CN115850338A - Compound containing diazirine nucleosides as well as preparation method and application thereof - Google Patents
Compound containing diazirine nucleosides as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a compound containing diazirine nucleoside and a preparation method and application thereof, wherein one of Reidesciclovir, acyclovir, vidarabine and stavudine reacts with succinic anhydride in dichloromethane to obtain an intermediate product containing monocarboxylic acid; or one of the Ruidecy Wer, acyclovir, vidarabine and stavudine and pimelic acid are condensed under EDC & HCl to obtain an intermediate product containing monocarboxylic acid; the diazirine-containing linker and the intermediate product containing monocarboxylic acid are condensed under EDC & HCl to obtain the diazirine-containing nucleoside compound. The compound can form a covalent bond with target protein, realize irreversible combination, provide a new action mechanism and a new combination mode, improve the combination efficiency and action endurance of the small molecule inhibitor, and improve the action strength and time of the medicament.
Description
Technical Field
The invention belongs to the technical field of medicine preparation, and particularly relates to a compound containing diazirine nucleoside and a preparation method and application thereof.
Background
In the face of sudden outbreaks of new coronavirus infections, new drug development cannot be carried out in the traditional mode as scheduled. The early rapid spread of SARS-CoV-2 and the lack of specific therapy for COVID-19 encourages approved drugs to be tested beyond their original indications. Although this strategy is feasible and has greater early clinical benefit, it has been discovered after significant resources have been devoted to the study that the "old drug new" strategy lacks target specificity and does not achieve ideal performance in preventing COVID-19. Aiming at the potential targets of coronaviruses, the medicine which is directly and covalently combined with the target protein of SARS-CoV-2 is reasonably designed and developed, is expected to solve the problems of lack of specificity and poor drug effect, and is an intelligent strategy for realizing effective COVID-19 therapeutic medicine discovery.
Photoaffinity labeling is the formation of a stable covalent binding complex with a target protein by introducing a photosensitive group into the active molecule. The drug is irreversibly bound to the target protein residue through a covalent bond, thereby exerting more efficient antiviral activity. Compared with the traditional reversible inhibitor antiviral drugs, the covalent inhibitor has the characteristics of higher biochemical efficiency, stronger and durable effect, reduced administration dosage and administration frequency, separated pharmacodynamics and pharmacokinetics, quick elimination of the drugs, capability of maintaining the efficacy, prevention of drug resistance, targeting of rare and non-conservative residues of specific proteins, higher selectivity and the like, and particularly has very important application prospect in the research field of antiviral and antitumor drugs.
Disclosure of Invention
The invention aims to provide a compound containing diazirine nucleoside, a preparation method and application thereof, wherein the compound has lower cytotoxicity, is applied to preparation of antiviral drugs, and improves the action intensity and time of the drugs.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a compound containing diazirine nucleosides, which has the structural formula:
wherein R is Rudexilvir, acyclovir, vidarabine or stavudine.
A preparation method of a compound containing diazirine nucleosides comprises the following steps:
1) Reacting one of Reidesciclovir, acyclovir, vidarabine and stavudine with succinic anhydride in dichloromethane to obtain an intermediate product containing monocarboxylic acid; or
Under the condensation action of EDC & HCl, one of Ruidecy Wer, acyclovir, vidarabine and stavudine and pimelic acid are obtained as an intermediate product containing monocarboxylic acid;
2) The diazirine-containing linker and the intermediate product containing monocarboxylic acid are condensed under EDC & HCl to obtain the diazirine-containing nucleoside compound.
Further, one of the Ruidexilvir, the acyclovir, the vidarabine and the stavudine and succinic anhydride react in dichloromethane to obtain an intermediate product containing monocarboxylic acid, and the method comprises the following steps: dissolving 1.00mmol of one of Reidesciclovir, acyclovir, vidarabine and stavudine and 1.20mmol of succinic anhydride in 10mL of anhydrous dichloromethane, and reacting for 8h to obtain an intermediate product containing monocarboxylic acid;
under the condensation effect of EDC & HCl, one of Reidcisvir, acyclovir, vidarabine and stavudine and pimelic acid are obtained as an intermediate product containing monocarboxylic acid, and the method comprises the following steps:
dissolving pimelic acid, EDC & HCl and HOBt in dichloromethane, adding DIPEA at 0 ℃, reacting for 1h to generate active ester, adding one of Reidesciclovir, acyclovir, vidarabine and stavudine, and stirring for 10h to obtain an intermediate product containing monocarboxylic acid.
Further, the bisaziridine-containing linker is 2-amino-N- (4- (3- (trifluoromethyl) -3H-diaza-3-yl) benzyl) pent-4-ynylamide.
Further, the bisaziridine-containing linker was prepared by the following procedure:
condensation of 4- [3- (trifluoromethyl) -3H-bisaziridin-3-yl ] benzylamine hydrochloride with 2- ((tert-butoxycarbonyl) amino) -4-pentynoic acid in EDC · HCl gives a bisaziridine-containing linker intermediate with a Boc protecting group; and (3) removing the protecting group of the linker intermediate containing the bis-aziridine with the Boc protecting group under the action of trifluoroacetic acid to obtain the linker containing the bis-aziridine.
Further, 2- ((tert-butoxycarbonyl) amino) -4-pentynoic acid, EDC. HCl and HOBt were dissolved in dichloromethane, DIPEA was added dropwise with stirring at 0 ℃ for 1 hour, after reaction, 4- [3- (trifluoromethyl) -3H-bisaziridin-3-yl ] benzylamine hydrochloride was added to conduct reaction to obtain a bisaziridine-containing linker intermediate with a Boc protecting group, and the bisaziridine-containing linker intermediate with a Boc protecting group was dissolved in dichloromethane and trifluoroacetic acid was added dropwise at 0 ℃ for 6 hours to obtain a bisaziridine-containing linker.
Further, the intermediate product containing the monocarboxylic acid obtained in the step 1) is dissolved in anhydrous dichloromethane, then EDC & HCl and HOBt are added to the chain body containing the diazirine obtained in the step 2), then DIPEA is added under the condition of 0 ℃, and the reaction is carried out for 10h, so that the nucleoside compound containing the diazirine is obtained.
The application of the compound containing the diazirine nucleoside in preparing the anti-new coronavirus medicine.
The application of the compound containing diazirine nucleoside in preparing the antiviral drug taking RdRp as a binding target.
The application of the compound containing the diazirine nucleoside in the aspect of confirming the protein target.
Compared with the prior art, the invention has the following beneficial effects:
the invention utilizes condensation and other reactions to synthesize a target compound and constructs a compound library, wherein the compound is a SARS-CoV-2 small molecule inhibitor with a novel nucleoside molecular structure of diazirine. The invention discovers that the photoaffinity reaction group biaziridine can form covalent binding with target protein based on earlier-stage research in earlier-stage laboratories, and more efficient antiviral activity is exerted. Based on the structure of the nucleoside antiviral drug, photosensitive groups are introduced by adopting an in-situ assembly strategy, so that the nucleoside antiviral drug which can be covalently combined with target protein is synthesized, and the cytotoxicity is low. Compounds and RdRp, 3CL of SARS-CoV-2 pro Protein incubation, ultraviolet irradiation, covalent bonding with target protein, marking by click reaction, screening nucleoside compounds capable of marking protein, determining active sites acting with the target protein by mass spectrometry, and determining affinity and bonding mode of the two by computer simulation molecular dynamics. Further examining the safety of the candidate drug, evaluating the potential biological activity and safety of the nucleoside compound of the diazirine, and selecting 3CL with potential binding site pro A preliminary search for the mechanism was performed with ACE2 target protein. Wherein partial compounds have no inhibiting effect on the pseudo virus infection of ACE 2; and partThe compounds not only bind and label RdRp proteins, but also 3CL pro The hydrolase has certain inhibiting effect. The compound can form a covalent bond with target protein, realize irreversible combination, provide a new action mechanism and a new combination mode, improve the combination efficiency and action endurance of the small molecule inhibitor, and improve the action strength and time of the medicament.
Drawings
FIG. 1 is a diagram showing the synthetic scheme of a covalently bound nucleoside compound RD-1 containing diazirine according to the present invention;
wherein, the compound 1 is Reidexilvir, the compound 2 is succinic anhydride, the compound 4 is 4- [3- (trifluoromethyl) -3H-bisaziridin-3-yl ] benzylamine hydrochloride, the compound 5 is 2- ((tert-butoxycarbonyl) amino) -4-pentenoic acid, and the RD-1 position contains covalent bonding nucleoside compounds of bisaziridine.
The labels in the figures are:
a.DIPEA,DCM,rt,6h；b.EDC,HOBt,DIPEA,DCM,rt,12h；c.TFA,DCM,rt,2h；d.EDC,HOBt,DIPEA,DCM,rt,10h。
FIG. 2 is a diagram of the target protein confirmation and competition experiment of RD-1 and Reidesciclovir.
FIG. 3 is a graph showing the safety evaluation of bisaziridine-containing nucleosides (no inhibition on HEK293 cells); wherein, (a) is SD-1, (b) RD-1, and (c) AD-1.
FIG. 4 is a graph showing the results of 1, 5, 20. Mu. Mol nucleoside compounds on the treatment of ACE2 cells with SARS-CoV-2 pseudovirus.
FIG. 5 is a synthetic route diagram of a nucleoside compound RG-1 containing diazirine provided by the invention;
wherein, the compound 1 is ReideCivir, the compound 2 is pimelic acid, the compound 4 is 4- [3- (trifluoromethyl) -3H-bisaziridin-3-yl ] benzylamine hydrochloride, the compound 5 is 2- ((tert-butoxycarbonyl) amino) -4-pentenoic acid, and the nucleoside compound RG-1 containing bisaziridine.
The labels in the figures are:
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。
FIG. 6 is a diagram of the confirmation and competition experiment of the target proteins of RG-1 and Reidesciclovir.
FIG. 7 is a safety assessment of bisaziridine-containing nucleosides (no inhibition on HEK293 cells); wherein, (a) is SG-1, (b) is RG-1, (c) is AG-1, (d) is AG-2, and (e) is NP-1.
FIG. 8 is a graph showing the results of 1, 5, 20. Mu.M nucleoside compounds on treatment of ACE2 cells with SARS-CoV-2 pseudovirus.
Detailed Description
The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.
In the invention, the room temperature is 25 ℃, EDC & HCl is 1- (3-dimethylaminopropyl) ethyl carbodiimide hydrochloride, HOBt is 1-hydroxybenzotriazole, DIPEA is N, N-diisopropylethylamine.
The structural formula of the compound containing the diazirine nucleoside is as follows:
wherein R is Rudexilvir, acyclovir, vidarabine or stavudine.
Application of compound containing diazirine nucleoside in preparing medicine for resisting new coronavirus is disclosed.
Application of the compound containing diazirine nucleoside in preparing antiviral drugs taking RdRp as a binding target.
Application of compound containing diazirine nucleoside in confirming protein target. The protein targets were confirmed for: binding to computer molecule docking corroborates the target protein of the active molecule.
In the invention, through HRMS, 1 H NMR and other means characterize the structure of the target compound.
The linker containing diazirine is 2-amino-N- (4- (3- (trifluoromethyl) -3H-diaza-3-yl) benzyl) pent-4-ynylamide, and the structural formula is as follows:
the preparation and activity screening method of nucleoside compounds with diazirine provided by the invention are described in detail below with reference to the accompanying drawings and specific examples.
Example 1
Referring to fig. 1, the preparation method of the compound RD-1 containing diazirine nucleoside comprises the following steps:
dissolving Reidesciclovir and succinic anhydride in anhydrous dichloromethane, reacting for 8h at room temperature, and treating to obtain an intermediate product containing monocarboxylic acid; the specific process is as follows:
dissolving 0.332mmol of Reidesciclovir and 0.398mmol of succinic anhydride in 10mL of anhydrous dichloromethane solution, slowly dropwise adding DIPEA1.328mmol at the temperature of 0 ℃, and after dropwise adding, reacting for 8h at room temperature; the organic solvent was removed at low pressure to give a crude product, which was separated by column chromatography using ethyl acetate to give an intermediate product containing monocarboxylic acid, weighing 0.15g, with a yield of 64.4%.
LC-MS(ESI,m/z):701.65[M+H] + ，703.65[M-H] - 。
Dissolving 0.488mmol of 2- ((tert-butoxycarbonyl) amino) -4-pentenoic acid, 0.716mmol of EDC. HCl and 0.573mmol of HOBt in 10mL of anhydrous dichloromethane, stirring at 0 ℃ for a period of time after the system is cooled, slowly dropwise adding DIPEA3.975mmol, reacting for 1H, adding 0.397mmol of 4- [3- (trifluoromethyl) -3H-bisaziridin-3-yl ] benzylamine hydrochloride to react to obtain a crude product containing the bisaziridine intermediate, washing twice with saturated sodium bicarbonate, washing with saturated anhydrous sodium chloride, drying an organic phase with sodium sulfate, evaporating the solvent under reduced pressure to obtain a crude product, separating and purifying the crude product by using a chromatographic column, eluting with petroleum ether/ethyl acetate (v/v = 1:1) to obtain a bisaziridine-containing linker intermediate with a Boc protecting group, weighing 0.04g, and obtaining a yield of 41.7%;
LC-MS(ESI,m/z):413.43[M+H] + ，411.37[M-H] - 。
removing the protecting group of the linker intermediate containing the diazirine with the Boc protecting group under the action of trifluoroacetic acid to obtain the target photolinker, wherein the specific process comprises the following steps of:
dissolving 0.0971mmol of a linker intermediate containing bisaziridine with a Boc protecting group in 10mL of anhydrous dichloromethane at 0 ℃, stirring at 0 ℃ until the system is cooled, slowly dropwise adding trifluoroacetic acid (2 mL), reacting at 0 ℃ for 1h, reacting at room temperature for 6h to obtain a crude linker containing bisaziridine, adjusting the pH of the system to be neutral by saturated sodium bicarbonate, extracting and collecting an organic phase by dichloromethane, drying and washing the organic phase by saturated sodium chloride and anhydrous sodium sulfate respectively, evaporating the solvent under reduced pressure to obtain a crude product, separating and purifying the crude product by a chromatographic column to obtain the linker containing bisaziridine, weighing 0.065g, and obtaining the yield of 74.7%.
LC-MS(ESI,m/z):311.10[M+H] + ，309.20[M-H] - 。
The specific process for preparing the nucleoside compound containing diazirine is as follows:
dissolving 0.387mmol of intermediate product containing monocarboxylic acid, 0.225mmol of EDC. HCl and 0.17mmol of HOBt in 10mL of anhydrous dichloromethane solution, stirring uniformly at 0 ℃, dropwise adding DIPEA3.325mmol under ice bath, stirring for 1h, adding 0.323mmol of linker containing diaziridine, stirring for 10h at room temperature, after the reaction is finished, removing the organic solvent at low pressure to obtain a crude product, separating the crude product by a chromatographic column, and eluting by using petroleum ether/ethyl acetate (V/V = 1/10) to obtain nucleoside compound RD-1 containing diaziridine, weighing 0.027g and having the yield of 7.0%.
The structure of the obtained target nucleoside compound RD-1 is as follows:
the nucleoside compound with diazirine has the name: 2-ethylbutyl ((((2R, 3S,4R, 5R) -5-cyano-3,4-dihydroxy-5- (4- (4-oxo-4- ((1-oxo-1- ((4- (3- (trifluoromethyl) -3H-diazoxazin-3-yl) benzyl) amino) -2-yl) amino) butyrylamino) pyrroloindole [2,1-f ] [1,2,4] triazin-7-yl) tetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) -L-alanine, abbreviated as RD-1.
The hydrogen spectrum nuclear magnetic resonance data is as follows:
1 H NMR(400MHz,DMSO-d 6 )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)。
LC-MS(ESI,m/z):995.34[M+H] + 。
example 2
A nucleoside compound having a diazirine prepared by the steps of:
dissolving acyclovir and succinic anhydride in anhydrous dichloromethane, and reacting for 10h at room temperature to obtain an intermediate product containing monocarboxylic acid; the specific process is as follows:
dissolving acyclovir 0.888mmol and succinic anhydride 0.888mmol in 10mL of anhydrous dichloromethane solution, slowly dropwise adding DIPEAA 1.328mmol at the temperature of 0 ℃, reacting at the room temperature after dropwise adding is finished, and reacting for 8h; the organic solvent was removed at low pressure to give a crude product which was separated on a chromatographic column and eluted with ethyl acetate to give an intermediate product containing monocarboxylic acid in a weight of 0.16g in 55.2% yield.
LC-MS(ESI,m/z):326.27[M+H] + ，324.34[M-H] - 。
Dissolving 0.488mmol of 2- ((tert-butoxycarbonyl) amino) -4-pentenoic acid, 0.716mmol of EDC. HCl and 0.573mmol of HOBt in 10mL of anhydrous dichloromethane, stirring at 0 ℃ for a period of time after the system is cooled, slowly dropwise adding 0.397mmol of DIPEA3.975, reacting for 1H, adding 0.397mmol of 4- [3- (trifluoromethyl) -3H-bisaziridin-3-yl ] benzylamine hydrochloride, reacting to obtain a crude product containing the bisaziridine, washing twice with saturated sodium bicarbonate, washing with saturated sodium chloride, drying an organic phase with anhydrous sodium sulfate, evaporating the solvent under reduced pressure to obtain the crude product, separating and purifying the crude product by using a chromatographic column, eluting with petroleum ether/ethyl acetate (v/v = 1:1) to obtain a bisaziridine-containing linker intermediate with a Boc protecting group, weighing 0.04g, and obtaining the yield of 41.7%;
LC-MS(ESI,m/z):413.43[M+H] + ，411.37[M-H] - 。
removing the protecting group of the linker intermediate containing the bisaziridine with the Boc protecting group under the action of trifluoroacetic acid to obtain the target bisaziridine linker, wherein the specific process is as follows:
dissolving 0.0971mmol of a linker intermediate containing bisaziridine with a Boc protecting group in 10mL of anhydrous dichloromethane at 0 ℃, stirring at 0 ℃ until the system is cooled, slowly dropwise adding trifluoroacetic acid (2 mL), reacting at 0 ℃ for 1h, reacting at room temperature for 6h to obtain a crude product of the linker containing bisaziridine, adjusting the pH of the system to be neutral by saturated sodium bicarbonate, extracting and collecting an organic phase by dichloromethane, drying and washing the organic phase by saturated sodium chloride and anhydrous sodium sulfate respectively, evaporating the solvent under reduced pressure to obtain a crude product, separating and purifying the crude product by a chromatographic column to obtain the linker containing bisaziridine, weighing 0.065g, and obtaining the yield of 74.7%.
LC-MS(ESI,m/z):311.10[M+H] + ，309.20[M-H] - 。
The specific process for preparing the diazirine-containing nucleoside compound is as follows:
dissolving intermediate product 0.163mmol containing monocarboxylic acid, EDC & HCl 0.244mmol and HOBt 0.196mmol in 10mL of anhydrous dichloromethane solution, stirring uniformly at 0 ℃, dropwise adding DIPEA 1.358mmol under ice bath, stirring for 1h, adding linker 0.136mmol containing diaziridine, stirring for 10h at room temperature, after the reaction is finished, removing organic solvent at low pressure to obtain crude product, separating the crude product by chromatographic column, and eluting with petroleum ether/ethyl acetate (V/V = 10/1) to obtain nucleoside compound AD-1 based on the diaziridine, weighing 0.031g and obtaining yield of 31.0%.
The structure of the obtained target compound AD-1 is as follows:
the hydrogen spectrum nuclear magnetic resonance data is as follows: 1 H NMR(400MHz,DMSO-d 6 )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)。
LC-MS(ESI,m/z):618.20[M+H] + 。
example 3
A nucleoside compound having a diazirine prepared by the steps of:
dissolving stavudine and succinic anhydride in anhydrous dichloromethane, and reacting for 10h at room temperature to obtain an intermediate product containing monocarboxylic acid; the specific process is as follows:
dissolving 2.230mmol of stavudine and 2.676mmol of succinic anhydride in 10mL of anhydrous dichloromethane solution, slowly dropwise adding DIPEAA 1.328mmol at 0 ℃, reacting at room temperature after dropwise adding, and reacting for 8h; the organic solvent was removed by low pressure rotary column to give crude product, which was separated by column chromatography eluting with petroleum ether/ethyl acetate (v/v = 1:5) to give an intermediate product containing monocarboxylic acid, weighing 0.16g, yield 48.6%.
LC-MS(ESI,m/z):325.28[M+H] + ，323.28[M-H] - 。
Dissolving 0.488mmol of 2- ((tert-butoxycarbonyl) amino) -4-pentenoic acid, 0.716mmol of EDC. HCl and 0.573mmol of HOBt in 10mL of anhydrous dichloromethane, stirring at 0 ℃ for a period of time until the system is cooled, slowly adding 0.39mmol of DIPEA3.975mmol, reacting for 1H, adding 0.39mmol of 4- [3- (trifluoromethyl) -3H-bisaziridin-3-yl ] benzylamine hydrochloride, reacting to obtain a crude product containing the bisaziridine intermediate, washing twice with saturated sodium bicarbonate, washing with saturated sodium chloride, drying an organic phase with anhydrous sodium sulfate, evaporating the solvent under reduced pressure to obtain a crude product, separating and purifying the crude product with a chromatographic column, eluting with petroleum ether/ethyl acetate (v/v = 1:1) to obtain a bisaziridine-containing linker intermediate with a Boc protecting group, weighing 0.04g, and obtaining a yield of 41.7%.
LC-MS(ESI,m/z):413.43[M+H] + ，411.37[M-H] - 。
Removing the protecting group of the linker intermediate containing the diazirine with the Boc protecting group under the action of trifluoroacetic acid to obtain the target linker, wherein the specific process is as follows:
dissolving 0.0971mmol of a bisaziridine-containing linker intermediate with a Boc protecting group in 10mL of anhydrous dichloromethane at 0 ℃, stirring at 0 ℃ until the system is cooled, slowly dropwise adding trifluoroacetic acid (2 mL), reacting at 0 ℃ for 1h, reacting at room temperature for 6h to obtain a crude bisaziridine-containing linker product, adjusting the pH of the system to be neutral by saturated sodium bicarbonate, extracting by dichloromethane to collect an organic phase, drying and washing the organic phase by saturated sodium chloride and anhydrous sodium sulfate respectively, evaporating the solvent under reduced pressure to obtain a crude product, and separating and purifying the crude product by a chromatographic column to obtain the bisaziridine-containing linker, wherein the weight of the crude product is 0.065g, and the yield of the crude product is 74.7%.
LC-MS(ESI,m/z):311.10[M+H] + ，309.20[M-H] - 。
The specific process for preparing the nucleoside compound containing diazirine is as follows:
dissolving 0.387mmol of intermediate product containing monocarboxylic acid, 0.580mmol of EDC. HCl and 0.464mmol of HOBt in 10mL of anhydrous dichloromethane solution, stirring uniformly at 0 ℃, dropwise adding DIPEA 3.225mmol under ice bath, stirring for 1h, adding 0.323mmol of linker containing diazirine, stirring for 10h at room temperature, after the reaction is finished, removing the organic solvent at low pressure to obtain a crude product, separating the crude product by a chromatographic column, and eluting by using petroleum ether/ethyl acetate (V/V = 10/1) to obtain the nucleoside compound SD-1 containing diazirine, weighing 0.015g and obtaining the yield of 10.4%.
The structure of the obtained target compound SD-1 is as follows:
the hydrogen spectrum nuclear magnetic resonance data is as follows: 1 H NMR(400MHz,DMSO-d 6 )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,1H),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)。
LC-MS(ESI,m/z):603.21[M+H] +
example 4
Determination of binding site of RD-1 and target protein RdRp.
The binding target protein of the nucleoside compound RD-1 containing diazirine is determined to be RdRp by a gel imaging method.
(1) Liquid I: RD-1 (1. Mu.M); and II, liquid: rdRp protein (1. Mu.g)
(2) Adding solution I and solution II into 1.5mL EP tube, reacting for 1h in dark condition, placing the reaction system at a distance of 365nm and a wavelength of 3cm, performing photo-crosslinking for 30min, and adding CuSO 4 ·H 2 O0.006 mM, sodium ascorbate 0.005mM, TBTA 0.001mM, azido Cy 3. Mu.M, incubated at 90rpm for 2h in a constant temperature shaker at 37 ℃.
(3) After completion of the reaction, the reaction mixture was centrifuged by a low-temperature high-speed centrifuge (10000rpm 20min), the supernatant was discarded, 80. Mu.L of PBS was added, and 8. Mu.L of the supernatant was taken and added to 2. Mu.L of Loading Buffer (5X), and the mixture was loaded.
(4) SDS-PAGE separation
1) Preparation of separation gel
Preparing 10mL of 6% separation gel according to the table 1, uniformly mixing by vortex, quickly introducing into a gel preparation mold, adding 1mL of isopropanol to remove bubbles so as to enable a gel surface to be smooth and flat, standing at room temperature for 30min, slowly removing the isopropanol along one side of the gel preparation surface, carefully sucking residual isopropanol by using filter paper, and taking care that the residual isopropanol cannot touch the gel surface.
TABLE 1 preparation of 6% separation gel reagent and dosage
2) Preparation of concentrated gum
Preparing 5mL of 5% glass plate concentrated gel according to Table 2, mixing by vortexing, pouring into a gel mold quickly, inserting a comb of concentrated gel of a size corresponding to the glass plate carefully (taking care not to generate any bubbles in the comb), standing at room temperature for 30min, separating the comb from the concentrated gel slowly and uniformly, adding electrophoresis buffer (1X)
TABLE 2 preparation of 5% concentrated gum reagent and amounts
3) Sample loading
The Loading of crosslinked sample was 8. Mu.L, and the Loading Buffer (5X) was 2. Mu.L.
4) Electrophoretic separation
The gel was run at 90V to the vicinity of the concentrated gel and then stopped at 120V to the bottom of the gel.
Referring to FIG. 2, the nucleoside compound RD-1 containing diazirine does not alter the target protein SARS-CoV-2RdRp of Reidesvir.
Example 5
(1) HEK293 cells in exponential growth phase were diluted 10 with DMEM medium 4 Cell solutions of the order of one/mL were plated in parallel in 96-well plates (2000-4000/well) at a volume of 180. Mu.L/well and were 5% CO at 37 ℃% 2 Culturing for 12h;
(2) 20 μ L of test compound was added to each well at different concentrations to give final concentrations of compound in the wells: 0.001 μ M,0.01 μ M,0.1 μ M,1 μ M,10 μ M,100 μ M, 3 duplicate wells for each concentration, 6 duplicate wells for negative control, no compound added to each well, and 5% CO at 37 deg.C 2 Culturing for 48h;
(3) mu.L of MTT (5 mg/mL) was added to each well to give a final concentration of 0.5mg/mL MTT in each well at 37 ℃ with 5% CO 2 Culturing for 4 hr, carefully removing supernatant, adding DMSO 150 μ L into each well, shaking for 15min, measuring ultraviolet absorption (OD) at 490nm of each well with ELISA detector, calculating cell inhibition rate, and calculating IC of compound according to the inhibition rate by linear regression method 50 The value, the calculation formula of the cell inhibition rate is:
inhibition% = (control well mean OD value-drug use group mean OD value)/control well mean OD value × 100%;
the detection result shows that compared with a negative control group, the nucleoside compound containing the diazirine has almost no in vitro inhibition effect on the HEK293 normal cell, as shown in (a), (b) and (c) in figure 3.
Example 6
(1) Diluting virus susceptible cells Vero cells, 293T cells, hela cells and RD cells in growth index stage into 10 with DMEM medium 4 Cell solutions of the order of one/mL were plated in parallel in 96-well plates (2000-4000/well) at a volume of 180. Mu.L/well and 5% CO at 37 ℃ in a culture medium 2 Culturing for 12h;
(2) 20 μ L of test compound was added to each well at different concentrations to give final concentrations of compound in the wells: 0.001 μ M,0.01 μ M,0.1 μ M,1 μ M,10 μ M,100 μ M, 3 duplicate wells for each concentration, 6 duplicate wells for negative control, no compound added to each well, and 5% CO at 37 deg.C 2 Culturing for 48h;
(3) mu.L of MTT (5 mg/mL) was added to each well to give a final concentration of 0.5mg/mL MTT in each well at 37 ℃ with 5% CO 2 Culturing for 4 hr, carefully removing supernatant, adding DMSO 150 μ L into each well, shaking for 15min, measuring ultraviolet absorption (OD) at 490nm of each well with ELISA detector, calculating cell inhibition rate, and calculating IC of compound according to the inhibition rate by linear regression method 50 The value, the calculation formula of the cell inhibition rate is:
inhibition% = (control well mean OD value-drug use group mean OD value)/control well mean OD value × 100%;
the detection result shows that compared with a negative control group, the nucleoside compound containing diazirine has little in vitro inhibition effect on virus susceptible cells except RD-1 on RD cells, and is shown in Table 3.
TABLE 3 IC of diazirine-containing nucleosides to cells susceptible to viruses 50
Example 7
Effect of compounds containing bisaziridine nucleosides on the entry of SARS-CoV-2 spike pseudovirus into ACE2 cells.
(1) After ACE2 cells were seeded into 96-well plates (200. Mu.L) and cultured for 24h, 20. Mu.L of cell culture medium was aspirated, and 20. Mu.L of medium containing various concentrations of nucleosides (0.001. Mu.M, 0.01. Mu.M, 0.1. Mu.M, 1. Mu.M, 10. Mu.M, 100. Mu.M) was added and cultured.
(2) After 48h the medium was aspirated and 22. Mu.L MTT was added to each well for incubation.
(3) After 4h the MTT was aspirated, 200. Mu.L DMSO shaker was added to each well and incubated at room temperature for 10min, and the absorbance was measured at 490 nm.
(4) After ACE2 cells were seeded into 96-well plates (100. Mu.L) and cultured for 24h, 50. Mu.L of cell culture medium was aspirated and incubated with 50. Mu.L of medium containing nucleoside compounds at different concentrations (1. Mu.M, 5. Mu.M, 20. Mu.M). After 2h incubation, 10. Mu.L of SARS-Cov-2 spike pseudovirus was added and cultured.
(5) After 10h, the pseudovirus-containing medium was aspirated and 200. Mu.L of fresh medium was added for culture.
(6) After 48h, the medium was aspirated, and 20. Mu.L of cell lysate and 100. Mu.L of luminescence were added to each well using the Luciferase Assay System for absorbance measurement at 561 nm.
The results in FIG. 4 and Table 4 show that the bisaziridine-containing nucleosides do not inhibit pseudoviral infection.
TABLE 4 cytotoxicity of RD-1, SD-1 and AD-1 against ACE2
Example 8
Compound containing diazirine nucleoside against SARS-CoV-2 3CL pro The effect of activity.
(1) Wild type SARS-CoV-2 3CL pro The gene was constructed in pEGX-6P vector (Novagen). The constructed plasmid was transferred into E.coli BL21 cells, and the target egg was induced with 0.25mM isopropyl beta-d-1-thiogalactoside (IPTG) at 16 deg.CAnd (5) white for 18h. The collected cells were placed in a medium containing 20mM Tris-HCl (pH 8), 150mM NaCl, 4mM MgCl 2 And 5% glycerol lysis buffer solution, and carrying out low-temperature ultrasonic cell lysis homogenization. After centrifugation at 12 000rpm for 40min at 4 ℃ to remove cell debris, the supernatant was loaded onto a Ni-nitrilotriacetic acid (Ni-NTA) column. Washing of SARS-CoV-2 3CL with a wash solution containing 200mM imidazole (pH 8) pro Adding SUMO protease to generate MERS-CoV-2 3CL pro . The crude protein was purified by Superdex 75 gel filtration chromatography (GE Healthcare) and finally the target protein was concentrated to 30mg/mL and stored at-80 ℃.
(2) The mutant proteins were prepared using a Rapid mutation System kit (Transgen Biotech). After mutagenesis, the mutated recombinant plasmid was verified by gene sequencing and the mutated protease was expressed by an enzyme preparation method.
(3) The FRET-based polypeptide NMATSAVLQSGFRK (DNP) M was synthesized by the solid phase method, and used as a substrate, and the 3CL was used pro Cleavage of the Gln-Ser bond generates fluorescence. 2.0. Mu.M SARS-CoV 3CL pro Incubate with 6 different concentrations of inhibitor (DMSO alone as blank) for 30min at 37 ℃ in 50 μ L buffer (pH =8.0,20mm Tris-HCl,150mM NaCl). The reaction started after the addition of 30. Mu.M substrate solution (50. Mu.L). The change in relative fluorescence units, lambda, was determined with a microplate reader ex Is 340nm, lambda em Is 440nm.
The results in Table 5 show that nucleosides containing diaziridine are active against SARS-CoV-2 3CL pro All have certain inhibiting effect.
TABLE 5 SARS-CoV-2 3CL pro IC 50 /μM
Example 9
Referring to FIG. 5, the bisaziridine-containing nucleoside compound RG-1, was prepared by the following steps:
under the condensation action of EDC & HCl, intermediate products containing monocarboxylic acid are obtained; the specific process is as follows:
dissolving 0.444mmol of pimelic acid, 0.294mmol of EDC & HCl and 0.232mmol of HOBt in anhydrous methane, slowly dropwise adding 0.332mmol of TEA at 0 ℃, adding 0.088mmol of Reidesciclovir after finishing dropwise adding, reacting for 8h at room temperature, and finishing the reaction; the organic solvent was removed at low pressure to give a crude product, which was separated on a chromatographic column and eluted with petroleum ether/ethyl acetate (v/v = 1) to give an intermediate product containing monocarboxylic acid, weighing 0.18g, yield 72.0%.
LC-MS(ESI,m/z):745.75[M+H] + ，743.75[M-H] - 。
Dissolving 0.488mmol of 2- ((tert-butoxycarbonyl) amino) -4-pentenoic acid, 0.716mmol of EDC. HCl and 0.573mmol of HOBt in 10mL of anhydrous dichloromethane, stirring at 0 ℃ for a period of time after the system is cooled, slowly dropwise adding 0.397mmol of DIPEA3.975mmol, reacting for 1H, adding 0.397mmol of 4- [3- (trifluoromethyl) -3H-bisaziridin-3-yl ] benzylamine hydrochloride, reacting to obtain a crude intermediate containing the bisaziridine, washing twice with saturated sodium bicarbonate, washing with saturated sodium chloride, drying an organic phase with anhydrous sodium sulfate, evaporating the solvent under reduced pressure to obtain a crude product, separating and purifying the crude product by using a chromatographic column, eluting with petroleum ether/ethyl acetate (v/v = 1:1) to obtain an intermediate containing the bisaziridine with a Boc protecting group, weighing 0.04g, and obtaining the yield of 41.7%;
LC-MS(ESI,m/z):413.43[M+H] + ，411.37[M-H] - 。
removing the protecting group of the linker intermediate containing the diazirine with the Boc protecting group under the action of trifluoroacetic acid to obtain the target linker, wherein the specific process comprises the following steps of:
dissolving 0.0971mmol of a linker intermediate containing bisaziridine with a Boc protecting group in 10mL of anhydrous dichloromethane at 0 ℃, stirring at 0 ℃ until the system is cooled, slowly dropwise adding trifluoroacetic acid (2 mL), reacting at 0 ℃ for 1h, reacting at room temperature for 6h to obtain a crude product of the photoaffinity linker containing the bisaziridine, adjusting the pH of the system to be neutral by saturated sodium bicarbonate, extracting and collecting an organic phase by dichloromethane, drying and washing the organic phase by saturated sodium chloride and anhydrous sodium sulfate respectively, evaporating the solvent under reduced pressure to obtain a crude product, separating and purifying the crude product by using a chromatographic column to obtain the linker containing the bisaziridine, wherein the weight of the linker is 0.065g, and the yield of the linker is 74.7%.
LC-MS(ESI,m/z):311.10[M+H] + ，309.20[M-H] - 。
The specific process for preparing the nucleoside compound RG-1 containing diazirine is as follows:
dissolving 0.387mmol of intermediate product containing monocarboxylic acid, 0.580mmol of EDC. HCl, 0.464mmol of HOBt and 0.322mmol of linker containing diaziridine in 10mL of anhydrous dichloromethane solution, stirring uniformly at 0 ℃, dropwise adding DIPEA3.325mmol under ice bath, stirring for 10h at room temperature after dropwise adding is completed, after the reaction is finished, removing the organic solvent at low pressure to obtain a crude product, separating the crude product by a chromatographic column, and eluting by using petroleum ether/ethyl acetate (V/V = 1/10) to obtain the nucleoside compound RG-1 containing the diaziridine, weighing 0.028g and having the yield of 7.0%.
The structure of the obtained diazirine-containing nucleoside compound RG-1 is as follows:
nucleoside compound RG-1 containing diaziridine with covalent bonding, name:
2-ethylbutyl ((((2R, 3S,4R, 5R) -5-cyano-3,4-dihydroxy-5- (4- (7 oxo-7- ((2- ((4- (3-trifluoromethyl) -3H-diazozin-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.
The hydrogen spectrum nuclear magnetic resonance data is as follows: 1 H NMR(400MHz,DMSO-d 6 )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)。
LC-MS(ESI,m/z):1037.39[M+H] + 。
example 10
A bisaziridine-containing nucleoside compound AG-1 prepared by the steps of:
dissolving acyclovir, pimelic acid, 4-DMAP, EDC & HCl in anhydrous dichloromethane, reacting for 6h at room temperature, and treating to obtain an intermediate product containing monocarboxylic acid; the specific process is as follows:
dissolving 0.088mmol of acyclovir, 0.444mmol of pimelic acid, 0.088mmol of 4-DMAP and 0.261mmol of EDC & HCl in 10mL of anhydrous dichloromethane solution, reacting at room temperature, and finishing 8 hours; the organic solvent was removed at low pressure to give a crude product, which was separated on a chromatographic column and eluted with ethyl acetate/methanol (v/v = 5/1) to give an intermediate product containing monocarboxylic acid weighing 0.017g with a yield of 55.51%.
LC-MS(ESI,m/z):368.37[M+H] + ，366.37[M-H] - 。
Dissolving 0.488mmol of 2- ((tert-butoxycarbonyl) amino) -4-pentenoic acid, 0.716mmol of EDC. HCl and 0.573mmol of HOBt in 10mL of anhydrous dichloromethane, stirring at 0 ℃ for a period of time after the system is cooled, slowly dropwise adding 0.397mmol of DIPEA3.975mmol, reacting for 1H, adding 0.397mmol of 4- [3- (trifluoromethyl) -3H-bisaziridin-3-yl ] benzylamine hydrochloride, reacting to obtain a crude intermediate product containing the bisaziridine, washing twice with saturated sodium bicarbonate, washing with saturated sodium chloride, drying an organic phase with anhydrous sodium sulfate, evaporating the solvent under reduced pressure to obtain a crude product, separating and purifying the crude product by using a chromatographic column, eluting with petroleum ether/ethyl acetate (v/v = 1:1) to obtain a linking body intermediate containing the bisaziridine with a Boc protecting group, weighing 0.04g and obtaining the yield of 41.7%;
LC-MS(ESI,m/z):413.43[M+H] + ，411.37[M-H] - 。
removing the protecting group of the linker intermediate containing the diazirine with the Boc protecting group under the action of trifluoroacetic acid to obtain the target linker, wherein the specific process is as follows:
dissolving 0.0971mmol of a linker intermediate containing bisaziridine with a Boc protecting group in 10mL of anhydrous dichloromethane at 0 ℃, stirring at 0 ℃ until the system is cooled, slowly dropwise adding trifluoroacetic acid (2 mL), reacting at 0 ℃ for 1h, reacting at room temperature for 6h to obtain a crude linker containing bisaziridine, adjusting the pH of the system to be neutral by saturated sodium bicarbonate, extracting by dichloromethane to collect an organic phase, drying and washing the organic phase by saturated sodium chloride and anhydrous sodium sulfate respectively, evaporating the solvent under reduced pressure to obtain a crude product, separating and purifying the crude product by a chromatographic column to obtain the linker containing bisaziridine, wherein the weight of the linker is 0.065g, and the yield of the linker is 74.7%.
LC-MS(ESI,m/z):311.10[M+H] + ，309.20[M-H] - 。
The specific process of the diazirine-containing nucleoside compound AG-1 is as follows:
dissolving 0.637mmol of intermediate product containing monocarboxylic acid, 1.044mmol of EDC & HCl, 0.763mmol of HOBt and 0.530mmol of linker containing diaziridine in 10mL of anhydrous dichloromethane solution, stirring uniformly at 0 ℃, dropwise adding 5.300mmol of DIPEA under ice bath, stirring for 10h at room temperature after dropwise adding, after the reaction is finished, removing the organic solvent at low pressure to obtain a crude product, separating the crude product by a chromatographic column, and eluting by ethyl acetate to obtain nucleoside compound AG-1 containing diaziridine, wherein the weight is 0.039g, and the yield is 9.3%.
The structure of the obtained nucleoside compound AG-1 containing diazirine is as follows:
the hydrogen spectrum nuclear magnetic resonance data is as follows: 1 H NMR(400MHz,DMSO-d 6 )δ10.72(s,1H),8.63(s,1H),8.17(d,J＝8.0Hz,2H),7.81(s,1H),7.38(d,J＝8.2Hz,2H),7.22(d,J＝7.9Hz,2H),6.59(s,2H),5.34(s,2H),4.47–4.39(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):618.20[M+H] + 。
example 11
The diazirine-containing nucleoside compound SG-1 is prepared by the following steps:
dissolving stavudine, pimelic acid, 4-DMAP and DCC in anhydrous dichloromethane, reacting for 8h at room temperature, and processing to obtain an intermediate product containing monocarboxylic acid; the specific process is as follows:
dissolving 0.892mmol of stavudine, 4.460mmol of pimelic acid, 0.892mmol of 4-DMAP and 5363 mmol of DCC 2.676 in 10mL of anhydrous dichloromethane solution, reacting at room temperature for 8h; filtering twice with qualitative filter paper, removing the organic solvent with low pressure rotary to obtain crude product, separating the crude product with chromatographic column, and eluting with petroleum ether/ethyl acetate (v/v = 1/5) to obtain intermediate product containing monocarboxylic acid with weight of 0.217g and yield of 65.8%.
LC-MS(ESI,m/z):368.37[M+H]+，366.37[M-H]-。
Dissolving 0.488mmol of 2- ((tert-butoxycarbonyl) amino) -4-pentenoic acid, 0.716mmol of EDC. HCl and 0.573mmol of HOBt in 10mL of anhydrous dichloromethane, stirring at 0 ℃ for a period of time after the system is cooled, slowly dropwise adding 0.397mmol of DIPEA3.975mmol, reacting for 1H, adding 0.397mmol of 4- [3- (trifluoromethyl) -3H-bisaziridin-3-yl ] benzylamine hydrochloride, reacting to obtain a crude intermediate containing the bisaziridine, washing twice with saturated sodium bicarbonate, washing with saturated sodium chloride, drying an organic phase with anhydrous sodium sulfate, evaporating the solvent under reduced pressure to obtain a crude product, separating and purifying the crude product by using a chromatographic column, eluting with petroleum ether/ethyl acetate (v/v = 1:1) to obtain an intermediate containing the bisaziridine linker with a Boc protecting group, weighing 0.04g, and obtaining the yield of 41.7%;
LC-MS(ESI,m/z):413.43[M+H] + ，411.37[M-H] - 。
removing the protecting group of the linker intermediate containing the diazirine with the Boc protecting group under the action of trifluoroacetic acid to obtain a target linker, wherein the specific process comprises the following steps of:
dissolving 0.0971mmol of a bisaziridine-containing linker intermediate with a Boc protecting group in 10mL of anhydrous dichloromethane at 0 ℃, stirring at 0 ℃ until the system is cooled, slowly dropwise adding trifluoroacetic acid (2 mL), reacting at 0 ℃ for 1h, reacting at room temperature for 6h to obtain a crude bisaziridine-containing linker product, adjusting the pH of the system to be neutral by saturated sodium bicarbonate, extracting by dichloromethane to collect an organic phase, drying and washing the organic phase by saturated sodium chloride and anhydrous sodium sulfate respectively, evaporating the solvent under reduced pressure to obtain a crude product, separating and purifying the crude product by a chromatographic column to obtain the bisaziridine-containing linker, weighing 0.065g, and obtaining the yield of 74.67%.
LC-MS(ESI,m/z):311.10[M+H] + ，309.20[M-H] - 。
The specific process for preparing the nucleoside compound SG-1 containing diazirine comprises the following steps:
dissolving 0.387mmol of intermediate product containing monocarboxylic acid, 0.580mmol of EDC. HCl, 0.464mmol of HOBt and 0.323mmol of linker containing diaziridine in 10mL of anhydrous dichloromethane solution, stirring uniformly at 0 ℃, dropwise adding DIPEA 3.225mmol under ice bath, stirring for 8h at room temperature after dropwise adding, after the reaction is finished, removing the organic solvent at low pressure to obtain a crude product, separating the crude product by a chromatographic column, eluting by using petroleum ether/ethyl acetate (V/V = 10/1) to obtain the nucleoside compound SG-1 containing the diaziridine, weighing 0.039g and obtaining the yield of 15.3%.
The structure of the obtained nucleoside compound SG-1 containing diaziridine is as follows:
the hydrogen spectrum nuclear magnetic resonance data are as follows: 1 H NMR(400MHz,DMSO-d 6 )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＝6.7Hz,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)。
LC-MS(ESI,m/z):659.48[M+H] +
example 12
The diazirine-containing nucleoside compound AG-2 is prepared by the following steps:
dissolving vidarabine, pimelic acid, 4-DMAP and DCC in anhydrous dichloromethane, and reacting for 6h at room temperature to obtain an intermediate product containing monocarboxylic acid; the specific process is as follows:
dissolving 0.784mmol of vidarabine, 3.742mmol of pimelic acid, 0.748mmol of 4-DMAP and 2.245mmol of DCC in 10mL of anhydrous dichloromethane solution, reacting at room temperature, and finishing 6 h; filtering twice with qualitative filter paper, removing organic solvent with low pressure cyclone to obtain crude product, separating the crude product with chromatographic column, eluting with ethyl acetate/methanol (v/v = 1/5) to obtain intermediate product containing monocarboxylic acid, weighing 0.189g, and yield 61.8%.
LC-MS(ESI,m/z):410.41[M+H] + ，408.37[M-H] - 。
Dissolving 0.488mmol of 2- ((tert-butoxycarbonyl) amino) -4-pentenoic acid, 0.716mmol of EDC. HCl and 0.573mmol of HOBt in 10mL of anhydrous dichloromethane, stirring at 0 ℃ for a period of time after the system is cooled, slowly dropwise adding 0.397mmol of DIPEA3.975mmol, reacting for 1H, adding 0.397mmol of 4- [3- (trifluoromethyl) -3H-bisaziridin-3-yl ] benzylamine hydrochloride, reacting to obtain a crude intermediate containing the bisaziridine, washing twice with saturated sodium bicarbonate, washing with saturated sodium chloride, drying an organic phase with anhydrous sodium sulfate, evaporating the solvent under reduced pressure to obtain a crude product, separating and purifying the crude product by using a chromatographic column, eluting with petroleum ether/ethyl acetate (v/v = 1:1) to obtain an intermediate containing the bisaziridine with a Boc protecting group, weighing 0.04g, and obtaining the yield of 41.7%;
LC-MS(ESI,m/z):413.43[M+H] + ，411.37[M-H] - 。
removing the protecting group of the linker intermediate containing the diazirine with the Boc protecting group under the action of trifluoroacetic acid to obtain the target photolinker, wherein the specific process is as follows:
dissolving 0.0971mmol of a bisaziridine-containing linker intermediate with a Boc protecting group in 10mL of anhydrous dichloromethane at 0 ℃, stirring at 0 ℃ until the system is cooled, slowly dropwise adding trifluoroacetic acid (2 mL), reacting at 0 ℃ for 1h, reacting at room temperature for 6h to obtain a crude bisaziridine-containing linker product, adjusting the pH of the system to be neutral by saturated sodium bicarbonate, extracting by dichloromethane to collect an organic phase, drying and washing the organic phase by saturated sodium chloride and anhydrous sodium sulfate respectively, evaporating the solvent under reduced pressure to obtain a crude product, and separating and purifying the crude product by a chromatographic column to obtain the bisaziridine-containing linker, wherein the weight of the crude product is 0.065g, and the yield of the crude product is 74.7%.
LC-MS(ESI,m/z):311.10[M+H] + ，309.20[M-H] - 。
The specific process of the diazirine-containing nucleoside compound AG-2 is as follows:
dissolving 0.244mmol of intermediate product containing monocarboxylic acid, 0.366mmol of EDC. HCl, 0.293mmol of HOBt and 0.204mmol of linker containing diaziridine in 10mL of anhydrous dichloromethane solution, stirring uniformly at 0 ℃, dropwise adding DIPEA 2.0355mmol under ice bath, stirring for 10h at room temperature after dropwise adding, removing organic solvent at low pressure after reaction is finished to obtain crude product, separating the crude product by a chromatographic column, eluting with ethyl acetate/methanol (V/V = 5/1) to obtain nucleoside compound AG-2 containing diaziridine, weighing 0.027g and having yield of 15.79%.
The structure of the obtained diazirine-containing nucleoside compound AG-2 is as follows:
the hydrogen spectrum nuclear magnetic resonance data is as follows: 1 HNMR(400MHz,DMSO-d 6 )δ10.72(s,1H),8.63(s,1H),8.17(d,J＝8.0Hz,2H),7.81(s,1H),7.38(d,J＝8.2Hz,2H),7.22(d,J＝7.9Hz,2H),6.59(s,2H),5.34(s,2H),4.47–4.39(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] +
example 13
The nucleoside compound RG-1 containing diazirine is combined with target protein RdRp.
The method adopts a gel imaging method to determine that the nucleoside compound RG-1 containing diazirine is combined with a target protein RdRp.
(1) Liquid I: RD-1 (1. Mu.M); and II, liquid: rdRp protein (1. Mu.g)
(2) Adding solution I and solution II into 1.5mL EP tube, reacting for 1h in dark condition, placing the reaction system at a distance of 3cm from 365nm, performing photo-crosslinking for 30min, and performing photo-crosslinkingAdding CuSO 4 ·H 2 O0.006 mM, sodium ascorbate 0.005mM, azide-Cy 3. Mu.M, incubated at 90rpm for 2h at 37 ℃ in a constant temperature shaker.
(3) After completion of the reaction, the reaction mixture was centrifuged at a low temperature and high speed centrifuge (10000rpm, 20min), the supernatant was discarded, 80. Mu.L of PBS was added, and 2. Mu.L of Loading Buffer (5X) was added to 8. Mu.L of the supernatant, and the mixture was applied.
(4) SDS-PAGE separation
1) Preparation of separation gel
Preparing 10mL of 6% separation gel according to the table 6, uniformly mixing by vortex, quickly introducing into a gel preparation mold, adding 1mL of isopropanol to remove bubbles so as to make the gel surface smooth and flat, standing at room temperature for 30min, slowly removing the isopropanol along one side of the gel preparation surface, carefully sucking residual isopropanol by using filter paper, and taking care that the residual isopropanol cannot touch the gel surface.
TABLE 6% preparation of separation gel reagent and dosage
2) Preparation of concentrated gum
5mL of 5% glass plate concentrated gel was prepared according to Table 7, vortexed, mixed well, poured quickly into a gel mold, a comb of concentrated gel of a size corresponding to the glass plate was carefully inserted immediately (care was taken not to generate any air bubbles in the comb during insertion), allowed to stand at room temperature for 30min, the comb was removed from the concentrated gel uniformly and slowly, separated from the comb, and run-on buffer (1X) was added
TABLE 7% concentrated gum preparation reagents and amounts
3) Sample loading
The Loading of crosslinked sample was 8. Mu.L, and the Loading Buffer (5X) was 2. Mu.L.
4) Electrophoretic separation
The gel was run at 90V to the vicinity of the concentrated gel and then stopped at 120V to the bottom of the gel.
Referring to Table 7 and FIG. 6, the nucleoside compound RG-1 containing diazirine did not alter the SARS-CoV-2RdRp, a target protein of Reidesvir.
Example 14
(1) HEK293 cells in exponential growth phase were diluted 10 in DMEM medium 4 Cell solutions of the order of one/mL were plated in parallel in 96-well plates (2000-4000/well) at a volume of 180. Mu.L/well and were 5% CO at 37 ℃% 2 Culturing for 12h;
(2) 20 μ L of test compound was added to each well at different concentrations to give final concentrations of compound in the wells: 0.001 μ M,0.01 μ M,0.1 μ M,1 μ M,10 μ M,100 μ M, 3 duplicate wells for each concentration, 6 duplicate wells for negative control, no compound added to each well, and 5% CO at 37 deg.C 2 Culturing for 48h;
(3) mu.L of MTT (5 mg/mL) was added to each well to give a final concentration of 0.5mg/mL MTT in each well at 37 ℃ with 5% CO 2 Culturing for 4 hr, carefully removing supernatant, adding DMSO 150 μ L into each well, shaking for 15min, measuring ultraviolet absorption (OD) at 490nm of each well with ELISA detector, calculating cell inhibition rate, and calculating IC of compound according to the inhibition rate by linear regression method 50 The value, the calculation formula of the cell inhibition rate is:
inhibition% = (control well mean OD value-drug use group mean OD value)/control well mean OD value × 100%;
the detection result shows that compared with the negative control group, the nucleoside compound containing diazirine has almost no in vitro inhibition effect on the HEK293 normal cells, as shown in (a), (b), (c), (d) and (e) in figure 7.
Example 15
(1) Diluting virus susceptible cells Vero cells, 293T cells, hela cells and RD cells in growth index stage into 10 with DMEM medium 4 Cell solutions of the order of magnitude of one/mL were plated in parallel in 96-well plates (2000-4000 cells/well)The volume of inoculation per well was 180. Mu.L, and 5% CO at 37 ℃ 2 Culturing for 12h;
(2) 20 μ L of test compound was added to each well at different concentrations to give final concentrations of compound in the wells: 0.001 μ M,0.01 μ M,0.1 μ M,1 μ M,10 μ M,100 μ M, 3 duplicate wells for each concentration, 6 duplicate wells for negative control, no compound added to each well, and 5% CO at 37 deg.C 2 Culturing for 48h;
(3) mu.L of MTT (5 mg/mL) was added to each well to give a final concentration of 0.5mg/mL MTT in each well at 37 ℃ with 5% CO 2 Incubating for 4h, carefully removing supernatant, adding DMSO 150 μ L into each well, shaking for 15min, measuring ultraviolet absorbance (OD) at 490nm of each well with ELISA detector, calculating cell inhibition rate, and calculating IC of compound according to inhibition rate by linear regression method 50 The value, the calculation formula of the cell inhibition rate is:
inhibition% = (control well mean OD value-drug use group mean OD value)/control well mean OD value × 100%;
the detection result shows that compared with a negative control group, the nucleoside compound containing diazirine has almost no in vitro inhibition effect on virus-susceptible cells except for the Vero cells by AG-2, and is shown in the table 8.
TABLE 8 IC of bisaziridine-containing nucleosides on cells susceptible to virus 50
Example 16
Inhibition of SARS-CoV-2 spike pseudovirus invasion of ACE2 cells by bisaziridine-containing nucleosides.
(1) After ACE2 cells were seeded into 96-well plates (200. Mu.L) and cultured for 24h, 20. Mu.L of cell culture medium was aspirated and added to the culture with different concentrations of compounds (0.001. Mu.M, 0.01. Mu.M, 0.1. Mu.M, 1. Mu.M, 10. Mu.M, 100. Mu.M).
(2) After 48h the medium was aspirated and 22. Mu.L of MTT was added to each well for incubation.
(3) After 4h the MTT was aspirated, 200. Mu.L DMSO shaker was added to each well and incubated at room temperature for 10min, and the absorbance was measured at 490 nm.
(4) After ACE2 cells were seeded into 96-well plates (100 μ L) and cultured for 24h, 50 μ L of cell culture medium was aspirated and incubated with 50 μ L of medium containing different concentrations of compounds (1 μ M, 5 μ M, 20 μ M). After 2h incubation, 10. Mu.L of SARS-Cov-2 spike pseudovirus was added and cultured.
(5) After 10h, the pseudovirus-containing medium was aspirated and 200. Mu.L of fresh medium was added for culture.
(6) After 48h, the medium was aspirated, and 20. Mu.L of cell lysate and 100. Mu.L of luminescence were added to each well using the Luciferase Assay System for absorbance measurement at 561 nm.
The results in Table 9 and FIG. 8 show that the bisaziridine-containing nucleoside compounds only inhibit infection by pseudoviruses by AG-1.
TABLE 9 cytotoxicity (IC) of NP-1, AG-1, RG-1, AG-2 and SG-1 against ACE2 50 /μM)
Example 17
Nucleoside compound containing diazirine for SARS-CoV-2 3CL pro The effect of activity.
(1) Wild type SARS-CoV-2 3CL pro The gene was constructed in the pEGX-6P vector (Novagen). The constructed plasmid was transferred into E.coli BL21 cells and the target protein was induced with 0.25mM isopropyl beta-d-1-thiogalactoside (IPTG) for 18h at 16 ℃. The collected cells were placed in a medium containing 20mM Tris-HCl (pH 8), 150mM NaCl, 4mM MgCl 2 And in a lysis buffer solution containing 5% of glycerol, performing low-temperature ultrasonic cell lysis and homogenization. After centrifugation at 12 000rpm for 40min at 4 ℃ to remove cell debris, the supernatant was loaded onto a Ni-nitrilotriacetic acid (Ni-NTA) column. Washing of SARS-CoV-2 3CL with a wash solution containing 200mM imidazole (pH 8) pro Adding SUMO protease to generate MERS-CoV-2 3CL pro . Purifying by Superdex 75 gel filtration chromatography (GE Healthcare) to obtain crude protein, and purifying to obtain the final productThe target protein was concentrated to 30mg/mL and stored at-80 ℃.
(2) The mutant proteins were prepared using a Rapid mutation System kit (Transgen Biotech). After mutagenesis, the mutated recombinant plasmid was verified by gene sequencing and the mutated protease was expressed by an enzyme preparation method.
(3) The FRET-based polypeptide NMATSAVLQSGFRK (DNP) M was synthesized by the solid phase method, and used as a substrate, and the 3CL was used pro Cleavage of the Gln-Ser bond generates fluorescence. 2.0 μ M SARS-CoV-2 CL 3CL pro Incubate with 6 different concentrations of inhibitor (DMSO alone as blank) for 30min at 37 ℃ in 50 μ L buffer (pH =8.0,20mm Tris-HCl,150mM NaCl). The reaction started after the addition of 30. Mu.M substrate solution (50. Mu.L). The change in relative fluorescence units, lambda, was determined with a microplate reader ex Is 340nm, lambda em Is 440nm.
Table 10 results show that nucleoside compounds containing diazirine are active against SARS-CoV-2 3CL pro All have certain inhibiting effect.
TABLE 10 nucleoside Compounds for SARS-CoV-2 3CL pro Hydrolase Inhibition (IC) 50 /μM)
The preparation method of the RD-1, AD-1, SD-1, RG-1, AG-1, SD-1 and AG-2 covalent binding nucleoside compounds is simple, easy to realize and high in yield. The covalently bound nucleoside compounds with brand-new structures, brand-new covalent binding modes and action modes of RD-1, AD-1, SD-1, RG-1, AG-1, SD-1 and AG-2 of the invention are covalently bound to prepare 3CL of the novel coronavirus SARS-CoV-2 pro Hydrolase and RdRp protein activity, wherein RD-1 not only binds to and labels the RdRp protein, but also binds to 3CL pro The hydrolase has certain inhibiting effect. In addition, covalently bound nucleoside anti-neocoronavirus drugs have low cytotoxicity. The covalent inhibitor can form covalent bond with target protein, realize irreversible combination, provide new action mechanism and combination mode, improve the combination efficiency and action endurance of small molecule inhibitor,the action intensity and time of the medicine are improved.
Claims (10)
1. A compound containing diazirine nucleoside is characterized in that the structural formula of the compound is as follows:
wherein R is Rudexilvir, acyclovir, vidarabine or stavudine.
2. A preparation method of a compound containing diazirine nucleoside is characterized by comprising the following steps:
1) Reacting one of Reideciclovir, acyclovir, vidarabine and stavudine with succinic anhydride in dichloromethane to obtain an intermediate product containing monocarboxylic acid; or
Under the condensation action of EDC & HCl, one of Ruidecy Wer, acyclovir, vidarabine and stavudine and pimelic acid are obtained as an intermediate product containing monocarboxylic acid;
2) The diazirine-containing linker and the intermediate product containing monocarboxylic acid are condensed under EDC & HCl to obtain the diazirine-containing nucleoside compound.
3. The method for preparing a compound containing diazirine nucleoside according to claim 2, wherein one of Reideciclovir, acyclovir, vidarabine and stavudine and succinic anhydride are reacted in dichloromethane to obtain an intermediate product containing monocarboxylic acid, comprising the steps of: dissolving 1.00mmol of one of Reideciclovir, acyclovir, vidarabine and stavudine and 1.20mmol of succinic anhydride in 10mL of anhydrous dichloromethane, and reacting for 8h to obtain an intermediate product containing monocarboxylic acid;
under the condensation action of EDC & HCl, one of RIDESICWER, acyclovir, vidarabine and stavudine and pimelic acid obtain an intermediate product containing monocarboxylic acid, which comprises the following steps:
dissolving pimelic acid, EDC & HCl and HOBt in dichloromethane, adding DIPEA at 0 ℃, reacting for 1h to generate active ester, adding one of Reidesciclovir, acyclovir, vidarabine and stavudine, and stirring for 10h to obtain an intermediate product containing monocarboxylic acid.
4. The method according to claim 2, wherein the diazirine-containing linker is 2-amino-N- (4- (3- (trifluoromethyl) -3H-diaza-3-yl) benzyl) pent-4-ynylamide.
5. The method of claim 2, wherein the linker containing diazirine is prepared by the following steps:
condensation of 4- [3- (trifluoromethyl) -3H-bisaziridin-3-yl ] benzylamine hydrochloride with 2- ((tert-butoxycarbonyl) amino) -4-pentynoic acid in EDC · HCl gives a bisaziridine-containing linker intermediate with a Boc protecting group; and (3) removing the protecting group of the linker intermediate containing the bis-aziridine with the Boc protecting group under the action of trifluoroacetic acid to obtain the linker containing the bis-aziridine.
6. The process for preparing a bisaziridine nucleoside compound according to claim 2, wherein 2- ((tert-butoxycarbonyl) amino) -4-pentynoic acid, EDC. HCl and HOBt are dissolved in dichloromethane, DIPEA is added dropwise with stirring at 0 ℃ to react for 1 hour, 4- [3- (trifluoromethyl) -3H-bisaziridin-3-yl ] benzylamine hydrochloride is added to react to obtain a bisaziridine-containing linker intermediate with a Boc protecting group, the bisaziridine-containing linker intermediate with a Boc protecting group is dissolved in dichloromethane, trifluoroacetic acid is added dropwise at 0 ℃ to react for 6 hours to obtain the bisaziridine-containing linker.
7. The method of claim 2, wherein the intermediate product containing the monocarboxylic acid obtained in step 1) is dissolved in anhydrous dichloromethane, EDC. HCl, HOBt and the linker containing the bisaziridine obtained in step 2) are added, DIPEA is added at 0 ℃, and the reaction is carried out for 10h to obtain the bisaziridine-containing nucleoside compound.
8. Use of the compound of claim 1 containing diaziridine nucleosides for the preparation of a medicament against neocoronaviruses.
9. Use of a compound as defined in claim 1 containing diazirine nucleosides for the preparation of an antiviral medicament targeting RdRp.
10. Use of the compound of claim 1 containing diazirine nucleosides for validating a protein target.
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