Pyrimido triazole compound as well as preparation method and application thereof

Abstract

The invention relates to the field of medicinal chemistry, and particularly discloses pyrimidino triazole compounds as well as a preparation method and application thereof. The pyrimido triazole compound provided by the invention is a compound as shown in formula (I) or a pharmaceutically acceptable salt thereof: # imgabs0, through biological activity evaluation, the compound shows a remarkable anti-herpes simplex virus type 1 (HSV-1) virus effect in vitro and in vivo, and the median inhibitory concentration (EC50) is reduced by nearly 5 times compared with that of a control drug acyclovir; and a better therapeutic index is achieved. The invention breaks through the limitation of the existing anti-HSV-1 drug structure type, and has good potential and value of being further developed into anti-HSV-1 virus drugs.

Description

Technical Field

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

[0002] Herpes simplex virus type 1 (HSV-1) is a widely spread pathogen worldwide, which can cause a variety of diseases such as oral herpes, keratitis, encephalitis, etc., seriously threatening human health. According to the data of the World Health Organization, about 3.7 billion people under the age of 50 are infected with HSV-1 worldwide, and the mortality rate caused by HSV-1 encephalitis is as high as 70% each year, and more than half of the survivors have 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, but they have low bioavailability (about 15%-30%), frequent drug resistance (the resistance rate in immunosuppressed patients is 5%-10%), and cannot eliminate the viral genome lurking in the 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. New compounds with both efficient antiviral activity and good central permeability are urgently needed in the clinic.

[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 focuses on the DNA replication stage in the virus life cycle, and inhibitors are designed for thymidine kinase (TK) and DNA polymerase, but the virus can quickly escape through gene mutations. With the in-depth analysis of the interaction mechanism between the virus and the host, research hotspots have expanded to viral adsorption, host immune regulation and other links. For example, for the entry of viral glycoprotein gD, inhibitors can block the binding of the virus to host cell receptors, and compounds targeting the host NF-κB pathway can reduce tissue damage by inhibiting the inflammatory storm. However, most candidate drugs stop at preclinical research due to single targets, insufficient efficacy or toxicity issues. Currently, there are less than 10 new HSV-1 inhibitors entering 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 is facing the dual pressures of increasing drug resistance and a lack of severe treatment methods. There are more than one million new cases of severe HSV-1 infection 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 immunodeficiency population and the acceleration of viral mutation, the development of such highly effective, broad-spectrum and safe anti-HSV-1 drugs has become an urgent need to ensure public health security. Summary of the invention

[0006] Based on the current state of the art, the present invention aims to provide a novel compound containing a pyrimidine triazole structure with good anti-herpes simplex virus type 1 (HSV-1) activity and low toxicity; another object is to provide a preparation method thereof and its use as an antiviral drug in inhibiting HSV-1.

[0007] To achieve the purpose of the present invention, the molecular structure of the pyrimidotriazole compound of the present invention is as shown in formula (I):

[0008]

[0009] Wherein, R1 is an aryl group or a heteroaryl group.

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

[0011] The heteroaryl group is any one of unsubstituted or substituted pyridyl and furyl groups; the substituent group may be any one of halogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, amino or nitro groups.

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

[0013] More preferably, R1 is pyridyl substituted by halogen.

[0014] Preferably, the pyrimidotriazole compound is selected from the following compounds or pharmaceutically acceptable salts thereof, which are sequentially denoted as compounds 5a-i:

[0015]

[0016]

[0017] The compounds developed by the present invention can 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. Further, 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 compounds, and an 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) Place the intermediate 2 in a reaction flask, add a mixed solvent of an organic acid and water, slowly add an aqueous sodium nitrite solution to the system, and stir to react. After the reaction is completed, remove the solvent by vacuum distillation, extract, and concentrate under reduced pressure to further purify the intermediate 3.

[0022] (3) Place intermediate 3, tert-butyl (4-methylpiperidin-4-yl)carbamate and an organic base in a reaction flask, add an organic solvent to dissolve, stir to react, and concentrate under reduced pressure after the reaction is completed. After extraction, concentrate under reduced pressure, and purify by recrystallization, intermediate 4 is obtained.

[0023] (4) The intermediate 4 is placed in a reaction flask, an organic solvent is added to dissolve it, and then an organic acid or a solvent that dissolves acidic gas is 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 solvents, which only need 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, 6-chloropyridin-3-ylmethylamine; the organic base in steps (1) (3) is one or more of triethylamine, pyridine, piperidine, 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, ethyl acetate, One or more of alcohol, ethylene glycol, isopropanol, tert-butyl alcohol, and n-butyl alcohol; the organic acid in steps (2) and (4) is one or more of formic acid, acetic acid, propionic acid, butyric acid, benzenesulfonic acid, p-toluenesulfonic acid, and trifluoroacetic acid, and 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 realize the 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 reaction 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-effect mechanism showed stronger viral inhibitory activity than acyclovir in in vitro experiments, and its half inhibitory concentration (EC 50) was 2.34. 50 ) was reduced by nearly 5 times. In the HSV-1 encephalitis mouse model, the survival rate of the compound-treated group was 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 the acute toxicity test. Its unique chemical skeleton design breaks through the structural limitations of traditional nucleoside analogs, has excellent blood-brain barrier penetration ability and drug resistance barrier, and provides important scientific value and clinical application prospects for the research and development of anti-HSV-1 drugs.

[0026] Advantages and innovations of the present invention: The small molecule compound developed by the present invention organically combines viral protein inhibition with host immune regulation for the first time, providing a "two-pronged" innovative strategy for the design of antiviral drugs. Among them, compound 5i shows excellent clinical transformation value. This compound not only has an antiviral efficacy significantly superior to the first-line drug acyclovir (EC 50=1.95vs 4.83μM), and achieves synergistic therapeutic effects through a dual mechanism of action: while directly inhibiting HSV-1 viral replication (in vitro inhibition rate>90%), it can effectively regulate the host immune response and significantly reduce the cytokine storm and cell apoptosis caused by infection. This "antiviral-immunomodulatory" dual-effect mechanism significantly reduced brain tissue damage and weight loss in HSV-1-infected mice, improved survival rate, and showed 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 This is the mouse survival curve. Figure 3 Scoring mouse behavioral experiments. Figure 4 This is the hematoxylin-eosin (HE) staining of mouse brain tissue pathological sections. Figure 5 Immunofluorescence staining of mouse brain tissue pathological sections using fluorescent antibodies 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. Fig. 9 This is Tunel fluorescence staining of mouse brain tissue pathological sections after continuous drug administration for 5 days. Fig.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. Fig.11 Routine blood test of mice after continuous administration for 5 days. Fig.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 in conjunction with 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, if not otherwise specified, the percentages are all mass percentages. 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 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) containing 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, 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 operations were approved by the Life Science Ethics Review Committee of Zhengzhou University.

[0033] The pyrimidotriazole compounds involved in the following examples are successively recorded as compounds 5a-i, and the structural formulas are as follows:

[0034]

[0035] Example 1

[0036] The preparation processes of compounds 5a~i are similar, and 5a is taken 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 bottle, dissolved with 30 mL of n-butanol, and refluxed at 120°C for 6 h. The reaction was monitored by TLC (PE:EA=10:7). After the reaction was completed, most of the n-butanol was removed by vacuum concentration, the crude product was fried, and the crude product was purified by column chromatography (PE:EA=2:1) ​​to finally obtain intermediate 2a.

[0040] (2) Preparation of intermediate 3a

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

[0042] (3) Preparation of intermediate 4a

[0043] Intermediate 3a (433 mg, 1.0 eq.), (4-methylpiperidin-4-yl)carbamic acid tert-butyl ester (424 mg, 1.3 eq.), N,N-diisopropylethylamine (295 mg, 1.5 eq.) were placed in a 100 mL eggplant-shaped bottle, 30 mL of acetonitrile was added to dissolve, and stirred at room temperature for 4 hours. The reaction was monitored by TLC (PE:EA=1:1). After the reaction was completed, it was concentrated under reduced pressure, the system was dissolved in ethyl acetate and extracted with water three times, and concentrated under reduced pressure to obtain a crude product. The crude product was recrystallized and purified using ethyl acetate to obtain intermediate 4a.

[0044] (4) Preparation of Compound 5a

[0045] The intermediate 4a (397 mg, 1.0 eq.) was placed in a 100 mL eggplant-shaped bottle, 5 mL of ethyl acetate was added to dissolve, and then 10 mL of hydrochloric acid-ethyl acetate solution was added, and stirred at room temperature for 4 hours. The reaction was monitored by TLC (PE:EA=1:4), and after the reaction was completed, it was concentrated under reduced pressure, and then the system was dissolved with ethyl acetate several times and the solvent was removed to finally obtain a crude product, which was purified by recrystallization from ethyl acetate to obtain 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 pyrimidine triazole 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 pyrimidine triazole 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 pyrimidine triazole compound of this example 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 pyrimidine triazole 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 pyrimidine triazole compound of this example 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 pyrimidine triazole compound of this example 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 pyrimidine triazole 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-furanmethylamine.

[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 test 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 the virus suspension. Afterwards, different concentrations of the compounds were added to the cells and incubated for 72 hours. The MTT assay was used to detect the expression of the compounds at a wavelength of 490 nm (OD 490 ) absorbance. The calculation formula of virus inhibition rate is as follows:

[0074] Inhibition rate (%) = (OD 490 - Virus control group OD 490 ) / (blank group OD 490 - Virus control group OD 490 )×100%

[0075] The inhibition rate and the corresponding compound concentration were input into SPSS26 software to calculate the EC 50 .

[0076] Cytotoxicity screening assay:

[0077] Cell viability was determined using tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Beijing Solebao Company). Hep-2 cells were plated at 1.5 × 10 4 The cells were seeded at a density of 100 cells / well in a 96-well plate. When the cells grew to 80% confluence, different concentrations of compounds were added to each well. After 72 hours of incubation in an incubator, the concentrations of the compounds were measured using MTT at 490 nm (OD 490 ) absorbance. The calculation formula of cell activity is as follows:

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

[0079] The cell activity and the corresponding compound concentration were input into SPSS26 software to calculate the CC of the compound. 50 .

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

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

[0082]

[0083] a CC 50 : Half cytotoxic concentration.

[0084] b EC 50 :50% effective inhibitory concentration.

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

[0086] As can be seen from the table above, compound 5i exhibits 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 twice that of the positive control drug acyclovir (SI=29.45), showing better development potential and clinical treatment prospects.

[0087] Example 11 Mouse Experiment of Compound 5i

[0088] Animal Experiment 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 0.05mL. The injection site was the midpoint of the line connecting the right corner of the eye and the base of the ear. The needle insertion depth was 2-3mm, and a sense of emptiness could be felt during the injection process. The mice were first gavaged 4 hours after virus infection, and then given once a day for 5 days. Since the beginning of the model, the health status of the mice was observed, the body weight was weighed and recorded daily 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 biochemical tests.

[0090] In vivo therapeutic effect of compound 5i

[0091] Starting from adaptive feeding, the mice were observed for symptom changes and weight changes every day. Since the third day of infection with HSV-1, the mice in the virus control group have successively developed symptoms such as obvious discomfort, weight loss, eye ulcers, erect hair and motor dysfunction. In severe cases, they may experience hemiplegia, epileptic 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 a certain extent. In the virus control group, the weight of mice continued to decrease, but compound 5i could effectively inhibit this downward trend ( Figure 1 ). During the 14-day observation period, the survival rate of the virus control group dropped to 20%. In contrast, the survival rates of the medium and high concentration groups of compound 5i reached 100% and 90%, respectively ( Figure 2 ). It is noteworthy that the therapeutic effect of compound 5i exceeded that of the positive control drug acyclovir.

[0095] The behavioral scores of all mice were performed on the 4th, 5th and 6th days after modeling, and the scoring criteria are shown in Table 2. The results showed that compared with the normal group, the behavioral scores of the mice in the model group increased significantly, showing abnormal behavioral performance and severe impairment of all aspects of physical function. In contrast, the behavioral scores of the mice in the drug-treated group were lower than those in the model group, indicating that the degree of physical impairment of the mice in the drug-treated group was relatively mild, and their behavioral performance was better than that of the model group ( Figure 3 ).

[0096] After 5 consecutive days of administration, the complete main organs were collected 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] Observation of HE-stained brain tissue sections revealed that the brain tissue of mice in the normal group showed a typical normal appearance. The cells were arranged neatly, and no obvious abnormalities were observed. In contrast, the virus group showed a variety of abnormal signs: cell accumulation, accompanied by disordered tissue structure and congestion. In addition, nerve cells underwent degeneration and the virus induced extensive cell death. Importantly, when treated with compound 5i and acyclovir, respectively, brain tissue damage 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 day 6 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 mouse brain tissue. Subsequently, qRT-PCR was used to detect the mRNA levels 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 concentration 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 ( Fig. 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 intragastric administration of the drug to healthy mice for 5 days, the brain, heart, liver, spleen, lung and kidney were obtained for HE staining. Fig.10 As shown, compound 5i had no obvious 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 index tests ( Fig.11 and Fig.12 The results showed that compound 5i had no significant effect on the main blood indicators of healthy mice, showing good biosafety.

[0103] The compound 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 and has excellent blood-brain barrier penetration and drug resistance barriers. As a potential drug for the treatment of HSV-1 related diseases, it not only provides a new strategy for the treatment of HSV-1, but also opens up a new direction for the development of herpes virus family drugs.

Claims

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

2. The pyrimidotriazole compound according to claim 1, characterized in that: 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, characterized in that: 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 claim 1; (1) Add the raw material 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 an aqueous sodium nitrite solution to the system, stirring for reaction, 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. Extracting, concentrating under reduced pressure, and purifying by recrystallization to obtain intermediate 4; (4) The intermediate 4 is placed in a reaction flask, an organic solvent is added to dissolve it, and then an organic acid or a solvent that dissolves acidic gas is 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, characterized in that: 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 drugs against herpes simplex virus type 1 (HSV-1).

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