Glucosylated ribavirin compound as well as preparation method and application thereof

Abstract

The invention provides a 3 '-O-beta-glucosyl-ribavirin compound and a preparation method and application thereof.The method comprises the steps that uridine diphosphate-glucose and ribavirin serve as raw materials, the 3'-O-beta-glucosyl-ribavirin compound is synthesized under the catalysis of glycosyl transferase, and the 3 '-O-beta-glucosyl-ribavirin compound is obtained. The method is mild in reaction condition, simple to operate, high in yield and suitable for industrial production. The 3 '-O-beta-glucosyl-ribavirin compound prepared by the invention has an obvious inhibition effect on influenza viruses and enteroviruses, has an antiviral effect higher than that of an existing clinical drug ribavirin, and can be used as an antiviral drug candidate.

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and in particular to a glucosylated ribavirin compound and a preparation method and application thereof. Background Art

[0002] Ribavirin, also known as virazole, has a chemical name of 1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide and a chemical formula of C8H 12 N4O5 is a purine nucleoside analog. Ribavirin has a broad-spectrum antiviral effect, including DNA viruses and RNA viruses. Initial reports showed that ribavirin has inhibitory effects on a total of 16 DNA and RNA viruses in cell culture medium and mice. Due to this significant antiviral effect, ribavirin is also widely used in clinical practice. As early as the mid-1980s, before HCV was discovered in 1989, ribavirin was approved for the treatment of respiratory syncytial virus infection. It has the effect of inhibiting the growth of multiple viruses in vitro, such as respiratory syncytial virus, influenza virus, hepatitis A virus, and adenovirus.

[0003] Enzymes that catalyze glycosyl transfer reactions are collectively called glycosyltransferases (GTs). Most glycosylation reactions in nature are mediated by glycosyltransferases, which can transfer sugar residues from activated sugar donors to various acceptors. Among them, uridine diphosphate-dependent glycosyltransferases (UGTs) use UDP-activated sugar donors and are the most numerous enzymes in the glycosyltransferase family. Glycosylation is a key modification in the biosynthesis process, and the modified products exist as glycoside compounds and exert biological activity.

[0004] Glycoside compounds have a variety of sugar substitution forms, and the number and position of substitutions vary, so the structural diversity of these compounds is extremely rich. Small molecule glycosylation modification has a significant impact on its solubility, stability or biological activity. This process can also reduce or eliminate the toxicity of endogenous and exogenous substances, making glycoside compounds widely attractive compounds such as food additives, drug therapeutic agents or health products.

[0005] Although ribavirin can be obtained by chemical synthesis, there is no report on the biological or chemical synthesis pathway of glycosylated ribavirin. Therefore, it is necessary to develop a glycosylated ribavirin compound and a preparation method thereof. Summary of the invention

[0006] The present invention aims to provide a glucosyl ribavirin compound and a preparation method thereof. The 3′-O-β-glucosyl-ribavirin compound prepared by the method using the antiviral nucleoside drug ribavirin as a precursor compound has obvious anti-influenza virus and enterovirus activity and can be used as an active ingredient candidate for antiviral drugs. The preparation method is simple and has a high yield, and is suitable for industrial production.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] In the first aspect of the present invention, a 3'-O-β-glucosyl-ribavirin compound is provided, and the structural formula of the 3'-O-β-glucosyl-ribavirin compound is as follows:

[0009]

[0010] In a second aspect of the present invention, a method for preparing a 3′-O-β-glucosyl-ribavirin compound is provided, the method comprising:

[0011] Using uridine diphosphate-glucose and ribavirin as raw materials, 3′-O-β-glucosyl-ribavirin compound is synthesized under the catalysis of glycosyltransferase, and its chemical reaction formula is:

[0012]

[0013] Furthermore, the molar ratio of ribavirin to UDP-glucose is 1:(5-20).

[0014] Furthermore, the reaction conditions include: 30-60°C for 0.5-8h.

[0015] In the third aspect of the present invention, provided is the use of the 3′-O-β-glucosyl-ribavirin compound in the preparation of antiviral drugs.

[0016] Furthermore, the antiviral drug includes an anti-influenza virus drug or an anti-enterovirus drug.

[0017] Furthermore, the drug includes pharmaceutically acceptable excipients.

[0018] Furthermore, the dosage form of the drug includes one of capsules, pills, powders, tablets, granules, oral liquids and injections.

[0019] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0020] 1. The method for preparing a 3′-O-β-glucosyl-ribavirin compound provided by the present invention uses uridine diphosphate-glucose and ribavirin as raw materials and synthesizes the 3′-O-β-glucosyl-ribavirin compound under the catalysis of glycosyltransferase. The reaction conditions are mild, the operation is simple, the yield is high, and the method is suitable for industrial production.

[0021] 2. The 3′-O-β-glucosyl-ribavirin compound prepared by the present invention has a significant inhibitory effect against influenza virus and enterovirus, and its antiviral effect is higher than that of the current clinical drug ribavirin, and can be used as an antiviral drug candidate. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 It is the structural formula of 3′-O-β-glucosyl-ribavirin compound.

[0024] Figure 2 This is the reaction formula of the 3′-O-β-glucosyl-ribavirin compound of the present invention.

[0025] Figure 3 High performance liquid chromatography (HPLC) and mass spectrometry (MS / MS) analysis of 3′-O-β-glucosyl-ribavirin, wherein Figure 3 a is the liquid chromatography result, Figure 3 b is the mass spectrometry result.

[0026] Figure 4 This is the hydrogen spectrum of 3′-O-β-glucosyl-ribavirin.

[0027] Figure 5 This is the two-dimensional spectrum of 3′-O-β-glucosyl-ribavirin. DETAILED DESCRIPTION

[0028] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0029] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0030] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0031] The present application will be described in detail below with reference to embodiments and experimental data.

[0032] Example 1: 3′-O-β-glucosyl-ribavirin

[0033] The avpGT gene (the avpGT gene is described in references Pasternak, ARO; Balunas, MJ; Zechel, DL Discovery of 3′-O-β-glucosyltubercidin and the nucleoside specific glycosyltransferase AvpGT through genome mining. ACS Chem. Biol. 2022, 17(12), 3507-3514. DOI: 10.1021 / acschembio.2c00707) was amplified by PCR and cloned into an expression vector, and then transformed into Escherichia coli BL21 (DE3), heterologously overexpressed and purified in Escherichia coli, and a relatively pure protein AvpGT was obtained after SDS-PAGE analysis. The specific operation was as follows: ① The positive single clone was picked and cultured in 5 mL LB medium at 37°C overnight, and then transferred to 500 mL LB at 1%, and cultured at 37°C until the bacterial OD 600② Add an appropriate amount (20mL~30mL) of lysis buffer to the collected bacteria, shake the mixed bacteria and use an ultrasonic disruptor to ultrasonically disrupt the E. coli cells, centrifuge at 4℃12000rpm for 20min, and take the supernatant. At 4℃, load the supernatant into a gravity column filled with nickel, and elute with Tris buffer containing different concentrations (20mM~300mM) of imidazole. Perform SDS-PAGE analysis on the samples eluted at different concentrations to collect the purer protein samples. The present invention uses ribavirin as a substrate, adds UDP-glucose and a catalyst AvpGT (in other embodiments, the catalyst can also be directly purchased commercially) under the condition of pH 8.0 Tris-HCl buffer to carry out an in vitro biochemical reaction, wherein the molar ratio of ribavirin to UDP-glucose is 1:(5-20), and the reaction conditions include: 0.5-8h at 30-60°C.

[0034] After the reaction was completed, the formation of 3′-O-β-glucosyl-ribavirin was analyzed by liquid chromatography and mass spectrometry. Figure 3 shown.

[0035] The obtained product was characterized by H NMR and C NMR spectra ( Figure 4 ), the result is as follows: 1 H NMR(600MHz, Deuterium Oxide)δ8.66(d,J=1.5Hz,1H),5.97(d,J=3.6Hz,1H),4.57(dd,J=5.0,3.5Hz,1H),4.41(t,J=5.3Hz,1H),4.27(td,J=5.6, 3.2Hz,1H),3.77(m,1H),3.68(m,2H),3.56(dd,J=11.8,4.4Hz,1H),3.43(m,1H),3.37(m,2H),3.30(dd,J=9.3,7.9Hz,1H). 13 CNMR(151MHz,D2O)δ162.72,156.42,146.13,102.50,91.48,75.80,75.48,74.50,74.17,72.77,70.04,69.30,62.45,61.14.

[0036] Example 2: Effect of glycosylation modification on the antiviral activity of compounds

[0037] The ability of ribavirin before modification and the glycosylated 3'-O-β-glucosyl-ribavirin compound of Example 1 of the present invention to inhibit influenza virus PR8 strain and enterovirus EVA71 and the drug toxicity in 293T cells were tested in 293T cells and RD cells, respectively. The results are shown in the following table.

[0038] Table 1

[0039]

[0040] As can be seen from the above table, compared with ribavirin before modification, the glycosylated 3'-O-β-glucosyl-ribavirin compound of the present invention has significantly improved antiviral ability, has obvious inhibitory effect against influenza virus and enterovirus, and can be used as an antiviral drug candidate.

[0041] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0042] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0043] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A 3′-O-β-glucosyl-ribavirin compound, characterized in that: The structural formula of the 3′-O-β-glucosyl-ribavirin compound is shown below:

2. A method for preparing a 3′-O-β-glucosyl-ribavirin compound, characterized in that: The method comprises: Using uridine diphosphate-glucose and ribavirin as raw materials, 3′-O-β-glucosyl-ribavirin compound is synthesized under the catalysis of glycosyltransferase, and its chemical reaction formula is:

3. The method of claim 2, wherein: The molar ratio of ribavirin to UDP-glucose is 1:(5-20).

4. The method of claim 2, wherein the 3′-O-β-glucosyl-ribavirin compound is characterized in that: The reaction conditions include: reacting at 30-60° C. for 0.5-8 h.

5. Use of the 3'-O-β-glucosyl-ribavirin compound according to claim 1 in the preparation of antiviral drugs.

6. The use according to claim 5, characterized in that: The antiviral drugs include anti-influenza virus drugs or anti-enterovirus drugs.

7. The use according to claim 5, characterized in that: The drug includes pharmaceutically acceptable excipients.

8. The use according to claim 5, characterized in that: The dosage form of the medicine includes one of capsule, pill, powder, tablet, granule, oral solution and injection.

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