WO2014008673A1

WO2014008673A1 - Allopurinol derivative and preparation method and application thereof

Info

Publication number: WO2014008673A1
Application number: PCT/CN2012/078732
Authority: WO
Inventors: 李财虎; 石万棋; 赵洪祥; 李剑忠; 李颖; 董林; 尹述凡
Original assignee: 四川国康药业有限公司
Priority date: 2012-07-09
Filing date: 2012-07-17
Publication date: 2014-01-16
Also published as: CN102746306B; CN102746306A

Abstract

The present invention provides an allopurinol derivative as shown in Formula I, and a preparation method and an application thereof. The allopurinol derivative may be used for treating cancer and gout.

Description

Allopurinol derivatives, preparation methods and uses thereof

The present invention relates to allopurinol derivatives and processes for their preparation and use.

Background technique

Tumor is one of the most common and serious diseases that endanger human life in today's world. Chemotherapy, radiotherapy, and surgical therapy are currently the main treatments for cancer. At present, the number of drugs used for tumor treatment is limited, and the price thereof is relatively high, which brings a large economic burden to tumor patients. Therefore, it is especially necessary to develop more effective anti-tumor drugs.

Allopurinol, chemical name: 4_hydroxy-I pyrazole [3, 4_d] pyrimidine, the structure is as follows:

It is an isomer of natural hypoxanthine, which inhibits the activity of xanthine oxidase, prevents the oxidation of jaundice and hypoxanthine to produce uric acid, thereby lowering the concentration of serum uric acid, mainly for the treatment of hyperuricemia and gout. . Since becoming a drug of choice for the treatment of gout in 1963, it has been the first line of medicine for the treatment of gout.

At present, allopurinol has not been used as a lead compound, and after structural modification and modification, it is used for anti-tumor reports.

Summary of the invention

It is an object of the present invention to provide an allopurinol derivative, a process for its preparation and use. The present invention also provides a pharmaceutical composition for antitumor and gout treatment.

Specifically, the present invention provides a structural formula of the formula I as follows:

I

Wherein, it is a linear thiol group of C1-4, or an ester group of C1-4; and R ₂ is an aromatic hydrocarbon group or a substituted aromatic hydrocarbon group of -NH ₂ or C6-10. Further, the ester group of C1-4 is -COOCH ₂ CH ₃ , -CH ₂ COOCH ₂ CH ₃ or -CH ₂ C00C3⁄4;

The C6~10

go a step further,

The C6-10 substituted aromatic hydrocarbon group is

Among them, Ri -COOCH ₂ CH ₃ ;

R ₂ is -NH ₂

Further preferably, the compound is ethyl-4-mercapto-1 -pyrazole[3,4-6]pyrimidine, or (N-(4-chloroacetylamino)phenyl)-4-amino-1 - Pyrazole [3,4-]pyrimidin-1-ethyl-2-acetate.

The invention also provides a preparation method of the above ethyl-4-mercapto-1 -pyrazole [3, pyrimidine, which comprises the following steps:

A, 4-chloro-1-pyrazolo [3, pyrimidine and bromoacetamidine, by electrophilic substitution reaction to produce 4-chloro-1-ethyl-1,3-pyrazole [3, 4-6] pyrimidine;

B. Taking 4-chloro-1-ethyl-1,3-pyrazole [3, pyrimidine and hydrazine hydrate, 1-ethyl-4-mercapto-l-pyrazole [3, pyrimidine is produced by nucleophilic substitution reaction.

Wherein, in step A, the molar ratio of 4-chloro-1-pyrazolo[3,4-6]pyrimidine to bromoethene is 1:1-2; in step B, 4-chloro-1-ethylpyrazole The molar ratio of [3, 4-6 ]pyrimidine to hydrazine hydrate is 1:1-2.

Further, in the step A, the molar ratio of 4-chloro-1-pyrazole [3, pyrimidine to bromoacetone is 1: 1.0-1.5; in step B, 4-chloro-1-ethylpyrazole [3, 4-6] The molar ratio of pyrimidine to hydrazine hydrate is 1: 1.0-1.5.

The present invention also provides a method for preparing the above (N-(4-chloroacetamido:)phenyl:)-4-amino-1-pyrazole[3,pyrimidin-^^-)-2-ethyl acetate, which Including the following steps:

A, taking 4-chloro-1-pyrazolo[3,4-6]pyrimidine, and electrophilic substitution reaction with ethyl bromoacetate (4-chloro-1-pyrazole[3,4-6]pyrimidine- -1-)-2-ethyl acetate;

B, taking (4-chloro-1-pyrazole[3,4-6]pyrimidin-1-)-2-acetic acid ethyl ester, and electrophilic substitution reaction with p-phenylenediamine (N-(4-aminobenzene) Ethyl 4-amino-1 -pyrazole [3,4-]pyrimidine-1-ethyl-2-acetate;

C, taking (N-(4-aminophenyl)-4-amino-1-pyrazolo[3,4-6]pyrimidin-1-ethyl-2-acetate, by nucleophilic substitution reaction with benzoyl chloride The target compound is produced.

Wherein, in step A, 4-chloro-1-pyrazolo[3, the molar amount of pyrimidine and ethyl bromoacetate The ratio is 1: 1-2; in step B, the molar ratio of (4-chloro-1-pyrazol[3,4-6]pyrimidin-1-)-2-ethyl acetate to p-phenylenediamine is 1 : 1-2; In step C, the molar ratio of (N-(4-aminophenyl)-4-aminopyrazole[3,pyrimidin-1-)-2-ethyl acetate to formyl chloride is 1:1 ~2.

Further, in step A, the molar ratio of 4-chloro-1-pyrazolo[3,pyrimidine to ethyl bromoacetate is 1:1.0-1.5; in step B, (4-chloropyrazole[3, 4 - The molar ratio of ethylpyrimidin--1-)-2-ethyl acetate to p-phenylenediamine is 1: 1.0-1.5; in step C, (N-(4-aminophenyl)-4-aminopyrazole [ 3. The molar ratio of ethyl pyrimidine- 1-)-2-ethyl acetate to formyl chloride is 1: 1.0-1.5.

The invention also provides the use of the above compounds in the manufacture of a medicament for the treatment of anti-tumor or gout. Further, the drug is an anti-hepatocarcinoma cell drug or a xanthine oxidase inhibitor.

The present invention also provides a pharmaceutical composition for antitumor and gout treatment, which is prepared by the above compound as an active ingredient together with a pharmaceutically usable adjuvant.

The allopurinol derivative of the invention can effectively inhibit tumor growth, and its activity is equivalent to 17-AAG which is undergoing phase 3 clinical trial, and has good anti-tumor effect; it can also effectively inhibit xanthine oxidase activity and can be used for gout. The treatment provides a new drug selection for clinical treatment of cancer and gout. Meanwhile, the preparation method of the compound of the invention is simple, the cost is low, the yield is high, and the invention has good industrial application prospect.

detailed description

Example 1 Synthesis of ethyl-4-mercapto-Ipyrazole [3, pyrimidine (referred to as compound 4)

(1) Synthesis of 4-chloro- 1-ethyl- 1 pyrazole [3, 4-pyrimidine (referred to as compound 3)

4-Chloro-1-pyrazolo[3,4-pyrimidine (O.lg, 0.65 mmol) was dissolved in 5 mL of dry DMF, and the reaction mixture was stirred at room temperature for 10 min, and slowly added dropwise with a dropping funnel. Triethylamine (0.20 g, 1.95 mmol), and the mixture was stirred at room temperature for 30 min. Further, ethidium bromide (0.084 g, 0.78 mmol) dissolved in 3 mL of dry DMF was slowly added dropwise to the mixture, and stirring was continued for 1 hour, and then a catalytic amount of KI was added. The reaction mixture was completely reacted by TLC, and the mixture was poured into 15 mL of water. The pH was adjusted to acidic with dilute hydrochloric acid, then ethyl acetate (4×20 mL) was added and the organic layer was washed with saturated sodium chloride. The mixture was dried over anhydrous magnesium sulfate and evaporated. The crude product was purified by chromatography EtOAcjjjjjj

Compound 3: white crystal, 76~77 ° C 3⁄4 NMR (400 MHz, DMSO-d ₆ ) δ: 8.87 (s, IH, CH), 8.49 (s, IH, CH), 4.51 (dd, J = 7.24 Hz , J = 14.52 Hz, 2H, CH ₂ ), 1.45 (t, J = 7.26 Hz, 3H, CH ₃ ); ¹³ C NMR (100 MHz, DMSO-d ₆ ) 156.65, 150.77, 150.35, 134.40, 106.12, 42.43 , 15.12; IR (KBr, v, cm" ¹ ): 3466, 3094, 2924, 2854, 1690, 1575, 1538, 1460, 1389, 1283, 1209, 1131, 1007, 956, 782, 680, 594, 533, 406. HRMS (ESI) calcd for C ₇ H ₇ C1N ₄ [M+H] ⁺ 182.0359 found 183.0535.

(2) Synthesis of 1-ethyl-4-mercapto- 1 pyrazole [3, 4-d-pyrimidine (referred to as compound 4)

Compound 3 (0.1 g, 0.56 mmol) was dissolved in 5 mL of acetonitrile and hydrazine hydrate (0.034 g,

0.68 mmol) was slowly added to the mixture, and the reaction was stirred at room temperature for 3 hr and then was then reacted with TLC. The reaction mixture was concentrated to give a crude solid. The crude product was washed with water and ethyl acetate.

Compound 4: White solid, 198~200 ° C ^ NMR (400 MHz, DMSO-d ₆ ) δ: 8.29 (s, IH, CH), 8.05 (s, IH, CH), 4.85 (s, 2H, NH ₂ ), 4.65 (s, IH, NH), 4.30 (dd, J = 7.18 Hz, J = 14.38 Hz, 2H, CH ₂ ), 1.36 (t, J = 7.22 Hz, 3H, CH ₃ ); ¹³ C NMR ( 100 MHz, DMSO-d ₆ ) 161.03, 155.41, 153.66, 135.16, 99.12, 41.50, 15.20; IR (KBr, v, cm" ¹ ): 1717, 1659, 1599, 1538, 1497, 1441, 1375, 1346, 1295 , 1251, 1182, 1091, 962, 913, 860, 785, 694, 620, 541. HRMS (ESI) calcd for C ₇ H ₁₀ N ₆ [M+H] ⁺ 178.0967 found 179.1044. Example 2 (N-( Preparation of (4-chloroacetamido)phenyl)-4-amino-IH-pyrazole [3, 4-pyrimidin-1-system-2-ethylethyl ester (abbreviated as compound 8e) '. -,,, ,,-hurt;

■:,,,,,·?

, ,

8e

(1) (4-Chloro-I pyrazole [3, 4-pyrimidin-1-(2-)-2-ethylacetate (referred to as compound 6) to make

TEA (1.78 g, 17.62 mmol) was slowly added to 4-chloro-1-pyrazolo[3,4-pyrimidine (0.81 g, 5.26 mmol) dissolved in 20 mL dry DMF and stirred at room temperature for 30 min. Ethyl bromoacetate (1.06 g, 6.39 mmol) dissolved in 5 mL of dry DMF was slowly added dropwise to the mixture and stirring was continued for 2 h. The reaction mixture was detected by TLC until the reaction was completed, and then poured into water (30 mL), and the pH was adjusted to acid with dilute hydrochloric acid, and then ethyl acetate (3×25 mL), and the organic layer was washed with saturated sodium chloride . The crude product was purified by silica gel column chromatography eluting eluting

Compound 6: white crystal, 83~84 ° C 3⁄4 NMR (400 MHz, DMSO-d ₆ ) δ: 8.91 (s, 1 Η, CH), 8.58 (s, 1H, CH), 5.45 (s, 2H, CH ₂ ), 4.17 (dd, J = 7.10 Hz, J = 14.2 Hz, 2H, CH ₂ ), 1.20 (t, J = 7.10 Hz, 3H, CH ₃ ); ¹³ C NMR (100 MHz, DMSO-d ₆ ) 167.90 , 156.53, 151.47, 151.09, 135.32, 106.29, 61.80, 48.86, 14.36; IR (KBr, v, cm" ¹ ): 3458, 3116, 2992, 2938, 1734, 1591, 1556, 1486, 1404, 1357, 1250, 1218, 1188, 1134, 1019, 950, 861, 786, 713, 568; HRMS (ESI) calcd for C ₉ H ₉ C1N ₄ 0 ₂ [M+H] + 240.0414 found 241.0492.

(2) Synthesis of (N-(4-aminophenyl)-4-amino- 1 pyrazole [3,4- /i-pyrimidine-1-ethyl-2-acetate (abbreviated as compound 7)

Compound 6 (1.73 g, 7.21 mmol) was dissolved in 25 mL of acetonitrile, then p-phenylenediamine (0.78 g, 7.21 mmol) was slowly added to the mixture. The mixture was warmed to 80 ° C and stirred for 3 h. To complete reaction. The reaction mixture was concentrated to give a crude solid. The crude product was purified by silica gel column chromatography eluting with EtOAc (EtOAc)

Compound 7: Gray solid, 179.8~181. 9 ° C 3⁄4 NMR (400 MHz, DMSO-d ₆ ) δ: 9.76 (br, 1H, NH), 8.27 (s, 2H, CH), 7.37 (s, 2H, ArH), 6. 62(s, 2H, ArH), 5·02~ 5. 18(m, 4H), 4.14(dd, J=7.04 Hz, J=14.16 Hz, 2H, CH ₂ ), 1.19(t , J=7.08 Hz, 3H, CH ₃ ); ¹³ C NMR (100 MHz, DMSO-d ₆ ) 167.80, 155.73, 154.30, 153.61, 132.52, 127.41, 123.34, 113.94, 100.76, 61.18, 47.80, 13.94; KBr, v, cm" ¹ ): 3476, 3381, 3200, 2940, 1735, 1591, 1514, 1440, 1329, 1291, 1254, 1212, 1019, 915, 795, 740, 706, 603, 516; HRMS (ESI Calcd for C ₁₅ H ₁₆ N ₆ 0 ₂ [M+H] ⁺ 312.1335 found 313.1439.

(3) Synthesis of (N-((4-chloroacetamido)phenyl)-4-amino- 1 pyrazole [3, 4-d-pyrimidin-1-)-2-ethylacetate (abbreviated as compound 8e)

Method 1: Compound 7 (0.50 g, 1.60 mmol) and TEA (0.50 g, 4.95 mmol) were added to 13 mL of dichloromethane in an ice bath, stirred for a while, and then dissolved in 2 mL of dichloromethane. The benzoyl chloride (0.25g, 1.79mmoi:> in the crucible was slowly added dropwise to the mixture. After the addition was completed, it was transferred to room temperature and heated to 50 ° C and stirred for 1 h. The reaction solution was detected by TLC until the reaction was completed. In 20 mL of water, the pH was adjusted to acidic with dilute hydrochloric acid, and then extracted with dichloromethane (3.times.sup.5mL). The organic layer was washed with saturated sodium chloride and concentrated to give a solid. The crude product was subjected to silica gel column chromatography eluting with petroleum ether: acetone:dichloromethane: chloroform (1 : 1 : 3 : 1) to give 8e.

Compound 8e: pink solid, 189·7~191. 5 ° C ^ NMR (400 MHz, DMSO-d ₆ ) δ: 10.34 (br, 1H, NH), 10.14 (br, 1H, NH), 8.40 (s , 1H, CH), 8.26(s, 1H, CH), 7.78(d, J=8.52 Hz, 2H, ArH), 7. 62(d, J=8.96 Hz, 2H, ArH), 5.23(s, 2H , CH ₂ ), 4.26(s, 2H, CH ₂ ), 4.15 (dd, J=7.10 Hz, J= 14.22 Hz, 2H, CH ₂ ), 1.20 (t, J=7.10 Hz, 3H, CH ₃ ); ¹³ C NMR (100 MHz, DMSO-d ₆ ) 168.23, 164.89, 156.00, 154.78, 154.05, 135.26, 134.86, 132.97, 122.40, 120.27, 101.29, 61.72, 48.40, 44.04, 14.43; IR (KBr, v, cm" ¹ ): 3627, 3265, 3197, 3110, 2992, 1742, 1667, 1572, 1513, 1434, 1294, 1209, 1095, 1019, 966, 919, 844, 785, 705, 656, 520; HRMS (ESI) calcd For Ci ₇ H ₁₇ ClN ₆ 0 ₃ [M+H] ⁺ 388.1051 found 389.1121.

Method 2 can also be used:

Compound 7 (0.50 g, 1.60 mmol) and TEA (0.50 g, 4.95 mmol) were sequentially added to 13 mL of THF under ice-cooling, stirred for a period of time, and then benzoyl chloride (0.25 g) dissolved in 2 mL of THF. 1.79mmoi:> slowly add dropwise to the mixture and continue stirring under ice bath. The reaction solution is detected by TLC until the reaction is completed, then poured into 20 mL of water, extracted with ethyl acetate (3 X i5 mL), and the organic layer is saturated. The sodium chloride was washed and condensed to obtain a solid. The crude product was recrystallized from anhydrous methanol to give 8e. The following describes the beneficial effects of the present invention by the test examples. Compounds 3, 4, 6, 7, 8e used in the following test examples , prepared by the examples 1 and 2, respectively.

Test Example 1 Antitumor effect of the compound of the present invention

Compounds 3, 4, 6, 7, 8e and the positive control 17-AAG (17-allyamino-17-demethoxygeldanamycin referred to as 17-AAG are first-generation geldanamycin derivatives) were subjected to Hsp90 inhibition experiments by MTT assay.

Well-grown human liver cancer cells 7402 and 7221 were seeded in 96-well plates at 7 x lO ³ /well. After 24 h, the concentration gradient (final concentration) of 2 g/ml, 4 g/ml, 8 g/ml, 16 g/ml, 32 g/ml and 64 g/ml were added to the plate and cultured for 72 hours. Thereafter, MTT solution 10 μl (; 5η3⁄4/ηι1) was added, and the mixture was applied at 37 ° C for 4 h, the supernatant was discarded, DMSO 150 μl/well was added, and the mixture was shaken at room temperature for 30 min to determine the OD570nm value. According to the cell proliferation inhibition rate formula: inhibition rate (%) = (control group OD value - experimental group OD value y control group OD value x l00%. IC ₅ . The value is calculated using statistical software. The experimental results are shown in Table 1. Table 1 Results of anti-tumor activity determination of target compounds

Compound IC ₅₀ value (μ g ■ ml ¹ ) Compound IC ₅₀ value (μ g . mL - No. 72h No. 72h

Bel-7402 SMMC-7221 Bel-7402 SMMC-7221

17-AAG 7. 27 5. 77 6 >100 >100 allopurinol 7 >64 >64

3 >100 >100 8e 7. 27 3. 51

4 4. 55 6. 28

" : no activity value; 1 ( _{5 ()} value > 64, indicating that the IC ₅ Q of the compound exceeds 64 μ _§ · ιη Ι The specific concentration is not clear, but still indicates that the compound has an inhibitory effect on the above tumor, only weak activity In a positive control; IC ₅ . Value > 100, supra.

It can be seen from the above test that allopurinol has no antitumor activity against Bel-7402 and S匪C-7221, and the compound of the present invention 4-8e has good tumor inhibitory activity, indicating that the compound of the formula I of the present invention has good antitumor activity. . Among them, the activity of the compounds 4 and 8e is particularly preferable, and not only is it superior to other allopurinol derivatives, but its activity is also comparable to that of 17-AAG. Test Example 2 Inhibition test of xanthine oxidase (X0D) by the compound of the present invention

Determination of X0D activity: Under the catalysis of xanthine oxidase, the substrate xanthine is oxidized to uric acid, ie the following reactions occur:

X0D

Xanthine + 0 ₂ ^ Uric acid + 0 ₂

The X0D activity is determined by the photometric value near the characteristic absorption peak (λ = 290ηπι) of the product uric acid.

The experimental method was measured spectrophotometrically at 290 nm. In the quartz cuvette, add buffer solution, substrate solution and enzyme solution, pre-warm at 25 °C for 2 min, measure the change in absorbance at 290 nm, record once every 5 sec, measure for 1 min, react during this time, The absorbance increases linearly with time, and the slope is the reaction rate (dA/min). The larger the slope, the stronger the activity of the enzyme.

Sample determination: In the same way, add the sample solution (5 L, lO L, 15 L, 20 L) to the enzymatic reaction system, pre-warm at 25 °C for 2 min, and record the absorbance increase at 290 nm every 5 sec. , a total of lmin. Each sample was operated in parallel three times, and the reaction rate was recorded separately, and the average was used to calculate the inhibition rate of the sample.

Table 2 Results of determination of target compound on xanthine oxidase inhibitory activity

It can be seen from the above experiments that the compound of the formula I of the present invention is effective in inhibiting xanthine oxidase and has good anti-gout activity. In summary, the allopurinol derivative of the present invention can effectively inhibit tumor growth, and its activity is comparable to that of 17-AAG which is undergoing phase 3 clinical trial, and has good antitumor effect; it can also effectively inhibit xanthine oxidase activity. It can be used for the treatment of gout, and provides a new drug selection for clinical treatment of cancer and gout. Meanwhile, the preparation method of the compound of the invention is simple, the cost is low, the yield is high, and the invention has good industrial application prospect.

Claims

demand for profit

1. The compound shown in formula I

I

Among them, is 11, C1-4 linear alkyl group or C1-4 ester group; R ₂ is -NH ₂ , C6~10 aromatic hydrocarbon group or substituted aromatic hydrocarbon group.

2. The compound according to claim 1, characterized in that: the ester group of C1-4 is -COOCH ₂ CH ₃ , -

The C6~10

3. The chemical according to claim 2, characterized in that:

The C6~10 substituted aromatic hydrocarbon group is

4. The compound according to any one of claims 1-3, characterized in that: is methyl or -C

5. The compound according to claim 4, characterized in that: the compound is -ethyl-4-hydrazinopyrazole [3, 4-pyrimidine, or (N-(4-chloroacetamido)phenyl) -4-Aminopyrazole[3, 4-ί]pyrimidine-1-)-2-ethyl acetate.

6. The preparation method of ethyl-4-hydrazinopyrazo[3,4-pyrimidine according to claim 5, characterized in that: it includes the following steps:

A. Take 4-chloropyrazolo[3,4-pyrimidine and bromoacetyl, and generate 4-chloroethylpyrazole[3,4-ί]pyrimidine through electrophilic substitution reaction;

B. Take 4-chloro-1-ethylpyrazole [3, 4-pyrimidine and hydrazine hydrate, and generate 1-ethyl-4-hydrazinopyrazole [3, 4-pyrimidine] through nucleophilic substitution reaction.

7. The preparation method according to claim 6, characterized in that: in step A, the molar ratio of 4-chloropyrazole [3, 4-pyrimidine and acetyl bromide is 1: 1-2; in step B, 4 -Chloro-1-ethylpyra The molar ratio of azole [3, 4-ί]pyrimidine and hydrazine hydrate is 1: 1-2.

8. The preparation method of (N-(4-chloroacetylamino)phenyl)-4-aminopyrazole [3, 4-pyrimidine-1-)-2-ethyl acetate according to claim 5, which is characterized in that: It includes the following steps:

A. Take 4-chloropyrazolo[3,4-ί]pyrimidine and react with ethyl bromoacetate to generate (4-chloropyrazolo[3,4-pyrimidine-1-)-2-ethyl acetate through electrophilic substitution reaction ester;

B. Take (4-chloropyrazole [3,4-ί]pyrimidine-1-)-2-ethyl acetate, and react with p-phenylenediamine through electrophilic substitution reaction to generate (N-(4-aminophenyl)- 4-Aminopyrazole [3,4-pyrimidine-1-)-2-ethyl acetate;

C. Take (N-(4-aminophenyl)-4-aminopyrazole[3,4-ί]pyrimidine-1-)-2-ethyl acetate and react with benzoyl chloride to generate the target compound through nucleophilic substitution reaction .

9. The preparation method according to claim 8, characterized in that: in step A, the molar ratio of 4-chloropyrazolo[3,4-pyrimidine and ethyl bromoacetate is 1: 1-2;

In step B, the molar ratio of (4-chloropyrazole [3, 4-pyrimidine-1-)-2-ethyl acetate to p-phenylenediamine is 1: 1-2;

In step C, the molar ratio of (N-(4-aminophenyl)-4-aminopyrazole [3, 4-ί]pyrimidine-1-)-2-ethyl acetate to formyl chloride is 1: 1~ 2.

10. The use of the compound according to any one of claims 1 to 5 in the preparation of anti-tumor or gout medicines.

11. The use according to claim 10, characterized in that: the drug is an anti-hepatoma cell drug or a xanthine oxidase inhibitor.

12. A pharmaceutical composition for anti-tumor and gout treatment, characterized in that: it is a preparation prepared from the compound described in any one of claims 1 to 5 as an active ingredient, plus pharmaceutically available excipients.