A compound containing bisacrididine nucleoside, its preparation method and application

Abstract

This invention discloses a compound containing diacylpropidine, its preparation method, and its applications. Remdesivir, acyclovir, vidarabine, and stavudine, along with succinic anhydride, are reacted in dichloromethane to obtain an intermediate containing a monocarboxylic acid; or remdesivir, acyclovir, vidarabine, and stavudine, along with pimelic acid, are condensed in EDC·HCl to obtain an intermediate containing a monocarboxylic acid; a diacylpropidine-containing linker is then condensed with the intermediate containing the monocarboxylic acid in EDC·HCl to obtain a compound containing diacylpropidine. This compound can form covalent bonds with target proteins, achieving irreversible binding, providing a new mechanism of action and binding mode, improving the binding efficacy and duration of action of small molecule inhibitors, and enhancing the intensity and duration of drug action.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically relating to a compound containing diaziridine nucleoside, its preparation method, and its application. Background Technology

[0002] Faced with the sudden outbreak of the novel coronavirus infection, new drug development cannot proceed in the conventional manner. The rapid early spread of SARS-CoV-2 and the lack of specific therapies for COVID-19 encouraged trials of approved drugs beyond their original indications. Although this strategy is feasible and yields greater early clinical benefits, after investing significant resources in research, it has been found that the "drug repurposing" strategy lacks target specificity and has not achieved ideal results in stopping COVID-19. Targeting potential targets of coronaviruses, the rational design and development of drugs that directly covalently bind to the target proteins of SARS-CoV-2 holds promise for addressing the problems of lack of specificity and poor efficacy, and represents a wise strategy for discovering effective COVID-19 treatments.

[0003] Photoaffinity labeling involves introducing photosensitive groups into active molecules to form stable covalently bound complexes with target proteins. Drugs irreversibly bind to target protein residues via covalent bonds, thereby exerting more efficient antiviral activity. Compared to traditional reversible inhibitor antiviral drugs, covalent inhibitors offer advantages such as higher biochemical efficiency, stronger and longer-lasting effects, reduced dosage and frequency, separation of pharmacodynamics and pharmacokinetics, maintenance of efficacy even after rapid drug clearance, prevention of drug resistance, and high selectivity by targeting rare, non-conserved residues in specific proteins. These advantages make them particularly promising for research in antiviral and antitumor drugs. Summary of the Invention

[0004] The purpose of this invention is to provide a compound containing a diazinonidine, its preparation method, and its application. This compound has low cytotoxicity and can be used in the preparation of antiviral drugs to enhance the drug's potency and duration of action.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A compound containing a diazinonidine nucleoside has the following structural formula:

[0007]

[0008] R represents remdesivir, acyclovir, vidarabine, or stavudine.

[0009] A method for preparing a compound containing a diazinonidine nucleoside includes the following steps:

[0010] 1) Remdesivir, acyclovir, vidarabine, and one of stavudine react with succinic anhydride in dichloromethane to give an intermediate containing a monocarboxylic acid; or

[0011] The intermediate containing a monocarboxylic acid was obtained by condensing remdesivir, acyclovir, vidarabine, and stavudine with pimelic acid in EDC·HCl.

[0012] 2) The linker containing diacylpropidine and the intermediate containing a monocarboxylic acid were condensed in EDC·HCl to obtain a nucleoside compound containing diacylpropidine.

[0013] Furthermore, one of remdesivir, acyclovir, vidarabine, and stavudine, along with succinic anhydride, is reacted in dichloromethane to obtain an intermediate containing a monocarboxylic acid, comprising the following steps: dissolving 1.00 mmol of one of remdesivir, acyclovir, vidarabine, and stavudine, along with 1.20 mmol of succinic anhydride, in 10 mL of anhydrous dichloromethane and reacting for 8 h to obtain an intermediate containing a monocarboxylic acid;

[0014] The intermediate product containing a monocarboxylic acid is obtained by condensing remdesivir, acyclovir, vidarabine, and stavudine with pimelic acid in EDC·HCl, including the following steps:

[0015] Pimelic acid, EDC·HCl, and HOBt were dissolved in dichloromethane, and then DIPEA was added at 0°C. After reacting for 1 hour to generate an active ester, one of Remdesivir, Acyclovir, Vidarabine, and Stavudine was added and stirred for 10 hours to obtain an intermediate containing a monocarboxylic acid.

[0016] Furthermore, the linker containing bisacrylidine is 2-amino-N-(4-(3-(trifluoromethyl)-3H-diaza-3-yl)benzyl)pent-4-yneamide.

[0017] Furthermore, the linker containing diazinonidine is prepared by the following process:

[0018] 4-[3-(trifluoromethyl)-3H-bisacryl-3-yl]benzylamine hydrochloride and 2-((tert-butoxycarbonyl)amino)-4-pentyneic acid were condensed in EDC·HCl to obtain a biacrylidine-containing linker intermediate with a Boc protecting group; the Boc-protected biacrylidine-containing linker intermediate was deprotected in the presence of trifluoroacetic acid to obtain the biacrylidine-containing linker.

[0019] Further, 2-((tert-butoxycarbonyl)amino)-4-pentyneic acid, EDC·HCl, and HOBt were dissolved in dichloromethane, and DIPEA was added dropwise with stirring at 0°C. After reacting for 1 h, 4-[3-(trifluoromethyl)-3H-bisacrididin-3-yl]benzylamine hydrochloride was added to react and obtain a Boc-protected intermediate containing bisacrididin. The Boc-protected intermediate containing bisacrididin was dissolved in dichloromethane, and trifluoroacetic acid was added dropwise at 0°C. After reacting for 6 h, a bisacrididin-containing intermediate was obtained.

[0020] Further, the intermediate product containing monocarboxylic acid obtained in step 1) was dissolved in anhydrous dichloromethane, and then EDC·HCl, HOBt and the linker containing diacylpropidine obtained in step 2) were added. Then DIPEA was added at 0°C and the reaction was carried out for 10 h to obtain a nucleoside compound containing diacylpropidine.

[0021] The application of a compound containing diazinonidine, as described above, in the preparation of an anti-COVID-19 drug.

[0022] The application of a bipropidine nucleoside compound as described above in the preparation of an antiviral drug targeting RdRp.

[0023] The application of a compound containing diazinonidine as described above in confirming protein targets.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] This invention utilizes condensation reactions to synthesize target compounds and constructs a compound library. These compounds are novel nucleoside inhibitors of SARS-CoV-2 with a diacillin-based molecular structure. Based on previous laboratory research, this invention demonstrates that the photoaffinity-reactive group diacillin can covalently bind to target proteins, exhibiting more efficient antiviral activity. Therefore, building upon the structure of nucleoside antiviral drugs, an in-situ assembly strategy was used to introduce photosensitive groups, synthesizing a nucleoside anti-SARS-CoV-2 drug capable of covalently binding to the target protein and exhibiting low cytotoxicity. The compounds are related to the RdRp and 3CL of SARS-CoV-2. pro After protein incubation and UV irradiation, the protein covalently binds to the target protein. Labeling is then performed via a click reaction, and nucleoside compounds capable of labeling the protein are screened. Mass spectrometry analysis is used to determine the active sites interacting with the target protein, and computer simulations of molecular dynamics are employed to determine the affinity and binding mode. Further investigation of the candidate drug's safety and bioactivity is conducted. The potential bioactivity and safety of biacpropidine nucleoside compounds are evaluated, and 3CL compounds with potential binding sites are selected. proPreliminary exploration of the mechanism by which these compounds target ACE2 proteins was conducted. Some compounds showed no inhibitory effect on pseudoviral infection by ACE2; while others not only bound to and labeled the RdRp protein but also targeted 3CL. pro Hydrolytic enzymes have a certain inhibitory effect. This compound can form covalent bonds with the target protein to achieve irreversible binding, providing a new mechanism of action and binding mode, improving the binding efficacy and duration of action of small molecule inhibitors, and enhancing the strength and duration of drug action. Attached Figure Description

[0026] Figure 1 Synthetic route diagram of RD-1, a covalently bound nucleoside compound containing diacpropidine, provided by the present invention;

[0027] Among them, compound 1 is remdesivir, compound 2 is succinic anhydride, compound 4 is 4-[3-(trifluoromethyl)-3H-bisacrylidine-3-yl]benzylamine hydrochloride, and compound 5 is 2-((tert-butoxycarbonyl)amino)-4-pentenoic acid, a covalently bound nucleoside compound containing bisacrylidine at the RD-1 position.

[0028] The image is labeled as follows:

[0029] a.DIPEA,DCM,rt,6h; b.EDC,HOBt,DIPEA,DCM,rt,12h; c.TFA,DCM,rt,2h; d.EDC,HOBt,DIPEA,DCM,rt,10h.

[0030] Figure 2 Figure 1 shows the target protein confirmation and competition experiments of RD-1 and remdesivir.

[0031] Figure 3 Safety evaluation of nucleoside compounds containing diazinon (no inhibitory effect on HEK293 cells); among which, (a) is SD-1, (b) RD-1, and (c) AD-1.

[0032] Figure 4 The figure shows the results of treating ACE2 cells with 1, 5, and 20 μMol nucleoside compounds for SARS-CoV-2 pseudovirus.

[0033] Figure 5 Synthetic route diagram of RG-1, a nucleoside compound containing diacpropidine, provided by the present invention;

[0034] Among them, compound 1 is remdesivir, compound 2 is pimelic acid, compound 4 is 4-[3-(trifluoromethyl)-3H-bisacryl-3-yl]benzylamine hydrochloride, compound 5 is 2-((tert-butoxycarbonyl)amino)-4-pentenoic acid, and RG-1 is a nucleoside compound containing bisacrylidine.

[0035] The image is labeled as follows:

[0036] a.EDC,HOBt,TEA,DCM,rt,5h; b.EDC,HOBt,DIPEA,DCM,rt,12h; c.TFA,DCM,rt,2h; d.EDC,HOBt,DIPEA,DCM,rt,10h.

[0037] Figure 6 Figure 1 shows the confirmation and competition experiments of RG-1 and remdesivir's target proteins.

[0038] Figure 7 Safety evaluation of nucleoside compounds containing diacylpropidine (no inhibitory effect on HEK293 cells); where (a) is SG-1, (b) is RG-1, (c) is AG-1, (d) is AG-2, and (e) is NP-1.

[0039] Figure 8 Figure 1 shows the results of treating ACE2 cells with SARS-CoV-2 pseudovirus using 1, 5, and 20 μM nucleoside compounds. Detailed Implementation

[0040] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. These descriptions are intended to explain the invention and not to limit it.

[0041] In this invention, room temperature refers to 25°C, EDC·HCl is 1-(3-dimethylaminopropyl)ethylcarbodiimide hydrochloride, HOBt is 1-hydroxybenzotriazole, and DIPEA is N,N-diisopropylethylamine.

[0042] The structural formula of the compound containing diazinonidine in this invention is as follows:

[0043]

[0044] R represents remdesivir, acyclovir, vidarabine, or stavudine.

[0045] Application of compounds containing diazinonidine in the preparation of drugs against COVID-19.

[0046] Application of diazinon-containing nucleoside compounds in the preparation of antiviral drugs targeting RdRp.

[0047] Applications of diazinon-nucleotide compounds in protein target confirmation. The purpose of protein target confirmation is to use computer molecular docking to confirm the target protein of an active molecule.

[0048] In this invention, HRMS, 1 The structure of the target compound was characterized by 1H NMR and other methods.

[0049] The linker containing bisacrylamide in this invention is 2-amino-N-(4-(3-(trifluoromethyl)-3H-diaza-3-yl)benzyl)pent-4-yneamide, with the following structural formula:

[0050] The preparation and activity screening method of nucleoside compounds containing diacylpropidine provided by the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0051] Example 1

[0052] See Figure 1 A method for preparing the compound RD-1 containing adipropidine nucleoside includes the following steps:

[0053] Remdesivir and succinic anhydride were dissolved in anhydrous dichloromethane and reacted at room temperature for 8 hours to obtain an intermediate product containing a monocarboxylic acid; the specific process is as follows:

[0054] Remdesivir 0.332 mmol and succinic anhydride 0.398 mmol were dissolved in 10 mL of anhydrous dichloromethane. DIPEA 1.328 mmol was slowly added dropwise at 0 °C. After the addition was completed, the reaction was carried out at room temperature for 8 h. The organic solvent was removed by low-pressure rotation to obtain the crude product. The crude product was separated by column chromatography and eluted with ethyl acetate to obtain an intermediate containing a monocarboxylic acid, weighing 0.15 g, with a yield of 64.4%.

[0055] LC-MS (ESI, m / z): 701.65 [M+H] + 703.65 [MH] - .

[0056] 0.488 mmol of 2-((tert-butoxycarbonyl)amino)-4-pentenoic acid, 0.716 mmol of EDC·HCl, and 0.573 mmol of HOBt were dissolved in 10 mL of anhydrous dichloromethane. The mixture was stirred at 0 °C for a period of time. After the system cooled, 3.975 mmol of DIPEA was slowly added dropwise. After reacting for 1 h, 0.397 mmol of 4-[3-(trifluoromethyl)-3H-bisacrididin-3-yl]benzylamine hydrochloride was added to react and give crude product containing bisacrididin. The crude product was washed twice with saturated sodium bicarbonate, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. The solvent was removed by vacuum distillation to give crude product. The crude product was purified by column chromatography and eluted with petroleum ether / ethyl acetate (v / v = 1:1) to give a 0.04 g intermediate containing bisacrididin with a Boc protecting group, yield 41.7%.

[0057] LC-MS (ESI, m / z): 413.43 [M+H] +411.37 [MH] - .

[0058] The Boc-protected intermediate containing bisacrylidine was deprotected under the action of trifluoroacetic acid to obtain the target photolinker. The specific process is as follows:

[0059] At 0 °C, 0.0971 mmol of a Boc-protected intermediate containing diacaridine was dissolved in 10 mL of anhydrous dichloromethane. The mixture was stirred at 0 °C until cooled, and then 2 mL of trifluoroacetic acid was slowly added dropwise. The reaction was carried out at 0 °C for 1 h, followed by 6 h at room temperature to obtain a crude diacaridine-containing intermediate. The pH of the system was adjusted to neutral by saturated sodium bicarbonate, and the organic phase was collected by dichloromethane extraction. The organic phase was dried and washed with saturated sodium chloride and anhydrous sodium sulfate, respectively. The solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by column chromatography to obtain a diacaridine-containing intermediate weighing 0.065 g, with a yield of 74.7%.

[0060] LC-MS (ESI, m / z): 311.10 [M+H] + 309.20 [MH] - .

[0061] The specific process for preparing nucleoside compounds containing diazinonidine is as follows:

[0062] 0.387 mmol of the intermediate containing a monocarboxylic acid, 0.225 mmol of EDC·HCl, and 0.17 mmol of HOBt were dissolved in 10 mL of anhydrous dichloromethane solution and stirred at 0 °C until homogeneous. 3.325 mmol of DIPEA was added dropwise under ice bath conditions, and the mixture was stirred for 1 h. Then, 0.323 mmol of the linker containing diacylpropidine was added, and the mixture was stirred at room temperature for 10 h. After the reaction was completed, the organic solvent was removed by low-pressure rotary evaporation to obtain the crude product. The crude product was separated by column chromatography and eluted with petroleum ether / ethyl acetate (V / V = 1 / 10) to obtain RD-1, a nucleoside compound containing diacylpropidine, weighing 0.027 g, with a yield of 7.0%.

[0063] The structure of the target nucleoside compound RD-1 is as follows:

[0064]

[0065] The nucleoside compound containing bisacrylidine is named 2-ethylbutyl((((2R,3S,4R,5R)-5-cyano-3,4-dihydroxy-5-(4-(4-oxo-4-((1-oxo-1-((4-(3-(trifluoromethyl)-3H-diazin-3-yl)benzyl)amino)-2-yl)amino)butyryl)pyrroloindole[2,1-f][1,2,4]triazin-7-yl)tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine, abbreviated as RD-1.

[0066] The proton NMR data are as follows:

[0067] 1 H NMR(400MHz, DMSO-d6)δ8.60(s,6H),8.30(d,J=7.7Hz,7H),7.92(s,2H),7.33(d,J=8.2Hz,4H),7.22 -7.13(m,3H),6.91–6.88(m,3H),6.83(t,J=4.9Hz,3H),6.32(d,J=6.0Hz,2H),6.03(dd ,J=16.1,6.8Hz,2H),5.36(d,J=5.7Hz,2H),4.99(s,1H),4.67–4.59(m,1H),4.23(d,J= 5.5Hz,2H),4.13–4.05(m,1H),3.97–3.90(m,1H),3.89–3.80(m,1H),3.16(d,J=5.1Hz, 4H), 2.50 (s, 2H), 2.49–2.48 (m, 1H), 1.22 (dd, J = 8.5, 4.9Hz, 1H), 0.79 (t, J = 6.8Hz, 1H).

[0068] LC-MS (ESI, m / z): 995.34 [M+H] + .

[0069] Example 2

[0070] Nucleoside compounds containing diacylpropidine are prepared by the following steps:

[0071] Acyclovir and succinic anhydride were dissolved in anhydrous dichloromethane and reacted at room temperature for 10 hours to obtain an intermediate containing a monocarboxylic acid; the specific process is as follows:

[0072] Acyclovir 0.888 mmol and succinic anhydride 0.888 mmol were dissolved in 10 mL of anhydrous dichloromethane. DIPEA 1.328 mmol was slowly added dropwise at 0 °C. After the addition was complete, the reaction was carried out at room temperature for 8 h. The organic solvent was removed by low-pressure rotation to obtain the crude product. The crude product was separated by column chromatography and eluted with ethyl acetate to obtain an intermediate containing a monocarboxylic acid, weighing 0.16 g, with a yield of 55.2%.

[0073] LC-MS (ESI, m / z): 326.27 [M+H] + 324.34 [MH] - .

[0074] 0.488 mmol of 2-((tert-butoxycarbonyl)amino)-4-pentenoic acid, 0.716 mmol of EDC·HCl, and 0.573 mmol of HOBt were dissolved in 10 mL of anhydrous dichloromethane. The mixture was stirred at 0 °C for a period of time. After the system cooled, 3.975 mmol of DIPEA was slowly added dropwise. After reacting for 1 h, 0.397 mmol of 4-[3-(trifluoromethyl)-3H-bisacrididin-3-yl]benzylamine hydrochloride was added to react and give a crude product containing bisacrididin. The crude product was washed twice with saturated sodium bicarbonate, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. The solvent was removed by vacuum distillation to give the crude product. The crude product was purified by column chromatography and eluted with petroleum ether / ethyl acetate (v / v = 1:1) to give a Boc-protected intermediate containing bisacrididin, weighing 0.04 g, with a yield of 41.7%.

[0075] LC-MS (ESI, m / z): 413.43 [M+H] + 411.37 [MH] - .

[0076] The Boc-protected intermediate containing bisacrylidine was deprotected with trifluoroacetic acid to obtain the target bisacrylidine linker. The specific process is as follows:

[0077] At 0 °C, 0.0971 mmol of a Boc-protected intermediate containing diacaridine was dissolved in 10 mL of anhydrous dichloromethane. The mixture was stirred at 0 °C until cooled, and then 2 mL of trifluoroacetic acid was slowly added dropwise. The reaction was carried out at 0 °C for 1 h, followed by 6 h at room temperature to obtain a crude diacaridine-containing intermediate. The pH of the system was adjusted to neutral by saturated sodium bicarbonate, and the organic phase was collected by dichloromethane extraction. The organic phase was dried and washed with saturated sodium chloride and anhydrous sodium sulfate, respectively. The solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by column chromatography to obtain a diacaridine-containing intermediate weighing 0.065 g, with a yield of 74.7%.

[0078] LC-MS (ESI, m / z): 311.10 [M+H] + 309.20 [MH] - .

[0079] The specific process for preparing nucleoside compounds containing diazinonidine is as follows:

[0080] 0.163 mmol of the intermediate containing a monocarboxylic acid, 0.244 mmol of EDC·HCl, and 0.196 mmol of HOBt were dissolved in 10 mL of anhydrous dichloromethane solution and stirred at 0 °C until homogeneous. 1.358 mmol of DIPEA was added dropwise under ice bath conditions, and the mixture was stirred for 1 h. Then, 0.136 mmol of the linker containing bisacrylidine was added, and the mixture was stirred at room temperature for 10 h. After the reaction was completed, the organic solvent was removed by low-pressure rotation to obtain the crude product. The crude product was separated by column chromatography, and eluted with petroleum ether / ethyl acetate (V / V = 10 / 1) to obtain AD-1, a nucleoside compound based on bisacrylidine, weighing 0.031 g, with a yield of 31.0%.

[0081] The structure of the target compound AD-1 is as follows:

[0082]

[0083] The proton NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.82–10.70(m,1H),8.61(s,1H),8.47(s,1H),8.29(d,J=8.3Hz ,1H),7.81(s,1H),7.67(d,J=3.9Hz,1H),7.47–7.44(m,1H),7.15(d,J=4.0Hz,2H),6.59( s,1H),5.35(s,1H),4.29(dd,J=11.0,6.0Hz,1H),4.07(s,2H),3.64(s,3H),3.58(s,1H) ,3.46(s,4H),2.90(s,1H),1.89(s,1H),1.23(s,1H),1.19(d,J=6.4Hz,1H),1.06(s,1H).

[0084] LC-MS (ESI, m / z): 618.20 [M+H] + .

[0085] Example 3

[0086] Nucleoside compounds containing diacylpropidine are prepared by the following steps:

[0087] Stavudine and succinic anhydride were dissolved in anhydrous dichloromethane and reacted at room temperature for 10 hours to obtain an intermediate containing a monocarboxylic acid; the specific process is as follows:

[0088] Stavudine 2.230 mmol and succinic anhydride 2.676 mmol were dissolved in 10 mL of anhydrous dichloromethane. DIPEA 1.328 mmol was slowly added dropwise at 0 °C. After the addition was completed, the reaction was carried out at room temperature for 8 h. The organic solvent was removed by low-pressure rotation to obtain the crude product. The crude product was separated by column chromatography and eluted with petroleum ether / ethyl acetate (v / v = 1:5) to give an intermediate containing a monocarboxylic acid, weighing 0.16 g, with a yield of 48.6%.

[0089] LC-MS (ESI, m / z): 325.28 [M+H] + 323.28 [MH] - .

[0090] 0.488 mmol of 2-((tert-butoxycarbonyl)amino)-4-pentenoic acid, 0.716 mmol of EDC·HCl, and 0.573 mmol of HOBt were dissolved in 10 mL of anhydrous dichloromethane. The mixture was stirred at 0 °C for a period of time. After the system cooled, 3.975 mmol of DIPEA was slowly added dropwise. After reacting for 1 h, 0.39 mmol of 4-[3-(trifluoromethyl)-3H-bisacrididin-3-yl]benzylamine hydrochloride was added to react and give crude product containing bisacrididin. The crude product was washed twice with saturated sodium bicarbonate, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. The solvent was removed by vacuum distillation to give crude product. The crude product was purified by column chromatography and eluted with petroleum ether / ethyl acetate (v / v = 1:1) to give a 0.04 g intermediate containing bisacrididin with a Boc protecting group, yielding 41.7%.

[0091] LC-MS (ESI, m / z): 413.43 [M+H] + 411.37 [MH] - .

[0092] The Boc-protected intermediate containing diazinonidine was deprotected with trifluoroacetic acid to obtain the target linked form. The specific process is as follows:

[0093] At 0 °C, 0.0971 mmol of a Boc-protected intermediate containing diacaridine was dissolved in 10 mL of anhydrous dichloromethane. The mixture was stirred at 0 °C until cooled, and then 2 mL of trifluoroacetic acid was slowly added dropwise. After reacting at 0 °C for 1 h, the mixture was reacted at room temperature for 6 h to obtain a crude diacaridine-containing intermediate. The pH of the system was adjusted to neutral by saturated sodium bicarbonate, and the organic phase was collected by dichloromethane extraction. The organic phase was dried and washed with saturated sodium chloride and anhydrous sodium sulfate, respectively. The solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by column chromatography to obtain a diacaridine-containing intermediate weighing 0.065 g, with a yield of 74.7%.

[0094] LC-MS (ESI, m / z): 311.10 [M+H] + 309.20 [MH] - .

[0095] The specific process for preparing nucleoside compounds containing diazinonidine is as follows:

[0096] 0.387 mmol of the intermediate containing a monocarboxylic acid, 0.580 mmol of EDC·HCl, and 0.464 mmol of HOBt were dissolved in 10 mL of anhydrous dichloromethane solution and stirred at 0 °C until homogeneous. 3.225 mmol of DIPEA was added dropwise under ice bath conditions, and the mixture was stirred for 1 h. Then, 0.323 mmol of the linker containing diacylpropidine was added, and the mixture was stirred at room temperature for 10 h. After the reaction was completed, the organic solvent was removed by low-pressure rotary evaporation to obtain the crude product. The crude product was separated by column chromatography and eluted with petroleum ether / ethyl acetate (V / V = 10 / 1) to obtain SD-1, a nucleoside compound containing diacylpropidine, weighing 0.015 g, with a yield of 10.4%.

[0097] The structure of the target compound SD-1 is as follows:

[0098]

[0099] The proton NMR data are as follows: 1H NMR (400MHz, DMSO-d6) δ11.38(s,1H),8.59(s,2H),8.28(d,J=7.5Hz,1H),7.39(d,J=7.7Hz,5H),7.31(d,J=8.2Hz ,2H),7.23(d,J=7.4Hz,1H),6.81(s,2H),6.38(d,J=6.2Hz,1H),5.99(s,1H),4.96(s,1H),4.41(d,J=6.7Hz,1H),4 .32(dd,J=1.6,9.8Hz,1H),4.28–4.20(m,1H),4.17–4.12(m,1H),4.03(dd,J=12.6,7.0Hz,1H),3.32(t,J=7.0Hz,1 H), 2.90 (s, 1H), 2.60–2.51 (m, 1H), 2.48 (d, 1H), 1.99 (d, J = 1.4Hz, 1H), 1.77 (s, 1H), 1.19 (dd, J = 4.9, 17.9Hz, 1H).

[0100] LC-MS (ESI, m / z): 603.21 [M+H] +

[0101] Example 4

[0102] Determination of the binding site between RD-1 and the target protein RdRp.

[0103] The gel imaging method was used to determine that the binding target protein of the nucleoside compound RD-1 containing diacpropidine is RdRp.

[0104] (1) Solution I: RD-1 (1 μM); Solution II: RdRp protein (1 μg)

[0105] (2) Add solution I and solution II to a 1.5 mL EP tube and react for 1 h under light-protected conditions. Then place the reaction system at a distance of 3 cm from the wavelength of 365 nm for photocrosslinking for 30 min. Then add CuSO4·H2O 0.006 mM, sodium ascorbate 0.005 mM, TBTA 0.001 mM, and Cy3 azide 3 μM. Incubate in a constant temperature shaker at 37 °C and 90 rpm for 2 h.

[0106] (3) After the reaction is completed, centrifuge at low temperature and high speed (10000 rpm for 20 min), discard the supernatant, add 80 μL of PBS, take 8 μL of it and add 2 μL of Loading Buffer (5×), and load the sample.

[0107] (4) SDS-PAGE separation

[0108] 1) Preparation of separating gel

[0109] Prepare 10 mL of 6% separating gel according to Table 1, vortex mix well, quickly pour into the gel mold and add 1 mL of isopropanol to remove air bubbles and make the gel surface smooth and flat. Let it stand at room temperature for 30 minutes, slowly discard the isopropanol along one side of the gel surface, and carefully absorb the residual isopropanol with filter paper, being careful not to touch the gel surface.

[0110] Table 1. Reagents and dosages for preparing 6% separating gel

[0111]

[0112] 2) Preparation of concentrated gel

[0113] Prepare 5 mL of 5% glass plate stacking gel according to Table 2. Vortex mix well and quickly pour into the gel casting mold. Immediately and carefully insert a stacking gel comb of the corresponding size to the glass plate (be careful not to create any air bubbles in the comb when inserting). Let stand at room temperature for 30 min. Slowly and evenly remove the comb from the stacking gel and separate it from the glass plate. Add electrophoresis buffer (1×).

[0114] Table 2. Reagents and dosages for preparing 5% stacking gel

[0115]

[0116] 3) Sample loading

[0117] The cross-linked sample loading volume was 8 μL, and the loading buffer (5×) was 2 μL.

[0118] 4) Electrophoretic separation

[0119] First, run at 90V until you reach the vicinity of the stacking gel, then switch to 120V and run until you reach the bottom layer of the gel.

[0120] See Figure 2 The nucleoside compound RD-1, which contains diacylpropidine, does not alter the target protein SARS-CoV-2RdRp of remdesivir.

[0121] Example 5

[0122] (1) HEK293 cells in the growth exponential phase were diluted to 10⁻⁶ cells in DMEM medium. 4 Cell solutions at the level of cells / mL were seeded in parallel into 96-well culture plates (2000-4000 cells / well), with an inoculation volume of 180 μL per well, and cultured at 37°C and 5% CO2 for 12 h.

[0123] (2) Add 20 μL of different concentrations of the test compound to each well to obtain the final concentrations of the compound in the wells: 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM. Set up 3 replicates for each concentration and 6 replicates for the negative control. Add cells to each well but do not add the compound. Continue to culture at 37℃ and 5% CO2 for 48 h.

[0124] (3) Add 20 μL of MTT (5 mg / mL) to each well to obtain a final concentration of 0.5 mg / mL of MTT in each well. Incubate at 37℃ and 5% CO2 for 4 h. Carefully aspirate the supernatant, add 150 μL of DMSO to each well, shake for 15 min, and measure the UV absorbance (OD value) at 490 nm in each well using an enzyme-linked immunosorbent assay (ELISA) reader. Then calculate the cell inhibition rate and use linear regression to calculate the IC50 of the compound based on the inhibition rate. 50 The formula for calculating the cell inhibition rate is:

[0125] Inhibition rate % = (average OD value of control wells - average OD value of drug-treated group) / average OD value of control wells × 100%;

[0126] The test results showed that, compared with the negative control group, nucleoside compounds containing diazinon had almost no inhibitory effect on normal HEK293 cells in vitro. Figure 3 As shown in (a), (b) and (c).

[0127] Example 6

[0128] (1) Virus-susceptible cells Vero cells, 293T cells, HeLa cells, and RD cells in the growth exponential phase were diluted with DMEM medium to a concentration of 10⁻⁶ cells / mL. 4 Cell solutions at the level of cells / mL were seeded in parallel into 96-well culture plates (2000-4000 cells / well), with an inoculation volume of 180 μL per well, and cultured at 37°C and 5% CO2 for 12 h.

[0129] (2) Add 20 μL of different concentrations of the test compound to each well to obtain the final concentrations of the compound in the wells: 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM. Set up 3 replicates for each concentration and 6 replicates for the negative control. Add cells to each well but do not add the compound. Continue to culture at 37℃ and 5% CO2 for 48 h.

[0130] (3) Add 20 μL of MTT (5 mg / mL) to each well to obtain a final concentration of 0.5 mg / mL of MTT in each well. Incubate at 37℃ and 5% CO2 for 4 h. Carefully aspirate the supernatant, add 150 μL of DMSO to each well, shake for 15 min, and measure the UV absorbance (OD value) at 490 nm in each well using an enzyme-linked immunosorbent assay (ELISA) reader. Then calculate the cell inhibition rate and use linear regression to calculate the IC50 of the compound based on the inhibition rate. 50 The formula for calculating the cell inhibition rate is:

[0131] Inhibition rate % = (average OD value of control wells - average OD value of drug-treated group) / average OD value of control wells × 100%;

[0132] The test results showed that, compared with the negative control group, nucleoside compounds containing diazinon had almost no in vitro inhibitory effect on virus-susceptible cells except for RD-1 on RD cells, as shown in Table 3.

[0133] Table 3. Nucleoside compounds containing diacylpropidine and their effects on IC50 in virus-susceptible cells. 50

[0134]

[0135] Example 7

[0136] The effect of diazinon-containing nucleoside compounds on the entry of SARS-CoV-2 spike pseudovirus into ACE2 cells.

[0137] (1) ACE2 cells were seeded into 96-well plates (200 μL). After 24 h of cell culture, 20 μL of cell culture medium was aspirated and 20 μL of nucleoside compounds (0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM) were added to the culture.

[0138] (2) After 48 hours, the culture medium was aspirated and 22 μL of MTT was added to each well for incubation.

[0139] (3) After 4 hours, MTT was aspirated, and 200 μL of DMSO was added to each well. The mixture was then incubated at room temperature for 10 minutes and its absorbance was measured at a wavelength of 490 nm.

[0140] (4) After ACE2 cells were seeded into 96-well plates (100 μL) and cultured for 24 h, 50 μL of cell culture medium was aspirated and 50 μL of culture medium containing different concentrations of nucleoside compounds (1 μM, 5 μM, 20 μM) was added for incubation. After incubation for 2 h, 10 μL of SARS-CoV-2 spike pseudovirus was added for culture.

[0141] (5) After 10 hours, the culture medium containing the pseudovirus was aspirated and 200 μL of fresh culture medium was added for further culture.

[0142] (6) After 48 hours, the culture medium was aspirated, and the absorbance was measured at 561 nm using the Luciferase Assay System with 20 μL of cell lysis buffer and 100 μL of luminescent solution added to each well.

[0143] Figure 4 The results in Table 4 show that nucleoside compounds containing diazinon have no inhibitory effect on pseudovirus infection.

[0144] Table 4. Cytotoxicity of RD-1, SD-1 and AD-1 against ACE2 cells Example 8

[0145] Contains diazinon nucleoside compounds against SARS-CoV-2 3CL pro Effects on activity.

[0146] (1) Wild-type SARS-CoV-2 3CL pro The gene was constructed in the pEGX-6P vector (Novagen). The constructed plasmid was transformed into *E. coli* BL21 cells, and the target protein was induced at 16°C for 18 h with 0.25 mM isopropyl β-d-1-thiogalactoside (IPTG). The collected cells were placed in a lysis buffer containing 20 mM Tris-HCl (pH 8), 150 mM NaCl, 4 mM MgCl2, and 5% glycerol, and homogenized by low-temperature sonication. After centrifugation at 12,000 rpm for 40 min at 4°C to remove cell debris, the supernatant was loaded onto a Ni-nitrilotriacetic acid (Ni-NTA) column. The SARS-CoV-2 3CL column was washed with wash buffer containing 200 mM imidazole (pH 8). pro Adding SUMO protease generates MERS-CoV-2 3CL pro The crude protein was purified by Superdex 75 gel filtration chromatography (GE Healthcare), and the target protein was finally concentrated to 30 mg / mL and stored at -80°C.

[0147] (2) The mutant protein was prepared using a rapid mutagenesis system kit (Transgen Biotech). After mutagenesis, the mutated recombinant plasmid was verified by gene sequencing, and the mutated protease was expressed using an enzyme preparation method.

[0148] (3) The FRET-based peptide NMATSAVLQSGFRK(DNP)M was synthesized by solid-phase method and used as a substrate by 3CL.pro Fluorescence is produced by cleaving the Gln-Ser bond. 2.0 μM SARS-CoV 3CL pro Six different concentrations of inhibitors (DMSO alone served as a blank control) were incubated in 50 μL buffer (pH 8.0, 20 mM Tris-HCl, 150 mM NaCl) at 37 °C for 30 min. The reaction began after the addition of 30 μM substrate solution (50 μL). Changes in relative fluorescence units (λ) were measured using a microplate reader. ex 340nm, λ em It is 440nm.

[0149] The results in Table 5 show that nucleoside compounds containing diaproidine are effective against SARS-CoV-2 3CL. pro All of them have a certain inhibitory effect.

[0150] Table 5 SARS-CoV-2 3CL pro IC 50 / μM

[0151]

[0152] Example 9

[0153] See Figure 5 The nucleoside compound RG-1, containing diacpropidine, is prepared by the following steps:

[0154] The intermediate containing a monocarboxylic acid was obtained by condensing remdesivir and pimelic acid in EDC·HCl; the specific process is as follows:

[0155] 0.444 mmol of pimelic acid, 0.294 mmol of EDC·HCl, and 0.232 mmol of HOBt were dissolved in anhydrous methane. 0.332 mmol of TEA was slowly added dropwise at 0 °C. After the addition was complete, 0.088 mmol of remdesivir was added, and the reaction was carried out at room temperature for 8 h. After the reaction was completed, the organic solvent was removed by low-pressure rotation to obtain the crude product. The crude product was separated by column chromatography, and eluted with petroleum ether / ethyl acetate (v / v = 1:10) to obtain an intermediate containing a monocarboxylic acid, weighing 0.18 g, with a yield of 72.0%.

[0156] LC-MS (ESI, m / z): 745.75 [M+H] + 743.75 [MH] - .

[0157] 0.488 mmol of 2-((tert-butoxycarbonyl)amino)-4-pentenoic acid, 0.716 mmol of EDC·HCl, and 0.573 mmol of HOBt were dissolved in 10 mL of anhydrous dichloromethane. The mixture was stirred at 0 °C for a period of time. After the system cooled, 3.975 mmol of DIPEA was slowly added dropwise. After reacting for 1 h, 0.397 mmol of 4-[3-(trifluoromethyl)-3H-bisacrididin-3-yl]benzylamine hydrochloride was added to react and give a crude intermediate containing bisacrididin. The crude intermediate was washed twice with saturated sodium bicarbonate, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. The solvent was removed by vacuum distillation to give the crude product. The crude product was purified by column chromatography and eluted with petroleum ether / ethyl acetate (v / v = 1:1) to give a 0.04 g intermediate containing bisacrididin with a Boc protecting group, yielding 41.7%.

[0158] LC-MS (ESI, m / z): 413.43 [M+H] + 411.37 [MH] - .

[0159] The Boc-protected intermediate containing diazinonidine was deprotected with trifluoroacetic acid to obtain the target linked form. The specific process is as follows:

[0160] At 0 °C, 0.0971 mmol of a Boc-protected intermediate containing diacaridine was dissolved in 10 mL of anhydrous dichloromethane. The mixture was stirred at 0 °C until cooled, and then 2 mL of trifluoroacetic acid was slowly added dropwise. After reacting at 0 °C for 1 h, the mixture was reacted at room temperature for 6 h to obtain a crude photoaffinity linker containing diacaridine. The pH of the system was adjusted to neutral by saturated sodium bicarbonate, and the organic phase was collected by dichloromethane extraction. The organic phase was dried and washed with saturated sodium chloride and anhydrous sodium sulfate, respectively. The solvent was removed by vacuum evaporation to obtain the crude product. The crude product was purified by column chromatography to obtain a 0.065 g diacaridine-containing linker, with a yield of 74.7%.

[0161] LC-MS (ESI, m / z): 311.10 [M+H] + 309.20 [MH] - .

[0162] The specific process for preparing the nucleoside compound RG-1 containing diacpropidine is as follows:

[0163] 0.387 mmol of the intermediate containing a monocarboxylic acid, 0.580 mmol of EDC·HCl, 0.464 mmol of HOBt, and 0.322 mmol of the linker containing diacylpropidine were dissolved in 10 mL of anhydrous dichloromethane. The mixture was stirred at 0 °C until homogeneous. 3.325 mmol of DIPEA was added dropwise under an ice bath. After the addition was complete, the mixture was stirred at room temperature for 10 h. After the reaction was complete, the organic solvent was removed by low-pressure rotary evaporation to obtain the crude product. The crude product was separated by column chromatography and eluted with petroleum ether / ethyl acetate (V / V = 1 / 10) to give RG-1, a nucleoside compound containing diacylpropidine, weighing 0.028 g, with a yield of 7.0%.

[0164] The structure of the obtained nucleoside compound RG-1 containing diacpropidine is as follows:

[0165]

[0166] The name of the covalently bonded nucleoside compound RG-1 containing diacpropidine is:

[0167] 2-Ethylbutyl((((2R,3S,4R,5R)-5-cyano-3,4-dihydroxy-5-(4-(7-oxy-7-((2-((4-(3-trifluoromethyl)-3H-diazinyl-3-yl)benzyl)carbamoyl)-4-yl-1-yl)amino)heptanoylamino)pyrroloindole[2,1-f][1,2,4]triazin-7-yl)tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine.

[0168] The proton NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ8.36(s,1H),7.67(d,J=1.0Hz,2H),7.07(dd,J=12.8,7.2Hz,4H),6.91(d,J=8.7Hz,4H),6 .65(t,J=5.5Hz,4H),6.61–6.56(m,3H),6.38(d,J=6.0Hz,3H),5.84–5.76(m,2H),4.93–4.90(m,1H),4.74(s,1H), 4.20(d,J=3.8Hz,1H),3.98(d,J=8.0Hz,2H),3.71–3.67(m,2H),3.61(dd,J=6.7,4.0Hz,1H),2.29(s,1H),2.27(s, 1H),2.25–2.24(m,3H),2.15–2.09(m,4H),1.89(s,2H),1.33(s,1H),1.23(s,3H),0.98(s,8H),0.55–0.54(m,4H).

[0169] LC-MS (ESI, m / z): 1037.39 [M+H] + .

[0170] Example 10

[0171] The nucleoside compound AG-1, containing diacpropidine, was prepared through the above steps:

[0172] Acyclovir, pimelic acid, 4-DMAP, and EDC·HCl were dissolved in anhydrous dichloromethane and reacted at room temperature for 6 hours to obtain an intermediate containing a monocarboxylic acid; the specific process is as follows:

[0173] Acyclovir 0.088 mmol, pimelic acid 0.444 mmol, 4-DMAP 0.088 mmol, and EDC·HCl 0.261 mmol were dissolved in 10 mL of anhydrous dichloromethane solution and reacted at room temperature for 8 h. After the reaction was completed, the organic solvent was removed by low-pressure rotation to obtain the crude product. The crude product was separated by column chromatography and eluted with ethyl acetate / methanol (v / v = 5 / 1) to give an intermediate containing a monocarboxylic acid, weighing 0.017 g, with a yield of 55.51%.

[0174] LC-MS (ESI, m / z): 368.37 [M+H] + 366.37 [MH] - .

[0175] 0.488 mmol of 2-((tert-butoxycarbonyl)amino)-4-pentenoic acid, 0.716 mmol of EDC·HCl, and 0.573 mmol of HOBt were dissolved in 10 mL of anhydrous dichloromethane. The mixture was stirred at 0 °C for a period of time. After the system cooled, 3.975 mmol of DIPEA was slowly added dropwise. After reacting for 1 h, 0.397 mmol of 4-[3-(trifluoromethyl)-3H-bisacrididin-3-yl]benzylamine hydrochloride was added to react and give a crude intermediate containing bisacrididin. The crude intermediate was washed twice with saturated sodium bicarbonate, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. The solvent was removed by vacuum distillation to give the crude product. The crude product was purified by column chromatography and eluted with petroleum ether / ethyl acetate (v / v = 1:1) to give a Boc-protected intermediate containing bisacrididin, weighing 0.04 g, with a yield of 41.7%.

[0176] LC-MS (ESI, m / z): 413.43 [M+H] + 411.37 [MH] - .

[0177] The Boc-protected intermediate containing diazinonidine was deprotected with trifluoroacetic acid to obtain the target linked form. The specific process is as follows:

[0178] At 0 °C, 0.0971 mmol of a Boc-protected intermediate containing diacaridine was dissolved in 10 mL of anhydrous dichloromethane. The mixture was stirred at 0 °C until cooled, and then 2 mL of trifluoroacetic acid was slowly added dropwise. After reacting at 0 °C for 1 h, the mixture was reacted at room temperature for 6 h to obtain a crude diacaridine-containing intermediate. The pH of the system was adjusted to neutral by saturated sodium bicarbonate, and the organic phase was collected by dichloromethane extraction. The organic phase was dried and washed with saturated sodium chloride and anhydrous sodium sulfate, respectively. The solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by column chromatography to obtain a diacaridine-containing intermediate weighing 0.065 g, with a yield of 74.7%.

[0179] LC-MS (ESI, m / z): 311.10 [M+H] + 309.20 [MH] - .

[0180] The specific process for the nucleoside compound AG-1 containing diazinon is as follows:

[0181] 0.637 mmol of the intermediate containing a monocarboxylic acid, 1.044 mmol of EDC·HCl, 0.763 mmol of HOBt, and 0.530 mmol of the linker containing diacylpropidine were dissolved in 10 mL of anhydrous dichloromethane. The mixture was stirred at 0 °C until homogeneous. 5.300 mmol of DIPEA was added dropwise under an ice bath. After the addition was complete, the mixture was stirred at room temperature for 10 h. After the reaction was complete, the organic solvent was removed by low-pressure rotary evaporation to obtain the crude product. The crude product was separated by column chromatography and eluted with ethyl acetate to obtain AG-1, a nucleoside compound containing diacylpropidine, weighing 0.039 g, with a yield of 9.3%.

[0182] The structure of the obtained nucleoside compound AG-1 containing diacpropidine is as follows:

[0183]

[0184] The proton NMR data are as follows: 1H NMR (400MHz, DMSO-d6) δ10.72(s,1H),8.63(s,1H),8.17(d,J=8.0Hz,2H),7.81(s,1 H),7.38(d,J=8.2Hz,2H),7.22(d,J=7.9Hz,2H),6.59(s,2H),5.34(s,2H),4.47–4.3 9(m,2H),4.31(t,J=5.3Hz,2H),4.08(s,1H),3.65(s,2H),3.57(s,1H),2.88(s,1H), 2.20(t,J=7.4Hz,2H),2.13(d,J=7.3Hz,2H),1.47(s,2H),1.24(s,4H),0.85(s,1H).

[0185] LC-MS (ESI, m / z): 618.20 [M+H] + .

[0186] Example 11

[0187] The nucleoside compound SG-1 containing diacaridine was prepared by the following steps:

[0188] Stavudine, pimelic acid, 4-DMAP, and DCC were dissolved in anhydrous dichloromethane and reacted at room temperature for 8 hours to obtain an intermediate containing a monocarboxylic acid; the specific process is as follows:

[0189] Stavudine 0.892 mmol, pimelic acid 4.460 mmol, 4-DMAP 0.892 mmol, and DCC 2.676 mmol were dissolved in 10 mL of anhydrous dichloromethane solution and reacted at room temperature for 8 h. After the reaction was completed, the mixture was filtered twice with qualitative filter paper, and the organic solvent was removed by low-pressure rotary evaporation to obtain the crude product. The crude product was separated by column chromatography, and the intermediate containing monocarboxylic acid was obtained by elution with petroleum ether / ethyl acetate (v / v = 1 / 5), weighing 0.217 g, with a yield of 65.8%.

[0190] LC-MS (ESI, m / z): 368.37[M+H]+, 366.37[MH]-.

[0191] 0.488 mmol of 2-((tert-butoxycarbonyl)amino)-4-pentenoic acid, 0.716 mmol of EDC·HCl, and 0.573 mmol of HOBt were dissolved in 10 mL of anhydrous dichloromethane. The mixture was stirred at 0 °C for a period of time. After the system cooled, 3.975 mmol of DIPEA was slowly added dropwise. After reacting for 1 h, 0.397 mmol of 4-[3-(trifluoromethyl)-3H-bisacrididin-3-yl]benzylamine hydrochloride was added to react and give a crude intermediate containing bisacrididin. The crude intermediate was washed twice with saturated sodium bicarbonate, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. The solvent was removed by vacuum distillation to give the crude product. The crude product was purified by column chromatography and eluted with petroleum ether / ethyl acetate (v / v = 1:1) to give an intermediate containing a bisacrididin linker with a Boc protecting group, weighing 0.04 g, with a yield of 41.7%.

[0192] LC-MS (ESI, m / z): 413.43 [M+H] + 411.37 [MH] - .

[0193] The Boc-protected intermediate containing bisacrylidine was deprotected under the action of trifluoroacetic acid to obtain the target linker. The specific process is as follows:

[0194] At 0 °C, 0.0971 mmol of a Boc-protected intermediate containing diacaridine was dissolved in 10 mL of anhydrous dichloromethane. The mixture was stirred at 0 °C until cooled, and then 2 mL of trifluoroacetic acid was slowly added dropwise. After reacting at 0 °C for 1 h, the mixture was reacted at room temperature for 6 h to obtain a crude diacaridine-containing intermediate. The pH of the system was adjusted to neutral by saturated sodium bicarbonate, and the organic phase was collected by dichloromethane extraction. The organic phase was dried and washed with saturated sodium chloride and anhydrous sodium sulfate, respectively. The solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by column chromatography to obtain a diacaridine-containing intermediate weighing 0.065 g, with a yield of 74.67%.

[0195] LC-MS (ESI, m / z): 311.10 [M+H] + 309.20 [MH] - .

[0196] The specific process for preparing the nucleoside compound SG-1 containing diacpropidine is as follows:

[0197] 0.387 mmol of the intermediate containing a monocarboxylic acid, 0.580 mmol of EDC·HCl, 0.464 mmol of HOBt, and 0.323 mmol of the linker containing diacylpropidine were dissolved in 10 mL of anhydrous dichloromethane. The mixture was stirred at 0 °C until homogeneous. 3.225 mmol of DIPEA was added dropwise under an ice bath. After the addition was complete, the mixture was stirred at room temperature for 8 h. After the reaction was complete, the organic solvent was removed by low-pressure rotary evaporation to obtain the crude product. The crude product was separated by column chromatography and eluted with petroleum ether / ethyl acetate (V / V = 10 / 1) to obtain SG-1, a nucleoside compound containing diacylpropidine, weighing 0.039 g, with a yield of 15.3%.

[0198] The structure of the obtained nucleoside compound SG-1 containing diacaridine is as follows:

[0199]

[0200] The proton NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ11.38(s,1H),8.59(s,1H),8.28(d,J=7.5Hz,1H),7.39(d,J=7.7Hz,1H),7.31(d ,1H),7.23(d,J=7.4Hz,1H),6.81(s,1H),6.38(d,J=6.2Hz,1H),5.99(s,6H),4.96(s,1H),4.41(d,J=6.7 Hz,1H),4.32(dd,1H),4.28–4.20(m,1H),4.17–4.12(m,1H),4.03(dd,J=12.6,7.0Hz,1H),3.32(t,J=7. 0Hz, 1H), 2.90 (s, 2H), 2.60–2.51 (m, 1H), 2.48 (d, 1H), 1.99 (d, J = 1.4Hz, 2H), 1.77 (s, 5H), 1.19 (dd, 1H).

[0201] LC-MS (ESI, m / z): 659.48 [M+H] +

[0202] Example 12

[0203] The nucleoside compound AG-2 containing diacpropidine was prepared by the following steps:

[0204] Adenosine arabinoside, pimelic acid, 4-DMAP, and DCC were dissolved in anhydrous dichloromethane and reacted at room temperature for 6 hours to obtain an intermediate containing a monocarboxylic acid. The specific process is as follows:

[0205] 0.784 mmol of vidarabine, 3.742 mmol of pimelic acid, 0.748 mmol of 4-DMAP, and 2.245 mmol of DCC were dissolved in 10 mL of anhydrous dichloromethane and reacted at room temperature for 6 h. After the reaction was completed, the mixture was filtered twice with qualitative filter paper, and the organic solvent was removed by low-pressure rotary evaporation to obtain the crude product. The crude product was separated by column chromatography and eluted with ethyl acetate / methanol (v / v = 1 / 5) to give an intermediate containing a monocarboxylic acid, weighing 0.189 g, with a yield of 61.8%.

[0206] LC-MS (ESI, m / z): 410.41 [M+H] + 408.37 [MH] - .

[0207] 0.488 mmol of 2-((tert-butoxycarbonyl)amino)-4-pentenoic acid, 0.716 mmol of EDC·HCl, and 0.573 mmol of HOBt were dissolved in 10 mL of anhydrous dichloromethane. The mixture was stirred at 0 °C for a period of time. After the system cooled, 3.975 mmol of DIPEA was slowly added dropwise. After reacting for 1 h, 0.397 mmol of 4-[3-(trifluoromethyl)-3H-bisacrididin-3-yl]benzylamine hydrochloride was added to react and give a crude intermediate containing bisacrididin. The crude intermediate was washed twice with saturated sodium bicarbonate, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. The solvent was removed by vacuum distillation to give the crude product. The crude product was purified by column chromatography and eluted with petroleum ether / ethyl acetate (v / v = 1:1) to give a 0.04 g intermediate containing bisacrididin with a Boc protecting group, yielding 41.7%.

[0208] LC-MS (ESI, m / z): 413.43 [M+H] + 411.37 [MH] - .

[0209] The Boc-protected intermediate containing diazinonidine was deprotected under the action of trifluoroacetic acid to obtain the target photolinker. The specific process is as follows:

[0210] At 0 °C, 0.0971 mmol of a Boc-protected intermediate containing diacaridine was dissolved in 10 mL of anhydrous dichloromethane. The mixture was stirred at 0 °C until cooled, and then 2 mL of trifluoroacetic acid was slowly added dropwise. After reacting at 0 °C for 1 h, the mixture was reacted at room temperature for 6 h to obtain a crude diacaridine-containing intermediate. The pH of the system was adjusted to neutral by saturated sodium bicarbonate, and the organic phase was collected by dichloromethane extraction. The organic phase was dried and washed with saturated sodium chloride and anhydrous sodium sulfate, respectively. The solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by column chromatography to obtain a diacaridine-containing intermediate weighing 0.065 g, with a yield of 74.7%.

[0211] LC-MS (ESI, m / z): 311.10 [M+H] + 309.20 [MH] - .

[0212] The specific process for the nucleoside compound AG-2 containing diazinon is as follows:

[0213] 0.244 mmol of the intermediate containing a monocarboxylic acid, 0.366 mmol of EDC·HCl, 0.293 mmol of HOBt, and 0.204 mmol of the linker containing diacylpropidine were dissolved in 10 mL of anhydrous dichloromethane solution. The mixture was stirred at 0 °C until homogeneous. 2.0355 mmol of DIPEA was added dropwise under an ice bath. After the addition was complete, the mixture was stirred at room temperature for 10 h. After the reaction was complete, the organic solvent was removed by low-pressure rotary evaporation to obtain the crude product. The crude product was separated by column chromatography and eluted with ethyl acetate / methanol (V / V = 5 / 1) to obtain AG-2, a nucleoside compound containing diacylpropidine, weighing 0.027 g, with a yield of 15.79%.

[0214] The structure of the obtained nucleoside compound AG-2 containing diacpropidine is as follows:

[0215]

[0216] The proton NMR data are as follows: 1 HNMR(400MHz,DMSO-d6)δ10.72(s,1H),8.63(s,1H),8.17(d,J=8.0Hz,2H),7.81(s,1 H),7.38(d,J=8.2Hz,2H),7.22(d,J=7.9Hz,2H),6.59(s,2H),5.34(s,2H),4.47–4.3 9(m,2H),4.31(t,J=5.3Hz,2H),4.08(s,1H),3.65(s,2H),3.57(s,1H),2.88(s,1H), 2.20(t,J=7.4Hz,2H),2.13(d,J=7.3Hz,2H),1.47(s,2H),1.24(s,4H),0.85(s,1H). LC-MS(ESI,m / z):718.28[M+H] +

[0217] Example 13

[0218] The nucleoside compound RG-1, containing diazinon, binds to the target protein RdRp.

[0219] The gel imaging method was used to determine that the target protein of the nucleoside compound RG-1 containing diacpropidine is RdRp.

[0220] (1) Solution I: RD-1 (1 μM); Solution II: RdRp protein (1 μg)

[0221] (2) Add solution I and solution II to a 1.5 mL EP tube and react for 1 h under light-protected conditions. Then place the reaction system at a distance of 3 cm from the wavelength of 365 nm for photocrosslinking for 30 min. Then add CuSO4·H2O 0.006 mM, sodium ascorbate 0.005 mM, and azide-Cy3 3 μM and incubate at 37 °C and 90 rpm for 2 h in a constant temperature shaker.

[0222] (3) After the reaction is completed, centrifuge at low temperature and high speed (10000 rpm for 20 min), discard the supernatant, add 80 μL of PBS, take 8 μL of it and add 2 μL of Loading Buffer (5×), and load the sample.

[0223] (4) SDS-PAGE separation

[0224] 1) Preparation of separating gel

[0225] Prepare 10 mL of 6% separating gel according to Table 6, vortex mix well, quickly pour into the gel mold and add 1 mL of isopropanol to remove air bubbles and make the gel surface smooth and flat. Let it stand at room temperature for 30 minutes, slowly discard the isopropanol along one side of the gel surface, and carefully absorb the residual isopropanol with filter paper, being careful not to touch the gel surface.

[0226] Table 6. Reagents and dosages for preparing 6% separating gel

[0227]

[0228]

[0229] 2) Preparation of concentrated gel

[0230] Prepare 5 mL of 5% glass plate stacking gel according to Table 7. Vortex mix well and quickly pour into the gel casting mold. Immediately and carefully insert a stacking gel comb of the corresponding size to the glass plate (be careful not to create any air bubbles in the comb when inserting). Let stand at room temperature for 30 minutes. Slowly and evenly remove the comb from the stacking gel and separate it from the glass plate. Add electrophoresis buffer (1×).

[0231] Table 7. Reagents and Dosage for Preparing 5% Stacking Gel

[0232]

[0233] 3) Sample loading

[0234] The cross-linked sample loading volume was 8 μL, and the loading buffer (5×) was 2 μL.

[0235] 4) Electrophoretic separation

[0236] First, run at 90V until you reach the vicinity of the stacking gel, then switch to 120V and run until you reach the bottom layer of the gel.

[0237] See Table 7 and Figure 6 The nucleoside compound RG-1, which contains diacylpropidine, does not alter the target protein SARS-CoV-2 RdRp of remdesivir.

[0238] Example 14

[0239] (1) HEK293 cells in the growth exponential phase were diluted to 10⁻⁶ cells in DMEM medium. 4 Cell solutions at the level of cells / mL were seeded in parallel into 96-well culture plates (2000-4000 cells / well), with an inoculation volume of 180 μL per well, and cultured at 37°C and 5% CO2 for 12 h.

[0240] (2) Add 20 μL of different concentrations of the test compound to each well to obtain the final concentrations of the compound in the wells: 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM. Set up 3 replicates for each concentration and 6 replicates for the negative control. Add cells to each well but do not add the compound. Continue to culture at 37℃ and 5% CO2 for 48 h.

[0241] (3) Add 20 μL of MTT (5 mg / mL) to each well to obtain a final concentration of 0.5 mg / mL of MTT in each well. Incubate at 37℃ and 5% CO2 for 4 h. Carefully aspirate the supernatant, add 150 μL of DMSO to each well, shake for 15 min, and measure the UV absorbance (OD value) at 490 nm in each well using an enzyme-linked immunosorbent assay (ELISA) reader. Then calculate the cell inhibition rate and use linear regression to calculate the IC50 of the compound based on the inhibition rate. 50 The formula for calculating the cell inhibition rate is:

[0242] Inhibition rate % = (average OD value of control wells - average OD value of drug-treated group) / average OD value of control wells × 100%;

[0243] The test results showed that, compared with the negative control group, nucleoside compounds containing diazinon had almost no inhibitory effect on normal HEK293 cells in vitro. Figure 7 As shown in (a), (b), (c), (d) and (e).

[0244] Example 15

[0245] (1) Virus-susceptible cells Vero cells, 293T cells, HeLa cells, and RD cells in the growth exponential phase were diluted with DMEM medium to a concentration of 10⁻⁶ cells / mL. 4Cell solutions at the level of cells / mL were seeded in parallel into 96-well culture plates (2000-4000 cells / well), with an inoculation volume of 180 μL per well, and cultured at 37°C and 5% CO2 for 12 h.

[0246] (2) Add 20 μL of different concentrations of the test compound to each well to obtain the final concentrations of the compound in the wells: 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM. Set up 3 replicates for each concentration and 6 replicates for the negative control. Add cells to each well but do not add the compound. Continue to culture at 37℃ and 5% CO2 for 48 h.

[0247] (3) Add 20 μL of MTT (5 mg / mL) to each well to obtain a final concentration of 0.5 mg / mL of MTT in each well. Incubate at 37℃ and 5% CO2 for 4 h. Carefully aspirate the supernatant, add 150 μL of DMSO to each well, shake for 15 min, and measure the UV absorbance (OD value) at 490 nm in each well using an enzyme-linked immunosorbent assay (ELISA) reader. Then calculate the cell inhibition rate and use linear regression to calculate the IC50 of the compound based on the inhibition rate. 50 The formula for calculating the cell inhibition rate is:

[0248] Inhibition rate % = (average OD value of control wells - average OD value of drug-treated group) / average OD value of control wells × 100%;

[0249] The test results showed that, compared with the negative control group, nucleoside compounds containing diazinon had almost no in vitro inhibitory effect on virus-susceptible cells except for AG-2 on Vero cells, as shown in Table 8.

[0250] Table 8. Nucleoside compounds containing diazinon and their effects on IC50 in virus-susceptible cells. 50

[0251]

[0252] Example 16

[0253] Nucleoside compounds containing diazinon inhibit the invasion of SARS-CoV-2 spike pseudovirus into ACE2 cells.

[0254] (1) ACE2 cells were seeded into 96-well plates (200 μL). After culturing the cells for 24 h, 20 μL of cell culture medium was aspirated and 20 μL of compounds containing different concentrations (0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM) were added to the culture.

[0255] (2) After 48 hours, the culture medium was aspirated and 22 μL of MTT was added to each well for incubation.

[0256] (3) After 4 hours, MTT was aspirated, and 200 μL of DMSO was added to each well. The mixture was then incubated at room temperature for 10 minutes and its absorbance was measured at a wavelength of 490 nm.

[0257] (4) After ACE2 cells were seeded into 96-well plates (100 μL) and cultured for 24 h, 50 μL of cell culture medium was aspirated and 50 μL of culture medium containing different concentrations of compounds (1 μM, 5 μM, 20 μM) was added for incubation. After incubation for 2 h, 10 μL of SARS-CoV-2 spike pseudovirus was added for culture.

[0258] (5) After 10 hours, the culture medium containing the pseudovirus was aspirated and 200 μL of fresh culture medium was added for further culture.

[0259] (6) After 48 hours, the culture medium was aspirated, and the absorbance was measured at 561 nm using the Luciferase Assay System with 20 μL of cell lysis buffer and 100 μL of luminescent solution added to each well.

[0260] Table 9 and Figure 8 The results showed that among the nucleoside compounds containing diazinon, only AG-1 had a certain inhibitory effect on pseudovirus infection.

[0261] Table 9. Cytotoxicity of NP-1, AG-1, RG-1, AG-2, and SG-1 against ACE2 cells (IC50). 50 / μM)

[0262]

[0263] Example 17

[0264] Nucleoside compounds containing diazinon against SARS-CoV-2 3CL pro Effects on activity.

[0265] (1) Wild-type SARS-CoV-2 3CL pro The gene was constructed in the pEGX-6P vector (Novagen). The constructed plasmid was transformed into *E. coli* BL21 cells, and the target protein was induced at 16°C for 18 h with 0.25 mM isopropyl β-d-1-thiogalactoside (IPTG). The collected cells were placed in a lysis buffer containing 20 mM Tris-HCl (pH 8), 150 mM NaCl, 4 mM MgCl2, and 5% glycerol, and homogenized by low-temperature sonication. After centrifugation at 12,000 rpm for 40 min at 4°C to remove cell debris, the supernatant was loaded onto a Ni-nitrilotriacetic acid (Ni-NTA) column. The SARS-CoV-2 3CL column was washed with wash buffer containing 200 mM imidazole (pH 8). proAdding SUMO protease generates MERS-CoV-2 3CL pro The crude protein was purified by Superdex 75 gel filtration chromatography (GE Healthcare), and the target protein was finally concentrated to 30 mg / mL and stored at -80°C.

[0266] (2) The mutant protein was prepared using a rapid mutagenesis system kit (Transgen Biotech). After mutagenesis, the mutated recombinant plasmid was verified by gene sequencing, and the mutated protease was expressed using an enzyme preparation method.

[0267] (3) The FRET-based peptide NMATSAVLQSGFRK(DNP)M was synthesized by solid-phase method and used as a substrate by 3CL. pro Fluorescence is produced by cleaving the Gln-Ser bond. 2.0 μM SARS-CoV-2 3CL pro Six different concentrations of inhibitors (DMSO alone served as a blank control) were incubated in 50 μL buffer (pH 8.0, 20 mM Tris-HCl, 150 mM NaCl) at 37 °C for 30 min. The reaction began after the addition of 30 μM substrate solution (50 μL). Changes in relative fluorescence units (λ) were measured using a microplate reader. ex 340nm, λ em It is 440nm.

[0268] Table 10 shows the results of nucleoside compounds containing diaproidine against SARS-CoV-2 3CL. pro All of them have a certain inhibitory effect.

[0269] Table 10 Nucleoside compounds for SARS-CoV-2 3CL pro Hydrolytic enzyme inhibition (IC50) 50 / μM)

[0270]

[0271] The method for preparing the covalently bound nucleoside compounds RD-1, AD-1, SD-1, RG-1, AG-1, SD-1, and AG-2 of the present invention is simple, easy to implement, and has a high yield. The covalently bound nucleoside compounds RD-1, AD-1, SD-1, RG-1, AG-1, SD-1, and AG-2 of the present invention possess novel structures, novel covalent binding modes, and novel modes of action. These covalently bound nucleoside compounds can be used to prepare the 3CL of SARS-CoV-2. pro The activities of hydrolases and RdRp proteins were studied, with RD-1 not only binding to and labeling RdRp proteins but also exhibiting activity against 3CL. proHydrolytic enzymes exhibit some inhibitory activity. Furthermore, covalently bound nucleoside analogues against COVID-19 exhibit lower cytotoxicity. Covalent inhibitors can form covalent bonds with target proteins, achieving irreversible binding, providing novel mechanisms of action and binding modes, improving the binding efficacy and duration of action of small molecule inhibitors, and enhancing the strength and duration of drug action.

Claims

1. A class of compounds containing diaziridine nucleosides, characterized in that, The structural formula of the compound containing diazinonidine is shown below: 、 、 、 、 、 、 。 2. The use of the type of compound containing diazinonidine as described in claim 1 in the preparation of anti-COVID-19 drugs.

3. The use of the type of diazinon nucleoside compound described in claim 1 in the preparation of antiviral drugs targeting RdRp.

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