3,128.8,128.4,127.1,123.3,123.0,120.4,118.5,118.4,115.5,112.4,112.1,111.8,83.4,51.5 ppm.HRMS(ESI)[M+H]-Calcd forC 22 H 12 FN4O3 399.0899,found399.0898.

[0069] 7'-fluoro-1',3'-dimethyl-1'H-spiro[benzo[e]indole-1,5'-pyrimido[4,5-b]quinoline]-2,2',4'(3H,3'H,10'H)-trione(A11)

[0070] White solid,m.p.>300℃,yield 80%,342.504 mg. 1 H NMR(400 MHz,

[0071] DMSO-d6)δ=10.71(s,1H),9.62(s,1H),7.83-7.84(m,2H),7.35-7.53(m,2H),7.20-7.35(m,3 H),6.98-7.12(m,1H),6.29-6.31(m,1H),3.61(s,3H),2.97(s,3H)ppm. 13 CNMR(100 MHz,DMSO-d6)δ=181.1,159.6,150.5,146.0,139.4,132.1,130.0,129.5,129.2,129.0,128.4,127.2,123.2,122.9,120.8,119.1,115.8,115.6,112.0,111.8,84.0,52.4,30.3,27.4 ppm.HRMS(ESI)[M+H]+ Calcd forC 24 H 18 FN4O3 429.1357,found 429.1359.

[0072] 7-fluoro-3,4-dihydro-2H-spiro[acridine-9,1'-benzo[e]indole]-1,2'(3'H,10H)-dione(A12)

[0073] White solid,m.p.>300℃,yield 83%,318.828 mg. 1 H NMR(400 MHz,

[0074] DMSO-d6)δ=10.55-10.71(m,1H),9.87-10.01(m,1H),7.78(s,3H),7.23-7.40(m,3H),6.70-6.90(m,3H),2.64(s,2H),2.14(s,2H),1.86(s,2H)ppm. 13 C NMR(100MHz,DMSO-d6)δ=192.4,180.8,152.6,138.7,134.5,131.9,130.8,130.1,129.1,126.8,123.5,122.4,117.7,113.7,111.6,109.1,107.7,52.8,37.6,27.6,20.8 ppm.HRMS(ESI)[M+H]-Calcd for C 24 H 16 FN2O2 383.1201,found 383.1208.

[0075] 7-fluoro-3,3-dimethyl-3,4-dihydro-2H-spiro[acridine-9,1'-benzo[e]indole]-1,2'(3'H,10H)-dione(A13)

[0076] White solid,m.p.>300℃,yield 82%,337.971 mg. 1 H NMR(400 MHz,

[0077] DMSO-d6)δ=10.55-10.73(m,1H),9.78-9.96(m,1H),7.79(s,3H),6.29-7.24(m,6H),6.37-6.88(m,3H),2.50-2.54(s,2 H),1.98-2.10(m,2H),0.97-1.03(m,6H)ppm. 13 CNMR(100 MHz,DMSO-d6)δ=192.0,180.7,157.1,150.8,138.7,138.2,134.7,131.9,130.8,130.1,129.1,126.8,123.5,122.4,117.7,113.7,111.1,109.2,106.3,99.5,52.7,51.1,40.8,31.7,28.3,26.7 ppm.HRMS(ESI)[M+H] + Calcd forC 26 H 22 FN2O2 413.1660,found413.1656.

[0078] 9'-fluorospiro[benzo[e]indole-1,7'-chromeno[4,3-b]quinoline]-2,6'(3H,12'H)-dione(A14)

[0079] White solid,m.p.>300℃,yield 84%,364.652 mg. 1 H NMR(400 MHz,

[0080] DMSO-d6)δ=10.87(s,1H),10.38(s,1H),8.43-8.45(m,1H),7.84-7.90(m,2H),7.65-7.67(m,1 H),7.50-7.51(m,2H),7.33-7.39(m,3H),7.18-7.24(m,2H),7.04-7.11(m,1H),6.38-6.40(m,1H)ppm. 13C NMR(100 MHz,DMSO-d6)δ=180.9,159.7,159.0,157.4,152.2,144.7,139.5,132.7,131.7,130.0,129.6,128.8,128.3,127.3,124.2,123.1,120.2,118.9,117.0,116.2,115.9,112.5,112.2,111.9,93.8,52.3 ppm.HRMS(ESI)[M+H] + Calcd for C 27 H 16 FN2O3 435.1139,found435.1140.

[0081] 7'-ethyl-2'-thioxo-2',3'-dihydro-1'H-spiro[benzo[e]indole-1,5'-pyrimido[4,5-b]quinoline]-2,4'(3H,10'H)-dione9(A15)

[0082] White solid,m.p.>300℃,yield 81%,345.157mg. 1 H NMR(400 MHz,

[0083] DMSO-d6)δ=11.96-12.00(s,2 H),10.69(s,1H),9.07(s,1H),7.83(d,J=8 Hz,2H),7.36-7.38(m,1H),7.26-7.32(m,2H),7.18-7.22(m,1H),6.99-7.04(m,2H),6.41(s,1H),2.23-2.28(m,2H),0.88-0.91(m,3H)ppm. 13 C NMR(100 MHz,DMSO-d6)δ=180.9,173.7,159.0,145.6,139.5,139.2,132.1,129.9,129.6,129.2,128.6,128.4,127.9,127.1,125.3,122.9,121.2,120.6,117.0,111.7,88.4,51.1,27.3,15.2 ppm.HRMS(ESI)[M+H] + Calcd for C 24 H 18 N4O2S 410.1571,found 410.1572.

[0084] 7'-ethyl-1'H-spiro[benzo[e]indole-1,5'-pyrimido[4,5-b]quinoline]-2,2',4'(3H,3'H,10'H)-trione(A16)

[0085] White solid,m.p.>300℃,yield 86%,352.720 mg. 1 H NMR(400 MHz,

[0086] DMSO-d6)δ=10.51-10.61(s,3 H),9.28(s,1H),7.81(t,J=8 Hz,2H),7.38-7.40(m,1H),7.25-7.30(m,2H),7.17-7.21(m,1H),6.97-7.05(m,2H),6.40(s,1H),2.22-2.26(m,2H),0.90(t,J=8 Hz,3H)ppm. 13 C NMR(100 MHz,DMSO-d6)δ=181.5,161.8,150.0,146.0,139.4,138.6,132.9,129.9,129.5,129.3,128.9,128.5,127.7,126.9,125.2,122.8,121.5,120.7,116.6,111.7,84.1,51.2,27.3,15.2 ppm.HRMS(ESI)[M+H] + Calcdfor C 24 H 18 N4O3 410.1571,found 410.1572.

[0087] 7'-ethyl-1',3'-dimethyl-1'H-spiro[benzo[e]indole-1,5'-pyrimido[4,5-b]quinoline]-2,2',4'(3H,3'H,10'H)-trione(A17)

[0088] White solid,m.p.>300℃,yield 82%,359.299 mg. 1 H NMR(400 MHz,

[0089] DMSO-d6)δ=10.63(s,1H),9.50(s,1H),7.80(s,2H),7.18-740(m,5H),7.00(s,1H),6.42(s,1 H),3.59(s,3H),2.96(s,3H),2.26(s,2H),0.91(s,3H)ppm. 13 CNMR(100 MHz,DMSO-d6)δ=181.6,159.5,150.4,145.9,139.4,138.9,133.1,129.9,129.4,128.8,128.4,127.5,126.9,124.8,122.6,121.3,120.9,117.2,111.6,84.8,52.1,30.2,27.3,27.2,15.0ppm.HRMS(ESI)[M+H]-Calcd for C 26 H 21 N4O3437.1619,found 437.1611.

[0090] 7-ethyl-3,4-dihydro-2H-spiro[acridine-9,1'-benzo[e]indole]-1,2'(3'H,10H)-dione(A18)

[0091] White solid,m.p.>300℃,yield 86%,338.986 mg. 1 H NMR(400 MHz,

[0092] DMSO-d6)δ=10.56(s,1H),9.79(s,1H),7.75(s,3H),6.68-7.21(m,6H),2.64(s,2H),2.36(s,2 H),1.80-2.15(m,2H),0.98(s,3H)ppm. 13 C NMR(100 MHz,DMSO-d6)δ=192.3,181.0,152.3,140.0,137.1,136.7,134.5,132.0,130.8,129.8,129.0,126.7,126.5,123.4,122.8,117.6,114.6,108.4,108.3,52.4,37.8,27.6,20.9,15.5ppm.HRMS(ESI)[M+H] + Calcd for C 26 H 22N2O2 410.1571,found 410.1572.9'-ethylspiro[benzo[e]indole-1,7'-chromeno[4,3-b]quinoline]-2,6'(3H,12'H)-dione(A19)

[0093] White solid,m.p.>300℃,yield 84%,373.086mg. 1 H NMR(400MHz,

[0094] DMSO-d6)δ=10.76(s,1H),10.27(s,1H),8.38-8.47(m,1H),7.84(s,2H),7.68(s,1H),7.52(s,1H),7.08-7.36(m,7H),6.49(s,1H),2.29-2.31(m,2H),0.94(s,3H)ppm. 13 CNMR(100MHz,DMSO-d6)δ=181.4,159.0,152.2,144.6,139.5,139.4,132.8,132.5,129.9,129.6,129.4,129.3,128.4,128.0,127.1,125.2,124.1,123.1,122.8,121.4,120.5,117.1,116.9,112.7,111.7,94.5,52.1,27.3,15.1ppm.HRMS(ESI)[M+H] + Calcd forC 29 H 22 N2O3 410.1571,found 410.1572.

[0095] spiro[benzo[e]indole-1,7'-chromeno[4,3-b]quinoline]-2,6'(3H,12'H)-dione(A20)

[0096] White solid,m.p.>300℃,yield 82%,341.218mg. 1 H NMR(400MHz,

[0097] DMSO-d6)δ=10.76(s,1H),10.28(s,1H),8.47-8.49(m,1H),7.77-7.93(m,2H),7.64-7.74(m,1H),7.48-7.79(m,1H),7.30-7.45(m,4H),7.07-7.26(m,3H),6.84(s,1H),6.62-6.72(m,1H)ppm. 13 C NMR(100MHz,DMSO-d6)δ=181.3,158.9,152.2,144.7,139.4,134.9,132.6,129.9,129.6,129.4,129.3,128.5,128.4,127.1,126.5,124.3,124.1,123.1,122.8,121.5,120.4,116.9,112.6,111.7,94.8,52.1ppm.HRMS(ESI)[M+H] - Calcdfor C 27 H 15 N2O3 415.1088,found 415.1080.

[0098] 10'-fluorospiro[benzo[e]indole-1,7'-chromeno[4,3-b]quinoline]-2,6'(3H,12'H)-dione(A21)

[0099] White solid,m.p.>300℃,yield 85%,368.991mg. 1 H NMR(400MHz,

[0100] DMSO-d6)δ=10.84(s,1H),10.39(s,1H),8.38(d,J=8Hz,1H),7.88-7.82(m,3H),7.69-7.65(m,1H),7.53-7.19(m,6H),6.675(d,J=4Hz,2H)ppm. 13C NMR (100MHz, DMSO-d6)δ=181.2,160.3,158.9,152.2,144.6,139.5,132.8,130.0,129.7,129.2,128.7,128.6,128.3, 127.3,124.3,123.0,122.9,120.3,117.7,117.0,112.4,111.8,111.4,111.2,103.5,95.5,51.7ppm.HRMS(ESI)[M+H] - Calcd for C 27 H 15 FN2O3 434.1067, found 435.1133.

[0101] This embodiment optimized the conditions for the green synthesis of this type of spiroheterocyclic skeleton. Ultimately, a one-pot green method using water as a solvent at 80°C was employed to efficiently synthesize the 1'H-spiro[benzo[e]indole-1,4'-quinolin]-2(3H)-one skeleton. Furthermore, under these green synthetic conditions, 21 1H-Benzo[e]indole-2(3H)-one spirocyclic derivatives were synthesized, with all compounds yielding over 80%. Therefore, this synthetic method is green, efficient, atom-economical, and has a good substrate expansion range.

[0102] Example 2: In vitro antitumor activity detection:

[0103] (1) Cell Culture

[0104] Human gastric cancer cells AGS, BGC, and SGC were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Human ovarian cancer cells CP70, A2780, SKOV3, and OVCAR3 were purchased from the Cell Bank of the Treasure Trove of Typical Cultures, Chinese Academy of Sciences. The culture medium for A2780 cells consisted of 10% FBS (Gibco) and 1% penicillin antibiotic (Gibco) in DMEM. The remaining cells were cultured in 10% FBS (Gibco) and 1% penicillin antibiotic (Gibco) in RPMI 1640 medium. Cells were incubated at 37°C in a 5% CO2 incubator.

[0105] (2) MTT method

[0106] The MTT assay was used to assess cell viability. CP70 and A2780 cells were cultured at 4 × 10⁶ cells / year. 3Tumor cells were seeded at a density of 10 cells / well in 96-well plates. After 24 hours, tumor cells were treated with different concentrations of compounds for 72 hours. 20 μl of MTT reagent (5 mg / ml, Solarbio Science & Technology Co., Ltd., Beijing, China) was added to each well, and the cells were incubated at 37°C for 4 hours. After incubation, the culture medium was discarded, and the resulting crystals were dissolved in 150 μl of dimethyl sulfoxide (DMSO, Energy Chemical, Shanghai, China). The optical density (OD) was measured at 490 nm using a microplate spectrophotometer (MD, USA). The IC50 values ​​for each compound were determined. 50 The values ​​were calculated using GraphPad Prism 7.0 (San Diego, CA).

[0107] First, the in vitro antitumor activity of the compounds against CP70 and AGS tumor cells was evaluated using the MTT assay, with fluorouracil (5-FU) and oxaliplatin being the positive control drugs. Figure 2 As shown, preliminary screening revealed that among the six skeletons A1, A2, A3, A4, A5, and A20, skeletons A4 and A20 exhibited antitumor activity, with A20 derivatives generally superior to A4 derivatives. Further analysis revealed that among the derivatives of skeleton A4, A12 (R1=F) substituted with an electron-withdrawing group (EWG) showed increased activity compared to A4, while A18 (R1=-CH2CH3) substituted with an electron-donating group (EDG) showed significantly decreased activity compared to A4. A similar pattern was observed with skeleton A20, where compounds A21 (R1=F) and A9 (R1=Cl) were superior to skeleton A20 in CP70 cells (A), while compounds A14 (R1=F) and A9 (R1=Cl) showed better activity than skeleton A20 in the AGS cell line (B). However, compared to skeleton A20, the activity of EDG-substituted A19 was reduced. In summary, the introduction of EDG and EDW showed detrimental and beneficial effects on antitumor activity, respectively, and the introduction of Cl atoms significantly enhanced their antitumor activity. Ultimately, only the A4 and A20 skeletons and their derivatives exhibited better antitumor activity; therefore, we will continue to modify these two skeletons based on existing structure-activity relationship (SAR) analyses in future studies to obtain even more superior molecules.

[0108] Subsequently, the half-maximal inhibitory concentration (IC50) of several active compounds was determined using the MTT assay. Compound A9, the optimal compound, exhibited dose-dependent anti-proliferative activity against both ovarian and gastric cancer cell lines, as shown in Table 3. Therefore, compound A9 was selected as the optimal compound for further investigation of its biological activity.

[0109] Table 3. IC50 of active compounds on cancer cell lines 50 Value (μM)

[0110]

[0111] (3) Quantitative structure-activity relationship (QSAR)

[0112] The calculation of QSAR models requires molecular descriptors to describe various properties of molecules. Commonly used types of molecular descriptors are 2D and 3D molecular descriptors. Molecular descriptors for all spirocyclic compounds were calculated using the cheminformatics open-source toolkit RDKit. The results include 208 dimensions of 2D molecular descriptors based on physicochemical properties and molecular structure, such as molecular weight, topological polar surface area, and lipid-water partition coefficient.

[0113] A QSAR model was constructed using the Random Forest algorithm, with computed and dimensionality-reduced molecular descriptors as input. The Random Forest algorithm (RF) in this embodiment is a decision tree-based learning method. It generates several different subsets by sampling the total dataset, trains a decision tree for each subset, and finally combines the predictions of each decision tree to form the final RF prediction. RF improves prediction accuracy without significantly increasing computational cost. RF is insensitive to multicollinearity and still exhibits good regression performance with high-dimensional input data, demonstrating strong robustness even with missing data or imbalanced datasets. RF is also one of the most suitable machine learning algorithms for building QSAR models. Therefore, this study uses the RF algorithm to construct the QSAR model. Drug activity data from two tumor cell lines were used as the basis for model construction. Training was performed on a personal computer. Due to the characteristics of the Random Forest algorithm, the test and training sets are not distinguished. The default number of sub-decision trees is 800. The QSAR model was obtained through computer training. The R-values ​​of the CP70 and AGS models in the Random Forest method are compared. 2 The values ​​were 0.9656 and 0.9747 respectively, and this model can serve as a guide for future research.

[0114] (4) Colony cloning experiment

[0115] CP70 cells were seeded at a concentration of 2 × 10³ cells per well in 6-well plates and then treated with the specified drugs (DMSO, 0.625, 1.25, 2.5 μM A9, 2.5 μM oxaliplatin). When the cell colonies in the DMSO group reached 70%, they were fixed with 4% paraformaldehyde for 15 min and stained with crystal violet for 20 min. Visualized colonies were processed using ImageJ (National Institutes of Health, Bethesda, USA), and colonies containing more than 50 cells were counted.

[0116] Colony cloning assays were used to evaluate the antiproliferative activity of compound A9 against CP70 ovarian cancer cells. Cells were incubated with different concentrations of A9 and oxaliplatin, and the results are as follows: Figure 5 As shown in Figures AB. The results showed that A9 inhibited tumor cell growth in a dose-dependent manner compared to the solvent group. Encouragingly, 1.25 μM A9 exhibited anti-ovarian cancer growth activity comparable to 2.5 μM oxaliplatin. Furthermore, the effects of A9 on the migration and invasion abilities of CP70 cells were investigated using cell scratch assays and Transwell assays. The results showed that A9 inhibited cell migration and invasion in a dose-dependent manner. Figure 4 It (CF) and its ability to inhibit cell migration is significantly better than that of the positive drug oxaliplatin.

[0117] (5) Apoptosis experiment

[0118] CP70 cells were seeded in 6-well plates (1.5 × 10⁵ cells / well) and incubated with A9, oxaliplatin, and DMSO at specific concentrations for 30 hours each in a 37°C, 5% CO₂ incubator. Cells were then digested with EDTA-free trypsin and transferred to 4 ml centrifuge tubes. Apoptosis was detected using the FITC Annexin V Apoptosis Detection Kit (Cat.#BA11100, Enogen, China) according to the manufacturer's protocol. Cells were stained with annexin V-FITC and propidium iodide (PI) at room temperature in the dark for 15 minutes. Cell analysis was then performed by flow cytometry (FACS Calibur, BD, USA). Data were analyzed using FlowJo (BD, USA).

[0119] Annexin / PI double staining results showed that A9 induced apoptosis in a dose-dependent manner, exhibiting significant apoptosis-inducing activity against ovarian cancer cells at a concentration of 5 μM, and its activity was significantly superior to the positive control drug oxaliplatin. Figure 5 ).

[0120] (6) Pyroptosis experiment

[0121] CP70 and A2780 cells were cultured in 96-well plates (3.0 × 10⁻⁶). 3 Cells were cultured overnight in cells / wells, then treated with specific concentrations of A9, oxaliplatin, and DMSO at 37°C and 5% CO2. DMSO was used as a negative control. Cell pyroptosis was observed and photographed using an inverted fluorescence microscope (ECLIPSE TI-S, Nikon, Japan). Continuous observation after drug administration was performed, such as... Figure 6 As shown, CP70 and A2780 cells treated with A9 exhibited cytoplasmic swelling and large air bubbles within the plasma membrane, and the concentration that induced pyroptosis was significantly lower than that of oxaliplatin. Compounds A14 and A20 were also found to induce pyroptosis.

[0122] Example 3: In vivo antitumor activity test:

[0123] Four-week-old BALB / c nude mice were provided by the Experimental Animal Center of Wenzhou Medical University (Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing). Logarithmic growth phase A2780 cells were digested with trypsin and suspended in a cell suspension. The cell suspension was then resuspended in DMEM medium and subcutaneously injected into the mice (1×10⁻⁶). 7 (cells / 100μl). Tumors reached 50–100 mm. 3 Subsequently, the animals were randomly assigned to different treatment groups and received either 30% castor oil or A9 (5 mg / kg / day). Tumor growth was recorded every other day using the formula V = π / 6 × (length × width). 2 The tumor volume (V) was calculated. Mice were sacrificed 18 days after treatment.

[0124] Given the good intracellular anticancer activity of A9, the in vivo anti-ovarian cancer activity of A9 was further evaluated using the A2780 nude mouse xenograft tumor model. Figure 7 As shown in AC, A9 exhibited effective antitumor activity; 5 mg / kg A9 significantly reduced tumor volume and weight, but A9 had no significant effect on the body weight of nude mice. Figure 7 D). In addition, the expression levels of total GSDME protein, GSDME-N, and Caspase 3 in tumor tissue were assessed. Figure 7 As shown in E, A9 at a dose of 5 mg / kg effectively upregulated the expression of the pyroptosis-related protein GSDME-N, while inhibiting the expression level of GSDME, consistent with the in vitro results.

[0125] In summary, this invention provides a novel class of spiroheterocyclic compounds, which have not been reported in the prior art. Furthermore, this invention provides a green synthetic method for this class of spiroheterocyclic skeletons, and the conditions have been optimized. Finally, using a one-pot green method with water as the solvent, the 1'H-spiro[benzo[e]indole-1,4'-quinolin]-2(3H)-one skeleton was efficiently synthesized at 80°C. Moreover, under these green synthetic conditions, 21 1H-Benzo[e]indole-2(3H)-one spirocyclic derivatives were synthesized, with all compounds yielding over 80%. Therefore, this synthetic method is green, efficient, atom-economical, and has a good substrate expansion range.

[0126] The synthesized compounds (10 μM) were screened for in vitro antitumor activity. The results showed that compounds A9, A14, A19, A20, and A21 exhibited good inhibitory activity against tumor cell growth. Furthermore, a QSAR model was constructed using the random forest algorithm of artificial intelligence. In the two tumor cell lines used for screening, CP70 and AGS, the model R... 2 The values ​​were 0.9656 and 0.9747 respectively, indicating that both models showed good fit and performance.

[0127] In recent years, it has been discovered that inducing pyroptosis in tumor cells holds promise as a new direction for anti-tumor drug development. Further investigation revealed that several compounds (A9, A14, A20) can induce pyroptosis in tumor cells. Among them, the optimal compound, A9, exhibits significantly stronger activity than 5-FU and oxaliplatin. A9 also showed significant activity against IC50 in CP70 and AGS cell lines. 50 The effective concentrations were 2.71±0.61 μM and 1.21±0.23 μM, respectively, therefore A9 was chosen for further investigation. In ovarian cancer cells CP70, compound A9 significantly inhibited cell colony formation, migration, and invasion, all in a concentration-dose dependent manner. A9 concentration-dependently reduced Caspase-3 expression, thereby inducing the production of the pyroptosis-related protein GSDME-N and the expression of apoptosis-related proteins (Cleaved-PARP, BAX, Bcl-2). All of these activities of A9 were significantly superior to the positive control drug oxaliplatin. In the A2780 tumor-bearing nude mouse model, A9 at a low dose of 5 mg / kg / day significantly reduced tumor volume and weight, decreased Caspase-3 expression in tumor tissue, and induced GSDME-N production, while having no significant effect on the body weight of the nude mice.

[0128] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A 1H-benzo[e]indole-2(3H)-ketone spirocyclic derivative, the chemical formula of which is shown in general formula (VII): ； in, R1 is H, or a meta-substituted halogen, or the general formula (VII) is a compound as shown below: 。 2. The 1H-benzo[e]indole-2(3H)-ketone spirocyclic derivative according to claim 1, characterized in that: R1 is a meta-substituted halogen.

3. The method for synthesizing the 1H-benzo[e]indole-2(3H)-one spirocyclic derivative as described in claim 1 or 2, characterized in that, Its synthesis reaction formula is as follows: 。 4. The method for synthesizing the 1H-benzo[e]indole-2(3H)-ketone spirocyclic derivative according to claim 3, characterized in that: The solvent used in the reaction is water with added surfactant.

5. The method for synthesizing the 1H-benzo[e]indole-2(3H)-ketone spirocyclic derivative according to claim 4, characterized in that: The solvent used in the reaction was 10 wt% SDS / H2O, the reaction temperature was 80 °C, and the SDS was sodium dodecyl sulfate.

6. The use of the 1H-benzo[e]indole-2(3H)-ketone spirocyclic derivative as described in claim 1 or 2 in the preparation of an antitumor drug, wherein the tumor is a gastric cancer or ovarian cancer tumor.

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