Triazolopyrimidine antitumor compounds and their preparation methods and applications

Abstract

The present invention belongs to the field of medicine and specifically relates to a novel triazolopyrimidine antitumor compound, a preparation method, and an application thereof. The present invention adopts a simple and efficient synthesis method to prepare a novel triazolopyrimidine compound, the specific general structure of which is as follows: In vitro antitumor activity studies of the compound of the present invention have shown that the novel triazolopyrimidine compound has a significant inhibitory effect on the growth of human pancreatic cancer cells (PANC-1), human gastric cancer cells (SGC7901), and human breast cancer cells (MDA-MB-231), indicating that the triazolopyrimidine compound can be used as a lead compound or candidate compound for the development of antitumor drugs.

Description

Technical Field

[0001] The present 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 worldwide. Currently, cancer treatments include targeted therapy, chemotherapy, radiotherapy, and surgical intervention, with chemotherapeutic drugs playing a key role. However, existing chemotherapeutic drugs suffer from limitations such as limited efficacy, poor selectivity, high toxicity, and drug resistance. Therefore, developing novel antitumor drugs with superior efficacy and minimal side effects has become a key strategy in antitumor drug research. Triazolopyrimidine heterocyclic structures, as bioisosteres of the purine ring, possess antiviral, antibacterial, anti-inflammatory, and antitumor pharmacological activities. To identify novel, safe, and highly effective antitumor drugs, we designed and synthesized a series of triazolopyrimidine compounds based on computer-assisted drug molecular screening and piezosynthesis, and investigated their antitumor activity. This research is of great research value and will contribute to the development of independently developed drugs 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-mentioned purpose, the triazolopyrimidine compound of the present invention has the following general structural formula:

[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: first, 5-amino-1,2,4-triazolethiol (Q1) and methyl 4-(bromomethyl)benzoate undergo a nucleophilic substitution reaction in dichloromethane to produce an intermediate Q2; second, intermediate Q2 undergoes a cyclization reaction with methyl benzoylacetate to produce an intermediate Q3; intermediate Q3 undergoes a substitution reaction with phosphorus oxychloride to produce an intermediate Q4; and intermediate Q4 undergoes a substitution reaction with various amines to produce a target product, a triazolopyrimidine compound;

[0009] The synthetic route is as follows:

[0010]

[0011] The reaction conditions for 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 present 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 have strong inhibitory activity against human breast cancer cells (MDA-MB-231), with an inhibition rate of 79% at 20 μM and an 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 any 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 a clear and complete description of the technical solutions in the specific embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is in no way 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 making creative efforts are all within the scope of protection of the present invention.

[0023] The structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR measurements are performed using a Bruker AV-500 NMR spectrometer. The solvents used are deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), with tetramethylsilane (TMS) as the internal standard. Chemical shifts (δ) are reported in ppm.

[0024] Examples of compound synthesis

[0025] Example 1: Preparation of methyl 4-(((5-phenyl-7-(phenylamino))-[1,2,4]triazol-[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), cooled to 0°C with an ice-water bath, and 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 reaction was allowed to proceed at room temperature for 8 h. Thin-layer chromatography monitored 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. The target compound Q2 was then obtained as a fine white solid by flash column chromatography using a mixture of petroleum ether and ethyl acetate in a volume ratio of 3:1 as the mobile phase.

[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 mixture was heated and refluxed for 24 h. TLC monitored the complete conversion of the reaction raw materials. The mixture 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, 15 ml of phosphorus oxychloride was added to a 25 ml round-bottom flask, followed by the slow addition of compound Q3 (1.00 g, 2.55 mmol). The temperature was raised to 90°C and the reaction was allowed to proceed for 5 h. Complete conversion of the starting materials was monitored by thin-layer chromatography, and the mixture was cooled to room temperature. The mixture was then slowly added dropwise to a 100 ml ice-water bath and stirred for 30 min. The pH of the solution was adjusted to 7 with aqueous ammonia, filtered, and dried. The target compound Q4 was then purified by flash column chromatography using a 40:1 volume ratio of dichloromethane to methanol as the mobile phase to obtain the desired product, a white solid.

[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 h. The reaction raw materials were completely converted as monitored by thin-layer chromatography. The solution was cooled to room temperature, filtered, and recrystallized from 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, 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] 4-Fluoroaniline was used instead of aniline, and the preparation method was the same as that in 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-morpholino-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl))thio)methyl)benzoate (2-3)

[0041] Using morpholine instead of aniline, the preparation method is the same as that in Example 1.

[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 that in 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] Use p-bromoaniline to replace aniline, and 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] Use p-chloroaniline to replace aniline, and 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-anisidine instead of aniline, the preparation method is the same as that in 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 instead of 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 instead of 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] Use 4-hydroxyaniline to replace aniline, and 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] 2-fluoroaniline was used instead of aniline, and the preparation method was the same as that in 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: The CCK8 assay was used with three cell lines: 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 holes of 96-well plates with PBS. Incubate for 24 hours under 5% CO2, 37 ° C, and 90% humidity conditions 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 replicates, incubate for 48 hours, observe under an inverted microscope, add 10 μL CCK8 solution to each well, and continue to culture for 2 hours. Measure the absorbance of each well at 450 nm with a microplate reader. Use GraphPad Prism 9.5 software to statistically analyze 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 merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the 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 scope of protection determined by the claims of the present invention.

Claims

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

2. The method for preparing the triazolopyrimidine compound according to claim 1, wherein: 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. Then, intermediate Q2 reacts with methyl benzoyl acetate to produce intermediate Q3. Intermediate Q3 reacts with phosphorus oxychloride to produce intermediate Q4 by chlorination reaction. Intermediate Q4 reacts with various amines to produce target product Q5. The various amines are: 4-fluoroaniline, morpholine, 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, wherein The invention is used as an active ingredient in preparing medicines for treating breast cancer, gastric cancer or pancreatic cancer.

4. An anti-tumor pharmaceutical 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, pharmaceutical salt or excipient.

Images