CN107056680B - Spiro[cyclopropane-1,3'-indoline]-2'-one compound containing difluoromethyl group and its medicinal use - Google Patents

Abstract

The invention discloses a kind of spiral shell [cyclopropane -1,3'- indoline] -2'- ketone compounds, preparation method and its medicinal usage containing difluoromethyl.Such compound contains the active difluoromethyl group of potential source biomolecule and 3, and 3'- Oxoindole loop coil cyclopropane skeleton can provide chemical combination material resource for bioactivity screening, and the screening and pharmaceutical industry to drug have important application value.Meanwhile the present invention has carried out tumor growth inhibiting activity screening to two kinds of tumor cell lines such as human prostate (PC-3) and human lung carcinoma cell (A549) for such compound.Result of study finds that such compound has certain inhibition tumor cell growth activity, it is contemplated that as antineoplastic use.

Description

Spiro[cyclopropane-1,3'-indoline]-2'-one compounds and drugs containing difluoromethyl material use

technical field

The invention relates to the technical field of chemistry, in particular to a difluoromethyl-containing spiro[cyclopropane-1,3'-indoline]-2'-one compound and its medicinal use.

Background technique

The spiro-ring oxindole structure unit widely exists in many natural molecules and clinical drug molecules. Among the oxindole compounds that have been found to contain a 3,3'-spiro ring structure, many of them have high biological activity. Such as anti-tumor, anti-virus, anti-HIV, anti-oxidation, etc. Among them, molecules containing oxindole spirocyclopropane structural units are more attractive, because such compounds can be used as progesterone receptor antagonists, RSV inhibitors, kinase inhibitors, antitumor drugs, etc.

On the other hand, difluoromethyl (-CF ₂ H), as an important fluorine-containing group, contains a weakly acidified CH bond, which is an isostere of hydroxyl, mercapto and hydroxymethyl, and can regulate molecular The biological activity, metabolic stability and lipophilicity of fluoroalkyl play the same important role as other fluoroalkyl groups in the fields of medicine and pesticides. Therefore, the introduction of difluoromethyl into the molecule can endow it with many excellent properties, while introducing or maintaining the key recognition elements of biological targets. These determine its great potential and scientific research value in the fields of biomedicine and bioimaging.

In view of the potential biological activity of oxindole spirocyclopropane compounds and the important role of difluoromethyl in medicinal chemistry, difluoromethyldiazomethane was used as a building block to synthesize the compound by [3+2] reaction and condensation The ring reaction strategy reacted with 3-alkenyloxindoles to synthesize 3,3'-oxindole spirocyclopropanes containing difluoromethyl groups may lead to a series of structurally and reactively interesting new compounds, which The synthesis can establish a material basis for the application of spiro[cyclopropane-1,3'-indoline]-2'-one compounds containing difluoromethyl in drug discovery and druggability evaluation.

Contents of the invention

The object of the present invention is to provide a spiro[cyclopropane-1,3'-indoline]-2'-one compound containing difluoromethyl group and its application in the preparation of drugs for preventing and treating tumor diseases. This kind of important drug active molecule has important application value for drug screening and pharmaceutical industry, and the synthesis of this kind of molecule has the advantages of mild reaction conditions, simple operation and good substrate universality.

The present invention is achieved as follows: in-situ generated difluoromethyldiazomethane and isatin-derived 3-alkenyl oxide indole carry out [3+2] reaction and ring condensation in an organic solvent under catalyst-free conditions reaction to obtain spiro[cyclopropane-1,3'-indoline]-2'-one compounds containing difluoromethyl. Such compounds have the structure shown in general formula (I):

In the ^formula , R1 is ^an alkyl group, an alkoxy group or a halogen, R2 is an alkyl group, and ^R3 is an alkyl group, an aryl group or an acyl group.

Described organic solvent is dichloromethane, chloroform, carbon tetrachloride, acetonitrile, tetrahydrofuran, benzene, toluene, xylene, mesitylene, 1,4-dioxane, ethylene glycol dimethyl ether, Ethylene glycol diethyl ether or methyl tert-butyl ether.

The reaction temperature of the difluoromethyldiazomethane generated in situ and the isatin-derived 3-alkenyloxindole is 25-130° C., and the reaction time is 1-50 hours.

The application of the spiro[cyclopropane-1,3'-indoline]-2'-one compound containing difluoromethyl group in the preparation of drugs for preventing and treating tumor diseases.

The reaction principle of the present invention is as follows:

Wherein, R ¹ , R ² , R ³ and the organic solvent are as above.

By adopting the above-mentioned technical scheme, difluoroethylamine (1) is placed in a reaction flask, organic solvent, tert-butyl nitrite and acetic acid are added in sequence, and reacted at 70°C for 10 minutes, and heating is stopped to obtain in situ Difluoromethyldiazomethane (2). Then the isatin-derived 3-alkenyloxindole (3) was added into the reaction flask, and a series of difluoromethyl-containing spiro[cyclopropane-1, 3'-indoline]-2'-ketone compound (4). This type of compound contains a potentially biologically active difluoromethyl group and a 3,3'-oxindole spirocyclopropane skeleton, which can establish a material basis for drug discovery and drugability evaluation, and has important application value. The raw materials of the invention are easy to obtain, can be carried out in various organic solvents, and have the advantages of mild reaction conditions, simple operation and good substrate universality.

Description of drawings

Accompanying drawing 1 is the single crystal-X-ray diffraction structure of the embodiment compound 4a of the present invention.

Accompanying drawing 2 is the single crystal-X-ray diffraction structure of the embodiment compound 4g of the present invention.

Accompanying drawing 3 is the single crystal-X-ray diffraction structure of the embodiment compound 4r of the present invention.

Accompanying drawing 4-6 is the nuclear magnetic resonance spectrum of the embodiment compound 4a of the present invention.

Detailed ways

The present invention will be further described below in conjunction with the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments, and it can be foreseen that those skilled in the art may produce various changes in the implementation situation in combination with the prior art.

Example 1 of the present invention: preparation of difluoromethyl-containing spiro[cyclopropane-1,3'-indoline]-2'-one (4); compound 4a: in a round bottom flask, add two Chloromethane (15mL), difluoroethylamine (1,97.3mg, 1.2mmol), tert-butyl nitrite (148.5mg, 1.44mmol), acetic acid (14.5mg, 0.24mmol), react at 70°C for 10 minutes , the heating is stopped, and difluoromethyldiazomethane (2) can be prepared in situ. Then add isatin-derived 3-alkenyloxindole (3a, 92.5mg, 0.4mmol), and stir well at room temperature for 48 hours, concentrate, then add toluene (15mL), react at 120°C for 2 hours, thin After the reaction was detected by layer chromatography, it was separated by silica gel column chromatography (300-400 mesh) (petroleum ether: ethyl acetate = 5:1), and 103.9 mg of a light yellow solid was obtained, with a yield of 88%. Melting point: 88.9-90.0°C, NMR and high-resolution mass spectrometry analysis results are as follows: 94:6trans/cis(determined by ¹⁹ F NMR analysis); ¹ H NMR(400MHz, CDCl ₃ )δ(major)7.36-7.31(m,2H),7.06(t ,J=7.8Hz,1H),6.91(d,J=7.8Hz,1H),6.40(td,J=6.0,56.0Hz,1H),4.19-4.11(m,2H),3.28(s,3H) ,2.99-2.95(m,2H),1.22(t,J=7.2Hz,3H); ¹³ C NMR(100MHz,CDCl ₃ )δ(major+minor)173.1,172.3,167.4,166.8,144.8,144.2,132.5 ,128.7,128.6,124.3,124.0,122.9,122.8,113.4(t,J=236.3Hz),108.9,108.5,108.2,62.0,61.9,35.8(d,J=8.6Hz),34.9(d,J=9.7 Hz), 34.6(t, J=33.7Hz), 33.3(t, J=33.9Hz), 32.8(d, J=7.9Hz), 27.0, 27.9, 25.9, 14.2, 14.1; ¹⁹ F NMR (376MHz, CDCl ₃ ) δ(major+minor)-111.04(ABd, J=54.2,293.9Hz, major),-112.94(ABd,J=55.4,297.4Hz,minor),-114.43(ABd,J=54.1,297.6Hz, minor), -115.88 (ABd, J＝54.1, 293.4Hz, major); HRMS (ESI-TOF) Calcd. for C ₁₅ H ₁₆ F ₂ NO ₃ [M+H] ⁺ :296.1093; found: 296.1101.

The single crystal-X-ray diffraction structure diagram and nuclear magnetic resonance spectrum diagram in the accompanying drawings are generated by prior art software. Those skilled in the art can understand the properties of each compound through the characteristics in the diagram. The numbers in the diagram may not be correct due to the reason of submission. It is clear, but does not affect the complete disclosure of the present invention.

The preparation method of compounds 4b-4s prepared by the examples is the same as that of compound 4a, and the feeding ratio is the same as that of compound 4a. Compounds 4b-4s can be obtained. The reaction yields are shown in Table 1, but it should be emphasized that the compounds of the present invention are not limited to Table 1 the content represented.

Table 1 shows the chemical structures of spiro[cyclopropane-1,3'-indoline]-2'-ones containing difluoromethyl groups

Table 1:

Compound 4b prepared in this example: red oil, yield 91%; NMR and high-resolution mass spectrometry analysis results are as follows: 93:7trans/cis (determined by ¹⁹ F NMR analysis); ¹ H NMR (400MHz, CDCl ₃ ) δ(major+minor)7.18(s,0.07H),7.16(s,0.93H),7.12(d,J＝7.9Hz,0.93H),6.83(d,J＝7.9Hz,0.07H),6.79( d,J=7.9Hz,0.93H),6.68(d,J=7.9Hz,0.07H),6.40(td,J=7.2,55.8,Hz,1H),4.36-4.30(m,0.14H),4.24 -4.07(m,1.86H),3.25(d,J＝1.6Hz,2.79H),3.20(d,J＝1.6Hz,0.21H),2.99-2.90(m,1.86H),2.86-2.83(m ,0.14H),2.34(s,0.21H),2.32(s,2.79H),1.39-1.35(m,0.21H),1.24-1.20(m,2.79H); ¹³ C NMR(100MHz,CDCl ₃ ) δ(major+minor)172.2,167.6,166.8(d,J=2.0Hz),165.9,141.8,132.9,132.4,129.4,128.9,125.0,124.0,123.6,122.2,113.4(t,J=236.3Hz), 108.2, 61.9, 61.2, 35.9(d, J=8.6Hz), 34.8(d, J=9.7Hz), 34.5(t, J=33.7Hz), 26.9, 26.3, 21.4, 21.3, 21.2, 14.3, 14.2; ¹⁹ F NMR (376MHz, CDCl ₃ ) δ(major+minor)-111.03(ABdd, J=5.9,54.7,294.3Hz, major),-112.87(ABdd,J=8.6,55.9,297.2Hz,minor)-114.51 (ABdd, J=7.1, 53.6, 297.3Hz, minor), -115.84 (ABdd, J=7.0, 56.1, 294.6Hz, major); HRMS (ESI-TOF) Calcd.for C ₁₆ H ₁₈ F ₂ NO ₃ [ M+H] ⁺ :310.1249; found: 310.1253.

Compound 4c prepared in this example: orange oil, yield 81%; NMR and high-resolution mass spectrometry analysis results are as follows: >99:1trans/cis (determined by ¹⁹ F NMR analysis); ¹ H NMR (400MHz, CDCl ₃ )δ7.16(dd, J=2.6,8.6Hz,1H),7.02(td,J=2.6,8.6Hz,1H),6.79(dd,J=8.0,8.6Hz,1H),6.38(td,J =7.4,55.2Hz,1H),4.25-4.09(m,2H),3.26(s,3H),3.02-3.01(m,1H),2.96-2.89(m,1H),1.23(t,J=7.2 Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ172.0, 166.7(d, J=2.0Hz, 1C), 159.2(d, J=239.2Hz, 1C), 140.2(d, J=1.8Hz, 1C ), 125.6(d, J=9.5Hz, 1C), 114.9(d, J=23.6Hz, 1C), 113.2(t, J=236.5Hz, 1C), 111.4(d, J=26.8Hz, 1C), 108.9(d, J=8.3Hz, 1C), 62.2, 35.9(dd, J=2.0, 8.6Hz, 1C), 34.9(d, J=4.5Hz, 1C), 34.9(t, J=28.9Hz, 1C ), 27.0, 14.1; ¹⁹ F NMR (376MHz, CDCl ₃ ) δ-111.07 (ABddd, J = 1.6, 5.9, 54.7, 295.3Hz, 1F), -116.01 (ABdd, J = 7.2, 55.6, 295.2Hz, 1F ), -119.47 (td, J＝4.1, 8.8Hz, 1F); HRMS (ESI-TOF) Calcd. for C ₁₅ H ₁₅ F ₃ NO ₃ [M+H] ⁺ : 314.0999; found: 314.1010.

Compound 4d prepared in this example: orange oil, yield 71%; NMR and high-resolution mass spectrometry analysis results are as follows: >99:1trans/cis (determined by ¹⁹ F NMR analysis); ¹ H NMR (400MHz, CDCl ₃ )δ(major) 7.37(d, J=2.0Hz, 1H), 7.02(dd, J=2.0, 8.4Hz, 1H), 6.82(d, J=8.4Hz, 1H), 6.37(td, J=7.2 ,55.2Hz,1H),4.26-4.10(m,2H),3.26(s,3H),3.02-2.91(m,2H),1.24(t,J＝7.2Hz,3H); ¹³ C NMR(100MHz, CDCl ₃ )δ(major+minor) 172.3, 166.7, 145.3, 134.7, 125.3, 124.0, 112.7, 122.3, 113.2 (t, J＝236.6Hz), 109.7, 109.3, 62.2, 35.6 (d, J＝8.6Hz) , 34.9 (d, J=8.6Hz), 34.7 (t, J=34.0Hz), 27.2, 27.0, 14.2; ¹⁹ F NMR (376MHz, CDCl ₃ ) δ(major)-111.06 (ABdd, J=5.6, 54.5 , 295.4Hz, major, 1F), -116.01 (ABdd, J＝7.0, 55.8, 295.6Hz, major, 1F); HRMS (ESI-TOF) Calcd.for C ₁₅ H ₁₅ ClF ₂ NO ₃ [M+H] ⁺ :330.0703; found: 330.0708.

Compound 4e was prepared in this example: yellow oil, yield 85%; NMR and high-resolution mass spectrometry analysis results are as follows: 97:3trans/cis (determined by ¹⁹ F NMR analysis); ¹ H NMR (400MHz, CDCl ₃ ) δ(major)7.49(s,1H),7.44(dd,J=1.9,8.4Hz,1H),6.77(d,J=8.4Hz,1H),6.36(td,J=7.2,55.3Hz,1H) ,4.26-4.10(m,2H),3.25(s,3H),3.01-2.93(m,2H),1.24(t,J=7.2Hz,3H); ¹³ C NMR(100MHz,CDCl ₃ )δ(major )171.7, 166.5, 143.2, 131.5, 126.2, 125.9, 115.5, 113.1 (t, J = 236.7Hz, 1C), 109.9, 62.2, 35.5 (d, J = 8.6Hz, 1C), 35.1 (d, J = 9.5 Hz, 1C), 35.0(t, J＝34.0Hz, 1C), 27.0, 14.2; ¹⁹ F NMR (376MHz, CDCl ₃ ) δ(major+minor)-111.02 (ABdd, J＝3.7, 54.6, 295.3Hz, major), -112.80 (ABdd, J＝8.2, 55.9, 298.9Hz, minor), -114.42 (ABdd, J＝7.2, 53.4, 298.9Hz, minor), -115.98 (ABdd, J＝6.7, 55.8, 295.7Hz , major); HRMS (ESI-TOF) Calcd. for C ₁₅ H ₁₅ BrF ₂ NO ₃ [M+H] ⁺ :374.0198; found: 374.0204.

Compound 4f prepared in this example: yellow oil, yield 80%; NMR and high-resolution mass spectrometry analysis results are as follows: 97:3trans/cis (determined by ¹⁹ F NMR analysis); ¹ H NMR (400MHz, CDCl ₃ ) δ(major)7.29(s,1H),7.20(d,J=8.2Hz,1H),6.89(d,J=8.4Hz,1H),6.38(td,J=7.0,55.2Hz,1H),4.20 -4.11(m,2H),3.28(s,3H),3.03-2.94(m,2H),1.23-1.19(m,3H); ¹³ C NMR(100MHz,CDCl ₃ )δ(major)172.1,166.5, 144.8, 142.9, 125.5, 121.9, 120.6(q, J=255.2Hz, 1C), 117.3, 113.1(t, J=236.2Hz), 108.9, 62.3, 35.7(d, J=8.6Hz), 35.1(d, J=9.5Hz), 34.9(t, J=34.1Hz), 27.0, 14.0; ¹⁹ F NMR (376MHz, CDCl ₃ ) δ(major+minor)-58.54(s, 3F),-111.14(ABdd, J= 5.6, 54.6, 295.2Hz, major), -113.01 (ABdd, J＝8.3, 55.9, 299.7Hz, minor), -114.46 (ABdd, J＝7.4, 53.4, 299.3Hz, minor), -116.14 (ABdd, J =7.1,55.6,295.6Hz, major); HRMS (ESI-TOF) Calcd. for C ₁₆ H ₁₅ F ₅ NO ₄ [M+H] ⁺ :380.0916; found: 380.0922.

Compound 4g prepared in this example: yellow solid, yield 92%; melting point: 102.0-103.8°C; NMR and high-resolution mass spectrometry analysis results are as follows: 89:11trans/cis (determined by ¹⁹ F NMR analysis); ¹ HNMR ( 400MHz, CDCl ₃ )δ(major+minor)7.39(d, J=8.2Hz,0.11H),7.28(d,J=8.2Hz,0.89H),7.05(d,J=1.9Hz,0.11H), 7.02(dd, J=1.8,8.2Hz,0.89H),6.94(d,J=1.8Hz,0.11H),6.90(d,J=1.8Hz,0.89H),6.37(td,J=7.1,55.2 Hz,1H),4.34-4.29(m,0.22H),4.23-4.07(m,1.78H),3.26(s,2.67H),3.22(s,0.33H)3.00-2.93(m,1.78H), 2.65-2.57(m,0.22H),1.36(t,J=7.2Hz,0.33H),1.22(t,J=7.2Hz,2.67H); ¹³ C NMR(100MHz,CDCl ₃ )δ(major+minor )172.3, 166.7, 145.3, 134.7, 125.3, 124.0, 122.7, 122.6, 122.3, 113.2(t, J=236.6Hz), 109.7, 109.3, 62.2, 62.1, 35.6(d, J=8.6Hz), 35.0(d , J=8.6Hz), 34.7(t, J=34.0Hz), 32.9(d, J=7.6Hz), 27.2, 27.0, 14.2, 14.1; ¹⁹ F NMR (376MHz, CDCl ₃ ) δ(major+minor) -111.03 (ABdd, J＝5.6, 54.6, 295.2Hz, major), -113.05 (ABdd, J＝8.2, 55.8, 298.8Hz, minor), -114.40 (ABdd, J＝7.4, 53.6, 298.7Hz, minor) ,-115.96 (ABdd, J=7.0, 55.6, 295.3Hz, major); HRMS (ESI-TOF) Calcd. for C ₁₅ H ₁₅ ClF ₂ NO ₃ [M+H] ⁺ :330.0703; found: 330.0713.

Compound 4h was prepared in this example: yellow solid, yield 83%; melting point: 103.7-105.2°C; NMR and high-resolution mass spectrometry analysis results are as follows: 92:8trans/cis (determined by ¹⁹ F NMR analysis); ¹ HNMR ( 400MHz, CDCl ₃ )δ(major+minor)7.32(d, J＝8.2Hz, 0.08H), 7.22(d, J＝8.2Hz, 0.92H), 7.18-7.16(m, 1H), 7.09(d, J＝1.6Hz, 0.08H), 7.04(d, J＝1.6Hz, 0.92H), 6.37(td, J＝7.2, 55.1Hz, 1H), 4.32-4.28(m, 0.16H), 4.23-4.06( m, 1.84H), 3.26(s, 2.76H), 3.21(s, 0.24H), 3.00-2.91(m, 1.84H), 2.65-2.57(m, 0.16H), 1.22(t, J＝7.2Hz ,2.76H),0.95(t,J=7.3Hz,0.24H); ¹³ C NMR(100MHz,CDCl ₃ )δ(major+minor)172.2,166.7,145.4,125.6,125.5,124.3,122.9,122.4,113.1 (t,J=236.6,Hz),112.4,112.1,62.1,35.6(d,J=8.7Hz),35.0(d,J=9.6Hz),34.7(t,J=33.9Hz),27.1,27.0, 14.2; ¹⁹ F NMR (376MHz, CDCl ₃ ) δ (major+minor) -111.03 (ABdd, J = 3.9, 54.6, 295.1 Hz, major), -113.05 (ABdd, J = 6.6, 55.8, 298.6 Hz, minor) , -114.40 (ABdd, J=7.1, 55.5, 298.8Hz, minor), -115.96 (ABdd, J=6.5, 55.6, 295.3Hz, major); HRMS (ESI-TOF) Calcd.for C ₁₅ H ₁₅ BrF ₂ NO ₃ [M+H] ⁺ :374.0198; found: 374.0210.

Compound 4i was prepared in this example: yellow solid, yield 96%; melting point: 83.3–85.0°C; NMR and high-resolution mass spectrometry analysis results are as follows: 92:8trans/cis (determined by ¹⁹ F NMR analysis); ¹ H NMR (400MHz, CDCl ₃ )δ(major+minor)7.05-7.03(m,0.16H),6.98-6.92(m,1.84H),6.90-6.87(m,0.92H),6.84-6.81(m,0.08H ),6.40(td,J＝7.2Hz,1H),4.34-4.28(m,0.16H),4.22-4.04(m,1.84H),3.86(s,3H),3.55(s,2.76H),3.50 (s,0.24H),2.99-2.83(m,1.84H),2.61-2.53(m,0.16H),1.36(t,J=7.1Hz,0.24H),1.21(t,J=7.2Hz,2.76 H); ¹³ C NMR (100MHz, CDCl ₃ ) δ(major+minor) 172.5, 166.7, 146.0, 145.6, 132.1, 125.6, 123.3, 122.9, 121.4, 116.7, 115.3, 113.4 (t, J=236.3Hz), 112.7, 112.5, 62.0, 61.9, 61.3, 56.1, 36.0(d, J=8.6Hz), 35.3(d, J=8.6Hz), 34.8(t, J=33.8Hz), 33.1(d, J=8.7Hz ), 30.6, 30.4, 14.3, 14.2; ¹⁹ F NMR (376MHz, CDCl ₃ ) δ (major+minor) -111.12 (ABdd, J = 5.8, 54.8, 294.4Hz, major), -112.85 (ABdd, J = 8.3 ,56.0,297.4Hz,minor),-114.53(ABdd,J＝7.2,53.5,297.3Hz,minor),-116.05(ABdd,J＝7.0,55.8,294.3Hz,major); HRMS(ESI-TOF) Calcd .forC ₁₆ H ₁₈ F ₂ NO ₄ [M+H] ⁺ :326.1198; found: 326.1202.

Compound 4j prepared in this example: yellow solid, yield 75%; melting point: 96.0–97.5°C; NMR and high-resolution mass spectrometry analysis results are as follows: 90:10trans/cis (determined by ¹⁹ F NMR analysis); ¹ H NMR (400MHz, CDCl ₃ )δ(major+minor)7.24(d, J=7.8Hz,0.10H),7.13(d,J=7.4Hz,0.90H),7.07-7.02(m,1H),6.99-6.95 (m,0.90H),6.81-6.79(m,0.10H),6.38(td,J＝6.9,55.2Hz,1H),4.23-4.07(m,2H),3.50(s,3H),3.01(d ,J=7.4Hz,0.90H),2.97-2.92(m,0.90H),2.87(d,J=9.3Hz,0.10H),2.65-2.57(m,0.10H),1.22(t,J=7.0 Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ(major+minor) 172.0, 170.9, 167.2, 166.5, 147.9(d, J=242.3Hz), 130.7(d, J=23.9Hz), 126.8( d,J=3.7Hz),123.3(d,J=6.6Hz),120.1(d,J=3.4Hz),118.7(d,J=3.3Hz),116.9,116.7(d,J=18.9Hz), 113.2(t, J=236.6Hz), 62.1, 62.0, 36.0(d, J=12.0Hz), 35.5(d, J=9.6Hz), 35.0(t, J=33.9Hz), 33.3(d, J= 7.9Hz), 29.7(d, J=6.1Hz), 29.5(d, J=6.0Hz), 14.2, 14.1; ¹⁹ F NMR (376MHz, CDCl ₃ ) δ(major+minor)-111.09(ABddd, J= 1.3,5.7,54.7,295.3Hz,major),-112.99(ABddd,J＝1.7,8.3,55.9,298.6Hz,minor),-114.60(ABdd,J＝7.3,53.5,298.5Hz,minor),-116.13 (ABdd,J=6.8,55.3,295.3Hz,major),(-135.65)–(-135.69)(m,minor),(-136.40)–(-136.45)(m,major); HRMS(ESI-TOF )Ca lcd.for C ₁₅ H ₁₅ F ₃ NO ₃ [M+H] ⁺ :314.0999; found: 314.1004.

Compound 4k was prepared in this example: white solid, yield 90%; melting point: 117.0-118.7°C; NMR and high-resolution mass spectrometry analysis results are as follows: 93:7trans/cis (determined by ¹⁹ F NMR analysis); ¹ HNMR ( 400MHz, CDCl ₃ )δ(major+minor)7.34(d, J＝7.8Hz, 0.07H), 7.29(d, J＝1.2Hz, 0.07H), 7.26-7.22(m, 1.86H), 6.95(t ,J=8.0Hz,1H),6.38(td,J=7.3Hz,1H),4.21-4.08(m,2H),3.67(s,0.21H),3.66(s,2.79H),3.01(d, J＝7.8Hz, 0.93H), 2.98-2.92(m, 0.93H), 2.88(d, J＝9.3Hz, 0.07H), 2.64-2.60(m, 0.07H), 1.22(t, J＝7.2Hz ,3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ(major+minor) 172.7, 166.4, 140.0, 131.2, 131.0, 126.7, 123.5, 123.4, 122.6, 121.3, 116.1, 113.2 (t, J＝236.7Hz) ,62.2,62.1,35.8(d,J=9.6Hz),35.6(d,J=8.6Hz),35.1(t,J=33.9Hz),33.5(d,J=7.6Hz),30.8,30.5,14.2 ; ¹⁹ F NMR (376MHz, CDCl ₃ ) δ(major+minor)-111.07(ABddd, J=2.3,5.4,54.6,295.5Hz, major),-112.87(ABdd,J=8.2,56.1,299.2Hz,minor )-114.52 (ABddd, J=3.0, 7.6, 53.4, 299.1Hz, minor), -116.08 (ABddd, J=2.3, 7.1, 55.7, 295.4Hz, major); HRMS (ESI-TOF) Calcd.for C ₁₅ H ₁₄ F ₂ NNaO ₃ [M+Na] ⁺ :352.0523; found: 352.0532.

Compound 4l prepared in this example: light brown solid, yield 97%; melting point: 104.3–105.7°C; NMR and high-resolution mass spectrometry analysis results are as follows: 89:11trans/cis (determined by ¹⁹ F NMR analysis); ¹ HNMR (400MHz, CDCl ₃ ) δ(major+minor)7.45(dd, J=1.0,8.2Hz,0.11H),7.42(dd,J=1.0,8.2Hz,0.89H),7.39(d,J=8.8Hz ,0.11H),7.27(d,J=7.6Hz,0.89H),6.90(t,J=7.9Hz,1H),6.38(td,J=7.2,55.2Hz,1H),4.23-4.06(m, 2H), 3.68(s, 0.33H), 3.66(s, 2.67H), 3.02(d, J=7.6Hz, 0.89H), 2.98-2.91(m, 0.89H), 2.88(d, J=9.4Hz ,0.11H),2.66-2.58(m,0.11H),1.21(t,J＝7.2Hz,3H); ¹³ C NMR(100MHz,CDCl ₃ )δ(major+minor)173.8,172.8,167.1,166.4, 141.4, 134.5, 134.3, 127.0, 125.2, 123.8, 123.7, 123.1, 121.8, 113.2(t, J=236.6Hz), 103.2, 102.8, 62.2, 62.1, 35.9(d, J=9.6Hz), 35.5(d, J=8.7Hz), 35.0(t, J=34.0Hz), 33.6(d, J=8.1Hz), 31.1, 30.7, 14.2; ¹⁹ F NMR(376MHz, CDCl ₃ ) δ(major+minor)-111.06( ABdd,J＝4.3,54.6,295.3Hz,major),-112.28(ABdd,J＝6.8,56.4,299.2Hz,minor),-114.49(ABdd,J＝7.2,53.5,299.2Hz,minor),-116.05 (ABdd, J=6.6, 54.6, 295.3Hz, major); HRMS (ESI-TOF) Calcd. for C ₁₅ H ₁₅ BrF ₂ NO ₃ [M+H] ⁺ :374.0198; found: 374.0202.

Compound 4m prepared in this example: yellow solid, yield 60%; melting point: 118.2–119.3°C; NMR and high-resolution mass spectrometry analysis results are as follows: >99:1trans/cis (determined by ¹⁹ F NMR analysis); ¹ HNMR (400MHz, CDCl ₃ )δ7.35-7.31(m,2H),7.08-7.04(m,1H),6.91(d,J＝7.5Hz,1H),6.40(td,J＝7.2,55.6Hz,1H ), 3.70 (s, 3H), 3.28 (s, 3H), 3.02-2.94 (m, 2H); ¹³ C NMR (100MHz, CDCl ₃ ) δ172.3, 167.3, 144.2, 128.7, 124.0, 123.0 (d, J＝ 2.7Hz), 122.0 113.3(t, J=236.4Hz), 108.6, 52.8, 35.9(d, J=8.5Hz), 34.7(d, J=9.5Hz), 34.6(t, J=33.7Hz), 26.9 ; ¹⁹ F NMR (376MHz, CDCl ₃ ) δ-111.09 (ABdd, J=5.3,54.9,295.1Hz, 1F), -115.91 (ABdd, J=5.9,55.5,294.2Hz, 1F); HRMS (ESI-TOF )Calcd.for C ₁₄ H ₁₄ F ₂ NO ₃ [M+H] ⁺ :282.0936; found: 282.0945.

Compound 4n prepared in this example: light yellow solid, yield 87%; melting point: 63.2–64.0°C; NMR and high-resolution mass spectrometry analysis results are as follows: >99:1trans/cis (determined by ¹⁹ F NMR analysis); ¹ HNMR (400MHz, CDCl ₃ ) δ7.36-7.31(m, 2H), 7.06(t, J=7.7Hz, 1H), 6.91(d, J=7.8Hz, 1H), 6.41(td, J=7.1, 55.3Hz, 1H), 4.12-3.98(m, 2H), 3.29(s, 3H), 3.02-2.95(m, 2H), 1.62-1.59(m, 2H) 0.86(t, J=7.4Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ172.4, 166.9, 144.2, 128.7, 124.1, 122.9, 122.8, 113.4 (t, J = 236.4Hz, 1C), 108.6, 67.6, 35.9 (d, J = 8.6Hz, 1C) , 34.9 (d, J=9.6Hz, 1C), 34.7 (t, J=33.7Hz, 1C), 26.9, 21.9, 10.3; ¹⁹ F NMR (376MHz, CDCl ₃ ) δ-111.04 (ABdd, J=5.6, 55.0, 294.6Hz, 1F), -115.89 (ABdd, J＝7.0, 55.6, 294.4Hz, 1F); HRMS (ESI-TOF) Calcd.for C ₁₆ H ₁₈ F ₂ NO ₃ [M+H] ⁺ :310.1249 ;found: 310.1255.

Compound 4o was prepared in this example: light yellow solid, yield 99%; melting point: 80.3–82.0°C; NMR and high-resolution mass spectrometry analysis results are as follows: 87:13trans/cis (determined by ¹⁹ F NMR analysis); ¹ HNMR (400MHz, CDCl ₃ )δ(major+minor)7.38-7.29(m,2H),7.08-7.02(m,1H),6.90(d,J＝8.2Hz,0.87H),6.79(d,J＝7.8 Hz,0.13H),6.38(td,J＝7.2,55.7Hz,1H),3.29(s,2.61H),3.23(s,0.39H),2.95-2.87(m,1.74H),2.81-2.78( m,0.13H),2.60-2.49(m,0.13H),1.41(s,1.17H),1.38(s,7.83H); ¹³ C NMR(100MHz,CDCl ₃ )δ(major+minor)172.6,165.7 ,144.1,132.2,128.7,128.6,128.5,125.0,124.2,124.1,122.9,122.7(d,J=5.9Hz),113.5(t,J=236.2Hz),108.9,108.5,108.2,82.9,35.9(d , J=9.0Hz), 35.6(d, J=9.0Hz), 34.3(t, J=33.6Hz), 33.8(d, J=8.0Hz), 28.2, 28.1, 26.9; ¹⁹ F NMR (376MHz, CDCl ₃ ) δ(major+minor)-110.97(ABdd,J=5.2,54.5,293.8Hz,major),-112.66(ABdd,J=8.8,56.1,296.4Hz,minor),-115.20(ABdd,J=6.9 ,53.5,296.3Hz,minor),-115.90(ABdd,J＝6.7,55.6,293.7Hz,major); HRMS(ESI-TOF) Calcd.for C ₁₇ H ₂₀ F ₂ NO ₃ [M+H] ⁺ : 324.1406; found: 324.1415.

Compound 4p prepared in this example: yellow solid, yield 93%; melting point: 70.3–72.3°C; NMR and high-resolution mass spectrometry analysis results are as follows: 85:15trans/cis (determined by ¹⁹ F NMR analysis); ¹ H NMR (400MHz, CDCl ₃ ) δ(major+minor)7.47(d, J=7.8Hz,0.15H),7.37(d,J=7.8Hz,0.85H),7.32(t,J=7.8Hz,1H), 7.04(t, J=6.0Hz, 0.85H), 6.97(d, J=8.0Hz, 0.15H), 6.93(d, J=7.8Hz, 0.85H), 6.83(t, J=8.2Hz, 0.15H ),6.40(td,J=8.0,56.0Hz,1H),4.35-4.30(m,0.3H),4.25-4.07(m,1.7H),3.86-3.80(q,J=7.2Hz,2H), 2.95(d,J=7.5Hz,0.85H),2.97-2.92(m,0.85H),2.85(d,J=9.4Hz,0.15H),2.65-2.56(m,0.15H),1.39-1.35( m,0.9H),1.32-1.21(m,5.1H); ¹³ C NMR(100MHz,CDCl ₃ )δ(major+minor)172.8,171.9,167.5,166.9,143.3,132.5,129.1,128.7,128.6,124.6 ,124.3,123.2,122.8,122.7,122.6,122.5,113.4(t,J=236.5Hz),109.0,108.7,108.4,62.0,61.3,35.9(d,J=9.0Hz),35.7,35.5,34.7(d , J=9.6Hz), 34.6(t, J=33.8Hz), 32.8(d, J=7.8Hz), 14.3, 14.2, 12.9, 12.8; ¹⁹ F NMR (376MHz, CDCl ₃ ) δ(major+minor) -111.06 (ABddd, J＝1.8, 5.7, 54.7, 294.5Hz, major), -113.01 (ABddd, J＝1.7, 8.3, 55.9, 297.6Hz, minor), -114.45 (ABdd, J＝7.3, 53.7, 297.6 Hz, minor), -113.01 (ABdd, J=7.3, 55.1, 294.3Hz, major); HRMS (ESI-TOF) Calcd.for C ₁₆ H ₁₈ F ₂ NO ₃ [M+H] ⁺ :310.1249; found: 310.1252.

Compound 4q prepared in this example: orange oil, yield 99%; NMR and high-resolution mass spectrometry analysis results are as follows: 85:15trans/cis (determined by ¹⁹ F NMR analysis); ¹ H NMR (400MHz, CDCl ₃ ) δ(major+minor)7.50(d,J=7.6Hz,0.15H),7.39(d,J=7.6Hz,0.85H),7.36-7.20(m,6H),7.07-7.01(m,1H), 6.87(d, J=7.8Hz,0.15H),6.81(d,J=7.8Hz,0.85H),6.47(td,J=7.4,55.2Hz,1H),5.08-4.90(m,2H),4.38 -4.33(m,0.30H),4.27-4.09(m,1.70H),3.13-3.11(m,0.85H),3.07-3.00(m,0.85H),2.98-2.95(m,0.15H),2.74 -2.66(m,0.15H),1.39(t,J=7.2Hz,0.45H),1.25(t,J=7.2Hz,2.55H); ¹³ C NMR(100MHz,CDCl ₃ )δ(major+minor) ( 236.5Hz), 109.9, 109.5, 62.1, 62.0, 44.6, 44.4, 35.90(d, J=9.0Hz), 35.0(d, J=9.7Hz), 34.9(t, J=33.8Hz), 33.5(t, J＝34.1Hz), 32.9(d, J＝7.7Hz), 14.1, 14.2; ¹⁹ F NMR (376MHz, CDCl ₃ ) δ(major+minor)-110.88 (ABdd, J＝5.5, 54.7, 294.6Hz, major ),-112.87(ABdd,J=8.3,55.9,297.8Hz,minor)-114.44(ABdd,J=7.3,53.7,297.6Hz,minor),-112.87(ABdd,J=7.6,55.7,294.5Hz,major ); HRMS (ESI-TOF) Calcd.for C ₂₁ H ₂₀ F ₂ NO ₃ [M +H] ⁺ :372.1406; found: 372.1418.

Compound 4r prepared in this example: yellow oil, yield 94%; NMR and high-resolution mass spectrometry analysis results are as follows: 92:8trans/cis (determined by ¹⁹ F NMR analysis); ¹ H NMR (400MHz, CDCl ₃ ) δ(major+minor)7.57-7.54(m,2H),7.46-6.43(m,4H),7.26(t,J=7.8Hz,1H),7.10(t,J=7.8Hz,1H),6.89( d,J=7.8Hz,1H),6.44(td,J=7.0,55.2Hz,1H),4.30-4.12(m,2H),3.13-3.03(m,1.84H),2.97(d,J=9.4 Hz, 0.08H), 2.76-2.68(m, 0.08H), 1.40(t, J=7.2Hz, 0.24H), 1.27(t, J=7.2Hz, 2.76H); ¹³ C NMR (100MHz, CDCl ₃ )δ(major+minor) 172.7, 171.9, 167.4, 166.8, 144.2, 134.1, 134.0, 129.8, 128.7, 128.6, 128.5, 126.8, 126.7, 124.6, 123.9, 123.3, 123.2, 113.3 (t, J = 236.3) ,110.2,109.9,62.1,62.0,36.1(d,J=8.5Hz),35.4(d,J=9.6Hz),35.2(t,J=33.9Hz),33.3(d,J=7.9Hz),14.2 ,14.1; ¹⁹ F NMR (376MHz, CDCl ₃ )δ(major+minor)-110.83(ABdd, J=3.6,54.7,295.2Hz, major),-115.18(ABdd,J=8.0,55.9,298.4Hz,minor ), -114.42 (ABdd, J=7.0, 53.6, 298.2Hz, minor), -115.94 (ABdd, J=6.8, 55.4, 294.9Hz, major); HRMS (ESI-TOF) Calcd.for C ₂₀ H ₁₈ F ₂ NO ₃ [M+H] ⁺ :358.1249; found: 328.1253.

Compound 4s prepared in this example: light yellow solid, yield 70%; melting point: 98.7-100.2°C; NMR and high-resolution mass spectrometry analysis results are as follows: 87:13trans/cis (determined by ¹⁹ F NMR analysis); ¹ HNMR (400MHz, CDCl ₃ ) δ(major+minor)7.99(d, J=8.2Hz,0.13H),7.89(d,J=8.2Hz,0.87H),7.44(d,J=7.8Hz,0.13H) ,7.38-7.32(m,1.87H),7.19-7.13(m,1H),6.36(td,J＝7.0,55.3Hz,1H),4.23-4.08(m,2H),3.04-3.00(m,1.74 H), 2.90(d, J＝9.4Hz, 0.13H), 2.66-2.61(m, 0.13H), 1.65(s, 7.83H), 1.59(s, 1.17H), 1.27-1.19(m, 3H) ; ¹³ C NMR (100MHz, CDCl ₃ ) δ(major+minor) 171.3, 166.2, 148.8, 140.3, 129.0, 125.0, 124.7, 122.9, 122.5, 115.2, 113.0 (t, J=236.8Hz), 85.3, 62.2, 36.8(d, J=9.6Hz), 36.3(d, J=9.6Hz), 35.6(t, J=34.1Hz), 28.2, 14.2; ¹⁹ F NMR(376MHz, CDCl ₃ )δ(major+minor)- 115.69(ABd,J=54.6,296.2Hz,major),-112.80(ABd,J=52.2,303.1Hz,minor),-114.78(ABd,J=53.3,297.6Hz,minor),-115.99(ABd,J =55.7, 296.2Hz, major); HRMS (ESI-TOF) Calcd. for C ₁₉ H ₂₂ F ₂ NO ₅ [M+H] ⁺ :382.1461; found: 382.1466.

The compound of formula (1) of the present invention has important biological activity, and the cytotoxicity test of two strains of tumor cells of human prostate (PC-3) and human lung cancer cell (A549) in vitro shows: the compound shown in this formula (1) Spiro[cyclopropane-1,3'-indoline]-2'-ketone compounds containing difluoromethyl group can inhibit the growth of tumor cells, and may be developed into new anti-tumor drugs.

The compound of formula (1) of the present invention or its pharmaceutically acceptable salt and its solvate can be combined with commonly used pharmaceutical adjuvants or carriers to prepare a pharmaceutical composition with tumor cell growth inhibitory activity and thus can be used for preventing and treating tumors. The various pharmaceutical compositions mentioned above can be in solid form, such as tablets, capsules, granules, powders, or in liquid form, such as injections, suspensions and emulsions. In addition, controlled release agents or sustained release agents or nano-agents known in the modern pharmaceutical industry can also be used.

The compound of formula (1) of the present invention or its pharmaceutically acceptable salt and solvate thereof can be combined with existing marketed antitumor drugs such as platinum drug cisplatin (DDP), deoxycytidine drug gemcitabine (Gemcitabine, Gemzar, Jianze ), paclitaxel (Paclitaxel), vinblastine (Vinorebine, NVB), camptothecin (Irinatecan, CPT-11), etoposide (Etoposide), etc. Used in combination, a cytotoxic composition with tumor growth inhibitory activity is prepared, which can be used for treating tumor diseases. Such pharmaceutical compositions can be in solid form, such as tablets, capsules, granules, and powders; or in liquid form, such as injections, suspensions, and emulsions. In addition, controlled release agents or sustained release agents or nano-agents known in the modern pharmaceutical industry can also be used.

Pharmacological Example 1: Cytotoxicity of Compound 4f, 4l, 4o or 4p to PC-3 Cells

PC-3 (human prostate cancer) cells were cultured with RPMI-1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin. Cells were added to 96 wells at a concentration of 5000 cells per well and cultured for 24 hours at 37 °C in an incubator with 5% _CO2 humidified air.

Cell viability was determined by the modified MTT method. After the cells were incubated for 24 hours, the newly prepared dimethyl sulfoxide solution of compound 4f, 4l, 4o or 4p was added to each well in a concentration gradient, so that the final concentration of the compound in the well was 6.25 μmol/L, respectively. 12.5μmol/L, 25μmol/L, 50μmol/L and 100μmol/L. After 48 hours, 10 μL of MTT (5 mg/mL) in phosphate buffer was added to each well, and after further incubation at 37° C. for 4 hours, unconverted MTT was removed by centrifugation for 5 minutes, and 150 μL of dimethyl sulfoxide was added to each well. The reduced MTT crystal formazan was dissolved, and the OD value was measured at a wavelength of 490 nm with a microplate reader. Among them, the half-inhibitory concentration IC ₅₀ of compound 4f, 4l, 4o or 4p on PC-3 cells was analyzed by spss software (version 19). The IC 50 of compound 4f against PC-3 tumor cells was 72.1 μmol/L; the IC ₅₀ of compound 4l against PC-3 tumor cells was 80.8 μmol/L _; the IC ₅₀ of compound 4o against PC-3 tumor cells was 25.7 μmol/L ; The IC ₅₀ of compound 4p on PC-3 tumor cells was 81.8 μmol/L; while the IC ₅₀ of positive control cisplatin on PC-3 tumor cells was 23.7 μmol/L.

Experimental conclusion: PC-3 cells are an effective tool and evaluation index for testing the cytotoxicity of compounds on tumor cells. This experiment shows that the difluoromethyl-containing spiro[cyclopropane-1,3'-indoline]-2'-ketone compound shown in this type of formula (1) has certain cytotoxicity to PC-3 cells, It is possible to develop new drugs with anti-tumor effects.

Pharmacological Example 2: Cytotoxicity of Compound 4n, 4o, 4q or 4r on A549 Cells

A549 (human lung cancer cells) were cultured with DMEM medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin. Cells were added to 96 wells at a concentration of 4000 cells per well and cultured for 24 hours at 37 °C in an incubator with 5% _CO2 humidified air.

Cell viability was determined by the modified MTT method. The specific method is as in Pharmacological Example 1. The IC ₅₀ of compound 4n against A549 tumor cells was 89.9 μmol/L; the IC ₅₀ of compound 4o against A549 tumor cells was 36.8 μmol/L; the IC ₅₀ of compound 4q against A549 tumor cells was 87.9 μmol/L; the compound 4r against A549 tumor cells The IC ₅₀ of the cells was 62.4 μmol/L; while the IC ₅₀ of the positive control cisplatin on A540 tumor cells was 23.0 μmol/L.

Experimental conclusion: A549 cells are an effective tool and evaluation index for testing the cytotoxicity of compounds on tumor cells. This experiment shows that the difluoromethyl-containing spiro[cyclopropane-1,3'-indoline]-2'-ketone compound shown in this type of formula (1) has certain cytotoxicity to A549 cells, and it is possible Developed into new drugs with anti-tumor effects.

From the above pharmacological examples, we can see that this type of compound has certain cytotoxicity to these two tumor cell lines. It can be seen that these compounds have the potential to be developed into antitumor drugs, and are worthy of further research.

Claims (3)

1. A difluoromethyl-containing spiro[cyclopropane-1,3'-indoline]-2'-ketone compound, characterized in that: the compound has a structure as shown in general formula (I): In the formula: R ¹ is methyl, methoxy, halogen; R ² is methyl, n-propyl, tert-butyl; R ³ is ethyl, phenyl, tert-butoxycarbonyl. 2. An antineoplastic pharmaceutical composition containing an effective amount of spiro[cyclopropane-1,3'-indoline]-2'-ketones containing difluoromethyl as claimed in claim 1 and pharmaceutical acceptable carrier. 3. The application of the difluoromethyl-containing spiro[cyclopropane-1,3'-indoline]-2'-one compound as claimed in claim 1 in the preparation of drugs for preventing and treating tumor diseases.