CN113929639A

CN113929639A - Antitumor compound with GSTP1 as target spot, and preparation method and application thereof

Info

Publication number: CN113929639A
Application number: CN202010602144.XA
Authority: CN
Inventors: 苟少华; 刘晴晴
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2020-06-29
Filing date: 2020-06-29
Publication date: 2022-01-14
Anticipated expiration: 2040-06-29
Also published as: CN113929639B

Abstract

The invention discloses an antitumor compound taking GSTP1 as a target spot and pharmaceutically acceptable salts thereof, which comprise a compound with a structure shown as a formula I, wherein n is 1-3; when R is¹When is H, R²Is aryl or alkyl; when R is²When the aryl is selected from phenyl, mono-substituted or di-substituted phenyl, and the substituent is CH₃、OCH₃、OCF₃F, Cl or Br, the substituents being in any of the ortho, meta or para positions; when R is²When it is an alkyl group, it is selected from C_1～4Dimethylamino radical of straight-chain alkyl, C_1～4Straight chain alkylDiethylamino group of (1), C_1～4Morpholinyl of straight-chain alkyl, C_1～4Piperidinyl or C of straight-chain alkyl groups_1～4Pyrrolidinyl of straight chain alkyl; or, R¹And R²Morpholine or piperidyl which forms a cyclic structure with the N atom.

Description

Antitumor compound with GSTP1 as target spot, and preparation method and application thereof

Technical Field

The invention relates to a compound, a preparation method and application thereof, in particular to an antitumor compound taking GSTP1 as a target spot, and a preparation method and application thereof.

Background

Glutathione mercaptotransferase (GSTs) belongs to II metabolic enzymes, is an extremely important exogenous chemical metabolic enzyme, is closely related to the generation and development of various tumors, and plays an important role in the process of cell detoxification. GSTs are a large super gene family, mainly including cytoplasmic, mitochondrial and microsomal families, wherein the cytoplasmic family is the most complex and most closely related to human diseases. The cytoplasm family mainly comprises seven subtypes of alpha, pi, mu, theta, kappa, omega and delta.

GSTP1(GST pi) has multiple biological functions, and is frequently overexpressed in tumor cells, such as human colon cancer, pancreatic cancer, uterine cancer, gastric cancer, breast cancer, lymphoma, liver cancer, melanoma, and osteosarcoma, but is lower in normal cells. Therefore, the GSTP 1-targeted drug plays an important role in tumor cells and has the lowest toxic and side effects on normal cells. Many exogenous chemicals are highly susceptible to the formation of reactive intermediates in biotransformation reactions, which can cause damage to the body by covalently bonding to important components of cellular biological macromolecules. GSTP1 can catalyze the combination of sulfhydryl of Glutathione (GSH) and various electrophilic compounds from in vivo and in vitro sources to form a compound with larger polarity, and Multidrug Resistance Protein (MRP) and P-glycoprotein (P-gp) can pump the compound out of the body, thereby realizing the detoxification of GSTP 1. Meanwhile, most chemotherapy drugs can be catalyzed by GSTP1 to combine with GSH to form a compound, and the compound is pumped out of the body by multi-drug resistant protein, so that the action time of the drugs in the body is shortened, the anti-tumor effect can not be effectively exerted, and the serious tumor cell multi-drug resistance is caused clinically. c-jun amino terminal kinase (JNK) plays an important role in various physiological and pathological processes such as cell cycle, reproduction, apoptosis and cell stress. Under a non-stress state, GSTP1 forms a complex with JNK through protein-protein interaction in a monomer form, so that the JNK is inactivated, and tumor cells are prevented from apoptosis. Under stress conditions, GSTP1 and JNK separate to form homodimers, JNK can mediate phosphorylation of c-jun protein to activate apoptosis pathway, but when GSTP1 expression is increased, apoptosis is inhibited, and tumor cells generate drug resistance. The GSTP1 can effectively promote tumor cell apoptosis, so GSTP1 has become one of the targets for researching antitumor drugs.

Currently, GSTP1 inhibitors are mainly advantageous for uric acid (EA) analogs, GSH-conjugates, GSH analogs, NBDHEX (6- (7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol) and analogs thereof, and the like. NBDHEX is a GSTP1 inhibitor reported by Duvicat university of Italy in 2005, the structure of which is shown as (2) in the following formula, and the structure of the derivative is shown as (3), and the derivative has strong antitumor activity on melanoma and osteosarcoma. Because of the high selectivity of GSTM2 subtype, the targeting property to tumor cells is low, and the poor water solubility also hinders the intensive research on the tumor cells. GSTM2 protein is expressed in many non-cancer tissues, and GSTP1 is expressed at high level in many solid tumors, which can promote the combination of several antitumor drugs and glutathione, so that the antitumor drugs are excreted from cells, and drug resistance is generated. The GSTP1 protein can effectively activate JNK pathway and promote tumor cell apoptosis.

Wherein n is 1, 2, 3.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to utilize hydroxyl in the structure of known compound NBDHEX to react with naphthenic acid anhydride to introduce carboxyl, and then introduce amine with different substituent groups through esterification reaction to obtain a compound with anti-tumor effect so as to obtain a high-efficiency low-toxicity medicament; the invention also aims to provide a preparation method of the compounds and discloses application of the compounds serving as GSTP1 inhibitors in preparing medicaments for treating lung cancer, melanoma and osteosarcoma.

The technical scheme is as follows: the antitumor compound taking GSTP1 as a target and pharmaceutically acceptable salts thereof comprise a compound with a structure shown in a formula I:

wherein, in formula I, n is 1-3;

when R is¹When is H, R²Is aryl or alkyl; when R is²When the aryl is phenyl, mono-substituted or di-substituted phenyl, the substituent is CH₃、OCH₃、OCF₃F, Cl or Br, the substituents may be in any of the ortho, meta or para positions; when R is²When it is an alkyl group, it may be C_1～4Dimethylamino radical of straight-chain alkyl, C_1～4Diethylamino of straight-chain alkyl, C_1～4Morpholinyl of straight-chain alkyl, C_1～4Piperidinyl or C of straight-chain alkyl groups_1～4Pyrrolidinyl of straight chain alkyl;

or, R¹And R²Morpholine or piperidine which may form a cyclic structure with the N atom.

Preferably, the GSTP 1-targeted antitumor compound and the pharmaceutically acceptable salt thereof are selected from the following compounds 4a-4 t:

TABLE 1 structures of representative Compounds 4a-4t

The preparation method of the antitumor compound taking GSTP1 as a target point and the pharmaceutically acceptable salt thereof comprises the following steps:

TBTU stands for condensing agent tetrafluoroboric acid-2- (1H-benzotriazole-1-yl) -1,1,3, 3-tetramethylammonium, Et₃N represents triethylamineAnd DMF represents the solvent N, N-dimethylformamide.

Further, the preparation method of the antitumor compound with GSTP1 as the target point and the pharmaceutically acceptable salt thereof specifically comprises the following steps:

dissolving compound 3 and TBTU in anhydrous DMF, stirring at room temperature, adding Et₃And N, adding different substituted secondary amines, stirring the reaction solution at 30-40 ℃ for 1-3h, then removing the solvent under reduced pressure, and separating the concentrated solution by silica gel column chromatography, wherein the eluent is a mixed solvent of dichloromethane and methanol to obtain a yellow or brown solid product, wherein the compounds 4o and 4r are oily substances.

Further, the preparation method of the antitumor compound with GSTP1 as the target and the pharmaceutically acceptable salt thereof comprises the following steps:

in formula III, n is 1-3; DMAP for 4-dimethylaminopyridine and DCM for CH₂Cl₂。

Further, the preparation method of the antitumor compound taking GSTP1 as a target and the pharmaceutically acceptable salt thereof specifically comprises the following steps:

1) synthesis of compound 2 (according to us patent: US 8796317): dissolving compound 1 named 4-chloro-7-nitro-2, 1, 3-benzoxadiazole in neutral ethanol and potassium phosphate buffer solution, stirring at room temperature for 5-15min, adding 6-mercapto-1-hexanol, reacting at room temperature for 20-28h, and regulating with KOH to keep the reaction neutral; after the reaction is finished, adding 3-bromopyruvic acid to consume unreacted 6-mercapto-1-hexanol, stirring for 1-2h, filtering, and washing a filter cake with water to obtain a dark yellow solid compound 2;

2) synthesis of Compound 3 (refer to invention patent application No.: 201811396573.5): dissolving 2 in CH₂Cl₂Adding naphthenic dianhydride and DMAP, reacting overnight at 40-50 ℃, cooling to room temperature, concentrating reaction liquid, and performing column chromatography separation to obtain yellowAnd 3, a color product, and an eluent is a mixed solvent of dichloromethane and methanol.

A pharmaceutical composition comprising an anti-tumour compound targeting GSTP1, according to formula I or any one of compounds 4a-4t, and pharmaceutically acceptable salts thereof.

The antitumor compound taking GSTP1 as a target and the pharmaceutically acceptable salt thereof are applied to a GSTP1 inhibitor.

The application of the antitumor compound taking GSTP1 as a target point and pharmaceutically acceptable salts thereof in a GSTM2 inhibitor.

The antitumor compound taking GSTP1 as a target spot and pharmaceutically acceptable salts thereof are applied to the preparation of medicines for treating lung cancer, melanoma and osteosarcoma.

Has the advantages that: the invention utilizes the hydroxyl in the structure of known compound NBDHEX to react with naphthenic acid anhydride to introduce carboxyl, and then introduces amine with different substituent groups through esterification reaction to obtain a novel compound with anti-tumor effect. The selective inhibition of glutathione transferase and in vitro antitumor activity test show that the compound of the present invention has obvious inhibition effect on some tumor cells and may be used in preparing medicine.

Detailed Description

Synthesis of compound 2 according to us patent: US8796317, the synthesis of Compound 3, is referred to the patent of the invention, application No. 201811396573.5. Reactant 1 was purchased from Shanghai Aladdin Biotechnology Ltd. The molecular structure of the compound prepared by the method is determined by nuclear magnetic hydrogen spectrum, carbon spectrum and high-resolution mass spectrum.

Example 1

Preparation of Compound 2(NBDHEX) and 3

(1) Preparation of Compound 2

4.0g (20mmol) of Compound 1 (4-chloro-7-nitro-2, 1, 3-benzoxadiazole) was dissolved in a buffer solution of ethanol and potassium phosphate at pH 7.0, stirred at room temperature for 10min, then 10.7g (40mmol) of 6-mercapto-1-hexanol was added, and the mixture was reacted at room temperature for 24h and adjusted with 1M KOH to keep the solution neutral. After completion of the reaction, 5.0g (30mmol) of 3-bromopyruvic acid was added, stirred for 1 hour, filtered, and the filter cake was washed with water (20 mL. times.3) to give Compound 2 as a dark yellow solid.

¹H NMR(600MHz,CDCl₃)δ8.39(d,J＝7.9Hz,1H),7.15(d,J＝7.9Hz,1H),3.65(t,J＝6.4Hz,2H),3.26(t,J＝7.4Hz,2H),1.88–1.84(m,2H),1.61–1.55(m,4H),1.47–1.42(m,2H).

HRMS(ESI)m/z calcd for C₁₂H₁₅N₃O₄S[M+Na]⁺ 320.0675,found 320.0684.

(2) Preparation of Compound 3

3.0g (10mmol) of 2 are dissolved in CH₂Cl₂Then, 3g (30mmol) succinic anhydride (n ═ 1) and 3.7g (30mmol) DMAP were added, and the mixture was reacted at 45 ℃ overnight, cooled to room temperature, concentrated, and subjected to column chromatography to give yellow product 3, which was a mixed solvent of dichloromethane and methanol at 10:1 as an eluent.

¹H NMR(600MHz,CDCl₃)δ8.41(d,J＝7.9Hz,1H),7.17(d,J＝7.9Hz,1H),4.11(t,J＝6.5Hz,2H),3.28(t,J＝7.3Hz,2H),2.68(t,J＝6.7Hz,2H),2.62–2.59(m,2H),1.88–1.83(m,2H),1.68–1.64(m,2H),1.59–1.54(m,2H),1.46–1.40(m,2H).

HRMS(ESI)m/z calcd for C₁₆H₁₉N₃O₇S[M+Na]⁺ 420.0836,found 420.0858.

Example 2

(II) preparation of target Compound

EXAMPLE 1 preparation of Compound 4a

189.6mg (0.5mmol) of Compound 3 and 192.7mg (0.6mmol) of TBTU were dissolved in 10mL of anhydrous DMF, stirred at room temperature for 5min, and 65.8mg (0.65mmol) of Et was added₃N, stirring was continued for 2min, then 55.9mg (0.6mmol) of aniline were added and the reaction was carried out at 35 ℃ for 2 h. The reaction solution was concentrated, and the concentrated solution was separated by silica gel column chromatography, and the eluent was a mixed solvent of dichloromethane and methanol (30:1), to give 164.1mg of a pale yellow solid, yield: 69.5 percent.

¹H NMR(600MHz,DMSO–d₆):δ9.96(s,1H),8.54(d,J＝7.9Hz,1H),7.55(d,J＝7.8Hz,2H),7.46(d,J＝8.0Hz,1H),7.26(t,J＝7.9Hz,2H),6.99(t,J＝7.4Hz,1H),4.03(t,J＝6.5Hz,2H),3.12(t,J＝7.4Hz,2H),2.59(s,4H),1.74–1.69(m,2H),1.60–1.55(m,2H),1.48–1.43(m,2H),1.39–1.34(m,2H)；¹³C NMR(150MHz,DMSO–d₆):δ172.9,170.3,149.6,143.1,140.5,139.7,132.8,132.5,129.1,123.4,122.5,119.3,64.3,31.4,31.0,29.2,28.5,28.3,27.8,25.3.

HRMS(ESI)m/z calcd for C₂₂H₂₅N₄O₆S[M+H]⁺ 473.1489,found 473.1498.

EXAMPLE 2 preparation of Compound 4b

Prepared as described in example 1, substituting 2-fluoroaniline for aniline, to give the yellow product in 71.3% yield.

¹H NMR(600MHz,CDCl₃):δ8.40[d,J＝7.9Hz,1H),8.25(t,J＝7.9Hz,1H),7.65(s,1H),7.13(d,J＝7.9Hz,1H),7.08–6.99(m,3H),4.14(t,J＝6.5Hz,2H),3.25(t,J＝7.4Hz,2H),2.76–2.71(m,4H),1.85–1.80(m,2H),1.69–1.65(m,2H),1.57–1.52(m,2H),1.46–1.41(m,2H)；¹³C NMR(150MHz,CDCl₃):δ172.8,169.7,153.1(d,J＝241.5Hz),149.1,142.4,141.8,132.4,130.7,126.2,124.5,124.3,121.6,120.3,114.8,64.6,32.0,31.5,29.3,28.4,28.3,27.7,25.3.

HRMS(ESI)m/z calcd for C₂₂H₂₄FN₄O₆S[M+H]⁺ 491.1395,found 491.1388.

EXAMPLE 3 preparation of Compound 4c

Prepared as described in example 1, substituting 2-chloroaniline for aniline, gave a light yellow product in 81.1% yield.

¹H NMR(600MHz,DMSO–d₆):δ9.52(s,1H),8.54(d,J＝8.1Hz,1H),7.70(d,J＝7.9Hz,1H),7.47(d,J＝8.0Hz,1H),7.45–7.43(m,1H),7.29(t,J＝7.3Hz,1H),7.14(t,J＝7.4Hz,1H),4.04(t,J＝6.5Hz,2H),3.33–3.31(m,2H),2.69–2.67(m,2H),2.60(t,J＝6.6Hz,2H),1.76–1.71(m,2H),1.6–1.56(m,2H),1.49–1.46(m,2H),1.40–1.35(m,2H)；¹³C NMR(150MHz,DMSO–d₆):δ172.7,170.7,149.6,143.1,140.5,135.4,132.8,132.5,129.9,127.7,126.6,126.4,126.3,122.6,64.2,31.0,30.9,29.3,28.5,28.3,27.8,25.3.

HRMS(ESI)m/z calcd for C₂₂H₂₄ClN₄O₆S[M+H]⁺ 507.1100,found 507.1120.

EXAMPLE 4 preparation of Compound 4d

Prepared as described in example 1, substituting 2-methylaniline for aniline, gave a pale yellow solid in 62.3% yield.

¹H NMR(600MHz,DMSO–d₆):δ9.31(s,1H),8.53(d,J＝7.9Hz,1H),7.47(d,J＝8.0Hz,1H),7.36(d,J＝7.9Hz,1H),7.16(d,J＝7.4Hz,1H),7.12(t,J＝7.5Hz,1H),7.03(t,J＝7.3Hz,1H),4.04(t,J＝6.6Hz,2H),3.33(t,J＝7.4Hz,2H),2.64–2.58(m,4H),2.17(s,3H),1.76–1.71(m,2H),1.61–1.56(m,2H),1.50–1.45(m,2H),1.40–1.35(m,2H)；¹³C NMR(150MHz,DMSO–d₆):δ172.9,170.2,149.6,143.1,140.5,136.8,132.8,132.5,132.0,130.7,126.3,125.4,125.3,122.6,64.2,31.0,30.8,29.5,28.5,28.3,27.8,25.3,18.3.

HRMS(ESI)m/z calcd for C₂₃H₂₇N₄O₆S[M+H]⁺ 487.1645,found 487.1649.

EXAMPLE 5 preparation of Compound 4e

Prepared as described in example 1, substituting 2-methoxyaniline for aniline, gave an orange solid in 75.5% yield.

¹H NMR(600MHz,DMSO–d₆):δ9.12(s,1H),8.54(d,J＝7.2Hz,1H),7.93(d,J＝6.0Hz,1H),7.46(d,J＝7.3Hz,1H),6.99(s,2H),6.84(s,1H),4.02(s,2H),3.81(s,3H),3.33(d,J＝9.9Hz,2H),2.68(s,2H),2.56(s,2H),1.77–1.68(m,2H),1.62–1.54(m,2H),1.51–1.43(m,2H),1.40–1.34(m,2H)；¹³C NMR(150MHz,DMSO–d₆):δ172.9,170.5,149.7,149.6,143.1,140.5,132.8,132.5,127.8,124.5,122.5,122.0,120.6,111.5,64.2,56.1,31.331.0,29.4,28.5,28.3,27.8,25.3.

HRMS(ESI)m/z calcd for C₂₃H₂₇N₄O₇S[M+H]⁺ 503.1595,found 503.1590.

EXAMPLE 6 preparation of Compound 4f

Prepared as described in example 1 using 3-chloroaniline instead of aniline, gave a light yellow solid in 75.5% yield.

¹H NMR(600MHz,DMSO–d₆):δ10.15(s,1H),8.54(d,J＝8.5Hz,1H),7.77(s,1H),7.46(d,J＝8.0Hz,1H),7.38(d,J＝8.2Hz,1H),7.29(t,J＝8.1Hz,1H),7.07(dd,J＝7.9,1.2Hz,1H),4.03(t,J＝6.5Hz,2H),3.32(t,J＝7.4Hz,2H),2.62–2.56(m,4H),1.74–1.69(m,2H),1.60–1.55(m,2H),1.48–1.43(m,2H),1.39–1.34(m,2H)；¹³C NMR(150MHz,DMSO–d₆):δ172.7,170.7,149.6,143.1,141.1,140.5,133.5,132.7,132.5,130.8,123.1,122.5,118.8,117.6,64.3,31.4,30.9,29.1,28.5,28.3,27.8,25.3.

HRMS(ESI)m/z calcd for C₂₂H₂₄ClN₄O₆S[M+Na]⁺ 507.1100,found 507.1109.

EXAMPLE 7 preparation of 4g Compound

Prepared as described in example 1, substituting 4-fluoroaniline for aniline, to give a yellow solid in 75.5% yield.

¹H NMR(600MHz,CDCl₃):δ8.40(d,J＝6.5Hz,1H),7.73(s,1H),7.44–7.42(m,2H),7.14(d,J＝6.4Hz,1H),6.96(t,J＝8.2Hz,2H),4.12(t,J＝6.2Hz,2H),3.25(t,J＝6.7Hz,2H),2.73(s,2H),2.66(s,2H),1.87–1.78(m,2H),1.69–1.62(m,2H),1.58–1.49(m,2H),1.47–1.38(m,2H)；¹³C NMR(150MHz,CDCl₃):δ173.2,169.7,160.0(d,J＝241.5Hz),149.1,142.4,141.9,133.8,132.4,130.8,121.5,121.4,120.3,115.6,115.4,64.6,31.8,31.6,29.3,28.3,28.2,27.6,25.3.

HRMS(ESI)m/z calcd for C₂₂H₂₄FN₄O₆S[M+H]⁺ 491.1395,found 491.1389.

EXAMPLE 8 preparation of Compound 4h

Prepared as described in example 1, substituting 4-bromoaniline for aniline, to give a yellow solid in 70.2% yield.

¹H NMR(600MHz,DMSO–d₆):δ10.10(s,1H),8.54(d,J＝7.9Hz,1H),7.52(d,J＝8.5Hz,2H),7.46(d,J＝7.9Hz,1H),7.42(d,J＝8.5Hz,2H),4.03(t,J＝6.2Hz,2H),3.31(t,J＝7.5Hz,2H),2.59(s,4H),1.74–1.68(m,2H),1.60–1.54(m,2H),1.48–1.42(m,2H),1.39–1.32(m,2H)；¹³C NMR(150MHz,DMSO–d₆):δ172.8,170.5,149.6,143.1,140.5,139.0,132.7,132.5,131.9,122.5,121.2,114.9,64.2,31.4,31.0,29.1,28.5,28.3,27.8,25.3.

HRMS(ESI)m/z calcd for C₂₂H₂₄BrN₄O₆S[M+H]⁺ 533.0576,found 533.0580.

Example 9 preparation of Compound 4i

Prepared as described in example 1, substituting 4-methylaniline for aniline, gave a yellow solid in 83.2% yield.

¹H NMR(600MHz,DMSO–d₆):δ9.85(s,1H),8.53(d,J＝8.0Hz,1H),7.45(d,J＝8.0Hz,1H),7.42(d,J＝8.3Hz,2H),7.04(d,J＝8.3Hz,2H),4.03(t,J＝6.5Hz,2H),3.31(t,J＝7.4Hz,2H),2.57(s,4H),2.20(s,3H),1.74–1.69(m,2H),1.60–1.55(m,2H),1.48–1.43(m,2H),1.39–1.34(m,2H)；¹³C NMR(150MHz,DMSO–d₆):δ172.9,170.0,149.6,143.1,140.5,137.2,132.8,132.5,132.2,129.4,122.5,119.3,64.2,31.3,31.0,29.3,28.5,28.3,27.8,25.3,20.9.

HRMS(ESI)m/z calcd for C₂₃H₂₇N₄O₆S[M+H]⁺ 487.1645,found 487.1640.

EXAMPLE 10 preparation of Compound 4j

Prepared as described in example 1 using 4-methoxyaniline instead of aniline to give a yellow solid in 81.0% yield.

¹H NMR(600MHz,DMSO–d₆):δ9.81(s,1H),8.54(d,J＝8.0Hz,1H),7.46–7.44(m,3H),6.82–6.80(m,2H),4.03(t,J＝6.5Hz,2H),3.67(s,3H),3.32(t,J＝7.4Hz,2H),2.57(s,4H),1.74–1.69(m,2H),1.60–1.55(m,2H),1.48–1.43(m,2H),1.39–1.34(m,2H)；¹³C NMR(150MHz,DMSO–d₆):δ172.9,169.7,155.4,149.6,143.1,140.5,132.9,132.8,132.5,122.5,120.8,114.2,64.2,55.6,31.2,31.0,29.3 28.5,28.3,27.8,25.3.

HRMS(ESI)m/z calcd for C₂₃H₂₇N₄O₇S[M+H]⁺ 503.1595,found 503.1580.

EXAMPLE 11 preparation of Compound 4k

Prepared as described in example 1 using 4- (3-fluoromethoxy) aniline instead of aniline to give a pale yellow solid in 86.7% yield.

¹H NMR(600MHz,CDCl₃):δ8.40(d,J＝7.9Hz,1H),7.85(d,J＝13.9Hz,1H),7.52(d,J＝8.9Hz,2H),7.14(t,J＝7.7Hz,3H),4.12(t,J＝6.5Hz,2H),3.26(t,J＝7.3Hz,2H),2.75(t,J＝6.2Hz,2H),2.68(t,J＝6.2Hz,2H),1.85–1.80(m,2H),1.68–1.64(m,2H),1.57–1.52(m,2H),1.45–1.40(m,2H)；¹³C NMR(150MHz,CDCl₃):δ173.3,169.9,149.1,145.0,142.4,141.9,136.6,132.4,130.8,121.6,121.2(d,J＝255.0Hz),120.8,120.3,64.7,31.8,31.5,29.2,28.3,28.2,27.6,25.31.

HRMS(ESI)m/z calcd for C₂₃H₂₄F₃N₄O₇S[M+H]⁺ 557.1312,found 557.1311.

EXAMPLE 12 preparation of Compound 4l

Prepared as described in example 1 using 3, 4-dimethoxyaniline instead of aniline to give a yellow solid in 84.8% yield.

¹H NMR(600MHz,DMSO–d₆):δ9.80(s,1H),8.53(d,J＝7.9Hz,1H),7.44(d,J＝8.0Hz,1H),7.25(d,J＝2.3Hz,1H),7.03–7.01(m,1H),6.81(d,J＝8.7Hz,1H),4.04(t,J＝6.5Hz,2H),3.68(s,3H),3.67(s,3H),3.31(t,J＝6.9Hz,2H),2.57(s,4H),1.74–1.69(m,2H),1.60–1.56(m,2H),1.48–1.43(m,2H),1.39–1.34(m,2H)；¹³C NMR(150MHz,DMSO–d₆):δ172.9,169.8,149.6,148.9,145.0,143.1,140.5,133.4,132.7,132.5,122.5,112.4,111.1,104.5,64.2,56.1,55.7,31.3,31.0,29.2,28.5,28.3,27.8,25.3.

HRMS(ESI)m/z calcd for C₂₄H₂₉N₄O₈S[M+H]⁺ 533.1701,found 533.1709.

Example 13 preparation of Compound 4m

Prepared as described in example 1, substituting 3-chloro-4-fluoroaniline for aniline, to give a yellow solid in 86.1% yield.

¹H NMR(600MHz,DMSO–d₆):δ10.19(s,1H),8.54(d,J＝8.0Hz,1H),7.87–7.86(m,1H),7.45(d,J＝8.0Hz,1H),7.39–7.37(m,1H),7.31(t,J＝9.1Hz,1H),4.03(t,J＝6.5Hz,2H),3.31(t,J＝7.4Hz,2H),2.59(s,4H),1.74–1.69(m,2H),1.60–1.55(m,2H),1.48–1.43(m,2H),1.38–1.33(m,2H)；¹³C NMR(150MHz,DMSO–d₆):δ172.8,170.6,154.1(d,J＝241.5Hz),149.6,143.1,140.6,136.9,132.8,132.5,122.5,120.6,119.5,117.4,117.3,64.3,31.3,31.0,29.1,28.5,28.3,27.8,25.3.

HRMS(ESI)m/z calcd for C₂₂H₂₃ClFN₄O₆S[M+H]⁺ 525.1005,found 525.0851.

EXAMPLE 14 preparation of Compound 4n

Prepared as described in example 1, substituting 3-dimethylaminopropylamine for aniline, gave a yellow solid in 79.0% yield.

¹H NMR(600MHz,DMSO–d₆):δ8.57(d,J＝8.0Hz,1H),8.14(t,J＝5.6Hz,1H),7.53(d,J＝8.0Hz,1H),4.01(t,J＝6.6Hz,2H),3.37–3.35(m,4H),3.11(q,J＝6.4Hz,2H),2.99–2.97(m,2H),2.69(s,6H),2.36(t,J＝6.9Hz,2H),1.80–1.74(m,4H),1.60–1.55(m,2H),1.51–1.46(m,2H),1.39–1.34(m,2H)；¹³C NMR(150MHz,DMSO–d₆):δ172.9,171.5,149.7,143.2,140.5,132.9,132.6,122.7,64.2,54.8,42.5,36.1,31.1,31.3,29.4,28.5,28.3,27.8,25.3,24.7.

HRMS(ESI)m/z calcd for C₂₁H₃₂N₅O₆S[M+H]⁺ 483.2096,found 483.2090.

EXAMPLE 15 preparation of Compound 4o

Prepared as described in example 1, substituting 3-diethylaminopropylamine for aniline, and gave a yellow oily liquid in 52.3% yield.

¹H NMR(600MHz,DMSO–d₆):δ8.56(d,J＝8.0Hz,1H),8.19(t,J＝5.6Hz,1H),7.52(d,J＝8.0Hz,1H),4.01(t,J＝6.6Hz,2H),3.37(t,J＝7.4Hz,2H),3.13(q,J＝6.3Hz,2H),3.07(q,J＝7.1Hz,4H),3.00(t,J＝8.1Hz,2H),2.37(t,J＝9.8Hz,2H),1.81–1.74(m,4H),1.60–1.55(m,2H),1.51–1.46(m,2H),1.40–1.35(m,2H),1.22(t,J＝7.3Hz,6H)；¹³C NMR(150MHz,DMSO–d₆):δ172.9,171.4,149.6,143.1,140.5,132.8,132.5,122.7,64.2,55.4,48.8,46.5,36.3,31.1,30.3,29.4,28.4,28.3,27.8,25.3,23.8,8.9.

HRMS(ESI)m/z calcd for C₂₂H₃₄N₅O₆S[M+H]⁺ 510.2381,found 510.2379.

EXAMPLE 16 preparation of Compound 4p

Prepared as described in example 1, substituting 2-dimethylaminoethylamine for aniline, to give a brown solid in 73.2% yield.

¹H NMR(600MHz,DMSO–d₆):δ8.57(d,J＝7.9Hz,1H),8.32-8.31(m,1H),7.52(d,J＝7.9Hz,1H),4.01(t,J＝6.4Hz,2H),3.41–3.38(m,2H),3.37(t,J＝7.2Hz,2H),3.09(t,J＝5.9Hz,2H),2.73(s,6H),2.52–2.51(m,2H),2.41(t,J＝6.8Hz,2H),1.79–1.74(m,2H),1.60–1.55(m,2H),1.51–1.46(m,2H),1.39–1.34(m,2H)；¹³C NMR(150MHz,DMSO–d₆):δ172.9,171.8,149.6,143.1,140.5,132.8,132.5,122.7,64.2,56.2,42.7,34.3,31.0,30.4,29.3,28.4,28.3,27.8,25.3.

HRMS(ESI)m/z calcd for C₂₀H₃₀N₅O₆S[M+H]⁺ 468.1911,found 468.1900.

EXAMPLE 17 preparation of Compound 4q

Prepared as described in example 1, substituting 4- (2-aminoethyl) morpholine for aniline, to give a brown solid in 73.2% yield.

¹H NMR(600MHz,DMSO–d₆):δ8.56(d,J＝8.0Hz,1H),7.79(t,J＝5.5Hz,1H),7.50(d,J＝8.0Hz,1H),4.00(t,J＝6.6Hz,2H),3.54(t,J＝4.5Hz,4H),3.37(t,J＝7.4Hz,2H),3.15(q,J＝6.7Hz,2H),2.48(t,J＝6.8Hz,2H),2.34–2.32(m,6H),2.30(t,J＝6.9Hz,2H),1.79–1.74(m,2H),1.60–1.55(m,2H),1.51–1.46(m,2H),1.40–1.35(m,2H)；¹³C NMR(150MHz,DMSO–d₆):δ172.9,171.0,149.6,143.1,140.5,132.8,132.6,122.6,66.6,64.1,57.9,53.8,36.4,31.0,30.3,29.5,28.4,28.3,27.8,25.3.

HRMS(ESI)m/z calcd for C₂₂H₃₂N₅O₇S[M+H]⁺ 510.2017,found 510.2013.

EXAMPLE 18 preparation of Compound 4r

Prepared as described in example 1, substituting 1- (2-aminoethyl) pyrrolidine for aniline, gave a yellow oily body in 51.2% yield.

¹H NMR(600MHz,DMSO–d₆):δ8.57(d,J＝7.9Hz,1H),8.27(t,J＝5.5Hz,1H),7.52(d,J＝8.0Hz,1H),4.01(t,J＝6.6Hz,2H),3.40–3.34(m,8H),3.14(t,J＝5.8Hz,2H),2.52–2.51(m,2H),2.41(t,J＝6.9Hz,2H),1.91(s,4H),1.79–1.74(m,2H),1.60–1.55(m,2H),1.51–1.46(m,2H),1.40–1.35(m,2H)；¹³C NMR(150MHz,DMSO–d₆):δ172.9,171.8,149.7,143.1,140.5,132.8,132.6,122.7,64.2,53.6,53.5,35.6,31.0,30.4,29.3,28.4,28.3,27.8,25.3,23.1.

HRMS(ESI)m/z calcd for C₂₂H₃₂N₅O₆[M+H]⁺ 494.2068,found 494.2073.

EXAMPLE 19 preparation of Compound 4s

Prepared as described in example 1, substituting morpholine for aniline, gave a pale yellow solid in 76.8% yield.

¹H NMR(600MHz,DMSO–d₆):δ8.56(d,J＝8.0Hz,1H),7.50(d,J＝8.0Hz,1H),4.01(t,J＝6.5Hz,2H),3.56(t,J＝4.7Hz,2H),3.51(t,J＝4.8Hz,2H),3.44(t,J＝4.6Hz,2H),3.40(t,J＝4.7Hz,2H),3.37(t,J＝7.4Hz,2H),2.57–2.55(m,2H),2.49–2.47(m,2H),1.79–1.74(m,2H),1.60–1.55(m,2H),1.51–1.46(m,2H),1.40–1.35(m,2H)；¹³C NMR(150MHz,DMSO–d₆):δ173.0,170.0,149.6,143.1,140.5,132.8,132.6,122.6,66.5,64.1,45.6,42.0,31.0,29.3,28.4,28.3,27.8,27.7,25.3.

HRMS(ESI)m/z calcd for C₂₀H₂₇N₄O₇S[M+H]⁺ 467.1595,found 467.1593.

EXAMPLE 20 preparation of Compound 4t

Prepared as described in example 1, substituting piperidine for aniline, to give a yellow solid in 77.5% yield.

¹H NMR(600MHz,DMSO–d₆):δ8.54(d,J＝8.0Hz,1H),7.49(d,J＝8.0Hz,1H),4.00(t,J＝6.6Hz,2H),3.38(t,J＝5.5Hz,4H),3.35–3.33(m,2H),2.54–2.52(m,2H),2.47–2.45(m,2H),1.79–1.74(m,2H),1.60–1.54(m,4H),1.51–1.46(m,4H),1.40–1.35(m,4H)；¹³C NMR(150MHz,DMSO–d₆):δ173.0,169.3,149.6,143.1,140.5,132.8,132.5,122.6,64.1,46.1,42.5,31.0,29.4,28.5,28.3,27.8,27.7,26.4,25.8,25.3,24.5.

HRMS(ESI)m/z calcd for C₂₁H₂₉N₄O₆S[M+H]⁺ 465.1802,found 465.1809.

(III) detection of GSTP1 and GSTM2 inhibition by Compounds

The experimental method comprises the following steps: the inhibition of GSTP1 and GSTM2 by compounds 4a-4t was tested by glutathione-S transferase kit.

The first part is an enzymatic reaction. 30 μ L of the matrix solution (prepared by mixing the working solution A and the standard solution GSH 1mmol/L at a ratio of 1: 1) was added into a 96-well plate, and a measurement group and a control group were provided, respectively. The compounds 4n,4o,4p and 4r to be tested were prepared as stock solutions with distilled water, the remaining compounds were prepared as stock solutions with DMSO solutions, and diluted with distilled water to a range of concentrations just before use, wherein the final concentration of DMSO was not more than 4 ‰ (the same applies to the experiments below). The compounds with different concentrations were mixed with 20nM GSTP1 protein and 5nM GSTM2 protein, respectively, and the total volume of 10. mu.L of the mixture of protein and compound was added to the assay group, and then the mixture of protein and compound was added to the control group after a water bath at 37 ℃ for 10 min.

The second part is a color reaction. 200. mu.L of the C working solution and 50. mu.L of the D working solution were added to the control group, the measurement group, the blank group and the standard group, respectively, for later use, 200. mu. L B of the working solution was added to the blank group, and 200. mu.L of the 20. mu. mol/L GSH standard solution was added to the standard group. Standing at room temperature for 15min, and detecting absorbance values of each group at 412nm with a microplate reader.

GST activity calculation formula:

the activity of GST under each concentration is calculated by the above formula, the inhibition rate is calculated, and a concentration-inhibition rate curve is made to calculate IC₅₀The value is obtained. Compound 2 and nbdheex were positive controls. The inhibition of glutathione transferase GSTP1 and GSTM2 at different concentrations of the compound was observed and the results are shown in Table 2.

TABLE 2 inhibition of GSTP1 and GSTM2 by compounds

As can be seen from the data in Table 2, although compound 2 and NBDHEX have good inhibitory effects on GSTP1, they have stronger inhibitory effects on GSTM 2. Compared with the parent compound NBDHEX, the introduction of the polyamino group in the invention leads the inhibition effect of partial compounds on GSTP1 to be improved, and simultaneously, the inhibition effect on GSTM2 is obviously reduced, wherein most of the polyamino compounds containing aliphatic rings have better inhibition effect on GSTP1 than the polyamino compounds containing aromatic rings. Notably, all representative compounds showed less inhibition of GSTM2 than NBDHEX. Of these, the two compounds 4n and 4s with the best activity, their IC for GSTP1₅₀The values were 0.16. mu.M and 0.33. mu.M, respectively, but the inhibition of GSTM2 was 4.5 times and 8.5 times lower than NBDHEX, respectively. NBDHEX is 73-fold selective for GSTM2 protein as compared to GSTP1, while compounds 4n and 4s are 1.8-fold and 1.9-fold selective for GSTM2 protein, respectively.

(IV) test of Compound cytotoxic Activity in vitro

The experimental method comprises the following steps: representative compounds of the invention were tested for cytotoxic activity using the MTT method. Cells in the logarithmic growth phase were counted and plated in 96-well plates at about 8000-10000 cells per well. Culturing overnight, and administering after cell adherence, wherein an administration group and a control group are respectively arranged. The compounds 4n,4o,4p and 4r to be tested were prepared as stock solutions in sterile water, and the remaining compounds wereStock solutions were prepared in DMSO and diluted in cell culture media to a range of concentrations just prior to use, with the final DMSO concentration not exceeding 4% o (the same experiment below). Each concentration was provided with 3 multiple wells. After adding the drug, the mixture is cultured for 72h, 20 mu L of MTT with the concentration of 5mg/mL is added, the mixture is incubated for 4h at 37 ℃, the supernatant is discarded, and 150 mu L of DMSO is added for dissolution. Measuring the OD value of each hole by using an enzyme-labeling instrument at 490 nm wavelength, calculating the inhibition rate, and calculating the IC by using a concentration-inhibition rate curve₅₀The value is obtained.

The in vitro antiproliferative activity of compounds 4a-4t on lung cancer cell a549, melanoma cell B16, osteosarcoma cell 143B, osteosarcoma cell HOS and human umbilical vein vascular endothelial cell HUVEC was tested, and compound 2 and nbdheex as positive controls. Observing the inhibition of the compound on the growth of tumor cells under different concentrations, and calculating the inhibition rate and IC thereof₅₀The cytotoxic activity of the drug was evaluated and the results are shown in table 3.

TABLE 3 cytotoxic Activity of Compounds

As can be seen from the data in table 3, although compound 2 and NBDHEX have good cytotoxic effects on tumor cells, they are also highly toxic to normal cells. The compounds 4a-4t have stronger antitumor activity on four cancer cells A549, B16, 143B and HOS, but the cytotoxicity on normal cells is lower than that of the compounds 2 and NBDHEX. IC of two compounds 4n and 4s with the best antitumor Activity₅₀The values are between 0.82 and 1.85. mu.M, and their toxicity to normal cells is 3.2 times lower than that of NBDHEX and 10.7 times lower than that of NBDHEX. The data in Table 3 show that the introduction of polyamino groups can improve the antitumor activity of part of compounds compared with NBDHEX and obviously reduce the toxicity to normal cells, wherein the polyamino compounds containing aliphatic rings have better antitumor activity than those containing aromatic rings.

Claims

1. An antitumor compound taking GSTP1 as a target spot and pharmaceutically acceptable salts thereof are characterized by comprising a compound with a structure shown as a formula I:

wherein n is 1-3;

when R is¹When is H, R²Is aryl or alkyl; when R is²When the aryl is selected from phenyl, mono-substituted or di-substituted phenyl, and the substituent is CH₃、OCH₃、OCF₃F, Cl or Br, the substituents being in any of the ortho, meta or para positions; when R is²When it is an alkyl group, it is selected from C_1～4Dimethylamino radical of straight-chain alkyl, C_1～4Diethylamino of straight-chain alkyl, C_1～4Morpholinyl of straight-chain alkyl, C_1～4Piperidinyl or C of straight-chain alkyl groups_1～4Pyrrolidinyl of straight chain alkyl;

or, R¹And R²Morpholine or piperidyl which forms a cyclic structure with the N atom.

2. The GSTP 1-targeted anti-tumor compound and pharmaceutically acceptable salts thereof according to claim 1, wherein the compound is selected from the group consisting of the following compounds 4a-4 t:

3. a method for preparing the GSTP 1-targeted anti-tumor compound and its pharmaceutically acceptable salt according to claim 1, comprising the steps of:

TBTU stands for condensing agent tetrafluoroboric acid-2- (1H-benzotriazole-1-yl) -1,1,3, 3-tetramethylammonium, Et₃N represents triethylamine, and DMF represents solvent N, N-dimethylformamide.

4. The method for preparing the antitumor compound targeting GSTP1 and its pharmaceutically acceptable salt according to claim 3, which comprises the following steps:

5. The process for preparing an antitumor compound targeting GSTP1 and its pharmaceutically acceptable salt according to claim 3, wherein the compound 3 is prepared according to the following reaction formula:

wherein n is 1-3; DMAP for 4-dimethylaminopyridine and DCM for CH₂Cl₂。

6. The method for preparing the antitumor compound targeting GSTP1 and its pharmaceutically acceptable salt according to claim 5, which comprises the following steps:

synthesis of Compound 3: dissolving 2 in CH₂Cl₂Adding naphthenic dianhydride and DMAP, reacting overnight at 40-50 ℃, cooling to room temperature, concentrating and reactingAnd (3) carrying out column chromatography separation on the solution to obtain a yellow product 3, wherein the eluent is a mixed solvent of dichloromethane and methanol.

7. A pharmaceutical composition comprising a compound according to any one of claims 1 to 2 and pharmaceutically acceptable salts thereof.

8. The GSTP 1-targeted anti-tumor compound of claim 1 and the pharmaceutically acceptable salt thereof for use in a GSTP1 inhibitor.

9. The GSTP 1-targeted anti-tumor compound of claim 1 and the pharmaceutically acceptable salt thereof for use in a GSTM2 inhibitor.

10. The use of an anti-tumor compound targeting GSTP1, according to claim 1, and pharmaceutically acceptable salts thereof, in the manufacture of a medicament for the treatment of lung cancer, melanoma, osteosarcoma.