Triazolopyrimidine antitumor compound as well as preparation method and application thereof

Abstract

The invention belongs to the field of medicines, and particularly relates to a novel triazolopyrimidine antitumor compound as well as a preparation method and application thereof. The triazolopyrimidine compound with a novel structure is prepared by adopting a simple and efficient synthesis method, and the specific structural general formula of the triazolopyrimidine compound is as follows: # imgabs0 #. The research on the in-vitro antitumor activity of the compound shows that the novel triazolopyrimidine compound has a good anti-tumor effect on human pancreatic cancer cells (PANC-1); the triazolopyrimidine compound has an obvious inhibition effect on the growth of human gastric cancer cells (SGC7901) and human breast cancer cells (MDA-MB-231), which indicates that the triazolopyrimidine compound can be used as a lead compound or a candidate compound for the development of antitumor drugs.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to a novel triazolopyrimidine anti-tumor compound and a preparation method and application thereof. Background Art

[0002] Cancer is a type of malignant tumor and one of the leading causes of death in the global population. At present, the treatment methods of cancer include targeted therapy, chemotherapy, radiotherapy and surgical intervention, among which chemotherapy drugs play an important role. However, existing chemotherapy drugs have limitations such as limited therapeutic effect, poor selectivity, high toxicity and drug resistance. Therefore, the development of new anti-tumor drugs with good efficacy and low toxicity has become one of the important strategies for anti-tumor drug research. As a bioelectronic isostere of the purine ring, the triazolopyrimidine heterocyclic structure has antiviral, antibacterial, anti-inflammatory and anti-tumor pharmacological activities. In order to seek novel, safe and efficient anti-tumor drugs, we designed and synthesized a series of triazolopyrimidine compounds based on computer-assisted drug molecular screening and splicing principles, and studied their anti-tumor activity. This study has important research value and is conducive to the development of drugs with independent intellectual property rights in my country. Summary of the invention

[0003] The present invention aims to provide a triazolopyrimidine compound with a novel structure and a simple preparation method; another object of the present invention is to provide the use of the triazolopyrimidine compound in anti-tumor drugs.

[0004] To achieve the above object, the general structural formula of the triazolopyrimidine compound of the present invention is as follows:

[0005]

[0006] In the structure shown in general formula I, R 1 and R 2 Selected from 4-fluorophenyl, morpholinyl, p-trifluoromethylphenyl, p-trifluoromethylphenyl, p-chlorophenyl, p-methoxyphenyl, 3-fluorophenyl, 3,4,5-trimethoxyphenyl, 4-hydroxyphenyl, 3-fluorophenyl and phenyl.

[0007]

[0008] The present invention also provides a method for preparing the above-mentioned triazolopyrimidine compound, comprising the following steps: firstly, 5-amino-1,2,4-triazolethiol (Q1) and methyl 4-(bromomethyl)benzoate undergo a nucleophilic substitution reaction in dichloromethane to generate an intermediate Q2; secondly, the intermediate Q2 undergoes a cyclization reaction with methyl benzoyl acetate to generate an intermediate Q3; the intermediate Q3 undergoes a substitution reaction with phosphorus oxychloride to generate an intermediate Q4; the intermediate Q4 undergoes a substitution reaction with various amines to generate a target product, the triazolopyrimidine compound;

[0009] The synthetic route is as follows:

[0010]

[0011] The reaction conditions of the above reaction are: a. triethylamine, N,N-dimethylformamide, room temperature; b. methanol, reflux; c. phosphorus oxychloride, 90°C; d. triethylamine, ethanol, reflux;

[0012] The various amines are: 4-fluoroaniline, morpholine, p-trifluoromethylaniline, p-bromoaniline, p-chloroaniline, p-methoxyaniline, 3-fluoroaniline, 3,4,5-trimethoxyaniline, 4-hydroxyaniline, 2-fluoroaniline, and aniline.

[0013] The present invention also provides the use of any one of the above compounds in the preparation of tumor drugs. Preferably, the tumor is selected from human pancreatic cancer, human gastric cancer and human breast cancer.

[0014] The present invention also provides an anti-tumor drug, comprising any one of the above triazolopyrimidine compounds and a pharmaceutically acceptable carrier, a pharmaceutically acceptable salt and an excipient.

[0015] The present invention has the following beneficial effects:

[0016] The invention provides a novel triazolopyrimidine compound.

[0017] The invention provides a method for synthesizing a triazolopyrimidine compound. The method is simple and safe to operate, has few reaction by-products, high yield, and is easy to separate and purify the product.

[0018] The in vitro anti-tumor activity study of the novel triazolopyrimidine compounds of the present invention found that the compounds had strong inhibitory activity against human breast cancer cells (MDA-MB-231), with an inhibition rate of 79% at 20 μM and IC 50 The value is 3.32μM, which has broad application prospects in the preparation of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 For compound 2-4 1 H-NMR

[0021] Figure 2 For compound 2-4 13C-NMR DETAILED DESCRIPTION

[0022] The following will be clearly and completely described in conjunction with the technical solutions in the specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, other embodiments obtained by ordinary technicians in this field without creative work are all within the scope of protection of the present invention.

[0023] The structure of the compound of the present invention is determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The NMR measurement uses a Bruker AV-500 nuclear magnetic spectrometer, the measurement solvent is deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3) and deuterated methanol (CD3OD), and the internal standard is tetramethylsilane (TMS). The chemical shift (δ) unit is ppm.

[0024] Examples of compound synthesis

[0025] Example 1: Preparation of methyl 4-(((5-phenyl-7-(phenylamino))-[1,2,4]triazole-[1,5-a]pyrimidin-2-yl)methyl)benzoate (2-1)

[0026] Preparation of methyl 4-(((5-amino-4H-1,2,4-triazol-3-yl))thio)methyl)benzoate (Q2)

[0027] Compound Q1 (1.00 g, 8.61 mmol) and potassium carbonate (2.35 ml, 17.22 mmol) were dissolved in N, N-dimethylformamide (15 ml), and the mixture was cooled to 0°C with an ice-water bath. 5 ml of N, N-dimethylformamide solution containing methyl 4-bromomethylbenzoate (2.17 g, 9.47 mmol) was added dropwise. After the addition was complete, the mixture was reacted at room temperature for 8 h. Thin layer chromatography was used to monitor the complete conversion of the reaction raw materials. Water (200 ml) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (30 ml). The organic phase was then dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to obtain a crude compound Q2. A mixture of petroleum ether and ethyl acetate in a volume ratio of 3:1 was used as the mobile phase, and the target compound Q2 was obtained as a fine white solid by rapid column chromatography.

[0028] Preparation of methyl 4-(((7-hydroxy-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl))thio)methyl)benzoate (Q3)

[0029] At room temperature, methyl 4-(((5-amino-4H-1,2,4-triazol-3-yl))thio)methyl)benzoate (Q2) (1.00 g, 3.78 mmol) and ethyl benzoylacetate (1.45 g, 7.57 mmol) were added to glacial acetic acid (15 ml), and the temperature was raised to reflux for 24 h. TLC monitored the complete conversion of the reaction raw materials. The reaction solution was cooled to room temperature, 5 ml of pure water was added to the reaction solution, stirred for 30 min, and filtered and dried to obtain the target compound Q3 as a white solid.

[0030] Preparation of methyl 4-(((7-chloro-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl))thio)methyl)benzoate (Q4)

[0031] At room temperature, add 15 ml of phosphorus oxychloride to a 25 ml round-bottom flask, then slowly add compound Q3 (1.00 g, 2.55 mmol), heat to 90 ° C, react for 5 hours, monitor the complete conversion of the reaction raw materials through thin layer chromatography, and cool to room temperature. Slowly add it dropwise to a 100 ml ice water bath, stir for 30 minutes, adjust the pH value of the solution to 7 with ammonia water, filter and dry, and then use a dichloromethane and methanol solution with a volume ratio of 40:1 as the mobile phase, and obtain the fine white solid target compound Q4 through rapid column chromatography.

[0032] Preparation of methyl 4-(((5-phenyl-7-(phenylamino))-[1,2,4]triazol-[1,5-a]pyrimidin-2-yl)methyl)benzoate (Q5)

[0033] At room temperature, the intermediate Q4 (250 mg, 608.46 μmol) was added to 15 ml of ethanol, and then aniline (111 μl, 1.22 mmol) was added to the reaction solution. The temperature was raised and refluxed for 5 hours. The reaction raw materials were completely converted as monitored by thin layer chromatography. The solution was cooled to room temperature, filtered, and recrystallized with ethanol solvent to obtain the target compound Q5 as a white solid.

[0034] Preparation of methyl 4-(((5-phenyl-7-(phenylamino))-[1,2,4]triazol-[1,5-a]pyrimidin-2-yl)methyl)benzoate (2-1)

[0035] At room temperature, the intermediate Q4 (250 mg, 608.46 μmol) was added to 15 ml of ethanol, and then aniline (111 μl, 1.22 mmol) was added to the reaction. The temperature was raised and refluxed for 5 h. The reaction raw materials were completely converted as monitored by thin layer chromatography. The mixture was cooled to room temperature, filtered, and recrystallized from ethanol solvent to obtain compound 2-1.

[0036] White solid, yield 77.69%; 1H NMR (500MHz, DMSO-d6) δ10.32(s,1H),8.01-7.97(m,2H),7.92(d,J=7.9Hz,2H),7.68(d,J=8.4 Hz,2H),7.56-7.46(m,7H),7.34(tt,J=6.5,2.1Hz,1H),6.85(s,1H),4.67(s,2H),3.83(s,3H). 13 C NMR(126MHz,DMSO-d6)δ165.94,165.00,160.19,156.15,145.68,143.89,137.19,136.69 ,130.42,129.60,129.30,129.24,128.47,127.13,126.20,124.39,86.36,52.05,34.08.

[0037] Example 2: Preparation of methyl 4-(((7-((4-fluorophenyl)amino)-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)thio)methyl)benzoate (2-2)

[0038] Use 4-fluoroaniline to replace aniline, and the preparation method is the same as Example 1

[0039] White solid, yield 68.04%; 1 H NMR(500MHz,DMSO-d6)δ10.29(s,1H),8.03-7.99(m,2H),7.92(d,J=8.4Hz,2H),7.68(d,J=8.3Hz,2H),7.6 0-7.54(m,2H),7.49(dd,J=5.0,1.9Hz,3H),7.35(t,J=8.8Hz,2H),6.77(s,1H),4.67(s,2H),3.83(s,3H). 13 C NMR (126MHz, DMSO-d6) δ166.44,165.51,161.61,160.68,159.67,156.59,146.47,144.38,137.63,133.43,130.93 ,129.79,129.74,129.73,129.70,129.23,128.97,127.67,127.45,127.39,116.98,116.80,86.72,52.54,34.58.

[0040] Example 3: Preparation of methyl 4-(((7-morpholine-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl))thio)methyl)benzoate (2-3)

[0041] The preparation method is the same as Example 1 except that morpholine is used to replace aniline.

[0042] White solid, yield 73.00%; 1 H NMR (500MHz, DMSO-d6) δ8.23-8.20(m,2H),7.92(d,J=8.3Hz,2H),7.63(d,J=8.3Hz,2H),7.55-7. 53(m,3H),7.04(s,1H),4.57(s,2H),3.89-3.87(m,4H),3.83(s,3H),3.80(dd,J=5.8,3.5Hz,4H). 13 C NMR(126MHz,DMSO-d6)δ165.96,164.38,159.98,157.35,149.40,143.82,136.89,1 30.66,129.25,129.05,128.72,128.41,127.48,91.25,65.59,52.08,48.00,34.00.

[0043] Example 4: Preparation of methyl 4-(((5-phenyl-7-((4-(trifluoromethyl))phenyl)amino)-[1,2,4]triazolyl)[1,5-a]pyrimidin-2-yl)thio)methyl)benzoate (2-4)

[0044] Using p-trifluoromethylaniline instead of aniline, the preparation method is the same as Example 1.

[0045] White solid, yield 78.87%; 1 H NMR(500MHz,DMSO-d6)δ10.58(s,1H),8.10-8.07(m,2H),7.93-7.90(m,2H),7.84(d,J=8.6Hz,2H),7.76 (d,J=8.4Hz,2H),7.68-7.64(m,2H),7.51(dd,J=5.1,1.9Hz,3H),7.14(s,1H),4.67(s,2H),3.82(s,3H). 13 C NMR (126MHz, DMSO-d6) δ165.94,165.21,160.44,156.11,144.78,143.82,140.98,136.99,130.59,129.31 ,129.22,128.76,128.50,127.36,126.72,126.69,125.55,125.29,123.48,123.12,87.53,52.06,34.13.

[0046] Example 5: Preparation of methyl 4-(((7-((4-bromophenyl)amino)-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)thio)methyl)benzoate (2-5)

[0047] Using p-bromoaniline instead of aniline, the preparation method is the same as Example 1.

[0048] White solid, yield 72.78%; 1 H NMR (500MHz, DMSO-d6) δ10.36 (s, 1H), 8.06-8.03 (m, 2H), 7.92 (d, J = 8.2Hz, 2H), 7.68(t,J=8.6Hz,4H),7.52-7.48(m,5H),6.92(s,1H),4.67(s,2H),3.83(s,3H). 13 C NMR(126MHz,DMSO-d6)δ165.95,165.07,160.27,156.10,145.33,143.86,137.08,136.27,13 2.45,130.51,129.31,129.23,128.76,128.48,127.27,126.23,118.26,86.71,52.07,34.09.

[0049] Example 6: 4-(((7-((4-chlorophenyl)amino)-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)thio)methyl)

[0050] Preparation of methyl benzoate (2-6)

[0051] Using p-chloroaniline instead of aniline, the preparation method is the same as Example 1.

[0052] White solid, yield 81.19%; 1 H NMR(500MHz,DMSO-d6)δ10.36(s,1H),8.07-8.01(m,2H),7.94-7.90(m,2H),7.69-7.64( m,2H),7.56(s,4H),7.50(dd,J=5.1,1.9Hz,3H),6.90(s,1H),4.67(s,2H),3.83(s,3H). 13C NMR(126MHz,DMSO-d6)δ165.94,165.07,160.25,156.10,145.43,143.86,137.08,135.81,13 0.49,130.06,129.53,129.31,129.23,128.74,128.48,127.25,125.97,86.65,52.06,34.09.

[0053] Example 7: Preparation of methyl 4-(((7-((4-methoxyphenyl)amino)-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)thio)methyl)benzoate (2-7)

[0054] Using p-methoxyaniline instead of aniline, the preparation method is the same as Example 1.

[0055] White solid, yield 83.57%; 1 H NMR (500MHz, DMSO-d6) δ10.16(s,1H),7.97(dd,J=6.7,3.0Hz,2H),7.92(d,J=8.3Hz,2H),7.68(d,J=8.3Hz,2H),7. 50-7.48(m,3H),7.43(d,J=8.9Hz,2H),7.07(d,J=8.9Hz,2H),6.66(s,1H),4.66(s,2H),3.83(s,3H),3.81(s,3H). 13 C NMR (126MHz, DMSO-d6) δ165.96,164.90,160.05,157.72,156.15,146.35,143.94,137.24,130.3 9,129.30,129.26,129.07,128.77,128.47,127.09,126.59,114.81,85.95,55.33,52.07,34.05.

[0056] Example 8: Preparation of methyl 4-(((7-((3-fluorophenyl)amino)-5-phenyl-[1,2,4]triazol-[1,5-a]pyrimidin-2-yl)thio)methyl)benzoate (2-8)

[0057] 3-fluoroaniline was used to replace aniline, and the preparation method was the same as that in Example 1.

[0058] White solid, yield 62.62%; 1H NMR(500MHz,DMSO-d6)δ10.41(s,1H),8.06-8.03(m,2H),7.92(d,J=8.2Hz,2H),7.67(d,J=8.0Hz,2H),7.57-7.53(m,1H), 7.52-7.50(m,3H),7.41(ddd,J=8.3,6.1,2.3Hz,2H),7.15(td,J=8.5,2.5Hz,1H),6.99(s,1H),4.67(s,2H),3.83(s,3H). 13 C NMR (126MHz, DMSO-d6) δ165.94,165.12,163.46,161.51,160.35,156.08,145.23,143.85,138.74,138.65,137.10,131.22 ,131.15,130.52,129.31,129.23,128.78,128.49,127.27,119.86,112.76,112.59,111.24,111.05,87.04,52.06,34.11.

[0059] Example 9: Preparation of methyl 4-(((5-phenyl-7-((3,4,5-trimethoxyphenyl))amino)-[1,2,4]triazolyl[1,5-a]pyrim-2-yl)thio)methyl)benzoate (2-9)

[0060] 3,4,5-trimethoxyaniline was used to replace aniline, and the preparation method was the same as that in Example 1.

[0061] White solid, yield 72.50%; 1 H NMR (500MHz, DMSO-d6) δ10.18(s,1H),8.04(dd,J=6.6,3.0Hz,2H),7.93(d,J=8.3Hz,2H),7.68(d,J=8.2H z,2H),7.53-7.49(m,3H),6.94(s,1H),6.87(s,1H),4.68(s,2H),3.84(s,3H),3.80(s,6H),3.72(s,3H). 13C NMR(126MHz,DMSO-d6)δ165.98,164.95,160.11,156.12,153.37,145.88,143.94,137.17,135.78,1 32.34,130.48,129.33,129.27,128.85,128.49,127.19,102.48,86.79,60.16,56.11,52.10,34.08.

[0062] Example 10: Preparation of methyl 4-(((7-((4-hydroxyphenyl)amino)-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)thio)methyl)benzoate (2-10)

[0063] Using 4-hydroxyaniline instead of aniline, the preparation method is the same as Example 1.

[0064] White solid, yield 61.52%; 1 H NMR(500MHz,DMSO-d6)δ10.06(s,1H),9.68(s,1H),7.97-7.94(m,2H),7.93-7.91(m,2H),7.69-7.67(m ,2H),7.51-7.47(m,3H),7.32-7.28(m,2H),6.91-6.88(m,2H),6.60(s,1H),4.66(s,2H),3.83(s,3H). 13 C NMR(126MHz,DMSO-d6)δ166.45,165.34,160.46,156.65,147.06,144.44,137.77,130.83,12 9.80,129.75,129.26,128.95,127.88,127.63,127.55,127.36,116.61,86.34,52.55,34.53.

[0065] Example 11: Preparation of methyl 4-(((7-((2-fluorophenyl)amino)-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)thio)methyl)benzoate (2-11)

[0066] Use 2-fluoroaniline to replace aniline, and the preparation method is the same as Example 1.

[0067] White solid, yield 65.57%; 1H NMR (500MHz, DMSO-d6) δ10.27(s,1H),7.99-7.97(m,2H),7.92(d,J=8.3Hz,2H),7.68(d,J=8.4Hz,2H),7.61(td,J=8. 0,1.5Hz,1H),7.50-7.46(m,5H),7.37(ddd,J=8.5,6.3,2.5Hz,1H),6.49(d,J=2.0Hz,1H),4.67(s,2H),3.84(s,3H). 13 C NMR (126MHz, DMSO-d6) δ166.45,165.79,160.73,158.35,156.52,156.38,146.40,144.36,137.47,131.03,129.78 ,129.75,129.32,129.30,128.98,127.65,125.92,125.90,124.35,124.26,117.46,117.31,87.02,52.56,34.53.

[0068] Application examples of compounds

[0069] In vitro anti-tumor activity test: Three cell lines were used using the CCK8 method, namely human pancreatic cancer cells (PANC-1), human breast cancer cells (MDA-MB-231), and human gastric cancer cells (SGC7901).

[0070] Collect logarithmic phase cells, adjust the concentration of cell suspension, add 100 μL to each well, adjust the cell density to be tested by plating, and fill the edge wells of 96-well plates with PBS. Incubate for 24 hours under 5% CO2, 37 ° C, and 90% humidity until the cell monolayer covers the bottom of the 96-well plate. Add the drug synthesized by the present invention with a concentration gradient, set 9 concentrations, 200 μL in the mother well, set 3 duplicate wells, incubate for 48 hours, observe under an inverted microscope, add 10 μL CCK8 solution to each well, and continue to culture for 2 hours. Use an enzyme marker to measure the absorbance of each well at 450nm. Use GraphPad Prism 9.5 software to count the experimental results when the compound concentration is 20 μM and calculate the inhibition rate. The results are shown in Table 1 below.

[0071] Table 1 In vitro antitumor activity test results

[0072]

[0073] As can be seen from Table 1, this type of novel triazolopyrimidine compound has a significant anti-tumor growth inhibitory effect on human pancreatic cancer cells (PANC-1), human breast cancer cells (MDA-MB-231), and human gastric cancer cells (SGC7901), indicating that this type of compound can be used as a candidate or lead compound for further development in the preparation of anti-tumor drugs.

[0074] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the claims of the present invention.

Claims

1. A novel triazolopyrimidine compound, characterized in that The structure is as follows:

2. The method for preparing the triazolopyrimidine compound according to claim 1, characterized in that: This is achieved through the following synthetic steps: First, 5-amino-1,2,4-triazolethiol (Q1) reacts with methyl 4-(bromomethyl)benzoate in dichloromethane to produce intermediate Q2 by nucleophilic substitution reaction, and then intermediate Q2 reacts with methyl benzoyl acetate to produce intermediate Q3 by cyclization reaction; intermediate Q3 reacts with phosphorus oxychloride to produce intermediate Q4; intermediate Q4 reacts with various amines to produce target product Q5 by substitution reaction; The various amines are: 4-fluoroaniline, morpholine, p-trifluoromethylaniline, p-trifluoromethylaniline, p-chloroaniline, p-methoxyaniline, 3-fluoroaniline, 3,4,5-trimethoxyaniline, 4-hydroxyaniline, aniline, and 2-fluoroaniline.

3. The use of the triazolopyrimidine compound according to claim 1 in the preparation of medicines, characterized in that: The invention is used as an active ingredient in the preparation of drugs for treating breast cancer, gastric cancer or pancreatic cancer.

4. An anti-tumor drug composition, characterized in that: The pharmaceutical composition uses the compound described in claim 1 as an active ingredient; the pharmaceutical composition also includes a pharmaceutically acceptable carrier, a pharmaceutically acceptable salt or an excipient.

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