CN114805369B - 2, 6-disubstituted purine compounds, preparation method and application thereof - Google Patents

Abstract

The present invention belongs to the technical field of antineoplastic pharmacy, and particularly relates to a 2,6-disubstituted purine compound and its application, and the compound or its pharmaceutically acceptable salt can be used as an inhibitor targeting EGFR L858R/T790M , has anti-tumor activity, can effectively inhibit the growth of cancer cells, and has a simple production method and low cost.

Description

A kind of 2,6-disubstituted purine compound and its preparation method and application

technical field

The invention belongs to the technical field of antitumor pharmacy, and in particular relates to a 2,6-disubstituted purine compound, a preparation method and application thereof.

Background technique

Epidermal growth factor receptor (EGFR) is a member of the HER family. EGFR is the expression product of the proto-oncogene c-erbB-1 and is a transmembrane tyrosine kinase receptor. EGFR plays an important role in physiological processes such as cell growth, proliferation and differentiation by affecting downstream signaling pathways such as PI3K/AKT. The loss of function or abnormal activity of EGFR will cause tumors, diabetes, immunodeficiency and cardiovascular diseases. In particular, EGFR has also been identified as a key target in non-small cell lung cancer.

The existing EGFR tyrosine kinase inhibitors have been developed to the third-generation inhibitors with the advantage of combining EGFR sensitive mutations and T790M mutation sites, but the emergence of drug resistance makes it difficult for them to exert ideal anti-tumor effects.

Contents of the invention

In order to solve the above technical problems, the present invention provides a 2,6-disubstituted purine compound and its preparation method and application, which can be used as an inhibitor targeting EGFR L858R/T790M, has anti-tumor activity, and can effectively inhibit the growth of cancer cells. The growth and production methods are simple and the cost is low.

A 2,6-disubstituted purine compound in the present invention that solves the above technical problems is characterized in that: the following compound or a pharmaceutically acceptable salt thereof:

F in the present invention represents a fluorine atom.

A kind of 2 in the present invention, the preparation method of 6-disubstituted purine compound comprises the following steps:

(1) Synthesis of Intermediate 1:

Dissolve 2,6-dichloro-9H-purine in tetrahydrofuran, add 1-fluoro-4-(2-bromoethyl)benzene and drop triethylamine, react at room temperature for 2 hours, concentrate under reduced pressure, and pass through Intermediate 1 was obtained after separation by column chromatography; wherein 2,6-dichloro-9H-purine: tetrahydrofuran: 1-fluoro-4-(2-bromoethyl) benzene: triethylamine = 9-11: 14- 16:8-9:22-24;

(2) Synthesis of intermediate 2:

Dissolve intermediate 1 in isopropanol and stir at room temperature, then add 4-(4-methylpiperazine)aniline and DIPEA in sequence. Then the reaction solution was heated to 80°C for 4 hours, and the yellow solid was obtained by suction filtration under reduced pressure. The crude product was separated by column chromatography to obtain intermediate 2; wherein, intermediate 1: isopropanol: 4-(4-methyl Piperazine) aniline: DIPEA = 10-12.5: 25-32: 5-6: 2-3;

(3) Synthesis of Intermediate 3:

Dissolve the intermediate 2, 3-nitroaniline and trifluoroacetic acid in sec-butanol, then raise the temperature of the reaction solution to 80°C and stir for 4 hours, then use column chromatography to separate the intermediate 3 to obtain the intermediate 3; among them, the intermediate 2: 3-nitroaniline: trifluoroacetic acid: sec-butanol = 13.5-15: 1-2: 0.6-1: 25-35;

(4) Synthesis of Intermediate 4:

Add intermediate 3, hydrazine hydrate and Raney nickel to methanol, react under ice bath conditions, filter under reduced pressure and concentrate, and obtain the target product intermediate 4 after column chromatography purification; among them, intermediate 3: hydrazine hydrate : Raney Nickel = 11-12: 0.2-0.4: 2-3;

(5) Synthesis of compound 1:

Add acryloyl chloride and TEA to the tetrahydrofuran solution in which intermediate 4 is dissolved, stir and react under ice-bath conditions, concentrate under reduced pressure, and then separate by column chromatography; wherein, intermediate 4: tetrahydrofuran: acryloyl chloride: TEA= 8-9: 18-22: 0.2-0.3: 0.5-0.7.

The column chromatography in the step (1) and step (2) is petroleum ether/ethyl acetate.

The column chromatography in the step (3) and step (5) is dichloromethane/methanol.

Use of the compound or a pharmaceutically acceptable salt thereof in the preparation of antitumor drugs.

The antitumor drug is an inhibitor drug targeting EGFR L858R/T790M.

The antitumor drug is a drug for treating tumors characterized by EGFR L858R/T790M overexpression.

A pharmaceutical composition of the present invention, comprising an effective dosage of the compound or pharmaceutically acceptable salt thereof according to claim 1. The compounds of the present invention may be prepared by methods known in the art in the following forms: tablets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, Suppositories, finely divided powders or aerosols or sprays for inhalation, sterile aqueous or oily solutions or suspensions or sterile emulsions for parenteral use (including intravenous, intramuscular or infusion). Liquid preparations can be prepared using sterile water or water-propylene glycol solutions as a solvent, or the active ingredient can be formulated in aqueous polyethylene glycol solution. Aqueous solutions for oral administration can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilizing and thickening agents as desired. Aqueous suspensions for oral use are obtained by dispersing finely divided active ingredients in water together with viscous substances such as natural synthetic gums, resins, methylcellulose, carboxymethylcellulose and other pharmaceutical fields known suspending agents.

Pharmaceutical compositions may be in unit dosage form. In such form, the composition is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, cachet, or tablet, or it can be the appropriate number of any of these in packaged form.

The active ingredient of the pharmaceutical composition of the present invention may be only the compound of the present invention, or it may be combined with other antitumor compounds as the active ingredient.

During the treatment of tumors, the pharmaceutical composition of the present invention can be combined with other antineoplastic drugs. For example, in combination with antiproliferative/antineoplastic agents, cytostatic agents, anti-invasion agents, growth factor function inhibitors, antiangiogenic agents, vascular injury agents, etc. used in medical oncology.

In the treatment of tumors, such combination therapy can be achieved by simultaneous, sequential or separate administration of the various therapeutic ingredients. Such combination products employ the compounds of this invention within an effective dosage range and the other pharmaceutically active agent within an approved dosage range.

The 2,6-disubstituted purine inhibitors targeting EGFR L858R/T790M in the present invention can selectively inhibit EGFR L858R/T790M protein, and show good anti-proliferation activity in corresponding tumor cells, so aiming at this The development of class inhibitors has very broad research prospects. Its selective inhibition shows better anti-proliferation activity for cells with high expression of EGFR L858R/T790M, specifically, the half-inhibitory concentration of H1975 is 6.33±0.57μM; while the anti-proliferation activity for cells expressing wild-type EGFR The activity is low, and the specific performance is that the half inhibitory concentration of A549 and A431 cells is 22.6±1.64μM and 23.9±1.24μM.

Description of drawings

Fig. 1 is a graph showing the test results of the anti-proliferation activity of the compound represented by formula I in the present invention on NCI-H1975, HCC827, A549, A431 cells.

Detailed ways

The specific implementation of the present invention will be further described below in conjunction with the examples, and the present invention is not limited to the scope of the examples.

Example 1

A 2,6-disubstituted purine compound, which provides the general structural formula of the compound as shown below or a pharmaceutically acceptable salt thereof:

Example 2

A 2,6-disubstituted purine compound, which is synthesized by the following reaction formula:

The compound is synthesized using the following reaction formula:

The conditions used: (i) TEA, THF; (ii) DIPEA, i-PrOH, 75-85°C; (iii) TFA, 2-butanol, 75-85°C; (iv) Ranny-Ni, Hydrazine hydrate, MeOH , 0-25°C; (v) TEA, THF, 0-25°C. The detailed steps are as follows:

(1) Synthesis of Intermediate 1:

Dissolve 2,6-dichloro-9H-purine in tetrahydrofuran, add 1-fluoro-4-(2-bromoethyl)benzene and drop triethylamine, react at room temperature for 1 hour, concentrate under reduced pressure, and pass through Intermediate 1 was obtained after separation by column chromatography; wherein 2,6-dichloro-9H-purine: tetrahydrofuran: 1-fluoro-4-(2-bromoethyl) benzene: triethylamine = 9: 14: 8: twenty two;

(2) Synthesis of Intermediate 2:

Dissolve intermediate 1 in isopropanol and stir at room temperature, then add 4-(4-methylpiperazine)aniline and DIPEA in sequence. Then the reaction solution was heated to 75°C for 5 hours, and a yellow solid was obtained by suction filtration under reduced pressure. The crude product was separated by column chromatography to obtain intermediate 2; wherein, intermediate 1: isopropanol: 4-(4-methyl Piperazine) aniline: DIPEA=10:25:5:2;

(3) Synthesis of intermediate 3:

Dissolve intermediate 2, 3-nitroaniline and trifluoroacetic acid in sec-butanol, then raise the temperature of the reaction solution to 75°C and stir for 5 hours, then use column chromatography to obtain intermediate 3; among them, intermediate 2: 3-nitroaniline: trifluoroacetic acid: sec-butanol = 13.5: 1: 0.6: 25;

(4) Synthesis of Intermediate 4:

Add intermediate 3, hydrazine hydrate and Raney nickel to methanol, react under ice bath conditions, filter under reduced pressure and concentrate, and obtain the target product intermediate 4 after column chromatography purification; among them, intermediate 3: hydrazine hydrate :Raney nickel=11:0.2:2;

(5) Synthesis of compound 1:

Add acryloyl chloride and TEA to the tetrahydrofuran solution in which intermediate 4 is dissolved, stir the reaction under ice-bath conditions, concentrate under reduced pressure and separate by column chromatography; wherein, intermediate 4: tetrahydrofuran: acryloyl chloride: TEA = 8 :18:0.2:0.5.

The column chromatography in step (1) and step (2) is petroleum ether/ethyl acetate.

The column chromatography in step (3) and step (5) is dichloromethane/methanol.

Example 3

(1) Synthesis of Intermediate 1:

Dissolve 2,6-dichloro-9H-purine in tetrahydrofuran, add 1-fluoro-4-(2-bromoethyl)benzene and drop triethylamine, react at room temperature for 3 hours, concentrate under reduced pressure, and pass through Intermediate 1 was obtained after separation by column chromatography; wherein 2,6-dichloro-9H-purine:tetrahydrofuran:1-fluoro-4-(2-bromoethyl)benzene:triethylamine=11:16:9: twenty four;

(2) Synthesis of intermediate 2:

Dissolve intermediate 1 in isopropanol and stir at room temperature, then add 4-(4-methylpiperazine)aniline and DIPEA in sequence. Then the reaction solution was heated to 85°C for 3 hours, and a yellow solid was obtained by suction filtration under reduced pressure. The crude product was separated by column chromatography to obtain intermediate 2; wherein, intermediate 1: isopropanol: 4-(4-methyl Piperazine) aniline: DIPEA=12.5:32:6:3;

(3) Synthesis of intermediate 3:

Dissolve intermediate 2, 3-nitroaniline and trifluoroacetic acid in sec-butanol, then raise the temperature of the reaction solution to 85°C and stir for 3 hours, then use column chromatography to obtain intermediate 3; among them, intermediate 2: 3-nitroaniline: trifluoroacetic acid: sec-butanol = 15:2:1:35;

(4) Synthesis of Intermediate 4:

Add intermediate 3, hydrazine hydrate and Raney nickel to methanol, react under ice bath conditions, filter under reduced pressure and concentrate, and obtain the target product intermediate 4 after column chromatography purification; among them, intermediate 3: hydrazine hydrate :Raney nickel=12:0.4:3;

(5) Synthesis of compound 1:

Add acryloyl chloride and TEA to the tetrahydrofuran solution in which intermediate 4 is dissolved, stir the reaction under ice-bath conditions, concentrate under reduced pressure and then separate it by column chromatography; wherein, intermediate 4: tetrahydrofuran: acryloyl chloride: TEA = 9 :22:0.3:0.7.

The column chromatography in step (1) and step (2) is petroleum ether/ethyl acetate.

The column chromatography in step (3) and step (5) is dichloromethane/methanol.

Example 4

A preparation method of 2,6-disubstituted purine compounds, the steps are as follows:

(1) Synthesis of Intermediate 1:

Dissolve 2,6-dichloro-9H-purine in tetrahydrofuran, add 1-fluoro-4-(2-bromoethyl)benzene and drop triethylamine, react at room temperature for 2 hours, concentrate under reduced pressure, and pass through Intermediate 1 was obtained after separation by column chromatography; wherein 2,6-dichloro-9H-purine: tetrahydrofuran: 1-fluoro-4-(2-bromoethyl) benzene: triethylamine = 10: 15: 8.6: 23.3;

(2) Synthesis of Intermediate 2:

Dissolve intermediate 1 in isopropanol and stir at room temperature, then add 4-(4-methylpiperazine)aniline and DIPEA in sequence. Then the reaction solution was heated to 80°C for 4 hours, and the yellow solid was obtained by suction filtration under reduced pressure. The crude product was separated by column chromatography to obtain intermediate 2; wherein, intermediate 1: isopropanol: 4-(4-methyl Piperazine) aniline: DIPEA=11.9:30:5.6:2.53;

(3) Synthesis of Intermediate 3:

Dissolve the intermediate 2, 3-nitroaniline and trifluoroacetic acid in sec-butanol, then raise the temperature of the reaction solution to 80°C and stir for 4 hours, then use column chromatography to separate the intermediate 3 to obtain the intermediate 3; among them, the intermediate 2: 3-nitroaniline: trifluoroacetic acid: sec-butanol = 14.3: 1.6: 0.740: 30;

(4) Synthesis of Intermediate 4:

Add intermediate 3, hydrazine hydrate and Raney nickel to methanol, react under ice bath conditions, filter under reduced pressure and concentrate, and obtain the target product intermediate 4 after column chromatography purification; wherein, sec-butanol: hydrazine hydrate : Raney nickel=11.4:0.248:2.6.

(5) Synthesis of compound 1:

Add acryloyl chloride and TEA to the tetrahydrofuran solution in which intermediate 4 is dissolved, stir the reaction under ice-bath conditions, concentrate under reduced pressure and separate it by column chromatography; wherein, intermediate 4: tetrahydrofuran: acryloyl chloride: TEA = 8.5 :20:0.241:0.657.

The column chromatography in step (1) and step (2) is petroleum ether/ethyl acetate.

The column chromatography in step (3) and step (5) is dichloromethane/methanol.

Example 5

A kind of preparation method of 2,6-disubstituted purine compound, concrete steps are as follows:

(1) Synthesis of Intermediate 1:

Dissolve 2,6-dichloro-9H-purine (1.00g, 5.3mmol) in 15mL of tetrahydrofuran, add 1-fluoro-4-(2-bromoethyl)benzene (0.86g, 4.2mmol), drop three Ethylamine (2.33 g, 10.6 mmol). The reaction was carried out at room temperature for 2 hours. After the completion of the reaction detected by TLC, the mixture was concentrated under reduced pressure. The crude product was separated by column chromatography (petroleum ether/ethyl acetate) to obtain intermediate 1 (1.19 g, yield 72%).

(2) Synthesis of intermediate 2:

Intermediate 1 (1.19g, 3.8mmol) was dissolved in 30mL of isopropanol and stirred at room temperature, followed by adding 4-(4-methylpiperazine) aniline (0.56g, 3.1mmol), DIPEA (2.53mL, 15.3mmol) . Then, the temperature of the reaction solution was raised to 80° C. for four hours. TLC detected that the reaction was complete and filtered under reduced pressure to obtain a yellow solid. The crude product was separated by column chromatography (petroleum ether/ethyl acetate) to obtain intermediate 2 (1.43 g, yield 81%).

(3) Synthesis of intermediate 3:

Intermediate 2 (1.43 g, 3.1 mmol), 3-nitroaniline (0.16 g, 2.5 mmol) and trifluoroacetic acid (740 μL, 10 mmol) were dissolved in sec-butanol (30 mL). Then the reaction solution was heated to 80° C. and stirred for 4 hours. When TLC showed that the reaction was complete, the intermediate 3 (1.14 g, yield 65%) was separated by column chromatography (dichloromethane/methanol).

(4) Synthesis of Intermediate 4:

Intermediate 3 (1.14g, 2.0mmol), hydrazine hydrate (248μL, 8.0mmol), and Raney nickel (0.26g, 4.0mmol) were added to methanol and reacted under ice bath conditions. TLC detection showed that the reaction of intermediate 3 was complete , filtered under reduced pressure, concentrated, and purified by column chromatography to obtain the target product intermediate 4 (0.85 g), a yellow solid, with a yield of 79%.

(5) Synthesis of compound 1:

Add acryloyl chloride (241 μL, 3.16 mmol) and TEA (657 μL, 4.74 mmol) to a solution of intermediate 4 (0.85 g, 1.58 mmol) in tetrahydrofuran (20 mL), stir the reaction under ice-bath conditions, TLC detection shows that the intermediate 4 The reaction is complete. It was concentrated under reduced pressure and separated by column chromatography (dichloromethane/methanol) to obtain a yellow solid (0.59 g, yield 63%). The above yield values are the yields in each step.

Compound 1, yellow solid, yield 63%; ¹ H NMR (400MHz, DMSO-d ₆ ) δ10.07(s, 1H), 9.32(s, 1H), 9.08(s, 1H), 8.34(s, 1H ),7.89–7.74(m,3H),7.46(d,J＝7.6Hz,1H),7.24(dd,J＝8.3,5.5Hz,2H),7.22–7.13(m,2H),7.07(t, J＝8.7Hz, 2H), 6.88(dd, J＝11.8, 7.8Hz, 2H), 6.49(dd, J＝16.9, 10.2Hz, 1H), 6.30–6.18(m, 1H), 5.74(d,J =12.1Hz, 1H), 4.40(t, J=7.3Hz, 2H), 3.17(t, J=7.3Hz, 2H), 3.07(t, J=5.0Hz, 4H), 2.45(q, J=9.0 , 6.9Hz, 4H), 2.23 (s, 3H). ¹³ C NMR (101MHz, DMSO) Δ163.4,160.2,160.2,152.5,151.1,147.1,142.2,139.2,134.8, 1322 .6, 132.4, 131.1, 131.0, 128.8, 126.9, 122.3, 120.8, 116.1, 115.7, 115.5, 115.2, 115.0, 112.8, 111.2, 55.2, 49.3, 46.3, 44.5, 34.8.

Test Example 1 Anti-proliferation test of compound 1 on NCI-H1975, HCC827, A549, A431 cells:

Anti-proliferation activity test in NCI-H1975, HCC827, A549, A431 cells,

H1975, HCC827, A549, and A431 cells in logarithmic growth phase were seeded in 96-well plates (approximately 5×10 ³ cells per well). After culturing for 24 hours, after the cells adhered to the wall, each compound was added at a specified concentration (0-40 μM) and incubated for 24 hours. MTT was dissolved using PBS to a final concentration of 0.5%, and then 20 μL of MTT solution was added to each well. Incubate at 37°C in a cell culture incubator with 5% CO ₂ in the dark for 3-4 hours, then discard the existing medium, add 150 μL of DMSO to each well, and detect the corresponding OD value at a wavelength of 490 nM with a microplate reader. The effect of each compound on cell proliferation was analyzed by SPSS, and the anti-proliferation activity of each compound was evaluated.

The results are shown in Figure 1. NCI-H1975, HCC827, A549, A431 cells were treated with compound 1, and the half inhibitory concentration of different tumor cells was determined. The corresponding OD value was measured by a microplate reader, and the anti-proliferation activity was evaluated by the MTT method.

It can be clearly seen from Figure 1 that the compound has significantly stronger antiproliferative activity on cells expressing EGFR L858R/T790M, and the half inhibitory concentrations on NCI-H1975, HCC827, A549, and A431 cells were 6.33±0.57μM, 6.10 ±0.80μM, 22.6±1.64μM, 23.9±1.24μM.

In another embodiment, the present invention also includes the use of a compound or a pharmaceutically acceptable salt thereof in the preparation of an antitumor drug.

In a further embodiment, the antitumor drug is an inhibitor drug targeting EGFR L858R/T790M.

Antineoplastic drugs are drugs for treating tumors characterized by EGFR L858R/T790M overexpression.

The present invention also provides a pharmaceutical composition, which is a preparation comprising an effective dose of the above-mentioned compound or a pharmaceutically acceptable salt thereof. The compounds of the present invention may be prepared by methods known in the art in the following forms: tablets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, Suppositories, finely divided powders or aerosols or sprays for inhalation, sterile aqueous or oily solutions or suspensions or sterile emulsions for parenteral use (including intravenous, intramuscular or infusion). Liquid preparations can be prepared using sterile water or water-propylene glycol solutions as a solvent, or the active ingredient can be formulated in aqueous polyethylene glycol solution. Aqueous solutions for oral administration can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilizing and thickening agents as desired. Aqueous suspensions for oral use are obtained by dispersing finely divided active ingredients in water together with viscous substances such as natural synthetic gums, resins, methylcellulose, carboxymethylcellulose and other pharmaceutical fields known suspending agents.

Pharmaceutical compositions may be in unit dosage form. In such form, the composition is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, cachet, or tablet, or it can be the appropriate number of any of these in packaged form.

The active ingredient of the pharmaceutical composition of the present invention may be only the compound of the present invention, or it may be combined with other antitumor compounds as the active ingredient.

In the process of treating tumors, the pharmaceutical composition of the present invention can be combined with other antineoplastic drugs. For example, in combination with antiproliferative/antineoplastic agents, cytostatic agents, anti-invasion agents, growth factor function inhibitors, antiangiogenic agents, vascular injury agents, etc. used in medical oncology.

In the treatment of tumors, such combination therapy can be achieved by simultaneous, sequential or separate administration of the various therapeutic ingredients. Such combination products employ the compounds of this invention within an effective dosage range and the other pharmaceutically active agent within an approved dosage range.

Based on the above experiments, an inhibitor that can effectively inhibit EGFR L858R/T790M from exerting its anti-cancer effect was obtained. This type of inhibitor can provide a good treatment for a variety of tumors that overexpress EGFR L858R/T790M, including non-small cell lung cancer. The method aimed at the development of such inhibitors has very broad research prospects.

The above implementation/test examples are merely examples for clear description, and are not intended to limit the implementation. For those of ordinary skill in the art, other changes or changes in different forms can be made on the basis of the above description. It is not necessary and impossible to exhaustively list all the implementation manners here. And the obvious changes or changes derived therefrom are still within the scope of protection of the present invention.

Claims (5)

1. A 2,6-disubstituted purine compound, characterized in that: a compound or a pharmaceutically acceptable salt thereof as shown below:

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of antitumor drugs. 3. The use according to claim 2, characterized in that: the antitumor drug is an inhibitor drug targeting EGFR L858R/T790M. 4. The use according to claim 2, characterized in that: the anti-tumor drug is a drug for treating tumors characterized by overexpression of EGFRL858R/T790M. 5. A pharmaceutical composition, characterized in that it is a preparation comprising an effective dose of the compound of claim 1 or a pharmaceutically acceptable salt thereof.

Images