CN107892691B

CN107892691B - 2,8, 9-trisubstituted-9H-purine compound and salt and application thereof

Info

Publication number: CN107892691B
Application number: CN201711378538.6A
Authority: CN
Inventors: 张三奇; 黑媛媛; 张�浩; 王瑾; 曹永孝; 辛敏行; 吕社民
Original assignee: Xian Jiaotong University
Current assignee: Xian Jiaotong University
Priority date: 2017-12-19
Filing date: 2017-12-19
Publication date: 2020-04-28
Anticipated expiration: 2037-12-19
Also published as: CN107892691A

Abstract

The invention provides a2, 8, 9-trisubstituted-9H-purine compound, a salt thereof and application thereof, belonging to the technical field of anti-cancer drugs. The compounds have novel structure, have the activity of inhibiting Epidermal Growth Factor Receptor (EGFR) tyrosine kinase, have obvious inhibitory activity on single-mutation (L858R) EGFR, have obvious in-vivo and in-vitro antitumor activity, can be used for treating cancers related to EGFR mutation, and have easily available synthetic raw materials and easily realized synthetic method.

Description

2,8, 9-trisubstituted-9H-purine compound and salt and application thereof

Technical Field

The invention belongs to the technical field of anti-cancer drugs, and particularly relates to a2, 8, 9-trisubstituted-9H-purine compound, and a salt and an application thereof.

Background

Cancer is one of the malignant diseases that seriously threatens human health. In recent 30 years, the incidence of cancer in China is in a rapid rise, the incidence of cancer is about 200/10 ten thousands, 320 new cases per year are more than 320 ten thousands, the death is about 270 more than ten thousands, and more than 700 thousands of patients are treated.

The main treatment means of cancer still include surgical treatment, radiation treatment and drug treatment, but the drug treatment is mainly used to a great extent. Therefore, research and development of new anticancer drugs are of great significance.

The traditional antitumor drug has strong activity, but lacks selectivity and has high toxicity. In recent years, with the progress of tumor molecular biology research, the pathogenesis of tumors is more understood, and a plurality of new targets of the action of anti-cancer drugs, such as Epidermal Growth Factor (EGFR) tyrosine kinase, PI3Ks, BTK and the like, are found.

In non-small cell lung cancer, about 50% of patients exhibit mutations in EGFR. In response to this feature, first generation EGFR tyrosine kinase inhibitors such as gefitinib, erlotinib, second generation EGFR tyrosine kinase inhibitor afatinib, third generation EGFR tyrosine kinase inhibitor oxitinib have been developed for use as anti-cancer drugs. However, after about 10 months of use of the oxitinib, the EGFR is found to have a mutation at a third site, namely C797S, so that acquired drug resistance is generated, and the effect of the oxitinib is obviously reduced. Therefore, the development of an EGFR single mutation (L858R) or EGFR triple mutation (L858R/T790M/C797S) inhibitor with novel structure and high activity for treating EGFR mutation cancer is of great significance.

Disclosure of Invention

The invention aims to provide a2, 8, 9-trisubstituted-9H-purine compound, a salt and an application thereof, the compound has a novel structure, has remarkable activity of inhibiting EGFR tyrosine kinase single mutation (L858R) and triple mutation (L858R/T790M/C797S), has obvious in-vivo and in-vitro antitumor activity, can be applied to preparation of an anticancer medicinal preparation, and has easily obtained synthetic raw materials and an easily realized synthetic method.

The invention is realized by the following technical scheme:

a2, 8, 9-trisubstituted-9H-purine compound has a structural formula as follows:

in the formula, R¹Is cycloalkyl, substituted cycloalkyl, heterocycloalkyl or substituted heterocycloalkyl;

R²is alkoxy, substituted alkoxy or substituted amino;

R³is phenyl, substituted phenyl or benzyl;

x is S or S ═ O; y is CH or N.

Preferably, said cycloalkyl is C₅-C₇A cycloalkyl group; substituted cycloalkyl being substituted C₅-C₇A cycloalkyl group; the heterocycloalkyl radical being an oxa or aza radical C₅-C₇A cycloalkyl group; substituted heterocycloalkyl being substituted oxaOr of aza C₅-C₇A cycloalkyl group.

Preferably, R¹Is 4-tetrahydropyranyl, 4-hydroxycyclohexyl or cyclopentyl.

Preferably, the substituted alkoxy is a tertiary amine substituted alkoxy and the substituted amino is a tertiary amine substituted alkylamino.

Preferably, R²Is 2- (dimethylamino) ethoxy, 1-methyl-4-piperidinyloxy, 4-methyl-1-piperazinyl, 4-ethyl-1-piperazinyl, N-methyl-N- (2-dimethylaminoethyl) amino or 4-dimethylamino-1-piperidinyl.

Preferably, the substituted phenyl group is a 3-fluorophenyl group or a 4-fluorophenyl group.

The pharmaceutically acceptable salt of the 2,8, 9-trisubstituted-9H-purine compound is hydrochloride, hydrobromide, nitrate, phosphate, sulfate, acetate, fumarate, malate, citrate, tartrate, maleate, lactate, citrate, camphorsulfonate, benzoate, gluconate, glutamate, isethionate, succinate or methanesulfonate.

The 2,8, 9-trisubstituted-9H-purine compound and the medicinal salt thereof are applied to the preparation of anti-cancer medicinal preparations.

Preferably, the anti-cancer pharmaceutical preparation is an anti-cancer pharmaceutical preparation capable of inhibiting single-mutated EGFR and/or capable of inhibiting triple-mutated EGFR.

Preferably, the single mutated EGFR is an EGFR which undergoes the L858R mutation; the triple-mutated EGFR is an EGFR which has undergone L858R/T790M/C797S mutation.

Preferably, the anti-cancer pharmaceutical preparation is tablets, capsules or injections, wherein each tablet, each grain or each preparation contains 30-500 mg of 8-phenylthio or 8-phenylsulfinyl or benzylthio-2, 9-disubstituted-9H-purine compounds or pharmaceutically acceptable salts thereof.

Preferably, the anticancer pharmaceutical preparation further comprises auxiliary materials, and the auxiliary materials comprise one or more of a stabilizer, a solubilizer, a lubricant and a disintegrant. Further preferably, the adjuvants include one or more of starch, dextrin, glucose, lactose, cellulose, polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone, pectin, cyclodextrin, Tween-80, polyvinyl alcohol, magnesium stearate and talc.

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

the 2,8, 9-trisubstituted-9H-purine compound or salt compound thereof provided by the invention has the activity of inhibiting Epidermal Growth Factor Receptor (EGFR) tyrosine kinase, has obvious inhibitory activity and IC (integrated Circuit) on single-mutation (L858R) EGFR₅₀The value is nanomolar, and can be used for treating cancers related to EGFR mutation. Experiments prove that the compounds have obvious in-vivo anti-tumor activity.

The 2,8, 9-trisubstituted-9H-purine compound and the salt compound thereof provided by the invention can be applied to the preparation of anti-cancer pharmaceutical preparations, wherein each tablet or granule or branch of the pharmaceutical preparation contains 30-500 mg. When the active compound provided by the invention is used for preparing an anticancer medicine preparation, the medicine can be prepared into tablets, capsules or injections. The pharmaceutical preparations can be prepared according to the conventional preparation process of various preparations. The preferable content is 30-300mg for the tablet or capsule. The oral preparation of the invention can contain pharmaceutic adjuvants including additives, stabilizers, solubilizers, lubricants, disintegrating agents and the like, such as starch, dextrin, glucose, lactose, cellulose, polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone, pectin, cyclodextrin, Tween-80, polyvinyl alcohol, magnesium stearate, talcum powder and the like.

Drawings

FIG. 1 shows a method for synthesizing 2,8, 9-trisubstituted-9H-purine compounds.

Fig. 2 is a tumor growth curve (. p < 0.01).

FIG. 3 is a graph showing the change in body weight of nude mice.

Detailed Description

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

The invention is further illustrated below by the synthetic procedures and evaluation of activity of some representative compounds of the invention. The structural formula and the number of the representative compound are shown as follows.

The synthetic method of the 2,8, 9-trisubstituted-9H-purine compound is shown in figure 1, and comprises the following steps:

the method comprises the following steps: reference (j.med.chem.2012,55,10685-10699) synthesizes compound a, which is refluxed with carbon disulfide and potassium hydroxide in ethanol/water (10:1) to give intermediate B;

step two: under the catalysis of the iodoidene iodide, the intermediate B and iodobenzene are stirred in dimethyl sulfoxide at 140 ℃ for 24 hours to obtain a product C, namely 8-thiophenyl-2, 9-disubstituted-9H-purine compounds; or, in the presence of potassium carbonate, stirring and refluxing the intermediate B and benzyl bromide in acetone for 4 hours to obtain a product D, namely the 8-benzylthio-2, 9-disubstituted-9H-purine compound;

step three: c is oxidized by m-chloroperoxybenzoic acid in dichloromethane to obtain a product D, namely the 8-benzene sulfinyl-2, 9-disubstituted-9H-purine compound.

Examples of the synthesis of the above compounds, the structures of which are given below¹And (4) HNMR characterization.

Example 1: synthesis of 2- (4- (4-methyl-1-piperazinyl) phenylamino) -8-thiophenyl-9- (2H-4-tetrahydropyranyl) -9H-purine (Structure 1):

the method comprises the following steps: synthesis of 2- (4- (4-methyl-1-piperazinyl) phenylamino) -8-mercapto-9- (2H-4-tetrahydropyranyl) -9H-purine (B1)

Reference j.med.chem.2012,55, 10685-. To a 100mL eggplant-shaped bottle, a1(0.40g), carbon disulfide (95mg), potassium hydroxide (70mg), ethanol (20mL) and water (2mL) were added, the mixture was refluxed for 3 hours, the solvent was removed under reduced pressure, and the residue was subjected to silica gel column chromatography (dichloromethane: methanol ═ 20:1) to give intermediate B1(0.40g) in 90.3% yield.

Step two: synthesis of 2- (4- (4-methyl-1-piperazinyl) phenylamino) -8-thiophenyl-9- (2H-4-tetrahydropyranyl) -9H-purine (Structure 1)

To a 25mL eggplant-shaped bottle was added B1(0.20g), iodobenzene (80 μ L), cuprous iodide (6.7mg), potassium carbonate (0.20g), 1, 10-phenanthroline (13.0mg), and DMSO (5mL), and the mixture was magnetically stirred at 140 ℃ for 24h under argon protection, cooled to room temperature, added 25mL dichloromethane, washed three times with water, and DMSO was removed. The organic phase was evaporated to dryness under reduced pressure, the residue was subjected to silica gel column chromatography (dichloromethane: methanol 20:1), and ethanol was recrystallized to give compound 1(60.3mg) with a yield of 56.7%.¹H NMR(400MHz,CDCl₃)δ8.70(s,1H,Ar-H),7.58(d,J＝8.9Hz,2H,Ar-H),7.50(dd,J＝7.9,1.6Hz,2H,Ar-H),7.44–7.31(m,3H,Ar-H),7.29(s,1H,NH),6.98(d,J＝9.0Hz,2H,Ar-H),4.72–4.62(m,1H,CH),4.12(dd,J＝11.5,4.2Hz,2H,CH×2),3.44(t,J＝11.4Hz,2H,CH×2),3.27–3.14(m,4H,CH₂×2),2.93(qd,J＝12.5,4.5Hz,2H,CH×2),2.69–2.55(m,4H,CH₂×2),2.38(s,3H,CH₃),1.55(dd,J＝12.4,2.5Hz,2H,CH×2).

Example 2: synthesis of 2- (4- (N-methyl-2-dimethylaminoethylamino) phenylamino) -8-thiophenyl-9- (2H-4-tetrahydropyranyl) -9H-purine (Structure 2):

reference j.med.chem.2012,55, 10685-. The synthesis of B2 is the same as B1. Synthesis of Compound 2 with B2 instead of B1 and Synthesis of Compound 1. The yield thereof was found to be 55.3%.¹H NMR(400MHz,CDCl₃)δ8.69(s,1H,Ar-H),7.52(s,1H,Ar-H),7.50(d,J＝2.3Hz,2H,Ar-H),7.49–7.47(m,1H,Ar-H),7.41–7.34(m,3H,Ar-H),6.77(d,J＝9.0Hz,2H,Ar-H),4.71–4.62(m,1H,CH),4.11(dd,J＝11.5,4.2Hz,2H,CH×2),3.48–3.45(m,2H,CH₂),3.42(d,J＝11.7Hz,2H,CH×2),2.96(s,3H,CH₃),2.95–2.86(m,2H,),2.55–2.47(m,2H,CH₂),2.31(s,6H,CH₃×2),1.55(dd,J＝12.4,2.5Hz,2H,CH×2).

Example 3: synthesis of 2- (4- (4-dimethylaminopiperidinyl) phenylamino) -8-thiophenyl-9- (2H-4-tetrahydropyranyl) -9H-purine (Structure 3):

reference j.med.chem.2012,55, 10685-. The synthesis of B3 is the same as B1. Synthesis of Compound 3 with B3 instead of B1 and Synthesis of Compound 1 as a process. The yield thereof was found to be 51.4%.¹H NMR(400MHz,CDCl₃)δ8.70(s,1H,Ar-H),7.57(d,J＝9.0Hz,2H,Ar-H),7.49(dd,J＝7.9,1.6Hz,2H,Ar-H),7.38(tdd,J＝7.0,5.0,1.9Hz,3H,Ar-H),7.31(s,1H,NH),6.98(d,J＝9.0Hz,2H,Ar-H),4.66(dd,J＝10.1,6.0Hz,1H,CH),4.12(dd,J＝11.5,4.2Hz,2H,CH×2),3.68(d,J＝12.3Hz,2H,CH×2),3.44(t,J＝11.4Hz,2H,CH×2),2.93(td,J＝12.4,7.9Hz,2H,CH×2),2.70(td,J＝12.1,1.9Hz,2H,CH×2),2.35(s,6H,CH₃×2),2.28(dd,J＝9.2,5.7Hz,1H,CH),1.95(d,J＝12.4Hz,2H,CH×2),1.69(tt,J＝12.0,6.1Hz,2H,CH×2),1.55(dd,J＝12.4,2.5Hz,2H,CH×2).

Example 4: synthesis of 2- (4- (2-dimethylaminoethoxy) phenylamino) -8-thiophenyl-9- (2H-4-tetrahydropyranyl) -9H-purine (Structure 4):

reference j.med.chem.2012,55, 10685-. The synthesis of B4 is the same as B1. Synthesis of Compound 4 with B4 instead of B1 and Synthesis of Compound 1. The yield thereof was found to be 60.4%.¹H NMR(400MHz,CDCl₃)δ8.70(s,1H,Ar-H),7.57(t,J＝6.1Hz,2H,Ar-H),7.50(dd,J＝7.9,1.6Hz,2H,Ar-H),7.42–7.32(m,4H,Ar-H,NH),6.95(d,J＝8.9Hz,2H,Ar-H),4.66(ddd,J＝12.1,7.9,4.2Hz,1H,CH),4.14–4.07(m,4H,CH×4),3.44(t,J＝11.6Hz,2H,CH₂),2.92(qd,J＝12.5,4.5Hz,2H,CH×2),2.75(t,J＝5.7Hz,2H,CH₂),2.37(s,6H,CH₃×2),1.56(dd,J＝12.4,2.4Hz,2H,CH×2).

Example 5: synthesis of 2- (2- (4-methyl-1-piperazinyl) -5-pyridylamino) -8-thiophenyl-9- (2H-4-tetrahydropyranyl) -9H-purine (Structure 5):

reference j.med.chem.2012,55, 10685-. The synthesis of B5 is the same as B1. Synthesis of Compound 5 with B5 instead of B1 and Synthesis of Compound 1. The yield thereof was found to be 51.6%.¹H NMR(400MHz,CDCl₃)δ9.04(s,1H,Ar-H),8.18(s,1H,NH),7.48(d,J＝8.9Hz,2H,Ar-H),7.44(dd,J＝7.9,1.6Hz,1H,Ar-H),7.38(m,2H,Ar-H),7.17(d,1H,Ar-H),6.79(d,J＝9.0Hz,1H,Ar-H),6.65(d,J＝9.0Hz,1H,Ar-H),4.72–4.62(m,1H,CH),4.12(dd,J＝11.5,4.2Hz,2H,CH×2),3.44(t,J＝11.4Hz,2H,CH×2),3.27–3.14(m,4H,CH₂×2),2.93(qd,J＝12.5,4.5Hz,2H,CH×2),2.69–2.55(m,4H,CH₂×2),2.38(s,3H,CH₃),1.55(dd,J＝12.4,2.5Hz,2H,CH×2)。

Example 6: synthesis of 2- (4- (2-dimethylaminoethoxy) phenylamino) -8- (3-fluorophenylthio) -9- (2H-4-tetrahydropyranyl) -9H-purine (Structure 6):

synthesis of Compound 6 Compound 1 was synthesized using B4 instead of B1, 3-fluoroiodobenzene instead of iodobenzene. The yield thereof was found to be 57.1%.¹H NMR(400MHz,CDCl₃)δ8.74(s,1H,Ar-H),7.58(d,J＝8.8Hz,2H,Ar-H),7.36(td,J＝8.0,5.9Hz,1H,Ar-H),7.29–7.18(m,3H,NH,Ar-H),7.12–7.02(m,1H,Ar-H),6.96(d,J＝8.9Hz,2H,Ar-H),4.74–4.60(m,1H,CH),4.13(dd,J＝12.1,6.7Hz,4H,CH₂,CH×2),3.47(t,J＝12.0Hz,2H,CH×2),3.02–2.88(m,2H,CH×2),2.81(t,J＝5.5Hz,2H,CH₂),2.41(s,6H,CH₃×2),1.62(d,J＝12.6Hz,2H,CH×2).

Example 7: synthesis of 2- (4- (2-dimethylaminoethoxy) phenylamino) -8- (4-fluorophenylthio) -9- (2H-4-tetrahydropyranyl) -9H-purine (Structure 7):

synthesis of Compound 7 Compound 1 was synthesized using B4 instead of B1, 4-fluoroiodobenzene instead of iodobenzene. The yield thereof was found to be 47.2%.¹H NMR(400MHz,CDCl₃)δ8.66(s,1H,Ar-H),7.62–7.53(m,4H,Ar-H),7.18(s,1H,NH),7.13(td,J＝8.5,1.5Hz,2H,Ar-H),7.00–6.92(m,2H,Ar-H),4.71–4.57(m,1H,CH),4.16(dd,J＝11.6,3.9Hz,2H,CH×2),4.10(t,J＝5.7Hz,2H,CH₂),3.51(t,J＝12.0Hz,2H,CH×2),2.95(qd,J＝12.4,4.2Hz,2H,CH×2),2.77(t,J＝5.6Hz,2H,CH₂),2.38(t,J＝3.8Hz,6H,CH₃×2),1.66(d,J＝12.2Hz,2H,CH×2).

Example 8: synthesis of 2- (4- (4-methyl-1-piperazinyl) phenylamino) -8-phenylthio-9- (1R, 4R-4-hydroxycyclohexyl) -9H-purine (formula 8)

Reference j.med.chem.2012,55, 10685-. The synthesis of B8 is the same as B1. Synthesis of Compound 8 with B8 instead of B1 and Synthesis of Compound 1. The yield thereof was found to be 55.6%.¹H NMR(400MHz,CDCl₃)δ8.68(s,1H,Ar-H),7.55(d,J＝6.8Hz,2H,Ar-H),7.49(s,2H,Ar-H),7.37(s,4H,Ar-H,NH),6.96(d,J＝6.6Hz,2H,Ar-H),4.46(s,1H,CH),3.80(s,1H,CH),3.21(s,4H,CH₂×2),2.63(s,4H,CH₂×2),2.39(s,3H,CH₃),2.14(s,4H,CH₂×2),1.67(d,J＝9.5Hz,2H,CH×2),1.40(d,J＝10.9Hz,2H,CH×2).

Example 9: synthesis of 2- (4- (2-dimethylaminoethoxy) phenylamino) -8-phenylthio-9-cyclopentyl-9H-purine (Structure 9):

reference j.med.chem.2012,55, 10685-. The synthesis of B9 is the same as B1. Synthesis of Compound 9 with B9 instead of B1 and Synthesis of Compound 1 as a process. The yield thereof was found to be 55.7%.¹H NMR(400MHz,CDCl₃)δ8.68(s,1H,Ar-H),7.61–7.53(m,2H,Ar-H),7.52–7.45(m,2H,Ar-H),7.38(dt,J＝7.6,4.4Hz,4H,Ar-H,NH),6.99–6.89(m,2H,Ar-H),4.96(dd,J＝17.3,8.6Hz,1H,CH),4.09(t,J＝5.7Hz,2H,CH₂),2.77(t,J＝5.7Hz,2H,CH₂),2.46–2.36(m,8H,CH×2,CH₃×2),2.05(d,J＝6.0Hz,2H,CH×2),1.89(d,J＝8.2Hz,2H,CH×2),1.79–1.61(m,2H,CH×2).

Example 10: synthesis of 2- (4- (2-dimethylaminoethoxy) phenylamino) -8-phenylsulfinyl-9-cyclopentyl-9H-purine (formula 10):

to a 25mL eggplant-shaped flask, Compound 9(60.4mg) and 10mL of methylene chloride were added, stirred, and cooled in an ice-water bath. 85% m-chloroperoxybenzoic acid (26.1mg) was dissolved in 10mL of dichloromethane, and added dropwise to an eggplant-shaped flask, and the mixture was stirred at room temperature for 30min after completion of the dropwise addition, and dichloromethane was removed, and the residue was subjected to column chromatography to obtain 50.1mg, yield 80.3%.¹H NMR(400MHz,CDCl₃)δ8.64(s,1H,Ar-H),7.69(s,1H,NH),7.55(d,J＝8.7Hz,2H,Ar-H),7.50–7.43(m,2H,Ar-H),7.35(q,J＝5.1Hz,3H,Ar-H),6.87(d,J＝8.8Hz,2H,Ar-H),5.03–4.84(m,1H,CH),4.54(d,J＝3.3Hz,2H,CH₂),3.70(s,2H,CH₂),3.32(s,6H,CH₃×2),2.53–2.31(m,2H,CH×2),2.01(s,2H,CH×2),1.88(s,2H,CH×2),1.66(d,J＝5.6Hz,2H,CH×2).

Example 11: synthesis of 2- (4- (2-dimethylaminoethoxy) phenylamino) -8-phenylsulfinyl-9- (2H-4-tetrahydropyranyl) -9H-purine (Structure 11):

synthesis of compound 11 was followed by synthesis of compound 10, replacing compound 9 with compound 4. The yield thereof was found to be 49.3%.¹H NMR(400MHz,CDCl₃)δ8.70(s,1H,Ar-H),7.61(d,J＝8.8Hz,2H,Ar-H),7.50(d,J＝6.3Hz,2H,Ar-H),7.39(d,J＝7.0Hz,3H,Ar-H),7.26(s,1H,NH),6.95(d,J＝8.7Hz,2H,Ar-H),4.67(t,J＝12.0Hz,1H,CH),4.59(s,2H,CH₂),4.12(dd,J＝11.3,3.5Hz,2H,CH×2),3.78(s,2H,CH×2),3.51–3.26(m,8H,CH₂,CH₃×2),2.92(tt,J＝12.1,6.2Hz,2H,CH×2),1.57(d,J＝12.2Hz,2H,CH×2).

Example 12: synthesis of 2- (4- (4-methyl-1-piperazinyl) phenylamino) -8-benzylthio-9- (2H-4-tetrahydropyranyl) -9H-purine (structural formula 12)

To a 25mL eggplant-shaped bottle, B1(100mg), acetone (10mL), potassium carbonate (81mg) and benzyl bromide (41.1mg) were added, stirred under reflux for 2h, cooled to room temperature, vacuum evaporated to dryness by organic phase, and the residue was subjected to silica gel column chromatography (dichloromethane: methanol ═ 20:1), and recrystallized from ethanol to give compound 12(70.2mg) with a yield of 56.7%.¹H NMR(400MHz,CDCl₃)δ8.65(s,1H,Ar-H),7.58(d,J＝8.9Hz,2H,Ar-H),7.43(d,J＝6.8Hz,2H,Ar-H),7.40–7.29(m,3H,Ar-H),7.27(s,1H,NH),6.98(d,J＝8.9Hz,2H,Ar-H),4.59(s,2H,CH₂),4.47–4.33(m,1H,CH),4.14(dd,J＝11.6,4.1Hz,2H,CH×2),3.49(t,J＝11.7Hz,2H,CH×2),3.30–3.18(m,4H,CH₂×2),2.89(qd,J＝12.5,4.6Hz,2H,CH×2),2.72–2.62(m,4H,CH₂×2),2.41(s,3H,CH₃),1.70(dd,J＝12.5,2.5Hz,2H,CH×2).

Example 13: synthesis of 2- (4- (2-dimethylaminoethoxy) phenylamino) -8-thiophenyl-9- (2H-4-tetrahydropyranyl) -9H-purine hydrochloride (Structure 13):

dissolving the compound 4 in tetrahydrofuran, adding concentrated hydrochloric acid in an amount which is 1.2 times of the molar weight of the compound, sealing, stirring the mixture at room temperature overnight, filtering, and drying to obtain a compound 12. EI-MS: 491.3(M + H)⁺)。

Example 14: synthesis of 2- (4- (2-dimethylaminoethoxy) phenylamino) -8- (4-fluorophenylthio) -9- (2H-4-tetrahydropyranyl) -9H-purine methanesulfonate (structural formula 14)

Dissolving the compound 7 in dichloromethane, adding 1.2 times of methanesulfonic acid in molar weight, stirring the mixture at room temperature overnight, performing suction filtration, and drying to obtain a compound 13. EI-MS:508.3(M+H⁺)。

example 15: synthesis of 2- (4- (4-dimethylamino-1-piperidinyl) phenylamino) -8-thiophenyl-9- (2H-4-tetrahydropyranyl) -9H-purine methanesulfonate (structural formula 15)

Dissolving the compound 3 in dichloromethane, adding methanesulfonic acid with the molar weight of 1.2 times, stirring the mixture at room temperature overnight, performing suction filtration, and drying to obtain a compound 14. EI-MS: 530.5(M + H)⁺)。

Example 16: assays for inhibiting EGFR-tyrosine kinase Activity

The method comprises the following steps: respectively dissolving a compound to be measured by DMSO to prepare 10mmol/L mother liquor, diluting step by step to obtain a solution with the concentration of 0.1 mu mol/L, and respectively adding 5 mu L of different drugs to be measured or DMSO into an enzyme reaction system. The composition of the enzyme reaction system is as follows: 40mmol/L Tris, pH 7.4, 10mmol/L MgCl₂0.1mg/mL BSA, 1mmol/L DTT, 10. mu. mol/L ATP, 25mg/L EGFR (L858R), 0.2mg/mL Poly (Glu, Tyr) as substrate, 50. mu.L of the final volume of the reaction system, and 10nmol/L of the drug. After the reaction system is placed at 30 ℃ for reaction for 40min, stop solution is added to stop the reaction, the ATP content in the system is detected by a luciferase method, a chemiluminescence signal is detected on an MD-SpectraMax M5 multifunctional microplate reader, and the intensity of the chemiluminescence signal is inversely proportional to the activity of enzyme in the reaction system. Substituting the detected chemiluminescent signal value into the formula:

inhibition rate ═ 1- ((Lu)_Enzyme-Lu_{Background of the invention})/Lu_Medicine-Lu_{Background of the invention})]×100％

In the formula: lu (Lu)_MedicineDenotes the administration group, Lu_{Background of the invention}Represents blank group (without enzyme or medicine), Lu_EnzymeThe solvent control group is shown.

The results of the experimental determination are shown in table 1.

TABLE 1 inhibition ratio (%) of EGFR (L858R) Activity at a concentration of 10nmol/L of the compound

Similarly, the inhibitory activity of the compounds on EGFR triple mutant kinase can be determined by substituting EGFR (L858 35 858R/T790M/C797S) for EGFR (L858R). Wherein, the compounds 3 and 7 have stronger inhibitory activity to EGFR triple mutation, and the inhibitory rates to the activity of EGFR (L858R/T790M/C797S) are respectively 48 percent and 55 percent when the concentration is 100 nmol/L.

Example 17: verification of in vitro anti-cancer Activity

In order to verify the anticancer activity of the synthesized 8-thiophenyl or phenylsulfinyl-2, 9-disubstituted-9H-purine compounds and salts thereof, AZD9291 is used as a positive control drug, and the growth inhibition effect of the compounds 1-14 on three human lung cancer cells HCC827 is determined by an in vitro MTT method.

The verification method comprises the following steps: tumor cell HCC827 is cultured in RPMI1640 medium containing 10% calf serum and containing penicillin 100 U.mL^-1Streptomycin 100. mu.g.mL^-1At 37 ℃ and 5% CO₂Subculturing in an incubator. Collecting 0.25% pancreatin digested adherent tumor cells, and preparing cell suspension with 10% calf serum-containing RPMI1640 culture solution, the concentration of which is 2.5 × 10⁴Individual cells/ml. mu.L (containing about 5000 tumor cells) per well of 96-well culture plate was inoculated and cultured at 37 ℃ for 24 hours. The administration groups were added with different concentrations of drugs, each set at 0.001, 0.01, 0.1, 1 and 10. mu. mol. L ^-15 concentration gradients, each set with 3 parallel wells. Adding solvent with the same volume as the medicine into the control group, placing at 37 deg.C and 5% CO₂Culturing for 72h in incubator, discarding culture solution, adding 20 μ L of 5 mg/mL^-1After incubation for 4h, the supernatant was discarded, 200. mu.L of DMSO was added to each well, and the Optical Density (OD) was measured at 490nm using a microplate reader after gentle shaking. And (4) calculating a result:

taking tumor cells treated by solvent control as a control group, and calculating the inhibition rate of the drug on the tumor cells according to the following formula:

and further using linear regression to determine the median Inhibitory Concentration (IC)₅₀)。

The results of the measurements are shown in Table 2.

TABLE 2 antiproliferative Inhibition (IC) of HCC827 cells by Compounds₅₀，μmol·L^-1)

The most active compound reported in document j.med.chem.2012,55,10685-10699 is 9 e. However, the CLogP of 9e is 6.40, and the water solubility and potency are poor.

The compound 9e and the compound disclosed by the invention have strong inhibitory activity on the proliferation of human lung cancer cell HCC 827. However, the present invention attaches 4-tetrahydropyranyl to the 9 position of the purine nucleus, providing compounds with relatively small Clog values. Multiple compounds at a concentration of 10^-6At mol/L, the growth inhibition rate of HCC827 cells is higher (E)_maxValue), the patent medicine property is better. A comparison of several representative compounds with 9e and the positive drug is shown in table 3.

The data in Table 3 show that the compounds provided by the present invention compare 9e and gefitinib at a concentration of 10^-6At mol/L, the growth inhibition rate (E) is higher_maxValue) while having a smaller ClogP value. As can be seen from Table 3, Compound 3 provided by the present invention has a smaller ClogP value and a smaller IC₅₀Value, higher growth inhibition rate (E) at equivalent concentration_maxValue), i.e. it presents better druggability, a significant improvement over 9 e.

Table 3 activity, molecular weight and CLog comparison of several compounds on HCC827 cells (n ═ 6)

Example 18: verification of in vivo anticancer Activity

In order to verify the in vivo anticancer activity of the compound provided by the invention, the in vivo anticancer activity of the compound 3 is investigated by intragastric administration by adopting a nude mouse HCC827 xenograft tumor model.

The verification method comprises the following steps: nude mice (BALB/c, 6 weeks old), female, 19-21g, purchased from Experimental animals technologies, Inc., Wei Tony, Beijing.

Cultured HCC827 cells were prepared to 1X 10 with PBS⁷cells/mL of cell suspension, 0.2mL of the cell suspension was subcutaneously inoculated into the right axilla of each nude mouse using a 1mL syringe, i.e., 2X 10 cells per nude mouse⁶And (4) cells. On day 18 post inoculation, mice were randomized into 3 groups of 6 mice each, each:

1) blank control group (NMP/PEG 400/H)₂O)

2) Compound 3 Low dose group (1.0 mg. kg)^-1)

3) Compound 3 high dose group (5.0 mg. kg)^-1)

Compound 3 was treated with NMP/PEG400/H₂O (volume ratio of 0.5:5:4.5) was dissolved and prepared to have concentrations of 0.5 and 0.1mg/mL^-1The sample solution of (1). The administration is carried out once a day for 14 days in a volume of 10 mL/kg^-1Body weight. The day of administration was d1, and mouse body weights were recorded before daily dosing and tumor volumes were measured every 3 days.

Tumor volume as a × b²X 0.50(a is tumor block length and b is tumor block width)

As a result: the dose of Compound 3 was 1.0 mg/kg^-1And 5.0mg kg^-1The growth inhibitory effect on HCC827 transplantable tumors in nude mice is shown in fig. 2; the effect on body weight of nude mice is shown in figure 3.

And (4) conclusion: the compound 3 provided by the invention has the dosage of 5.0 mg/kg^-1When the preparation is administrated by gastric lavage, the growth of HCC827 xenograft tumor can be remarkably inhibited.

The invention relates to an application of a composition of 8-benzene sulfinyl-2, 9-disubstituted-9H-purine compounds and salts thereof in preparing anticancer drugs, and the application can be capsules, oral liquid or granules or injections. The preparation can be prepared according to conventional preparation process of various preparations, wherein the content of effective components is 1-500mg, preferably 30-300 mg.

The oral preparation of the present invention may contain medicinal supplementary material including stabilizer, solubilizer, lubricant, etc. such as glucose, lactose, cellulose, polyvinyl pyrrolidone, cross-linked polyvinyl pyrrolidone, starch, pectin, cyclodextrin, Tween-80, polyvinyl alcohol, magnesium stearate, talcum powder, etc.

The test methods not described in detail in the present invention are those commonly used in the art or existing methods, and are not described herein.

Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes, modifications and equivalents may be made therein without departing from the scope of the invention.

Claims

1. A2, 8, 9-trisubstituted-9H-purine compound is characterized in that the structural formula is as follows:

in the formula, R³Is phenyl, substituted phenyl or benzyl;

x is S or S ═ O; y is CH or N;

R¹is 4-tetrahydropyranyl;

R²is 2- (dimethylamino) ethoxy, 1-methyl-4-piperidinyloxy, 4-methyl-1-piperazinyl, 4-ethyl-1-piperazinyl, N-methyl-N- (2-dimethylaminoethyl) amino or 4-dimethylamino-1-piperidinyl;

the substituted phenyl is 3-fluorophenyl or 4-fluorophenyl.

2. The pharmaceutically acceptable salt of the 2,8, 9-trisubstituted-9H-purine compound of claim 1, wherein the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, hydrobromide, nitrate, phosphate, sulfate, acetate, fumarate, malate, citrate, tartrate, maleate, lactate, citrate, camphorsulfonate, benzoate, gluconate, glutamate, isethionate, succinate and mesylate.

3. Use of a2, 8, 9-trisubstituted-9H-purine compound of claim 1 or a pharmaceutically acceptable salt of a2, 8, 9-trisubstituted-9H-purine compound of claim 2 in the manufacture of a pharmaceutical formulation against cancer; the anti-cancer pharmaceutical preparation can inhibit single-mutation EGFR and can inhibit triple-mutation EGFR.

4. The use of claim 3, wherein the single mutated EGFR is an EGFR that has undergone a L858R mutation; the triple-mutated EGFR is an EGFR which has undergone L858R/T790M/C797S mutation.