Pyrimidinotriazoles, processes for their preparation and use

Abstract

The application relates to the field of pharmaceutical chemistry, and particularly discloses pyrimidinotriazoles, a preparation method and application thereof. The pyrimidinotriazoles provided by the application are compounds shown in the following formula (I) or pharmaceutically acceptable salts thereof: the compounds are evaluated in terms of bioactivity, and the compounds show significant anti-HSV-1 virus effects in vitro and in vivo, the half maximal inhibitory concentration (EC 50 ) is reduced by nearly 5 times compared with that of the control drug acyclovir, and the compounds have a more excellent therapeutic index. The application breaks through the limitations of the structural types of existing anti-HSV-1 drugs, and has good potential and value for further development into anti-HSV-1 virus drugs.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and in particular to novel compounds containing pyrimidotriazole structures and their use as anti-herpes simplex virus type 1 (HSV-1) inhibitors in the preparation of antiviral drugs. Background Art

[0002] Herpes simplex virus type 1 (HSV-1) is a widespread pathogen worldwide, causing a variety of illnesses, including oral herpes labialis, keratitis, and encephalitis, posing a serious threat to human health. According to the World Health Organization, approximately 3.7 billion people under the age of 50 are infected with HSV-1. The annual mortality rate from HSV-1 encephalitis is as high as 70%, and over half of survivors suffer permanent neurological damage.

[0003] Although antiviral drugs have been used clinically for decades, existing treatment options still face severe challenges. Nucleoside analogs represented by acyclovir work by inhibiting viral DNA polymerase and have become the first-line treatment for HSV-1 infection. However, they have low bioavailability (about 15%-30%), frequent drug resistance (the resistance rate in immunosuppressed patients is 5%-10%), and are unable to eliminate viral genomes lurking in ganglia. What is even more difficult is that due to the limitations of the blood-brain barrier, it is difficult for conventional drugs to reach effective therapeutic concentrations in patients with HSV-1 encephalitis. Clinically, there is an urgent need for new compounds that have both efficient antiviral activity and good central penetrance.

[0004] In recent years, the development of HSV-1 antiviral drugs has gradually shifted from a single-target strategy to a multi-mechanism synergistic intervention. Traditional research has focused on the DNA replication stage of the viral life cycle, designing inhibitors against thymidine kinase (TK) and DNA polymerase, but the virus can rapidly escape through genetic mutations. With the in-depth understanding of the mechanisms of virus-host interaction, research has expanded to include viral adsorption and host immune regulation. For example, inhibitors targeting the entry of the viral glycoprotein gD can block the binding of the virus to host cell receptors, while compounds targeting the host NF-κB pathway can reduce tissue damage by inhibiting the inflammatory storm. However, most candidate drugs have been stopped in preclinical research due to single targets, insufficient efficacy, or toxicity issues. Currently, fewer than 10 new HSV-1 inhibitors have entered clinical trials, and no multi-target synergistic drugs have been approved for marketing, highlighting the urgency of innovative breakthroughs in this field. The current HSV-1 treatment field faces the dual pressures of increasing drug resistance and a lack of treatment options for severe cases. More than one million new cases of severe HSV-1 infection are reported worldwide each year, of which about 30% are resistant to acyclovir. Existing alternative drugs such as sodium foscarnet are limited in their long-term use due to severe nephrotoxicity.

[0005] With the expansion of the global immunodeficient population and the acceleration of viral mutation, the development of such efficient, broad-spectrum and safe anti-HSV-1 drugs has become an urgent need to ensure public health safety. SUMMARY

[0006] Based on the current technical status, the present application aims to provide a new type of compound containing a pyrimidine and triazole structure with good anti-HSV-1 virus activity and low toxicity; another purpose is to provide a preparation method thereof and its application as an antiviral drug in inhibiting HSV-1.

[0007] To achieve the purpose of the present application, the pyrimidine and triazole compound of the present application has a molecular structure as shown in formula (I):

[0008]

[0009] Among them, R1 is aryl or heteroaryl.

[0010] The aryl is unsubstituted, optionally substituted phenyl; the substituent can be any one of halogen, trifluoromethyl, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, amino or nitro.

[0011] The heteroaryl is any one of unsubstituted, optionally substituted pyridyl, furanyl; the substituent can be any one of halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, amino or nitro.

[0012] Preferably: R1 is phenyl, phenyl monosubstituted by halogen, trifluoromethyl, hydroxyl, C1-C3 alkyl; or R1 is furanyl or pyridyl, pyridyl substituted by halogen.

[0013] Further preferably: R1 is pyridyl substituted by halogen.

[0014] Preferably, the pyrimidine and triazole compound is selected from the following compounds or pharmaceutically acceptable salts thereof, in turn denoted as compounds 5a-i:

[0015]

[0016]

[0017] The compounds developed by the present invention may also be in the form of pharmaceutically acceptable salts during drug development and application, and those skilled in the art can make reasonable choices as needed. Furthermore, the pharmaceutically acceptable salt is one of hydrochloride, hydrobromide, sulfate, phosphate, borate, methanesulfonate, p-toluenesulfonate, naphthalenesulfonate, benzenesulfonate, citrate, lactate, pyruvate, tartrate, acetate, trifluoroacetate, maleate, succinate, mandelate, fumarate, salicylate, and phenylacetate.

[0018] Its synthetic route is as follows:

[0019]

[0020] (1) The raw materials 2,5-diamino-4,6-dichloropyrimidine (1), benzylamine or substituted benzylamine or amine compound, and organic base are added to a reaction flask, and an alcohol organic solvent is added to dissolve the mixture. The mixture is heated and refluxed to react. After the reaction is completed, the solvent is removed by concentration under reduced pressure, and the intermediate 2 is further purified.

[0021] (2) Intermediate 2 is placed in a reaction flask, a mixed solvent of an organic acid and water is added, and a sodium nitrite aqueous solution is slowly added to the system, and the reaction is stirred. After the reaction is completed, the solvent is removed by vacuum distillation, and the intermediate 3 is further purified by extraction and vacuum concentration.

[0022] (3) Intermediate 3, tert-butyl (4-methylpiperidin-4-yl)carbamate, and an organic base are placed in a reaction flask, an organic solvent is added to dissolve, and the mixture is stirred for reaction. After the reaction is completed, the mixture is concentrated under reduced pressure, extracted, concentrated under reduced pressure, and purified by recrystallization to obtain intermediate 4.

[0023] (4) The intermediate 4 is placed in a reaction flask, and an organic solvent is added to dissolve it. An organic acid or a solvent that dissolves acidic gas is then added, and the reaction is stirred. After the reaction is completed, the mixture is concentrated under reduced pressure to remove the solvent to obtain a crude product, which is then purified by recrystallization to obtain the target compound.

[0024] In the present invention, there is no special limitation on the selection of solvent, which only needs to ensure the dissolution of the reaction raw materials and the normal progress of the reaction. Preferably, the substituted benzylamine in step (1) is 2-chlorobenzylamine, 4-chlorobenzylamine, 3-bromobenzylamine, 4-trifluoromethylbenzylamine, 4-tert-butylbenzylamine; the amine compound is one of 4-aminomethylphenol, 2-furylmethylamine, and 6-chloropyridin-3-ylmethylamine; the organic base in steps (1) and (3) is one or more of triethylamine, pyridine, piperidine, and N,N-diisopropylethylamine; the alcohol organic solvent in step (1) is methanol, ethyl acetate, ethyl acetate, ethyl acetate, ethyl acetate, ethyl acetate, ethyl acetate, ethyl acetate, ethyl acetate, ethyl acetate, ethyl acetate, ethyl acetate, ethyl acetate, ethyl acetate, ethyl acetate, ethyl acetate, ethyl acetate, ethyl acetate, ethyl acetate, ethyl acetate, ethyl acetate, ethyl acetate, ethyl acetate, One or more of alcohol, ethylene glycol, isopropanol, tert-butanol, and n-butanol; one or more of the organic acids formic acid, acetic acid, propionic acid, butyric acid, benzenesulfonic acid, p-toluenesulfonic acid, and trifluoroacetic acid in steps (2) and (4); the acidic gas is one or more of hydrogen chloride and hydrogen bromide; the organic solvent in step (4) is one or more of methanol, ethanol, tetrahydrofuran, dichloromethane, chloroform, ether, ethyl acetate, acetone, acetonitrile, and methyl tert-butyl ether.

[0025] The present invention also provides the use of pyrimidine triazole compounds in the preparation of inhibitors or antiviral drugs based on HSV-1 targets. The small molecule compounds developed by the present invention achieve dual regulation of viral targets and host pathways for the first time: on the one hand, by specifically inhibiting the ICP4 immediate early protein and gD glycoprotein of HSV-1, the viral gene transcription cascade and cell invasion process are blocked; on the other hand, the expression of STING and NF-κB signaling pathways is downregulated, effectively controlling the cytokine storm and inhibiting infection-related cell apoptosis. This "antiviral-immunomodulatory" dual-action mechanism has shown stronger viral inhibitory activity than acyclovir in in vitro experiments, and its half inhibitory concentration (EC50) is 2.5. 50 ) was reduced by nearly 5-fold. In a mouse model of HSV-1 encephalitis, the survival rate of the group treated with the compound of the present invention increased to 90%, the viral load in brain tissue decreased by 3 orders of magnitude, and pathological analysis showed significantly reduced neuroinflammatory damage. In addition, the compound of the present invention did not show adverse reactions such as liver and kidney damage in acute toxicity tests. Its unique chemical skeleton design breaks through the structural limitations of traditional nucleoside analogs, possessing excellent blood-brain barrier penetration and drug resistance barriers, providing important scientific value and clinical application prospects for the development of anti-HSV-1 drugs.

[0026] Advantages and innovations of this invention: The small molecule compound developed by this invention organically combines viral protein inhibition with host immune regulation for the first time, providing a "two-pronged" innovative strategy for antiviral drug design. Among them, compound 5i has shown excellent clinical translation value. This compound not only has an antiviral efficacy significantly superior to the first-line drug acyclovir (EC 50=1.95 vs 4.83μM), achieving a synergistic therapeutic effect through a dual mechanism of action: while directly inhibiting HSV-1 viral replication (in vitro inhibition rate >90%), it also effectively modulates the host immune response, significantly alleviating infection-induced cytokine storms and cell apoptosis. This dual "antiviral-immunomodulatory" mechanism significantly reduced brain tissue damage and weight loss in HSV-1-infected mice, improved survival rates, and demonstrated good biosafety.

[0027] In addition, the synthesis method of the present invention has a simple route and a high yield of more than 65%. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1-6 This is an experiment on reducing clinical symptoms and brain damage associated with HSV-1 by compound 5i of the present invention. Figure 1 This is the weight change curve of mice. Figure 2 The mouse survival curve. Figure 3 Scoring of mouse behavioral experiments. Figure 4 Hematoxylin-eosin (HE) staining of mouse brain tissue pathological sections. Figure 5 Immunofluorescence staining of mouse brain tissue pathological sections using a fluorescent antibody against ICP4. Figure 6 The mRNA expression levels of virus-related genes (ICP4, ICP8, gD, UL30) in mouse brain tissue after 5 consecutive days of administration.

[0029] Figure 7-12 This is a safety evaluation of compound 5i of the present invention in mice. Figure 7 The mRNA expression levels of antiviral-related genes and inflammation-related genes in mouse brain tissue after 5 consecutive days of administration. Figure 8 The concentrations of inflammatory cytokines IFN-γ and TNF-α in mouse serum after 5 consecutive days of administration. Figure 9 Tunel fluorescence staining of mouse brain tissue pathological sections after 5 consecutive days of drug administration. Figure 10 Comparison of hematoxylin-eosin (HE) stained pathological sections of brain, heart, liver, spleen, lung and kidney between blank control group and drug control group after 5 consecutive days of administration. Figure 11 Routine blood test of mice after 5 consecutive days of administration. Figure 12 The effect of compound 5i on biochemical indicators of liver and kidney function in mice. DETAILED DESCRIPTION

[0030] The present invention is further described below with reference to examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0031] In the following examples, unless otherwise specified, the percentages are percentages by mass. The structures of the compounds were determined by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). The nuclear magnetic resonance instrument used was a Swedish Bruker DPX-400 superconducting nuclear magnetic resonance instrument, with tetramethylsilane (TMS) as the internal standard; the high-resolution mass spectrometer used was a Waters-Micromass Q-Tof mass spectrometer. The 16 compounds involved in the examples are all new compounds synthesized by the present invention, and their structures were determined by melting point, 1 HNMR, 13 It was confirmed by modern spectroscopic methods such as C NMR and HRMS.

[0032] Acyclovir (ACV) was purchased from GlpBio (Montclair, USA), primary antibodies against herpes simplex virus ICP4 and gD proteins were purchased from Abcam (UK), anti-cleaved PRAP, anti-cleaved Caspase-3, anti-Bax, anti-Bcl-2, anti-STING, and anti-Phospho-NF-κB (P65) were purchased from Cell Signaling Technology (CST; USA), anti-GAPDH was purchased from Abbkine (California, USA), APC anti-mouse CD206 and PE anti-mouse CD80 antibodies were purchased from Biolegend (California, USA), human laryngeal carcinoma epithelial cells (HEp-2) were purchased from Beijing Beina Chuanglian Biotechnology Research Institute (Beijing, China), and mouse BV2 microglial cell line (BV2) cells were purchased from Shanghai Qiyun Biotechnology Co., Ltd. (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) at 37°C and 5% CO. Herpes simplex virus type 1 KOS strain (HSV-1 / KOS) was purchased from the American Type Culture Collection (ATCC; Manassas, OH, USA) and cultured under CO₂. BALB / c mice (female, 5 weeks old, 16–18 g) were purchased from Beijing SPF Biotechnology Co., Ltd. (Beijing, China). All procedures were approved by the Life Science Ethics Review Committee of Zhengzhou University.

[0033] The pyrimidotriazole compounds involved in the following examples are sequentially referred to as compounds 5a-i, and have the following structural formulas:

[0034]

[0035] Example 1

[0036] The preparation processes of compounds 5a to 5i are similar, and 5a is used as an example for illustration.

[0037]

[0038] (1) Preparation of intermediate 2a

[0039] 2,5-Diamino-4,6-dichloropyrimidine (500 mg, 1.0 eq.), benzylamine (449 mg, 1.5 eq.), and N,N-diisopropylethylamine (469 mg, 1.3 eq.) were added to a 100 mL eggplant-shaped flask and dissolved in 30 mL of n-butanol. The mixture was then refluxed at 120°C for 6 h. The reaction was monitored by TLC (PE:EA = 10:7). After completion, the reaction was concentrated under reduced pressure to remove most of the n-butanol. The crude product was then dried and purified by column chromatography (PE:EA = 2:1) to obtain intermediate 2a.

[0040] (2) Preparation of intermediate 3a

[0041] Intermediate 2a (472 mg, 1.0 eq.) was placed in a 100 mL eggplant-shaped flask, and a mixture of 25 mL of acetic acid and 5 mL of water was added. After incubation at 0°C for 0.5 h, an aqueous sodium nitrite solution (652 mg, 5.0 eq., 7 M) was slowly added via a constant pressure dropping funnel. The mixture was stirred at 0°C for 1.5 h. The reaction was monitored by TLC (PE:EA = 1:1.5). After completion, most of the acetic acid was removed by vacuum distillation, followed by dissolution with ethyl acetate. The remaining acetic acid was neutralized by addition of saturated sodium bicarbonate aqueous solution, and the mixture was extracted 3-5 times with ethyl acetate and water. The mixture was concentrated under reduced pressure, and the crude product was fried and purified by column chromatography (PE:EA = 1.5:1) to obtain intermediate 3a.

[0042] (3) Preparation of intermediate 4a

[0043] Intermediate 3a (433 mg, 1.0 eq.), tert-butyl (4-methylpiperidin-4-yl)carbamate (424 mg, 1.3 eq.), and N,N-diisopropylethylamine (295 mg, 1.5 eq.) were placed in a 100 mL eggplant flask and dissolved in 30 mL of acetonitrile. The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC (PE:EA = 1:1). After completion of the reaction, the mixture was concentrated under reduced pressure, dissolved in ethyl acetate, and extracted three times with water. The crude product was concentrated under reduced pressure to obtain the crude product, which was then purified by recrystallization from ethyl acetate to obtain Intermediate 4a.

[0044] (4) Preparation of compound 5a

[0045] Intermediate 4a (397 mg, 1.0 eq.) was placed in a 100 mL eggplant-shaped flask and dissolved in 5 mL of ethyl acetate. Subsequently, 10 mL of hydrochloric acid-ethyl acetate solution was added and stirred at room temperature for 4 h. The reaction was monitored by TLC (PE:EA = 1:4). After completion of the reaction, the mixture was concentrated under reduced pressure and subsequently dissolved in ethyl acetate several times and the solvent removed to obtain a crude product. The crude product was then purified by recrystallization from ethyl acetate to afford compound 5a.

[0046] Compound 5a: 7-(4-amino-4-methylpiperidin-1-yl)-3-benzyl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine. Creamy white solid, yield 82%, melting point 196.9-198.2°C. 1 H NMR(400MHz,DMSO-d6)δ8.43(s,3H),7.38–7.29(m,5H),5.63(s,2H),5.00 (s,1H),4.43(s,1H),4.25(s,1H),3.75(s,1H),1.88(s,4H),1.42(s,3H). 13 C NMR(101MHz,DMSO-d6)δ135.88,129.30,128.65,128.25,121.29,52.14,50.06,23.38.HR-MS(ESI):Calcd.C 17 H 22 N8,[M+H] + m / z:339.2047,found:339.2049.

[0047] Example 2

[0048] The pyrimidotriazole compound of this embodiment has a structural formula as shown in Compound 5b, and its preparation process is basically the same as that of Example 1, except that the benzylamine in step (1) is replaced by 2-chlorobenzylamine.

[0049] Compound 5b: 7-(4-amino-4-methylpiperidin-1-yl)-3-(2-chlorobenzyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine. White solid, yield 79%, melting point 220.0-220.9°C. 1H NMR (400MHz, DMSO-d6) δ8.54(s,3H),7.53(dd,J=7.9,1.3Hz,1H),7.38(dd,J=7.8,1.8Hz,1H),7.34(dd,J=7.5, 1.4Hz,1H),7.07(dd,J=7.6,1.7Hz,1H),5.71(s,2H),5.00(s,1H),3.83(s,1H),1.99–1.86(m,4H),1.44(s,3H). 13 C NMR(101MHz,DMSO-d6)δ133.23,132.62,130.55,130.12,130.08,128.18,121.08,52.16,47.82,23.33.HR-MS(ESI):Calcd.C 17 H 21 ClN8,[M+H] + m / z:373.1658,found:373.1661.

[0050] Example 3

[0051] The pyrimidotriazole compound of this embodiment has a structural formula as shown in Compound 5c, and its preparation process is basically the same as that of Example 1, except that the benzylamine in step (1) is replaced by 4-chlorobenzylamine.

[0052] Compound 5c: 7-(4-amino-4-methylpiperidin-1-yl)-3-(4-chlorobenzyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine. White solid, yield 82%, melting point 263.5-266.0°C. 1 H NMR (400MHz, DMSO-d6) δ8.42(s,3H),7.43(d,J=8.3Hz,2H),7.32(d,J=8.4Hz,2H),5.63 (s,2H),5.00(s,1H),4.44(s,1H),4.22(s,1H),3.72(s,1H),1.87(s,4H),1.42(s,3H). 13 C NMR(101MHz,DMSO-d6)δ134.95,133.32,130.15,129.28,121.30,60.30,52.16,49.23,23.33,14.62.HR-MS(ESI):Calcd.C 17 H 21 ClN8,[M+H] + m / z:373.1658,found:373.1657.

[0053] Example 4

[0054] The pyrimidotriazole compound of this embodiment has a structural formula as shown in Compound 5d, and its preparation process is basically the same as that of Example 1, except that the benzylamine in step (1) is replaced by 3-bromobenzylamine.

[0055] Compound 5d: 7-(4-amino-4-methylpiperidin-1-yl)-3-(3-bromobenzyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine. White solid, yield 76%, melting point 232.4-234.1°C. 1 H NMR(400MHz,DMSO-d6)δ8.46(s,3H),7.56–7.51(m,2H),7.36–7.27(m,2H),5.65(s, 2H),4.99(s,1H),4.43(s,1H),4.26(s,1H),3.76(s,1H),1.89(s,4H),1.43(s,3H). 13 C NMR(101MHz,DMSO-d6)δ138.61,131.54,131.52,130.94,127.31,122.40,121.31,52.17,49.14,23.34.HR-MS(ESI):Calcd.C 17 H 21 BrN8,[M+H] + m / z:417.1153,found:417.1152.

[0056] Example 5

[0057] The pyrimidotriazole compound of this embodiment has a structural formula as shown in Compound 5e, and its preparation process is basically the same as that of Example 1, except that the benzylamine in step (1) is replaced by 4-trifluoromethylbenzylamine.

[0058] Compound 5e: 7-(4-amino-4-methylpiperidin-1-yl)-3-(4-trifluoromethylbenzyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine. White solid, yield 85%, melting point 166.0-168.2°C. 1H NMR (400MHz, DMSO-d6) δ8.52(s,3H),7.77(s,1H),7.71(d,J=7.3Hz,1H),7.64–7.57(m,2H),5.77(d ,J=5.3Hz,2H),5.00(s,1H),4.44(s,1H),4.30(s,1H),3.80(s,1H),1.97–1.83(m,4H),1.43(s,3H). 13 C NMR(101MHz,DMSO-d6)δ137.31,132.43,130.53,130.06,129.74,129.43,125.89,1 25.48,125.02,124.99,123.18,121.31,52.15,49.33,23.32.HR-MS(ESI):Calcd.C 18 H 21 F3N8,[M+H] + m / z:407.1921,found:407.1923.

[0059] Example 6

[0060] The pyrimidotriazole compound of this embodiment has a structural formula as shown in Compound 5f, and its preparation process is basically the same as that of Example 1, except that the benzylamine in step (1) is replaced by 4-tert-butylbenzylamine.

[0061] Compound 5f: 7-(4-amino-4-methylpiperidin-1-yl)-3-(4-tert-butylbenzyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine. White solid, yield 83%, melting point 253.5-255.7°C. 1 H NMR (400MHz, DMSO-d6) δ8.46 (s, 3H), 7.37 (d, J = 8.4Hz, 2H), 7.27 (d, J = 8.4Hz, 2H), 5.60 (s,2H),4.98(s,1H),4.28(s,2H),3.78(s,1H),1.89(s,4H),1.42(s,3H),1.24(s,9H). 13 C NMR(101MHz,DMSO-d6)δ151.15,132.96,128.13,126.06,121.32,52.16,34.81,31.56,23.37.HR-MS(ESI):Calcd.C 21 H 30 N8,[M+H] + m / z:395.2673,found:395.2679.

[0062] Example 7

[0063] The pyrimidotriazole compound of this embodiment has a structural formula as shown in Compound 5g, and its preparation process is basically the same as that of Example 1, except that the benzylamine in step (1) is replaced by 4-aminomethylphenol.

[0064] Compound 5g: 7-(4-amino-4-methylpiperidin-1-yl)-3-(4-hydroxybenzyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine. White solid, yield 65%, melting point 254.7-255.3°C. 1 H NMR (400MHz, DMSO-d6) δ8.44(s,3H),7.19(d,J=8.3Hz,2H),6.74(d,J=8.3Hz,2H),4.98(s,1H),4.40(s,2H),3.77(s,1H),1.88(s,4H),1.42(s,3H). 13 C NMR(101MHz,DMSO-d6)δ157.97,129.97,125.89,121.33,115.96,52.13,49.96,23.39.HR-MS(ESI):Calcd.C 17 H 22 N8O,[M+H] + m / z:355.1997,found:355.1999.

[0065] Example 8

[0066] The pyrimidotriazole compound of this embodiment has a structural formula as shown in compound 5h, and its preparation process is basically the same as that of Example 1, except that the benzylamine in step (1) is replaced by 2-furylmethylamine.

[0067] Compound 5h: 7-(4-amino-4-methylpiperidin-1-yl)-3-(furan-2-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine. White solid, yield 88%, melting point 288.4-290.1°C. 1 H NMR(400MHz, DMSO-d6)δ8.46(s,3H),7.64–7.62(m,1H),6.57(d,J=3.2Hz,1H),6.46–6.43(m,1 H),5.65(s,2H),4.96(s,1H),4.41(s,1H),4.26(s,1H),3.77(s,1H),1.88(s,4H),1.42(s,3H). 13C NMR(101MHz,DMSO-d6)δ136.99,131.83,131.52,131.38,130.33,128.60,121.34,52.17,48.65,23.31.HR-MS(ESI):Calcd.C 15 H 20 N8O,[M+H]+m / z:329.1840,found:329.1853.

[0068] Example 9

[0069] The pyrimidotriazole compound of this embodiment has a structural formula as shown in Compound 5i, and its preparation process is basically the same as that of Example 1, except that the benzylamine in step (1) is replaced by 6-chloropyridin-3-ylmethylamine.

[0070] Compound 5i: 7-(4-amino-4-methylpiperidin-1-yl)-3-(6-chloropyridin-3-ylmethyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine. White solid, yield 90%, melting point 232.4-234.1°C. 1 H NMR (400MHz, DMSO-d6) δ8.55–8.47(m,4H),7.81(dd,J=8.3,2.6Hz,1H),7.54(d,J=8.3Hz,1H) ,5.74(s,2H),4.99(s,2H),4.44(s,1H),4.30(s,1H),3.81(s,1H),1.91(s,4H),1.42(s,3H). 13 C NMR(101MHz,DMSO-d6)δ150.67,150.01,140.11,130.96,124.99,121.42,52.04,47.24,23.42.HR-MS(ESI):Calcd.C 16 H 20 ClN9,[M+H] + m / z:374.1610,found:373.1604.

[0071] Example 10 Anti-HSV-1 activity and cytotoxicity screening

[0072] Anti-HSV-1 activity experimental method:

[0073] Hep-2 cells were plated at 1.5 × 10 4 When the cells grew to 80% confluence, 100 TCID 50The cells were infected with virus suspension. Then, different concentrations of compounds were added to the cells and incubated for 72 hours. The absorbance at 490 nm wavelength (OD 490 ) was detected using MTT. The formula for calculating the virus inhibition rate is as follows:

[0074] Inhibition rate (%) = (compound group OD 490 - virus control group OD 490 ) / (blank group OD 490 - virus control group OD 490 ) x 100%

[0075] The inhibition rate and the corresponding compound concentration were input into SPSS 26 software to calculate the EC 50 of the compound.

[0076] Cell toxicity screening experiment method:

[0077] Cell activity was determined using tetrazolium 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Beijing Solaybao Company). Hep-2 cells were seeded into a 96-well plate at a density of 1.5 x 10 4 cells per well. When the cells grew to 80% confluence, different concentrations of compounds were added to each well. After incubation in an incubator for 72 hours, the absorbance at 490 nm wavelength (OD 490 ) was detected using MTT. The formula for calculating cell activity is as follows:

[0078] Cell activity (%) = compound group OD 490 / blank control group OD 490 x 100%

[0079] The cell activity and the corresponding compound concentration were input into SPSS 26 software to calculate the CC 50 of the compound.

[0080] The antiviral activity and cytotoxicity of the compounds in the present application against HSV are summarized in Table 1.

[0081] Table 1. Results of HSV-1 activity and cytotoxicity screening of compounds

[0082]

[0083] a CC 50 : half cytotoxic concentration.

[0084] b EC 50 : half effective inhibition concentration.

[0085] cSI: (Selectivity Index) = CC 50 / EC 50 .

[0086] As can be seen from the table above, compound 5i exhibited significantly better antiviral activity than the positive control drug acyclovir. 50 1.95±0.99μM) compared with acyclovir (EC 50 9.62±3.06μM) increased by nearly 5 times. It is worth noting that although the cytotoxicity of 5i (CC 50 102.52±14.88μM) was slightly higher than acyclovir (CC 50 283.34±22.82μM), but its therapeutic index (SI=52.57) is nearly 2 times that of the positive control drug acyclovir (SI=29.45), showing better development potential and clinical treatment prospects.

[0087] Example 11 Mouse Experiment with Compound 5i

[0088] Animal experimental methods:

[0089] The virus culture fluid was collected and inoculated intracranially into mice. The virus concentration was TCID 50 =10 -3 , each mouse was inoculated with 0.05 mL. The injection site was the midpoint of the line connecting the right corner of the eye and the base of the ear. The needle was inserted to a depth of 2-3 mm, and a sense of emptiness could be felt during the injection process. The mice were first gavaged 4 hours after viral infection, and then given once a day for 5 consecutive days. Since the beginning of the model, the health status of the mice was observed, their weight was weighed and recorded every day for 14 consecutive days. On the 6th day after the mice were infected with HSV-1, that is, 24 hours after the last dose, the complete main organs were taken for H&E, immunofluorescence, TUNEL, ELISA and qRT-PCR experiments. Whole blood and serum were collected for routine blood tests and blood biochemistry tests.

[0090] In vivo therapeutic effect of compound 5i

[0091] Beginning with adaptive feeding, mice were observed daily for changes in symptoms and weight. Starting on day 3 after HSV-1 infection, mice in the viral control group developed symptoms such as significant discomfort, weight loss, eye ulcers, piloerection, and motor dysfunction. In severe cases, they may experience hemiplegia, seizures, or even death.

[0092] Table 2. Animal behavior scoring criteria.

[0093]

[0094] After treatment with compound 5i, it was found that it alleviated the above symptoms to some extent. In the virus control group, the body weight of mice continued to decrease, but compound 5i effectively inhibited this decreasing trend Figure 1 ). During the 14-day observation period, the survival rate of the virus control group decreased to 20%. In contrast, the survival rates of the medium and high concentration groups of compound 5i reached 100% and 90%, respectively Figure 2 ). Notably, the therapeutic effect of compound 5i exceeded that of the positive control drug acyclovir.

[0095] On the 4th, 5th and 6th days after modeling, all mice were scored for behavior, and the scoring criteria are shown in Table 2. The results showed that the behavior scores of the model group mice were significantly increased compared with the normal group, showing abnormal behavior performance and severe impairment of body function in all aspects. In contrast, the behavior scores of the mice in the administration groups were lower than those in the model group, indicating that the degree of impairment of body function in the mice in the administration groups was relatively lighter, and their behavior performance was better than that in the model group Figure 3 ).

[0096] After 5 days of continuous administration, the complete main organs were taken for H&E, immunofluorescence, TUNEL, ELISA and qRT-PCR experiments. Whole blood and serum were collected for routine blood tests and blood biochemical tests.

[0097] Observing the HE-stained brain tissue sections, it was found that the brain tissue of the normal group mice presented a typical normal appearance. The cells were arranged in order, and no obvious abnormalities were observed. In contrast, the virus group presented a variety of abnormal signs: cell accumulation, accompanied by disordered tissue structure and hyperemia. In addition, the nerve cells underwent degeneration, and the virus induced extensive cell death. Importantly, when treated with compound 5i and acyclovir, respectively, the damage to the brain tissue was significantly reduced Figure 4 ).

[0098] Immunofluorescence analysis was used to detect the expression of ICP4 protein. The results showed that the virus control group showed obvious red fluorescence Figure 5 ). As shown in the figure, on the 6th day after infection, the ICP4 level of HSV-1 infected mice exceeded that of mice treated with compound 5i and acyclovir, further confirming the reduction of viral load in the brain tissue of mice. Subsequently, qRT-PCR was used to detect the mRNA level of virus-related genes in HSV-1 infected brain tissue. It was found that viral infection significantly increased the mRNA levels of ICP4, ICP8, gD and UL30, while compound 5i treatment could inhibit their expression Figure 6 ).

[0099] At the same time, compared with the virus control group, the expression levels of key pattern recognition receptors such as RIG-I, TLR3, NLRP3 and important pro-inflammatory cytokines such as IFN-γ, TNF-α, iNOS, IL-6, IRF3 and caspase-1 were significantly downregulated (e.g. Figure 7 ). We detected the concentrations of related cytokines in mouse serum 6 days after HSV-1 infection by ELISA. The results showed that the expression of TNF-α and IFN-γ increased significantly after HSV-1 infection, and compound 5i could inhibit the expression of these cytokines ( Figure 8 ).

[0100] In addition, TUNEL fluorescence showed that the green signal of apoptotic cells was widely detected in the virus control group, but was rarely observed in the compound 5i group ( Figure 9 ).

[0101] It can be seen from the above that compound 5i of the present invention can alleviate the clinical symptoms and tissue damage associated with herpes simplex encephalitis (HSE) caused by HSV-1 infection, and significantly improve the survival rate.

[0102] After continuous oral administration for 5 days in healthy mice, the brain, heart, liver, spleen, lung and kidney were harvested for HE staining. Figure 10 As shown in Figure 2, compound 5i had no significant side effects on the major organs of healthy mice. In addition, whole blood and serum were collected from mice for routine hematological and blood biochemical tests ( Figure 11 and Figure 12 The results showed that compound 5i had no significant effect on the main blood indicators of healthy mice, demonstrating good biosafety.

[0103] This compound showed no adverse reactions such as liver and kidney damage in acute toxicity testing. Its unique chemical backbone design transcends the structural limitations of traditional nucleoside analogs, demonstrating excellent blood-brain barrier penetration and resistance. Its potential as a therapeutic for HSV-1-related diseases not only provides a new strategy for HSV-1 treatment but also opens up new directions for the development of drugs targeting the herpes virus family.

Claims

1. A pyrimidotriazole compound, characterized in that: A compound represented by formula (I) or a pharmaceutically acceptable salt thereof: Wherein, R1 is phenyl, phenyl monosubstituted by halogen, trifluoromethyl, hydroxyl, or C1-C3 alkyl; or R1 is furyl, pyridyl, or pyridyl substituted by halogen.

2. The pyrimidotriazole compound according to claim 1, wherein R1 is pyridyl monosubstituted by halogen.

3. The pyrimidotriazole compound according to claim 1 or 2, characterized in that The pharmaceutically acceptable salt is one of hydrochloride, hydrobromide, sulfate, phosphate, borate, methanesulfonate, p-toluenesulfonate, naphthalenesulfonate, benzenesulfonate, citrate, lactate, pyruvate, tartrate, acetate, trifluoroacetate, maleate, succinate, mandelate, fumarate, salicylate, and phenylacetate.

4. The pyrimidotriazole compound according to claim 1, wherein Selected from the following compounds:

5. A method for preparing the pyrimidotriazole compound according to any one of claims 1 to 4, characterized in that: The following steps are involved: R1 is consistent with the statement in claim 1; (1) Add the raw materials 2,5-diamino-4,6-dichloropyrimidine (1), benzylamine or substituted benzylamine or amine compound, and organic base into a reaction flask, add an alcohol organic solvent to dissolve, heat and reflux to react, and after the reaction is completed, concentrate under reduced pressure to remove the solvent, and further purify to obtain intermediate 2; (2) placing intermediate 2 in a reaction flask, adding a mixed solvent of an organic acid and water, slowly adding a sodium nitrite aqueous solution to the system, stirring to react, and after the reaction is completed, removing the solvent by vacuum distillation, extracting, and concentrating under reduced pressure to further purify intermediate 3; (3) placing intermediate 3, tert-butyl (4-methylpiperidin-4-yl)carbamate, and an organic base in a reaction flask, adding an organic solvent to dissolve, stirring to react, and concentrating under reduced pressure after the reaction is completed. Extraction, concentrating under reduced pressure, and recrystallization purification are performed to obtain intermediate 4; (4) The intermediate 4 is placed in a reaction flask, and an organic solvent is added to dissolve it. An organic acid or a solvent that dissolves acidic gas is then added, and the reaction is stirred. After the reaction is completed, the mixture is concentrated under reduced pressure to remove the solvent to obtain a crude product, which is then purified by recrystallization to obtain the target compound.

6. The method for preparing the pyrimidotriazole compound according to claim 5, wherein: The substituted benzylamine in step (1) is 2-chlorobenzylamine, 4-chlorobenzylamine, 3-bromobenzylamine, 4-trifluoromethylbenzylamine or 4-tert-butylbenzylamine; the amine compound is one of 4-aminomethylphenol, 2-furylmethylamine and 6-chloropyridin-3-ylmethylamine.

7. Use of the compound according to any one of claims 1 to 4 in the preparation of antiviral drugs, characterized in that: The active ingredient is used for preparing anti-herpes simplex virus type 1 (HSV-1) medicine.

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