阅读说明：本技术 一种吲哚-2-酮brd4抑制剂及其制备与应用 (Indol-2-one BRD4 inhibitor and preparation and application thereof ) 是由 陈世武 徐宇 向蓉 于 2020-06-10 设计创作，主要内容包括：本发明公开一种吲哚-2-酮BRD4抑制剂及其制备与应用。本发明的吲哚-2-酮化合物由2-甲氧基-N-(2-氧吲哚-5-基)苯磺酰胺与酮或醛经克脑文格尔反应制备,或5-氨基-3-(丙烷-2-亚烷基)吲哚-2-酮与磺酰氯经磺酰胺化反应制备。本发明的用途是作为BRD4抑制剂,将所述的吲哚-2-酮化合物用于制备抗癌药物。(The invention discloses an indol-2-one BRD4 inhibitor and a preparation method and application thereof. The indole-2-ketone compound is prepared by performing a Kenawenger reaction on 2-methoxy-N- (2-oxyindole-5-yl) benzene sulfonamide and ketone or aldehyde, or performing a sulfonylation amination reaction on 5-amino-3- (propane-2-alkylidene) indole-2-ketone and sulfonyl chloride. The application of the invention is as BRD4 inhibitor, and the indole-2-ketone compound is used for preparing anticancer drugs.)

1. The invention provides indole-2-ketone shown as a structural formula I or pharmaceutically acceptable salt thereof:

wherein R is₁And R₂Is C1-C2 alkyl, and R₁And R₂Not hydrogen at the same time; or R₁And R₂The connection forms a quaternary ring structure; r₃Is substituted cyclohexyl, or substituted phenyl, or fused aryl;

preferably R₁Is hydrogen, or methyl, R₂Is C1-C2 alkyl; r₃Is 2-methoxyphenyl, or 4-methoxyphenyl, or 2-bromophenyl, or cyclohexyl, or 2-methoxy-5-bromophenyl, or 2-methoxy-5-fluorophenyl, or 2-methoxy-5-methylphenyl, or 2, 5-difluorophenyl, or 2-fluoro-5-methylphenyl, or 1-naphthyl, or 2-naphthyl, or 5-N, N-dimethyl-naphthyl-1, or 2, 3-dihydrobenzofuranyl-5。

More preferably R₁Is methyl, R₂A methyl group, R₃Is 2-methoxyphenyl, 4-methoxyphenyl or 2-methoxy-5-bromophenyl; or R₁And R₂Linked to form a cyclobutyl radical, R₃Is 2-methoxyphenyl.

2. The indol-2-ones of claim 1 can be prepared by reacting a compound of formula ii with a ketone or aldehyde directly via a knoevenagel reaction, or by reacting a compound of formula iii with a sulfonyl chloride via a sulfonylation reaction.

3. Indol-2-one according to claim 1 or a pharmaceutically acceptable salt thereof, as BRD4 inhibitor, for the preparation of an anticancer agent.

4. Indol-2-one according to claim 1 or a pharmaceutically acceptable salt thereof, for use in an anti-leukaemia medicament.

5. The indol-2-one or a pharmaceutically acceptable salt thereof of claim 1, for use in an anti-colon cancer medicament.

Technical Field

The invention relates to the field of organic chemistry and pharmaceutical chemistry, in particular to an indol-2-one BRD4 inhibitor and preparation and application thereof.

Background

The bromodomain and the super-terminal structure (BET) protein family, a class of proteins of the epigenetic code "read", able to specifically recognize acetylated lysine residues in histones H3 and H4 (Nature,2000,403(6765):41-45), consisting of bromodomains 2,3, 4 and T (BRD2, BRD3, BRD4 and BRDT) located on the nucleus and highly homologous in protein spatial structure (Cell,2012,149(1): 214-206231; Biological & Pharmaceutical Bulletin,2012,35(11): 2064-2068). Each protein comprises two subtypes, D1 and D2, and is located in the nucleus, wherein BRD2-4 is universally expressed in various tissue cells, while BRDT is specifically expressed only in tissues such as epithelial sperm cells, diploid epithelial sperm cells and round sperm (Journal of Medicinal Chemistry,2017,60(11), 4533-4558). The BRD4 can regulate and control the proliferation of cancer cells, influence the apoptosis and the transcription of the cells, participate in the processes of infiltration, metastasis and the like of tumor cells, and is one of important targets for research of antitumor drugs. In addition, BRD4 inhibitors can also be involved in the treatment of immunity, inflammation, diabetes, etc. by affecting inflammatory cytokines (IL-1 β, IL-6, IL-12 α, CXCL9 and CCL12) and nuclear factor (NF-kB).

At present, some BRD4 protein inhibitors enter clinical research, and show good treatment effects in clinical experimental research on treating hematoma such as Acute Myelogenous Leukemia (AML) and lymphoma and solid tumor such as breast cancer and prostate cancer. Among them, (+) -JQ1 was the first BRD4 inhibitor reported, and has demonstrated good inhibitory activity (IC) in the in vitro antiproliferative activity of leukemia AML and colon cancer HT-29 cells₅₀<0.5. mu.M) (Journal of Medicinal Chemistry,2016,59(4): 1565). (+) -JQ1 is able to inhibit the proliferation of tumor cells by down-regulating Bcl-2 and c-Myc expression as in Acute Myeloid Leukemia (AML). However, (+) -JQ1 was not studied clinically because of its rapid metabolism, high toxicity and poor pharmacokinetic profile in vivo (Nature,2010,468(7327): 1067). I-BET762 is a triazole BRD4 inhibitor, which has entered clinical stage in 2019 in the clinical treatment of lymphoma; ABBV-075 is another important class of BRD4 inhibitors, which have now entered clinical phase 2015 in clinical studies of breast cancer and AML. In view of potential application value of the BRD4 inhibitor, the invention discovers that an indol-2-one compound has higher inhibitory activity to BRD4 and also has better in-vitro proliferation inhibitory activity to leukemia and colon cancer cells; in terms of the preparation method, the invention can efficiently synthesize key intermediates and target compounds by a simple method.

Disclosure of Invention

The technical problem to be solved by the invention is to provide an indole-2-ketone compound as a BRD4 inhibitor.

The invention provides a compound shown as a structural formula I or a pharmaceutically acceptable salt thereof:

wherein R is₁And R₂Is C1-C2 alkyl, and R₁And R₂Not hydrogen at the same time; or R₁And R₂The connection forms a quaternary ring structure; r₃Is substituted cyclohexyl or substituted phenyl, or a fused aryl;

preferably R₁Is hydrogen or methyl, R₂Is an alkyl group; r₃Is 2-methoxyphenyl, or 4-methoxyphenyl, or 2-bromophenyl, or cyclohexyl, or 2-methoxy-5-bromophenyl, or 2-methoxy-5-fluorophenyl, or 2-methoxy-5-methylphenyl, or 2, 5-difluorophenyl, or 2-fluoro-5-methylphenyl, or 1-naphthyl, or 2-naphthyl, or 5-N, N-dimethyl-naphthyl-1, or 2, 3-dihydrobenzofuranyl-5.

More preferably R₁Is methyl, R₂A methyl group, R₃Is 2-methoxyphenyl, 4-methoxyphenyl or 2-methoxy-5-bromophenyl; or R₁And R₂Linked to form a cyclobutyl radical, R₃Is 2-methoxyphenyl.

The compound shown in the formula I can be prepared by directly performing a Kenaokuger reaction on a compound shown in the formula II and ketone or aldehyde, or performing a sulfonylation reaction on a compound shown in the formula III and sulfonyl chloride.

Some preferred structures of the present discovery:

the compound shown in the formula I or the pharmaceutically acceptable salt thereof is used as a BRD4 inhibitor and is used for preparing anticancer drugs.

Detailed Description

The following examples are provided to illustrate the present invention, but not to limit the present invention, and the simple modification of the preparation method of the present invention based on the idea of the present invention is within the protection scope of the present invention.

Example 1: preparation of 5-aminoindol-2-ones

5-Nitroindol-2-one (0.50g,2.81mmol) was taken in EtOH/H₂O-4: 1(10mL), slowly heated to 85 ℃, then added with iron powder and ammonium chloride, and further heated to react. After the reaction was completed, the reaction mixture was filtered while it was hot, and the filtrate was evaporated to dryness to obtain the product (0.41 g). Yellow solid, yield 99%.¹H NMR(600MHz,DMSO-d₆)δ9.95(s,1H),6.54(s,1H),6.51(d,J＝7.8Hz,1H),6.41(d,J＝7.8Hz,1H),5.03(s,2H),3.26(s,2H).

Example 2: preparation of 2-methoxy-N- (2-oxoindol-5-yl) benzenesulfonamide

Taking 5-aminoindole-2-ketone (0.70g,4.73mmol) and adding N, N-diisopropylethylamine (1.64mL, 9.46mmol) and o-methoxybenzenesulfonyl chloride (1.17g, 5.68mmol) into dichloromethane under the condition of stirring at room temperature, after the reaction is finished, carrying out suction filtration, and drying a filter cake to obtain a black solid (0.90 g). The yield is 60%;¹H NMR(600MHz,DMSO-d₆)δ10.24(s,1H),7.65(d,J＝7.2Hz,1H),7.54(d,J＝7.2Hz,1H),7.17(d,J＝8.4Hz,1H),7.00-6.95(m,2H),6.86(d,J＝8.4Hz,1H),6.61(d,J＝7.8Hz,1H),3.91(s,3H),3.37(s,2H).

example 3: preparation of 5-nitro-3- (propane-2-alkylidene) indol-2-ones

5-Nitroindol-2-one (2.00g,11.22mmol) was placed in a flask and 6mL of acetone solution was added at room temperature. Piperazine (0.67mL,6.73mmol) was added with stirring. After the reaction, the reaction mixture was filtered by suction and the filter cake was dried to give a yellow solid (2.10 g). The yield is 86%;¹H NMR(600MHz,DMSO-d₆)δ11.16(s,1H),8.27(d,J＝6.0Hz,1H),8.15-8.14(m,1H),6.99-6.97(m,1H),2.49(s,3H),2.42(s,3H).

example 4: preparation of 5-amino-3- (propane-2-alkylidene) indol-2-ones

Taking the prepared 5-nitro-3- (propane-2-alkylidene) indol-2-one (0.50g,2.81mmol) in EtOH/H₂O-4: 1(10mL), slowly heated to 85 ℃, then added with iron powder and ammonium chloride, and further heated to react. After the reaction was completed, the reaction mixture was filtered while it was hot, and the filtrate was evaporated to dryness to obtain a yellow solid (0.41 g). The yield is 90%;¹H NMR(600MHz,DMSO-d₆)δ9.98(s,1H),6.91(s,1H),8.50-8.37(m,2H),5.19(brs,2H),2.44(s,3H),2.21(s,3H).

example 5: synthesis of compound (Z) -N- (3-ethylidene-2-oxoindol-5-yl) -2-methoxybenzenesulfonamide

2-methoxy-N- (2-oxoindol-5-yl) benzenesulfonamide (0.10g,0.31mmol) was taken in a flask, and an appropriate amount of dichloromethane and piperazine (0.02mL,0.16mmol) were added under nitrogen protection at room temperature and stirred for two minutes. Continuing under nitrogen protection, acetaldehyde (3.10mmol) was slowly added dropwise. After the reaction, the solvent was evaporated to dryness, and column chromatography was performed to obtain 238mg of a yellow solid. The product detection data were as follows:

the yield is 40 percent; m.p.195-197 ℃; HPLC (MeOH: H)₂O＝85:15,1.0mL/min)t_R＝4.04min,99.3％.¹H NMR(400MHz,DMSO-d₆)δ10.28(s,1H),9.60(s,1H),7.63(d,J＝8.0Hz,1H),7.51(t,J＝8.0Hz,1H),7.26(s,1H),7.14(d,J＝8.4Hz,1H),6.95(t,J＝7.6Hz,1H),6.89(d,J＝8.4Hz,1H),6.79(q,J＝8.4Hz,1H),6.63(d,J＝8.4Hz,1H),3.88(s,3H),2.07(d,J＝8.4Hz,3H)；¹³C NMR(100MHz,DMSO-d₆)δ168.2,156.7,139.3,136.7,135.5,131.7,130.7,129.2,126.6,122.9,122.8,120.5,117.9,113.1,110.2,56.5,15.2.MS(ESI)m/z 343.2for[M-H]^-.

In the experiments for inhibiting enzymes and inhibiting cell growth described later, the sample number of this example was 1.

Example 6: synthesis of compound (Z) -2-methoxy-N- (2-oxo-3-propylidene indolin-5-yl) benzenesulfonamide

The procedure was the same as in example 5 except that propionaldehyde was used in place of acetaldehyde to obtain a yellow solid. The product detection data were as follows:

the yield is 31 percent; m.p.164-166 ℃; HPLC (MeOH: H)₂O＝85:15,1.0mL/min)t_R＝3.76min,99.1％.¹H NMR(600MHz,DMSO-d₆)δ10.32(s,1H),9.62(s,1H),7.66(d,J＝8.4Hz,1H),7.56(t,J＝7.8Hz,1H),7.24(s,1H),7.19(d,J＝8.4Hz,1H),6.99(t,J＝7.8Hz,1H),6.94(d,J＝8.4Hz,1H),6.73(t,J＝7.8Hz,1H),6.67(d,J＝8.4Hz,1H),3.92(s,3H),2.48-2.45(m,2H),1.13(t,J＝7.2Hz,3H)；¹³C NMR(150MHz,DMSO-d₆)δ156.3,142.5,134.9,131.3,130.2,127.5,122.4,122.1,112.6,109.8,55.9,21.8,18.5.MS(ESI)m/z 381.3for[M+Na]⁺.

In the experiments for inhibiting enzymes and inhibiting cell growth described later, sample No. 2 was used in this example.

Example 7: synthesis of the Compound 2-methoxy-N- (2-oxo-3- (propane-2-alkylene) indol-5-yl) benzenesulfonamide

The procedure was as in example 5 except that acetone was used instead of acetaldehyde to give a yellow solid. The product detection data were as follows:

the yield is 70 percent; m.p.180-182 ℃; HPLC (MeOH: H)₂O＝85:15,1.0mL/min)t_R＝3.72min,95.8％.¹H NMR(600MHz,DMSO-d₆)δ10.31(s,1H),9.56(s,1H),7.65(d,J＝8.4Hz,1H),7.54(d,J＝7.2Hz,1H),7.25(s,1H),7.17(d,J＝7.8Hz,1H),7.00-6.97(m,1H),6.91-6.89(m,1H),6.63(d,J＝8.4Hz,1H),3.92(s,3H),2.46(s,3H),2.17(s,3H)；¹³C NMR(150MHz,DMSO-d₆)δ168.4,156.3,154.6,137.3,134.9,130.8,130.2,126.3,123.7,122.5,121.5,120.0,117.9,112.6,108.9,56.0,24.4,22.2.MS(ESI)m/z 357.2for[M-H]^-.

In the experiments for inhibiting enzymes and inhibiting cell growth described later, the sample No. 3 was used in this example.

Example 8: synthesis of compound N- (3-cyclobutylidene-2-oxyindole-5-yl) -2-methoxybenzenesulfonamide

The procedure was as in example 5 except that cyclobutanone was used instead of acetaldehyde to give a yellow solid. The product detection data were as follows:

the yield is 60 percent; m.p.148-150 ℃; HPLC (MeOH: H)₂O＝85:15,1.0mL/min)t_R＝3.93min,98.8％.¹H NMR(600MHz,DMSO-d₆)δ10.20(s,1H),9.53(brs,1H),7.62(brs,1H),7.54(brs,1H),7.16(brs,1H),6.97(brs,1H),6.87(brs,2H),6.61(brs,1H),3.90(s,3H),3.21(brs,2H),3.07(brs,2H),2.25(brs,2H)；¹³C NMR(150MHz,DMSO-d₆)δ167.5,161.8,156.3,137.6,134.9,131.0,130.2126.3,123.0,121.5,120.0,115.7,112.6,109.3,55.9,33.4,32.5,18.9.MS(ESI)m/z 369.2for[M-H]^-.

In the experiments for inhibiting enzymes and inhibiting cell growth described later, the sample No. 4 was used in this example.

Example 9: synthesis of the Compound 2-bromo-N- (2-oxo-3- (propane-2-alkylene) indol-5-yl) benzenesulfonamide

The compound 5-amino-3- (propane-2-alkylene) indol-2-one (0.10g, 0.53mmol) was taken in a flask, and DCM (3mL), DIPEA (0.19mL, 1.06mmol) and o-bromobenzenesulfonyl chloride (0.64mmol) were added in this order to react at room temperature. After the reaction was completed, DCM: column chromatography with MeOH 150:1 gave a yellow solid. The product detection data were as follows:

the yield is 65 percent; m.p.145-147 ℃; HPLC (MeOH: H)₂O＝85:15,1.0mL/min)t_R＝4.19min,99.2％.¹H NMR(600MHz,DMSO-d₆)δ10.37(s,1H),10.19(s,1H),7.94(d,J＝7.2Hz,1H),7.81(d,J＝7.8Hz,1H),7.50(brs,2H),7.24(s,1H),6.90(d,J＝7.8Hz,1H),6.65(d,J＝9.0Hz,1H),2.45(s,3H),2.14(s,3H)；¹³C NMR(150MHz,DMSO-d₆)δ168.3,154.8,138.3,137.6,135.3,134.4,131.8,129.8,128.1,123.8,122.4,121.6,119.2,117.8,109.1,24.4,22.2.MS(ESI)m/z 405.1for[M-H]^-and 407.1for[M+H]⁺.

In the experiments for inhibiting enzymes and inhibiting cell growth described later, the sample No. 5 was used in this example.

Example 10: synthesis of the compound N- (2-oxo-3- (propane-2-alkylidene) indol-5-yl) cyclohexanesulfonamide

The procedure was as in example 9 except that o-bromobenzenesulfonyl chloride was replaced with cyclohexylsulfonyl chloride to give a yellow solid. The product detection data were as follows:

the yield is 28 percent; m.p.156-158 ℃; HPLC (MeOH: H)₂O＝85:15,1.0mL/min)t_R＝4.50min,95.5％.¹H NMR(600MHz,CDCl₃)δ10.40(s,1H),10.22(s,1H),7.78(s,1H),7.02(d,J＝7.8Hz,1H),6.95(d,J＝8.4Hz,1H),2.35-2.31(m,5H),1.85-1.79(m,5H),1.65-1.65(m,1H),1.45-1.39(m,3H),1.30-1.22(m,3H)；¹³C NMR(150MHz,CDCl₃)δ169.8,158.8,136.8,131.3,125.2,122.8,121.9,119.2,110.1,60.2,29.7,26.9,26.5,25.6,25.1,23.6.MS(ESI)m/z 333.2for[M-H]^-.

In the experiments for inhibiting enzymes and inhibiting cell growth described later, the sample No. 6 was used in this example.

Example 11: synthesis of the compound 5-bromo-2-methoxy-N- (2-oxo-3- (propane-2-alkylidene) indol-5-yl) benzenesulfonamide

The procedure was as in example 9 except that o-bromobenzenesulfonyl chloride was replaced with 5-bromo-2-methoxybenzenesulfonyl chloride to give a yellow solid. The product detection data were as follows:

the yield is 43 percent; m.p.143-145 ℃; HPLC (MeOH: H)₂O＝85:15,1.0mL/min)t_R＝4.39min,96.6％.¹H NMR(600MHz,DMSO-d₆)δ10.36(s,1H),9.78(s,1H),7.72(d,J＝8.4Hz,1H),7.68(s,1H),7.21(s,1H),7.16(d,J＝8.4Hz,1H),6.89(d,J＝7.8Hz,1H),6.65(d,J＝8.4Hz,1H),3.90(s,1H),2.46(s,3H),2.17(s,3H)；¹³C NMR(100MHz,DMSO-d₆)δ168.8,156.0,155.3,138.0,137.8,132.5,130.7,128.6,124.3,122.9,122.2,118.4,115.7,111.4,109.6,56.9,24.9,22.7.MS(ESI)m/z435.1for[M-H]^-and 437.1for[M+H]⁺.

In the experiments for inhibiting enzymes and inhibiting cell growth described later, sample No. 7 was used in this example.

Example 12: synthesis of the compound 5-fluoro-2-methoxy-N- (2-oxo-3- (propane-2-alkylene) indol-5-yl) benzenesulfonamide

The procedure was as in example 9 except that o-bromobenzenesulfonyl chloride was replaced with 5-fluoro-2-methoxybenzenesulfonyl chloride to give a yellow solid. The product detection data were as follows:

the yield is 17%; m.p.205-207 ℃; HPLC (MeOH: H)₂O＝85:15,1.0mL/min)t_R＝4.11min,99.9％.¹H NMR(600MHz,DMSO-d₆)δ10.35(s,1H),9.75(s,1H),7.43-7.39(m,2H),7.21(d,J＝6.0Hz,2H),6.89(d,J＝7.2Hz,1H),6.64(d,J＝8.4Hz,1H),3.90(s,3H),2.46(s,3H),2.17(s,3H)；¹³C NMR(150MHz,DMSO-d₆)δ168.3,153.9,152.8,137.5,130.3,123.7,122.5,121.7,117.9,116.7,114.4,109.0,56.6,24.4,22.2.MS(ESI)m/z 375.2for[M-H]^-.

In the experiments for inhibiting enzymes and inhibiting cell growth described later, the sample No. 8 was used in this example.

Example 13: synthesis of the Compound 2-methoxy-5-methyl-N- (2-oxo-3- (propane-2-alkylidene) indol-5-yl) benzenesulfonamide

The procedure was as in example 9 except that o-bromobenzenesulfonyl chloride was replaced with 2-methoxy-5-methylbenzenesulfonyl chloride to give a yellow solid. The product detection data were as follows:

the yield is 61%; m.p.231-233 ℃; HPLC (MeOH: H)₂O＝85:15,1.0mL/min)t_R＝3.95min,98.8％.¹H NMR(600MHz,DMSO-d₆)δ10.32(s,1H),9.51(brs,1H),7.45(s,1H),7.32(d,J＝6.6Hz,1H),7.22(s,1H),7.05(d,J＝8.4Hz,1H),6.89(d,J＝7.2Hz,1H),6.62(d,J＝7.8Hz,1H),3.86(s,1H),2.45(s,3H),2.19(s,3H),2.16(s,3H)；¹³C NMR(100MHz,DMSO-d₆)δ168.8,155.0,154.6,137.7,135.7,131.3,130.7,129.5,126.4,124.2,123.0,121.9,118.2,112.9,109.5,56.5,24.9,22.7,20.2.MS(ESI)m/z 371.2for[M-H]^-.

In the experiments for inhibiting enzyme and inhibiting cell growth described later, the sample No. 9 was used in this example.

Example 14: synthesis of the compound 2, 5-difluoro-N- (2-oxo-3- (propane-2-alkylene) indol-5-yl) benzenesulfonamide

The procedure was as in example 9, except that o-bromobenzenesulfonyl chloride was replaced with 2, 5-difluoro-benzenesulfonyl chloride to give a yellow solid. The product detection data were as follows:

the yield is 17%; m.p.238-240 ℃; HPLC (MeOH: H)₂O＝85:15,1.0mL/min)t_R＝4.22min,95.8％.¹H NMR(600MHz,DMSO-d₆)δ10.41(s,1H),10.39(brs,1H),7.56(brs,1H),7.51-7.48(m,2H),7.24(s,1H),6.89(d,J＝8.4Hz,1H),6.69(d,J＝8.4Hz,1H),2.46(s,3H),2.17(s,3H)；¹³C NMR(150MHz,DMSO-d₆)δ168.4,157.9,155.2,138.0,129.5,123.9,122.4,122.2,119.3,118.4,116.7,109.2,24.4,22.3.MS(ESI)m/z 363.2for[M-H]^-.

In the experiments for inhibiting enzymes and inhibiting cell growth described later, the sample number of this example was 10.

Example 15: synthesis of the Compound 2-fluoro-5-methyl-N- (2-oxo-3- (propane-2-alkylidene) indol-5-yl) benzenesulfonamide

The procedure was as in example 9 except that o-bromobenzenesulfonyl chloride was replaced with 2-fluoro-5-methylbenzenesulfonyl chloride to give a yellow solid. The product detection data were as follows:

the yield is 39%; m.p.240-242 ℃; HPLC (MeOH: H)₂O＝85:15,1.0mL/min)t_R＝4.18min,96.6％.¹H NMR(600MHz,DMSO-d₆)δ10.39(s,1H),10.17(s,1H),7.54(d,J＝5.4Hz,1H),7.46(d,J＝3.0Hz,1H),7.29(d,J＝8.4Hz,1H),7.25(s,1H),6.90(d,J＝7.2Hz,1H),7.68(d,J＝8.4Hz,1H),2.48(s,3H),2.28(s,3H),2.17(s,3H)；¹³C NMR(150MHz,DMSO-d₆)δ168.3,157.1,155.5,154.8,137.7,135.9,134.3,130.1,130.0,123.9,122.5,121.7,117.9,116.9,116.8,109.1,24.3,22.2,19.9.MS(ESI)m/z 359.2for[M-H]^-.

In the experiments for inhibiting enzyme and inhibiting cell growth described later, the sample No. of this example was 11.

Example 16: synthesis of the Compound N- (2-oxo-3- (propane-2-alkylene) indol-5-yl) -2, 3-dihydrobenzofuran-5-sulfonamide

The procedure was as in example 9 except that o-bromobenzenesulfonyl chloride was replaced with 2, 3-dihydrobenzofuran-5-sulfonyl chloride to give a yellow solid. The product detection data were as follows:

the yield is 61%; m.p.159-161 ℃; HPLC (MeOH: H)₂O＝85:15,1.0mL/min)t_R＝4.05min,98.1％.¹H NMR(600MHz,DMSO-d₆)δ10.35(s,1H),9.67(s,1H),7.54(s,1H),7.42(d,J＝8.4Hz,1H),7.21(s,1H),6.83(d,J＝7.2Hz,2H),6.65(d,J＝8.4Hz,1H),4.58(t,J＝8.4Hz,2H),3.17(t,J＝8.4Hz,2H),2.46(s,3H),2.16(s,3H)；¹³C NMR(100MHz,DMSO-d₆)δ168.9,163.5,155.2,137.9,131.5,131.4,129.1,128.7,124.6,124.3,122.9,122.4,118.8,109.6,109.4,72.6,55.4,28.8,24.9,22.7.MS(ESI)m/z 369.2for[M-H]^-.

In the experiments for inhibiting enzymes and inhibiting cell growth described later, the sample number of this example was 12.

Example 17: synthesis of the Compound N- (2-oxo-3- (propane-2-alkylene) indol-5-yl) naphthalene-2-sulfonamide

The procedure was as in example 9 except that o-bromobenzenesulfonyl chloride was replaced with naphthalene-2-sulfonyl chloride to give a yellow solid. The product detection data were as follows:

the yield is 60 percent; m.p.244-246 ℃; HPLC (MeOH: H)₂O＝85:15,1.0mL/min)t_R＝4.37min,97.0％.¹H NMR(600MHz,DMSO-d₆)δ10.35(s,1H),9.99(s,1H),8.32(s,1H),8.08(d,J＝8.4Hz,2H),7.99(d,J＝7.2Hz,1H),7.74(d,J＝8.4Hz,1H),7.66(d,J＝7.8Hz,1H),7.62(d,J＝7.2Hz,1H),7.20(s,1H),6.85(d,J＝8.4Hz,1H),6.61(d,J＝7.8Hz,1H),2.41(s,3H),2.05(s,3H)；¹³C NMR(150MHz,DMSO-d₆)δ168.3,154.7,137.7,136.5,134.1,131.5,130.4,129.2,129.1,128.8,127.9,127.7,127.6,123.8,122.4,122.2,118.6,109.5,109.0,24.3,22.1.MS(ESI)m/z 377.2for[M-H]^-.

In the experiments for inhibiting enzyme and inhibiting cell growth described later, the sample number of this example was 13.

Example 18: synthesis of the Compound N- (2-oxo-3- (propane-2-alkylene) indol-5-yl) naphthalene-1-sulfonamide

The procedure was as in example 9 except that o-bromobenzenesulfonyl chloride was replaced with naphthalene-1-sulfonyl chloride to give a yellow solid. The product detection data were as follows:

the yield is 50 percent; m.p.135-137 deg.c; HPLC (MeOH: H)₂O＝85:15,1.0mL/min)t_R＝4.27min,98.8％.¹H NMR(600MHz,DMSO-d₆)δ10.29(s,1H),10.23(s,1H),8.73(d,J＝8.4Hz,1H),8.18(d,J＝9.0Hz,1H),8.05(d,J＝6.0Hz,2H),7.72(d,J＝7.8Hz,1H),7.66(d,J＝7.2Hz,1H),7.55(d,J＝7.8Hz,1H),2.39(s,3H),1.92(s,3H)；¹³C NMR(100MHz,DMSO-d₆)δ168.8,155.2,137.9,134.8,134.7,134.1,130.7,130.5,129.5,128.5,128.0,127.4,124.9,124.2,122.8,122.3,118.4,109.5,55.4,24.7,22.6.MS(ESI)m/z 377.2for[M-H]^-.

In the experiments for inhibiting enzymes and inhibiting cell growth described later, the sample No. 14 was used in this example.

Example 19: synthesis of the compound 5- (dimethylamino) -N- (2-oxo-3- (propan-2-alkylene) indol-5-yl) naphthalene-1-sulfonamide

The procedure was as in example 9 except that o-bromobenzenesulfonyl chloride was replaced with 2,5- (dimethylamino) naphthalene-1-sulfonyl chloride to give a yellow solid. The product detection data were as follows:

the yield is 28 percent; m.p.220-222 ℃; HPLC (MeOH: H)₂O＝85:15,1.0mL/min)t_R＝4.70min,97.4％.¹H NMR(600MHz,DMSO-d₆)δ10.30(s,1H),10.17(s,1H),8.41-8.37(m,2H),8.04(d,J＝6.6Hz,1H),7.60(d,J＝7.8Hz,1H),7.17(t,J＝7.8Hz,1H),7.24(d,J＝7.2Hz,1H),6.96(s,1H),6.79(d,J＝7.8Hz,1H),6.58(d,J＝7.8Hz,1H),2.79(s,6H),2.39(s,3H),1.90(s,3H)；¹³C NMR(100MHz,DMSO-d₆)δ168.8,155.1,151.9,137.9,135.2,130.8,130.4,130.3,129.6,129.3,128.5,124.2,123.9,122.8,122.3,119.3,118.4,115.6,109.5,45.5,24.7,22.7.MS(ESI)m/z 420.3for[M-H]^-.

In the experiments for inhibiting enzyme and inhibiting cell growth described later, the sample number of this example was 15.

Example 20: BRD4 inhibitory Activity of Compounds 1-15

By adopting a TR-FRET method and selecting BRD4 inhibitors (+) -JQ1 and PFI-1 as positive compounds, the inhibitory activity of indole-2-ketone compounds 1-15 on BRD4(D1) in vitro is researched, and the experimental result is shown in Table 1.

TR-FRET detection: compound solutions of different concentration gradients were prepared and added to a 384-well Source plate for use, and equal amounts of positive control compound and DMSO were added. Then preparing a buffer solution and a protein solution; centrifuging and incubating; adding the prepared polypeptide solution, and centrifuging; then adding the solution to be tested, continuously centrifuging and incubating. To which a compound is reactedThereafter, EnVisio readings were taken and IC was calculated using GraphPad Prism 5₅₀The value is obtained.

Example 21: proliferation inhibitory Activity of Compounds 1-15

The in vitro cytotoxic activity of compounds against HT-29 or WI-38 cells was determined by MTT assay and against HL-60 cells by CCK8 assay, again with (+) -JQ1 and PFI-1 as a positive control, and the results are given in Table 1.

MTT method: HT-29 or WI-38 cells (human embryonic lung cells) (5X 10) in logarithmic growth phase³One cell/well) was added to a 96-well plate containing 10% FBS, and the mixture was incubated (37 ℃ C., 5.0% CO)₂) The culture was carried out for 24 hours. Then adding the medicines with different concentrations, and culturing for 72 hours. MTT solution (5mg/mL) was added, the mixture was incubated for 4 hours, the culture solution was discarded, DMSO was added, shaking was performed, and the absorbance was measured at 490nM using a microplate reader. IC was calculated using GraphPad Prism Software version 5.02₅₀The value is obtained.

CCK8 method: the culture and dosing method is similar to the MTT method. Proliferation inhibitory activity of HL-60 cells was measured using CCK8 solution instead of MTT solution in the above method.

The BRD4(D1) inhibitory and proliferation inhibitory activities of the compounds of Table 1

^aMean of two experiments

^bMean of three experiments, time 72 h

^cHL-60 was determined by the CCK8 method, HT-29 and WI-38 were determined by the MTT method

The experimental results show that most of the compounds of the present invention have potent inhibitory activity against BRD4(D1), wherein the inhibitory activity against BRD4(D1) is stronger for compound 1, compounds 3-11 and compound 14 than for positive control PFI-1, and IC is higher for compound 1, compounds 3-11 and compound 14 than for positive control PFI-1₅₀Values were all less than 70 nM. Meanwhile, the compounds have better antiproliferative activity on HL-60 and HT-29 cells and have lower toxicity on WI-38 cells. Wherein, the inhibitory activity of the compounds 2-5 and 7-9 on HL-60 is strongest; to be combined withThe compound 2, the compound 10 and the compound 15 have the strongest inhibitory activity on HT-29; the inhibitory activity of the compound 10, the compound 16, the compound 19 and the compound 21 on WI-38 is the weakest, and the toxicity on normal cells is lower. Especially, compound 3 and compound 7 not only have strong BRD4 inhibitory activity, but also have good proliferation inhibitory activity on HL-60 and HT-29 cancer cells, and have low toxicity on normal cells.

Comparative example

Comparison of therapeutic effects

Compared with (+) -JQ1 and PFI-1, the novel BRD4 antitumor active compounds find that the growth inhibition activity and the BRD4 inhibition activity of the compound 3 on human granulocyte leukemia cell HL-60 and human colon cancer cell HT-29 are both stronger than those of the PFI-1, and the toxicity on WI-38 cells is less than that of the (+) -JQ1 and the PFI-1. Wherein the inhibitory activity of the compound 3 on HL-60 and HT-29 is 2 times and 3 times of that of PFI-1, the toxicity of normal human embryonic lung fibroblast WI-38 is less than 1/2 of PFI-1 and less than 1/8 of (+) -JQ 1. And has an inhibitory activity against BRD4 of 4 times or more as high as PFI-1.