CN115650986A - Cinnamyl amino pyrazolo [3,4-d ] pyrimidine compound and preparation and application thereof - Google Patents

Cinnamyl amino pyrazolo [3,4-d ] pyrimidine compound and preparation and application thereof Download PDF

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CN115650986A
CN115650986A CN202211298291.8A CN202211298291A CN115650986A CN 115650986 A CN115650986 A CN 115650986A CN 202211298291 A CN202211298291 A CN 202211298291A CN 115650986 A CN115650986 A CN 115650986A
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pyrimidine
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pyrazolo
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赵鹏程
饶国武
张文
王举
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Zhejiang University of Technology ZJUT
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Zhejiang University of Technology ZJUT
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Abstract

The invention discloses a cinnamoylaminopyrazolo [3,4-d]Pyrimidine compounds and preparation and application thereof. The cinnamamidopyrazolo [3,4-d]The pyrimidine compounds have the following structural formula (I), wherein R is 2-Cl, 2-methyl, 3-methyl, 4-F or 3,4-dimethoxy. The invention discloses the cinnamoylaminopyrazolo [3,4-d]The pyrimidine compound or the pharmaceutically acceptable salt thereof is applied to the preparation of the medicine for treating or preventing cancer, wherein cancer cells of the cancer are human malignant melanoma cells A375, human liver cancer cells HepG2, human stomach cancer cells AGS, human breast cancer cells T47D, human breast cancer cells MDA-MB-231 and human colon cancer cells SW620, and the results show that the compound has good inhibition effect on the human malignant melanoma cells A375, the human liver cancer cells HepG2, the human stomach cancer cells AGS, the human breast cancer cells T47D, the human breast cancer cells MDA-MB-231 and the human colon cancer cells SW620.

Description

Cinnamyl amino pyrazolo [3,4-d ] pyrimidine compound and preparation and application thereof
Technical Field
The invention relates to a cinnamamide amino pyrazolo [3,4-d ] pyrimidine compound, a preparation method thereof and application of the compound in preparing a medicament for treating or preventing cancer.
Background
Pyrazolo [3,4-d ] pyrimidine is an important drug skeleton, and is similar to purine structure, and pyrazolo [3,4-d ] pyrimidine compounds play an important role in many pharmacological activities, such as anti-inflammatory activity, antibacterial activity, antiviral activity, antitubercular activity and the like. In 1955, skipper et al first conducted an investigation of the antitumor activity of pyrazolo [3,4-d ] pyrimidine compounds and found that 4-aminopyrazolo [3,4-d ] pyrimidines are lethal to experimental tumors, and thus, the investigation of pyrazolo [3,4-d ] pyrimidines has attracted extensive interest to researchers. Therefore, the pyrazolo [3,4-d ] pyrimidine compound has wide development prospect.
Disclosure of Invention
The first purpose of the invention is to provide a novel cinnamamide amino pyrazolo [3,4-d ] pyrimidine compound with good anticancer activity.
The second purpose of the invention is to provide a preparation method of the cinnamoylaminopyrazolo [3,4-d ] pyrimidine compound, which is simple and convenient, easy to operate, easy to obtain raw materials, low in production cost and suitable for industrial application.
The third purpose of the invention is to provide the application of the cinnamoylaminopyrazolo [3,4-D ] pyrimidine compound or the pharmaceutically acceptable salt thereof in preparing a medicament for treating or preventing cancers, wherein the cancers are human malignant melanoma, human lung cancer, human liver cancer, human stomach cancer, human breast cancer or human colon cancer, and the cancer cells of the cancers are human malignant melanoma cells A375, human liver cancer cells HepG2, human stomach cancer cells AGS, human breast cancer cells T47D, human breast cancer cells MDA-MB-231 and human colon cancer cells SW620.
The technical solution adopted by the present invention is specifically explained below.
In a first aspect, the present invention provides a cinnamoylaminopyrazolo [3,4-d ] pyrimidine compound having the following structural formula (i):
Figure BDA0003903553430000011
in the formula (I), R is 2-Cl, 2-methyl, 3-methyl, 4-F or 3,4-dimethoxy.
Particularly preferred cinnamoylaminopyrazolo [3,4-d ] pyrimidines in the present invention are selected from the group consisting of:
Figure BDA0003903553430000021
in a second aspect, the invention provides a preparation method of a cinnamamide pyrazolo [3,4-d ] pyrimidine compound shown in formula (I), wherein the preparation method comprises the following steps: adding 4-chloro-1H-pyrazolo [3,4-d ] pyrimidine (II) into an organic solvent A, dropwise adding hydrochloric acid under the stirring condition of-10-40 ℃, reacting for 5-50 minutes, dropwise adding an organic solvent A solution containing a compound (III), heating to a reflux temperature after dropwise adding, stirring and reacting for 0.5-5 hours, and separating and purifying the obtained reaction solution to obtain the cinnamamidopyrazolo [3,4-d ] pyrimidine compound shown in the formula (I);
Figure BDA0003903553430000022
in the formula (III), R is as defined in the formula (I).
Further, the ratio of the amount of the 4-chloro-1H-pyrazolo [3,4-d ] pyrimidine (II) to the amount of hydrochloric acid or compound (III) fed is 1: 0.1 to 1: 0.8 to 1.2, and preferably the ratio of the amount of the 4-chloro-1H-pyrazolo [3,4-d ] pyrimidine (II) to the amount of hydrochloric acid or compound (III) fed is 1: 0.1 to 0.5: 0.9 to 1.2.
Further, the organic solvent a is selected from one of the following: methanol, ethanol or isopropanol. The organic solvent A is used for dissolving the 4-chloro-1H-pyrazolo [3,4-d ] pyrimidine (II) and the compound (III). Preferably, the total volume of the organic solvent A is 10 to 50mL/mmol based on the amount of 4-chloro-1H-pyrazolo [3,4-d ] pyrimidine (II), wherein the volume of the organic solvent A used for dissolving 4-chloro-1H-pyrazolo [3,4-d ] pyrimidine (II) is 5 to 25mL/mmol based on the amount of 4-chloro-1H-pyrazolo [3,4-d ] pyrimidine (II), and the volume of the organic solvent A used for dissolving the compound (III) is 5 to 25mL/mmol based on the amount of 4-chloro-1H-pyrazolo [3,4-d ] pyrimidine (II).
Further, hydrochloric acid is added dropwise under the condition of stirring at the temperature of 0-30 ℃.
Further, under stirring at 0 to 30 ℃, a solution of the organic solvent A containing the compound (III) is added dropwise.
Further, the reaction process is followed by TLC (developing solvent is a mixed solution of dichloromethane and methanol with the volume ratio of 1-30: 1) to determine the reaction end point, and the reflux reaction time is generally 0.5-5 hours.
Further, the separation and purification adopts the following steps: after the reaction is finished, cooling the reaction liquid to room temperature, filtering, washing with water, drying a filter cake, and carrying out column chromatography to obtain the cinnamamide pyrazolo [3,4-d ] pyrimidine compound shown in the formula (I).
Furthermore, the column chromatography specifically comprises the following steps: adding an organic solvent B into a filter cake in a single-mouth bottle to dissolve the filter cake to obtain a dissolved solution, adding column chromatography silica gel (preferably 300-400-mesh coarse pore (zcx. II) type column chromatography silica gel) with the mass of 1-2 times of that of the filter cake into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of a dried filter cake and the silica gel, loading the mixture into a column, eluting by using a mixed solution of dichloromethane and methanol with a volume ratio of 1-30 as an eluent, performing TLC tracking detection (a developing agent is a mixed solution of dichloromethane and methanol with a volume ratio of 1-30), collecting an eluent containing the compound shown in the formula (I) according to TLC detection, concentrating and drying the eluent to obtain the compound shown in the formula (I); the organic solvent B is one of the following: petroleum ether, dichloromethane, methanol or ethyl acetate; the organic solvent B is used in an amount capable of dissolving the filter cake.
The organic solvent A and the organic solvent B are all indicated as organic solvents for reaction or column chromatography, and the letters in the invention do not refer to certain organic solvents, but are used for distinguishing the organic solvents appearing in different steps for the convenience of expression and clarity. The organic solvent A or B may be the same solvent or different solvents. .
In a third aspect, the invention provides an application of a cinnamoylaminopyrazolo [3,4-D ] pyrimidine compound shown as a formula (I) or a pharmaceutically acceptable salt thereof in preparation of a medicine for treating or preventing cancer, wherein cancer cells of the cancer are a human malignant melanoma cell A375, a human liver cancer cell HepG2, a human stomach cancer cell AGS, a human breast cancer cell T47D, a human breast cancer cell MDA-MB-231 and a human colon cancer cell SW620.
Preferably, the cancer cells are A375, hepG2, AGS, T47D, SW cells.
Preferably, in the cinnamamidopyrazolo [3,4-d ] pyrimidine compound shown in the formula (I), R is 2-Cl.
Preferably, the cancer cell is HepG2, and in the cinnamamidopyrazolo [3,4-d ] pyrimidine compound shown in the formula (I), R is 2-Cl, 4-F or 3,4-dimethoxy.
Preferably, the cancer cell is T47D, and in the cinnamamide pyrazolo [3,4-D ] pyrimidine compound shown in the formula (I), R is 2-Cl.
The term "pharmaceutically acceptable" refers herein to: the compounds are compatible chemically and/or toxicologically, with the other ingredients comprising the formulation and/or with the human or mammal with which the disease or condition is being prevented or treated.
The term "pharmaceutically acceptable salts" refers to relatively non-toxic, inorganic or organic acid addition salts of the compounds of the present invention. See, for example, S.M.Berge et al, "Pharmaceutical Salts", J.pharm.Sci.1977, 66,1-19. Among them, inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, nitric acid, or the like; organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) -benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, diglucosic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, and the like.
Compared with the prior art, the invention has the following beneficial effects: (1) Provides a novel cinnamoylaminopyrazolo [3,4-d ] pyrimidine compound with good anti-cancer (especially human liver cancer or human breast cancer) activity; (2) The preparation method of the cinnamamide amino pyrazolo [3,4-d ] pyrimidine compound is simple, easy to operate, easy to obtain raw materials, low in production cost, suitable for practicality and expected to be applied to preparation of medicines for preventing or treating tumor diseases; (3) Provides the application of a novel cinnamoylaminopyrazolo [3,4-D ] pyrimidine compound or pharmaceutically acceptable salt thereof in preparing a medicament for treating or preventing cancer, and the compound has good inhibitory action on human malignant melanoma cells A375, human liver cancer cells HepG2, human stomach cancer cells AGS, human breast cancer cells T47D, human breast cancer cells MDA-MB-231 and human colon cancer cells SW620.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions will be further clearly and completely described by embodiments. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Preparation of 4-chloro-1H-pyrazolo [3,4-d ] pyrimidine (II) is described in the literature (Organic Letters,2013,15 (8), 1882-1885).
Figure BDA0003903553430000041
The compounds (IIIa) to (IIIg) are prepared by the following reaction scheme, wherein R 1 The structures of (A) and (B) are as follows:
Figure BDA0003903553430000042
the structural formulae of the compounds (Ia) to (If) prepared in the examples are as follows:
Figure BDA0003903553430000043
example 1: preparation of Compound (IIIg)
7.4g (50 mmol) of cinnamic acid and 100mL of anhydrous dichloromethane are added into a three-neck flask, 100mL of anhydrous dichloromethane solution containing 70.0g (551 mmol) of oxalyl chloride is added dropwise under ice bath, 2.0g of DMF is added after dropwise addition, reaction is carried out for 12 hours at room temperature, and organic solvent is distilled off, so as to obtain 4.8g (29 mmol) of cinnamoyl chloride as light yellow oil.
Adding 4.8g (29 mmol) of cinnamoyl chloride and 100mL of anhydrous dichloromethane into a three-neck flask, dropwise adding 100mL of dichloromethane solution containing 5.5g (40 mmol) of m-nitroaniline and 4.2g (42 mmol) of triethylamine under the ice-bath condition, reacting at room temperature for 10 hours after dropwise adding, evaporating to remove the organic solvent, adding 100mL of dilute hydrochloric acid with the pH value of 1, filtering, washing a filter cake with 100mL of water multiplied by 3, and drying to obtain 5.6g (21 mmol) of N- (3-nitrophenyl) cinnamamide.
A three-necked flask was charged with 5.4g (20 mmol) of N- (3-nitrophenyl) cinnamamide, 15.2g (80.0 mmol) of tin dichloride and 1.1g (21 mmol) of ammonium chloride, and then 200mL of anhydrous ethanol was added thereto, and the mixture was refluxed for 1.5 hours with magnetic stirring, cooled to room temperature, the reaction solution was adjusted to a weakly alkaline pH with a saturated aqueous sodium bicarbonate solution, extracted with ethyl acetate (100 mL. Times.3), the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and the organic solvent was distilled off to obtain 3.2g (13 mmol) of the compound (III g).
Preparation of Compounds (IIIa) to (IIIf): compounds (IIIa) to (IIIf) were prepared by the method in reference to example 1, substituting cinnamic acid with the corresponding substituted cinnamic acid.
Example 2: preparation of Compound (Ia)
0.155g (1.0 mmol) of 4-chloro-1H-pyrazolo [3,4-d is dissolved in 25.0mL of ethanol]Pyrimidine (II), under the condition of magnetic stirring at 10 ℃, 1.0mL of hydrochloric acid (1.0 mol/L) is added dropwise, and then reaction is carried outAfter the reaction is finished, the reaction solution is heated up after the dripping, the reflux reaction is carried out for 0.5 hour (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of dichloromethane and methanol with the volume ratio of 1:1), the reaction solution is cooled to room temperature, the reaction solution is filtered, a filter cake is washed by (10 mL multiplied by 3) water and dried at 80 ℃, 5.0mL of petroleum ether solvent is added into the dried filter cake to dissolve the filter cake to obtain a dissolved solution, then 0.7 g of silica gel (300-400-mesh coarse pore (zcx.II) type column chromatography silica gel) is added into the dissolved solution, after the uniform mixing, the solvent is evaporated to obtain a mixture of the dried residue and the silica gel, the mixture is filled into a column, and then the volume ratio of the mixture is 1:1 as eluent, eluting, tracking and detecting by TLC (the developing solvent is mixed solution of dichloromethane and methanol with the volume ratio of 1:1), collecting eluent containing the compound shown in the formula (Ia) according to the TLC detection, evaporating the solvent from the collected eluent, and drying to obtain a white solid product, namely the compound (Ia), wherein the yield is 35 percent (calculated by the substance of the compound (IIIa) and the melting point is 287-290 ℃. 1 H NMR(500MHz,DMSO-d6)δ13.64(s,1H),10.38(s,1H),10.06(s,1H),8.39(s,1H),8.28(d,J＝9.5Hz,2H),7.87(d,J＝15.7Hz,1H),7.78-7.75(m,1H),7.62(d,J＝8.1Hz,1H),7.57-7.54(m,1H),7.46-7.42(m,3H),7.33(t,J＝8.1Hz,1H),6.93(d,J＝15.6Hz,1H). 13 C NMR(125MHz,DMSO-d 6 )δ163.17,155.27,154.82,154.41,139.78,139.45,135.43,133.56,132.66,131.30,130.18,129.07,127.97,127.81,125.58,116.69,114.61,112.27,100.73.ESI-MS:(m/z)calcd for C 20 H 15 ClN 6 O[M+H] + 391.1；found 391.2。
Example 3: preparation of Compound (Ia)
0.155g (1.0 mmol) of 4-chloro-1H-pyrazolo [3,4-d is dissolved in 5.0mL of methanol]Pyrimidine (II), 0.1mL hydrochloric acid (1.0 mol/L) is dripped under the condition of magnetic stirring at 40 ℃, the reaction is carried out for 50 minutes, 5.0mL methanol solution containing 0.327g (1.2 mmol) of compound (IIIa) is dripped, the temperature of the reaction solution is raised after the dripping, the reflux reaction is carried out for 5 hours (the reaction process adopts TLC tracking detection, a developing agent is dichloromethane and methanol mixed solution with the volume ratio of 30Washing, drying at 80 ℃, adding 5.0mL of dichloromethane solvent into the dried filter cake to dissolve the filter cake to obtain a dissolved solution, adding 0.8 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel) into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then mixing the mixture in a volume ratio of 30:1 as eluent, eluting, tracking and detecting by TLC (a developing solvent is a mixed solution of dichloromethane and methanol with a volume ratio of 30. 1 HNMR， 13 C NMR and MS were the same as in example 1.
Example 4: preparation of Compound (Ia)
0.155g (1.0 mmol) of 4-chloro-1H-pyrazolo [3,4-d is dissolved in 10.0mL of isopropanol]Pyrimidine (II), 0.5mL hydrochloric acid (1.0 mol/L) is dripped under the condition of magnetic stirring at 30 ℃ for reaction for 30 minutes, 10.0mL isopropanol solution containing 0.218g (0.8 mmol) of compound (IIIa) is dripped, the temperature of the reaction solution is raised after dripping, reflux reaction is carried out for 1 hour (the reaction process adopts TLC tracking detection, a developing agent is dichloromethane and methanol mixed solution with a volume ratio of 15:1 as eluent, eluting, tracing and detecting by TLC (developing solvent is dichloromethane and methanol mixed solution with volume ratio of 15. 1 HNMR， 13 C NMR and MS were the same as in example 1.
Example 5: preparation of Compound (Ia)
0.155g (1.0 mmol) of 4-chloro-1H-pyrazolo [3,4-d is dissolved in 20.0mL of ethanol]Pyrimidine (II), 0 ℃ under the condition of magnetic stirring, 0.2mL hydrochloric acid (1.0 mol/L) is dripped, reaction is carried out for 20 minutes, then 20.0mL ethanol solution containing 0.273g (1.0 mmol) of compound (IIIa) is dripped, the temperature of the reaction solution is raised after dripping, reflux reaction is carried out for 3 hours (the reaction process adopts TLC tracking detection, a developing agent is dichloromethane and methanol mixed solution with a volume ratio of 10:1 as eluent, eluting, tracking and detecting by TLC (a developing solvent is a mixed solution of dichloromethane and methanol with a volume ratio of 10. 1 HNMR， 13 C NMR and MS were the same as in example 1.
Example 6: preparation of Compound (Ib)
0.155g (1.0 mmol) of 4-chloro-1H-pyrazolo [3,4-d was dissolved in 20.0mL of ethanol]Pyrimidine (II), 0.2mL hydrochloric acid (1.0 mol/L) is dripped under the condition of magnetic stirring at the temperature of 0 ℃, the reaction is carried out for 20 minutes, then 20.0mL ethanol solution containing 0.307g (1.0 mmol) of compound (IIIb) is dripped, the temperature of the reaction solution is raised after the dripping, the reflux reaction is carried out for 2.5 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of dichloromethane and methanol with a volume ratio of 10The mixture of (a), packing the mixture into a column, and then mixing the mixture in a volume ratio of 10:1 as eluent, eluting, tracking and detecting by TLC (a developing solvent is a mixed solution of dichloromethane and methanol with a volume ratio of 10. ESI-MS m/z calcd for C 20 H 14 Cl 2 N 6 O[M+H] + 425.1；found425.2。
Example 7: preparation of Compound (ic)
0.155g (1.0 mmol) of 4-chloro-1H-pyrazolo [3,4-d is dissolved in 20.0mL of ethanol]Pyrimidine (ii), 0 ℃, under magnetic stirring, 0.2mL hydrochloric acid (1.0 mol/L) is added dropwise to react for 20 minutes, then 20.0mL ethanol solution containing 0.252g (1.0 mmol) of compound (iiic) is added dropwise, after the addition, the temperature of the reaction solution is raised, reflux reaction is performed for 3.5 hours (the reaction process is detected by TLC tracking, the developing agent is a mixed solution of dichloromethane and methanol with a volume ratio of 10:1 as eluent, eluting, tracking and detecting by TLC (a developing solvent is a mixed solution of dichloromethane and methanol with a volume ratio of 10. 1 H NMR(500MHz,DMSO-d 6 )δ13.63(s,1H),10.31(s,1H),10.06(s,1H),8.38(s,1H),8.27(d,J＝15.3Hz,2H),7.80(d,J＝15.6Hz,1H),7.60-7.57(m,2H),7.44(d,J＝8.4Hz,1H),7.33-7.23(m,4H),6.80(d,J＝15.6Hz,1H),2.39(s,3H). 13 C NMR(125MHz,DMSO-d 6 )δ163.69,155.22,154.91,154.44,139.72,139.61,137.71,137.02,133.65,132.46,130.85,129.55,128.98,126.52,126.20,123.64,116.53,114.57,112.29,100.69,19.51.ESI-MS:(m/z)calcd for C 21 H 18 N 6 O[M+H] + 371.2；found 371.2。
Example 8: preparation of Compound (Id)
0.155g (1.0 mmol) of 4-chloro-1H-pyrazolo [3,4-d is dissolved in 20.0mL of ethanol]Pyrimidine (II), 0 ℃ under the condition of magnetic stirring, 0.2mL hydrochloric acid (1.0 mol/L) is dripped, reaction is carried out for 20 minutes, then 20.0mL ethanol solution containing 0.252g (1.0 mmol) of compound (IIId) is dripped, the temperature of the reaction solution is raised after dripping, reflux reaction is carried out for 4 hours (the reaction process adopts TLC tracking detection, a developing agent is dichloromethane and methanol mixed solution with a volume ratio of 10:1 as eluent, eluting, tracking and detecting by TLC (a developing solvent is a mixed solution of dichloromethane and methanol with a volume ratio of 10. 1 H NMR(500MHz,DMSO-d 6 )δ13.62(s,1H),10.23(s,1H),10.04(s,1H),8.39(s,1H),8.28(d,J＝15.9Hz,2H),7.62(d,J＝7.9Hz,1H),7.55(d,J＝15.7Hz,1H),7.45-7.40(m,3H),7.35-7.30(m,2H),7.22(d,J＝7.5Hz,1H),6.87(d,J＝15.7Hz,1H),2.34(s,3H). 13 C NMR(125MHz,DMSO-d 6 )δ163.72,155.19,154.95,154.45,140.25,139.74,139.64,138.25,134.79,132.49,130.53,128.98,128.95,128.28,125.02,122.36,116.53,114.53,112.22,100.71,20.99.ESI-MS:(m/z)calcd for C 21 H 18 N 6 O[M+H] + 371.2；found 371.2。
Example 9: preparation of Compound (ie)
0.155g (1.0 mmol) of 4-chloro-1H-pyrazolo [3,4-d is dissolved in 20.0mL of ethanol]Pyridine (II), 0 ℃, under the condition of magnetic stirring, 0.2mL hydrochloric acid (1.0 mol/L) is dripped, the reaction is carried out for 20 minutes, then 20.0mL ethanol solution containing 0.256g (1.0 mmol) of compound (IIIe) is dripped, the temperature of the reaction solution is raised after the dripping, the reflux reaction is carried out for 4 hours (the reaction process adopts TLC tracking detection, a developing agent is dichloromethane and methanol mixed solution with a volume ratio of 10:1 as eluent, eluting, tracking and detecting by TLC (a developing solvent is a mixed solution of dichloromethane and methanol with a volume ratio of 10. 1 H NMR(500MHz,DMSO-d 6 )δ13.62(s,1H),10.30(s,1H),10.06(s,1H),8.37(s,1H),8.27(d,J＝23.2Hz,2H),7.67(dd,J＝8.6,5.6Hz,2H),7.56(s,2H),7.41(d,J＝8.4Hz,1H),7.28(dt,J＝24.4,8.5Hz,3H),6.84(d,J＝15.7Hz,1H). 13 C NMR(125MHz,DMSO-d 6 )δ163.92,163.61,161.95,155.19,154.97,154.44,139.74,139.61,138.98,132.48,131.51,131.49,130.01,129.95,128.96,122.42,116.54,116.17,115.99,114.55,112.23,100.72.ESI-MS:(m/z)calcdfor C 20 H 15 FN 6 O[M+H] + 375.1；found 375.4。
Example 10: preparation of Compound (if)
0.155g (1.0 mmol) of 4-chloro-1H-pyrazolo [3,4-d is dissolved in 20.0mL of ethanol]Pyrimidine (II), 0.2mL hydrochloric acid (1.0 mol/L) is added dropwise under the condition of magnetic stirring at 0 ℃, reaction is carried out for 20 minutes, 20.0mL ethanol solution containing 0.298g (1.0 mmol) of compound (IIIf) is added dropwise, and the temperature of reaction solution is raised after the dropwise addition is finishedAnd performing reflux reaction for 4.5 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of dichloromethane and methanol with a volume ratio of 10:1 as eluent, eluting, tracking and detecting by TLC (a developing solvent is a mixed solution of dichloromethane and methanol with a volume ratio of 10: 1), collecting eluent containing the compound shown in formula (if) according to TLC detection, evaporating the solvent from the collected eluent, and drying to obtain a white solid product, namely the compound (if), wherein the yield is 82% (based on the substance of the compound (II)) and the melting point is 260-263 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ13.62(s,1H),10.27(s,1H),10.03(s,1H),8.39(s,1H),8.28(d,J＝10.7Hz,2H),7.78(d,J＝15.8Hz,1H),7.60(d,J＝8.0Hz,1H),7.43(d,J＝8.7Hz,1H),7.32(t,J＝8.1Hz,1H),7.19(dd,J＝7.7,1.5Hz,1H),7.15-7.08(m,2H),6.91(d,J＝15.8Hz,1H),3.82(s,3H),3.77(s,3H). 13 C NMR(125MHz,DMSO-d 6 )δ163.85,155.28,154.84,154.44,153.00,147.69,139.73,139.65,134.81,132.65,129.02,128.42,124.57,123.65,118.87,116.56,114.59,114.24,112.29,100.71,60.82,55.90.ESI-MS:(m/z)calcd for C 22 H 20 N 6 O 3 [M+H] + 417.2；found417.2。
Example 11: in vitro test for anti-cancer Activity
The prepared compounds (Ia) to (if) are respectively subjected to biological activity tests on a human malignant melanoma cell A375, a human lung cancer cell A549, a human liver cancer cell HepG2, a human gastric cancer cell AGS, a human breast cancer cell T47D, a human breast cancer cell MDA-MB-231 and a human colon cancer cell SW620, wherein the compound (Ia) is prepared by the method of example 5.
The test method comprises the following steps: tetrazolium salt reduction (MTT process).
Cell lines: human malignant melanoma cells A375, human lung cancer cells A549, human liver cancer cells HepG2, human gastric cancer cells AGS, human breast cancer cells T47D, human breast cancer cells MDA-MB-231 and human colon cancer cells SW620. The cell lines were purchased from cell banks of Shanghai Life sciences of Chinese academy of sciences.
The experimental procedure was as follows:
(1) Culture of tumor cells
The cell generation number used in the experiment is within 5 generations. The consumables and reagents used for cell culture are subjected to rigorous sterilization procedures, and all experimental procedures are performed in a sterile operating station.
(a) Resuscitation of tumor cells
Before the experiment, the articles to be used, such as a culture bottle, a centrifuge tube, a liquid-transferring gun, a gun head, a waste liquid barrel and the like, are placed in a super clean bench, an ultraviolet lamp is turned on for sterilization, and the reagents used for cell culture are placed in a37 ℃ constant-temperature water bath kettle for preheating. And after all the work is ready, the ultraviolet lamp is turned off, and the super-clean bench fluorescent lamp and the ventilating fan are turned on. Removing the frozen tumor cells from the freezer at-80 deg.C, thawing rapidly in 37 deg.C water bath with shaking to ensure that the frozen solution in the frozen tube is still small and unfrozen, spraying alcohol, placing in a clean bench, immediately transferring the whole cells in the frozen tube to a 15mL centrifuge tube containing 10% FBS-containing DMEM (A375, hepg2, T47D, MDA-MB-231) or 10% FBS-containing 1640 (A549, AGS, SW 620), and gently beating and mixing with a pipette. Placing the centrifugal tube in a centrifuge, centrifuging at 1000rpm for 5min, and sucking off the supernatant in a super clean bench. Preparing a cell suspension by pipetting 2mL of a DMEM culture solution containing 10% FBS (A375, hepg2, T47D, MDA-MB-231) or 1640 culture solution containing 10% FBS (A549, AGS, SW 620) into a centrifuge tube containing a cell pellet with a pipette gun, and pipetting, transferring the cell suspension to a flask with a bottom area of 25cm 2 The air-permeable culture flask of (4) was supplemented with 4mL of a DMEM culture solution (A375, hepg2, T47D, MDA-MB-231) containing 10% FBS or a 1640 culture solution (A549, AGS, SW 620) containing 10% FBS, the cells were mixed by gently shaking the flask, and the flask was allowed to stand for 5% CO 2 And culturing in a constant temperature incubator at 37 ℃ for 2-3 days.
(b) Passage of tumor cells
When the cell isThe culture bottle can be subcultured when the culture bottle is in a good growth state and 70% -80% of the bottom of the culture bottle is fully paved. The passage operation steps are all completed in a super clean bench, and the passage can be started after the preparation work is done. Firstly, sucking the original culture solution in a culture bottle off by a pipette gun and beating the culture solution into a waste solution barrel, adding 2mL of PBS for repeated washing for several times, then adding about 600-700 mu L of trypsin (containing 0.02% of EDTA, phenol red and 0.25% of pancreatin) to ensure that the bottom of the bottle is completely covered by the trypsin, slightly shaking the culture bottle, putting the culture bottle into a constant-temperature incubator at 37 ℃ for incubation for 1-2 min, observing the cell shedding condition under a microscope, and slightly beating the bottle wall by using a finger belly if a small amount of cells are still attached to the wall until most of the cells can shed from the bottom of the bottle. After the digestion was terminated by adding 2mL of 10-vol FBS-containing DMEM medium (A375, hepg2, T47D, MDA-MB-231) or 10-vol FBS-containing 1640 medium (A549, AGS, SW 620), the cells were gently blasted with a pipette gun, the cells were completely blown off from the bottom of the flask, and the cell suspension was transferred to a 15mL centrifuge tube, 1000rpm, and centrifuged for 5min. The supernatant was aspirated off, diluted and beaten uniformly with a DMEM culture solution (A375, hepg2, T47D, MDA-MB-231) containing 10% FBS or a 1640 culture solution (A549, AGS, SW 620) containing 10% FBS, and then distributed evenly into 2 to 3 culture bottles to continue the 5% CO concentration 2 And culturing in a constant-temperature incubator at 37 ℃ for 2-3 days.
(c) Cryopreservation of tumor cells
The cell freezing solution is prepared in advance according to the volume ratio of FBS (fetal bovine serum) to DMSO =9:1, and is placed in a refrigerator at 4 ℃ for standby. And (4) freezing and storing operation steps are finished in the super clean bench, and freezing and storing can be started after preparation work is done. Firstly, sucking the original culture solution in a culture bottle, adding 2mL of PBS buffer solution for repeated washing for several times, pouring out, then adding about 600-700 mu L of trypsin (containing 0.02 percent of EDTA, phenol red and 0.25 percent of pancreatin), slightly shaking the culture bottle to ensure that the pancreatin can cover the bottom of the culture bottle, putting the culture bottle into a constant-temperature incubator at 37 ℃ for incubation for 1-2 min, observing the cell shedding condition under a microscope, and slightly knocking the bottle wall by using a finger belly if a small amount of cells are still attached to the wall until most of the cells can shed from the bottle bottom. After the digestion was terminated by adding 2mL of 10-vol FBS-containing DMEM medium (A375, hepg2, T47D, MDA-MB-231) or 10-vol FBS-containing 1640 medium (A549, AGS, SW 620), the cells were gently blasted with a pipette gun, the cells were completely blown off from the bottom of the flask, and the cell suspension was transferred to a 15mL centrifuge tube, 1000rpm, and centrifuged for 5min. The supernatant was decanted off. 1mL of the cryopreservation solution just taken out of the refrigerator at 4 ℃ was added, and the mixture was blown up uniformly to form a cell suspension, which was transferred into a cryopreservation tube. Standing at 4 deg.C for 30min after labeling cell type, cell generation number and freezing storage date, standing at-20 deg.C for 1 hr, and storing in-80 deg.C ultra-low temperature refrigerator.
(2) MTT experimental method
(a) Cell counting: after digesting and centrifuging tumor cells in a well-grown state in a culture flask, resuspending the cells in 4mL of 10-percent FBS-containing DMEM (A375, hepg2, T47D, MDA-MB-231) or 10-percent FBS-containing 1640 (A549, AGS, SW 620), taking 10. Mu.L of cell suspension to a cell counting plate, counting the cells, and diluting the cells to 5X 10 4 one/mL.
(b) Plate paving: the 96-well plate was used, 100. Mu.L of the diluted cell suspension was added to the experimental well, 100. Mu.L of 10-FBS-containing DMEM medium (A375, hepg2, T47D, MDA-MB-231) or 10-FBS-containing 1640 medium (A549, AGS, SW 620) was added to the blank well, 100. Mu.L of PBS buffer was added to the periphery, and CO was added 2 Culturing in a constant temperature incubator for 24h.
(c) Preparing a compound and adding medicine: the 10. Mu. Mol/mL compound prepared in DMSO and the positive control sorafenib were diluted to the prescribed concentrations of 20. Mu.M, 10. Mu.M, 5. Mu.M, 2.5. Mu.M and 1.25. Mu.M, respectively, in a 10-% FBS-containing DMEM medium (A375, hepg2, T47D, MDA-MB-231) or a 10-% FBS-containing 1640 medium (A549, AGS and SW 620), and the administration was carried out by the liquid replacement method. Discarding the original culture solution, adding 100 μ L of 10% FBS-containing DMEM culture solution containing compound or positive control (A375, hepg2, T47D, MDA-MB-231) or 10% FBS-containing 1640 culture solution (A549, AGS, SW 620) to the test well, adding 100 μ L of 10% FBS-containing DMEM culture solution (A375, hepg2, T47D, MDA-MB-231) or 10% FBS-containing 1640 culture solution (A549, AGS, SW 620) to the control and blank wells, adding the 96 well plate after adding the drug, and placing the 96 well plate in A5 CO 2 2 And continuously culturing in a constant-temperature incubator at 37 ℃.
(d) Adding MTT: after 48 hours, the 96-well plate is taken out and put into a super clean bench under the condition of keeping out of the sunAdd 10. Mu.L, 5mg/mL MTT solution per well, then put 5% CO 2 And continuously culturing in a constant-temperature incubator at 37 ℃.
(e) And (3) detection: the 96-well plate that adds MTT takes out in 3.5 ~ 4h of incubating in the incubator, with the solution in each hole aspirate carefully, the formazan that generates is dissolved to 150 uL's DMSO in each hole, then vibrates 10min on putting to the flat plate oscillator, uses the enzyme reader to detect the absorbance value under 490 nm.
(f) And (3) processing experimental data: the cell viability was calculated according to the following formula, and the value of 50% of the cell viability was IC 50 。
Cell survival (%) = [ (As-Ab)/(Ac-Ab) ]. Times.100%
As assay well (cell-containing culture solution, MTT, toxic substance)
Ac control well (cell culture, MTT, no toxic substance)
Ab blank well (MTT-containing, cell and toxic free medium culture).
The results of the test are shown in table 1:
TABLE 1 inhibition of cancer cell growth by Compound (I)
Figure BDA0003903553430000111
The above description is only an example of the present application and is not intended to limit the scope of the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims (10)

1. A cinnamamidopyrazolo [3,4-d ] pyrimidine compound having the following structural formula (I):
Figure FDA0003903553420000011
in the formula (I), R is 2-Cl, 2-methyl, 3-methyl, 4-F or 3,4-dimethoxy.
2. A process for preparing the cinnamoylaminopyrazolo [3,4-d ] pyrimidine compound of claim 1, said process comprising: adding 4-chloro-1H-pyrazolo [3,4-d ] pyrimidine (II) into an organic solvent A, dropwise adding hydrochloric acid under the stirring condition of-10-40 ℃, reacting for 5-50 minutes, dropwise adding an organic solvent A solution containing a compound (III), heating to a reflux temperature after dropwise adding, stirring and reacting for 0.5-5 hours, and separating and purifying the obtained reaction solution to obtain the cinnamamidopyrazolo [3,4-d ] pyrimidine compound shown in the formula (I);
Figure FDA0003903553420000012
in the formula (III), R is as defined in the formula (I).
3. The method of claim 2, wherein: the ratio of the amount of 4-chloro-1H-pyrazolo [3,4-d ] pyrimidine (II) to the amount of hydrochloric acid and compound (III) fed was 1: 0.1 to 1: 0.8 to 1.2.
4. The method of claim 2, wherein: the ratio of the amount of 4-chloro-1H-pyrazolo [3,4-d ] pyrimidine (II) to the amount of hydrochloric acid or compound (III) fed was 1: 0.1 to 0.5: 0.9 to 1.2.
5. The method of claim 2, wherein: the organic solvent A is selected from one of the following: methanol, ethanol or isopropanol.
6. The method of claim 2, wherein: under the condition of stirring at 0-30 ℃, hydrochloric acid is dripped.
7. The method of claim 2, wherein: under the condition of stirring at 0-30 ℃, dropwise adding an organic solvent A solution containing the compound (III).
8. The use of the cinnamoylaminopyrazolo [3,4-D ] pyrimidine compound or a pharmaceutically acceptable salt thereof according to claim 1, for the preparation of a medicament for the treatment or prevention of cancer, wherein the cancer cells of the cancer are human malignant melanoma cell a375, human hepatoma cell HepG2, human gastric cancer cell AGS, human breast cancer cell T47D, human breast cancer cell MDA-MB-231, human colon cancer cell SW620.
9. The use of claim 8, wherein: in the cinnamamide amido pyrazolo [3,4-d ] pyrimidine compounds shown in the formula (I), R is 2-Cl.
10. The use of claim 8, wherein: the cancer cell is HepG2, and in the cinnamamide pyrazolo [3,4-d ] pyrimidine compound shown in the formula (I), R is 2-Cl, 4-F or 3,4-dimethoxy.
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Citations (2)

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CN1390219A (en) * 1999-09-17 2003-01-08 艾博特股份有限两合公司 Pyrazolopyrimidines as therapeutic agents
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CN1390219A (en) * 1999-09-17 2003-01-08 艾博特股份有限两合公司 Pyrazolopyrimidines as therapeutic agents
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