Purine nucleoside phosphamide ester ZL-492 and application thereof in antiviral drugs

Abstract

The invention discloses a purine-like nucleoside phosphamide ester ZL-492 and an application of the purine-like nucleoside phosphamide ester ZL-492 in an antiviral drug. The purine-like nucleoside phosphamide ester ZL-492 is obtained by taking a compound 1 and a compound 2 as raw materials and reacting in an organic solvent in the presence of a magnesium catalyst, and the chemical structural formula of the purine-like nucleoside phosphamide ester ZL-492 is shown in the specification. Researches show that the purine-like nucleoside phosphamide ester ZL-492 has a good application prospect in the aspect of resisting HIV-1 virus infection, especially HIV-lucc / HBX2 and HIV-lucc / JRFL viruses, and the application range of the purine-like nucleoside phosphamide ester ZL-492 in the aspect of resisting HIV-lucc / HBX2 and HIV-lucc / JRFL viruses is widened. The compound has the advantages of remarkably improving the activity of nucleoside drugs, improving the oral absorption efficiency, reducing the administration dosage and frequency, reducing the blood toxicity and kidney toxicity and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to purine nucleoside phosphoramidate prodrug ZL-492 and its synthesis method and application in antiviral drugs, and belongs to the field of drug synthesis. BACKGROUND

[0002] The report released by the United Nations AIDS Programme and the World Health Organization in 2025 shows that AIDS is still a major global public health problem, and the number of global infections and deaths is still rising. Nucleoside drugs are important anti-HIV drugs, and their core principle of action is to simulate the structure of natural nucleosides to interfere with the synthesis and replication of viral RNA.

[0003] After nucleoside drugs enter the body, they need to undergo three core phosphate transformations to become substances with antiviral activity, namely monophosphorylation (NMP), diphosphorylation (NDP) and triphosphorylation (NTP) under the catalysis of kinase, and the final formed nucleoside triphosphate is the active form of the drug. In the above three transformation processes, the kinase catalyzing monophosphate has high selectivity and relatively low activity, and becomes the "rate-limiting step" in the conversion process.

[0004] Therefore, the present application introduces a prodrug strategy to design purine nucleosides as prodrugs, which enhances the water solubility of the drug and improves the oral bioavailability by introducing a phosphoramidate group and an ester bond side chain. The prodrug is only converted into the active form in the target tissue (virus-infected cells) after entering the body, and is rarely converted in normal cells or tissues, which can effectively reduce the damage to normal cells and reduce the toxic side effects of the drug. SUMMARY

[0005] In order to overcome the above technical defects, the present application provides a purine nucleoside phosphoramidate compound ZL-492, and studies its synthesis method and drug activity. With compound 1 and compound 2 as raw materials, under the catalysis of tert-butyl magnesium chloride, a one-pot synthesis method can be used to obtain the biologically active phosphoramidate purine nucleoside compound ZL-492. In the method of the present application, the synthesis steps are relatively simple, and the reaction conditions are relatively mild. The compound ZL-492 improves the low biological activity of nucleoside drugs, ensures that the drug is only activated in the target organ (liver), avoids interference with other organs, and significantly reduces the systemic toxic side effects.

[0006] The purine nucleoside phosphoramidate ZL-492 according to the present application has the following chemical structural formula: The present application also provides a synthesis method of the above-mentioned purine nucleoside phosphoramidate prodrug ZL-492, and the reaction equation is represented as follows: The synthesis method of the above-mentioned purine nucleoside phosphoramidate prodrug ZL-492 comprises the following steps: reacting compound 1 and compound 2 in an organic solvent in the presence of a magnesium catalyst to obtain purine nucleoside phosphoramidate ZL-492.

[0007] Further, in the above technical solution, the ratio of the compound 1, the compound 2 and the magnesium catalyst is 1.5:1:2.

[0008] Further, in the above technical solution, the organic solvent is selected from DMF.

[0009] Further, in the above technical solution, the magnesium catalyst is tert-butyl magnesium chloride.

[0010] Further, in the above technical solution, the reaction temperature is selected from 0-40℃.

[0011] Further, in the above technical solution, the reaction is carried out under the protection of nitrogen or argon.

[0012] Further, the application further provides the use of the aforementioned purine nucleoside phosphoramidate ZL-492 in the preparation of an antiviral drug.

[0013] Further, in the above technical solution, the virus is HIV-1 virus; the anti-HIV-1 virus includes HIV-luc / JRFL and HIV-luc / HBX2 virus.

[0014] The application further provides a pharmaceutical composition of the aforementioned purine nucleoside phosphoramidate prodrug ZL-492.

[0015] Advantages of the application 1. The application introduces a prodrug strategy, designs a purine nucleoside as a prodrug, enhances the water solubility of the drug, improves the biological oral availability, reduces the dosage and frequency of administration, and reduces the blood toxicity and kidney toxicity by introducing a phosphoramidate group and an ester bond side chain.

[0016] 2. The purine nucleoside phosphoramidate prodrug ZL-492 of the application shows good inhibition rate on HEK293T cells transfected with plasmid pJRFL / env and HEK293T cells transfected with plasmid HXB2 / env which can stably express HIV-1 virus. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The figure is the anti-HIV-1 activity graph of compound ZL-492 at a single concentration (5µM) and control drug FNC at concentrations of 10nM and 100nM; Figure 2 The figure is the HIV-luc / JRFL EC50 Test graph (EC 50 = 120.29 nM); Figure 3 The EC50of compound ZL-492 under different concentrations of HIV-luc / HBX2 50 Test graph (EC 50 = 96.2 nM); Figure 4 The CC50of compound ZL-492 was calculated 50 Graph (CC 50 = 81.37 μM). DETAILED DESCRIPTION

[0018] The application will be further described by specific examples. These examples are intended to be illustrative of the application and are not intended to limit the scope of the application. After reading the present disclosure, those skilled in the art will be able to affect various changes and modifications of the present application, and such equivalent changes and modifications are also intended to fall within the scope of the present application as defined by the claims.

[0019] The membrane permeability of the natural purine nucleoside analogs is poor, and in vivo metabolic studies show that the single phosphorylation (NTP) rate is the determining factor, and the prodrug strategy can greatly shorten the time required for this process, effectively avoiding the problems of poor antiviral efficacy, inactivation of the natural purine nucleoside analogs, and increasing the antiviral efficacy dose.

[0020] Example 1 General experimental procedure: under a nitrogen and anhydrous atmosphere, a 25 mL sealed tube was heated for 3 min with a baking gun and was aerated. The temperature was controlled at 0 o C, compound 1 (0.15 mml) and compound 2 (0.1 mml) were dissolved in 5 mL of super dry DMF, and tert-butyl magnesium chloride (0.4 ml) was slowly added dropwise, and the temperature was controlled at 0 o C reaction for 30 min, and was transferred to a 40 o C sand bath for stirring for 48 h. After TLC (thin layer chromatography) detection of the complete reaction, it was cooled to room temperature, and was quenched with ethyl acetate (4 mL) and saturated ammonium chloride solution (4 mL). The organic layer was separated, and the water layer was extracted with ethyl acetate (4 mL x 3). The combined organic phase was washed with saturated ammonium chloride solution (80 mL), dried over anhydrous Na2SO4, and the organic phase was concentrated, and was separated by silica gel column chromatography (dichloromethane / methanol 100-10 / 1) to obtain the purine nucleoside analog phosphoramidate ZL-492. The reaction equation is shown as follows: Isopropyl(((2-acetoxybenzyl)oxy)(((2R,3R,4S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)phosphoryl)-D-alaninate (34.42 mg); 52.8% yield; 1 H NMR (400 MHz, DMSO-d6): 8.08(dd, J = 4.2, 2.1 Hz, 1H), 7.98-7.73(m, 2H), 7.37 -7.22 (m, 3H), 7.15-7.05(m, 1H), 6.44 (dd, J = 18.7, 5.6 Hz, 1H), 6.38-6.26 (m, 1H), 6.09 (ddd, J = 13.3, 10.0, 6.5 Hz, 1H), 5.33(dtd, J = 53.4, 5.6, 1.8 Hz, 1H), 5.11-4.99(m, 2H), 4.81-4.59 (m, 2H), 4.26-4.14 (m, 2H), 3.82-3.67(m, 2H), 1.98 (d, J = 2.5Hz, 3H), 1.16-1.12 (m, 3H), 1.08-0.98 (m, 6H); 13 C NMR (151 MHz, DMSO-d6): 177.69, 175.40, 164.72, 163.52 -162.58 (m), 155.85 (d, J = 20.8 Hz), 154.16, 145.56, 134.86 (d, J = 82.6 Hz), 132.30, 125.18, 122.11, 100.46, 99.17, 86.14, 84.12 (d, J = 11.0 Hz), 83.43, 79.75, 73.22, 72.89, 66.12, 65.96, 55.13, 54.96, 26.56 (d, J = 4.7 Hz), 25.86, 24.90 (d, J = 6.0 Hz). HRMS (ESI) m / z: [M+Na] + Calcd for C 27 H 31 F2N6NaO9P 484.0456; Found 675.1748.

[0021] Example 2 This example is a preliminary test process and results of the compound ZL-492 inhibiting HIV-1 virus at the cellular level in Example 1. The materials and consumables listed in this example are available from commercial sources unless otherwise specified. Cells and viruses are from the cell and microbial resource library of CTCC or other relevant institutions. The experimental methods in this example are standard molecular biology, cell biology or virology procedures, which can be easily understood and operated by researchers in the field. The specific steps are as follows: 1. Cells: HEK 293T, culture conditions: DMEM medium 90% + 10% FBS + 1% Pen-Strep. CD4 + T cell Hut / CCR5, culture conditions: RPMI-1640 medium 90% + 10% FBS + 1% Pen-Strep, containing G418 (50 mg / mL), Puromycin (1 mg / mL).

[0022] 2. Virus Plasmid pLAI-Δenv-Luc2, plasmid pJRFL / env. pLAI-Δenv-Luc contains the HIV-1 backbone gene sequence, but deletes the env and vpr genes, and inserts the Luciferase reporter gene at the nef gene site; pJRFL contains the CCR5-tropic HIV-1 env gene.

[0023] Co-transfect HEK293T with plasmid pLAI-Δenv-Luc2 and plasmid pJRFL / env, co-transfect HEK293T with plasmid pLAI-Δenv-Luc2 and plasmid HXB2 / env, to obtain HIV-1 pseudotyped single-cycle infectious HIV-1 (HIV-luc / JRFL and HIV-luc / HBX2).

[0024] 3. Main reagents: DMEM (Gibco, cat: C11995500BT); RPMI-1640 medium (GIBCO, cat: C11875500BT); Fetal bovine serum FBS (Gibco, cat: 10099-141); Double-antibiotic Pen-Strep (10,000 U / mL) (Gibco, cat: 15140122); G418 (Millipore, cat: 345810); Puromycin Dihydrochloride (InvivoGen, cat: ant-pr-1); Luciferase Cell Culture Lysis 5X Reagent (promega, cat: E1531); Luciferase Assay Substrate (promega, cat: E1501); MTT: (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MCE, cat: HY-15924).

[0025] 4. Experimental procedure: Step 1, preliminary screening of compounds inhibiting HIV-1 activity: 1 x 10 5 Hut / CCR5 cells were seeded in 96-well plates, 50 μL of the compound to be tested was added (dissolved in cell culture medium, the final concentration of the compound was 5 μM), 50 μL of the virus HIV-luc / JRFL was added, and FNC was used as a compound control; 37°C incubation for 2 days, cell collection, lysis, supernatant collection, luciferase activity detection; virus replication calculation, and further inhibition rate calculation.

[0026] Step 2, dose-dependent anti-HIV-1 assay: 1 x 10 5 Hut / CCR5 cells were seeded in 96-well plates, 50 μM of the compound with good screening effect was added, the final concentration of the compound was 50 μM, 10 μM, 2 μM, 0.4 μM, 0.08 μM, and 0.016 μM; 50 μM of the virus HIV-luc / JRFL or HIV-luc / HBX2 was added; FNC (azvudine) was used as a compound control; 37°C incubation for 2 days, cell collection, lysis, supernatant collection, luciferase activity detection; virus replication calculation, and further inhibition rate calculation.

[0027] Step 3, MTT colorimetric method for detecting cytotoxicity: 1 x 10 5Hut / CCR5 cells were inoculated in 96-well cell culture plates. 100 μL (culture medium: RPMI-1640 / 10%FBS / 1% Pen / Strep); 100 μL compound solution was added; the final concentration of the compound was 250 μM, 50 μM, 10 μM, 2 μM, 0.4 μM; each concentration was repeated 3 times; a control without the compound was also set up; after 72 h of cell culture, 100 μL supernatant was discarded, 20 μL MTT (5 mg / mL) was added and incubated at 37°C for 4 h; centrifugation was performed, 100 μL supernatant was discarded, 100 μL DMSO was added, and the solution was shaken at room temperature in the dark for 15 min until the blue formazen was dissolved; the OD595, OD630 was detected by an enzyme-labeled instrument, and the cell survival rate was calculated.

[0028] Step 4, calculation of the drug antiviral activity selection index: Selective Index (SI) = CC 50 / EC 50 .

[0029] 5, detection results: Conclusion: Under the condition of 5 μM concentration, the compound ZL-492 has a significant inhibitory effect on HIV-luc / JRFL virus. The drug antiviral activity selection index SI (Selective Index) is 676.4486.

[0030] Under the condition of 5 μM concentration, the compound ZL-492 has a significant inhibitory effect on HIV-luc / HBX2 virus. The drug antiviral activity selection index SI (Selective Index) is 845.842.

[0031] The above merely describes the preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can make equivalent replacements or changes to the technical solutions and the inventive concept of the present application within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A purine nucleoside phosphoramidate ZL-492, having the chemical structure: .

2. The method of synthesis of the purine nucleoside analogue phosphoramidate ZL-492 according to claim 1, characterized by that, comprising the steps of: Compound 1 and compound 2 as raw material, in the presence of magnesium catalyst in organic solvent, to get purine nucleoside phosphoramidate ZL-492.

3. The method of synthesis of the purine nucleoside analogue phosphoramidate ZL-492 according to claim 2, characterized by the fact that: said magnesium catalyst is selected from tert-butylmagnesium chloride.

4. The method of synthesis of the purine nucleoside analogue phosphoramidate ZL-492 according to claim 2, characterized by that: molar ratio of compound 1, compound 2 and magnesium catalyst is 1.5:1:

2.

5. The method of synthesis of the purine nucleoside analogue phosphoramidate ZL-492 according to claim 2, characterized by that: the reaction is carried out under nitrogen or argon protection, the organic solvent is selected from DMF, and the reaction temperature is 0-40°C.

6. Use of the purine nucleoside analogue ZL-492 as claimed in claim 1 for the preparation of an antiviral drug.

7. Use of the purine nucleoside analogue ZL-492 as claimed in claim 6 for the preparation of an antiviral drug, said antiviral being against HIV-1 virus.

8. Use of the purine nucleoside analogue ZL-492 as claimed in claim 7 for the preparation of an antiviral drug, said antiviral against HIV-1 virus, said antiviral against HIV-1 virus including HIV-luc / JRFL and HIV-luc / HBX2 viruses.

9. A pharmaceutical composition comprising the purine nucleoside analogue prodrug ZL-492 as claimed in claim 1.

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