A glucosylated ribavirin compound, its preparation method and application

Abstract

The application provides a 3'-O-beta-glucosyl-ribavirin compound and a preparation method and application thereof, and the method comprises the following steps: taking uridine diphosphate-glucose and ribavirin as raw materials, and synthesizing the 3'-O-beta-glucosyl-ribavirin compound under the catalysis of a glycosyltransferase, the reaction condition is mild, the operation is simple, the yield is high, and the method is suitable for industrial production. The 3'-O-beta-glucosyl-ribavirin compound prepared by the application has obvious inhibitory effect on influenza virus and enterovirus, and the antiviral effect is higher than that of the present clinical drug ribavirin, and the 3'-O-beta-glucosyl-ribavirin compound can be used as an antiviral drug candidate.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparation technology, and in particular to a glucosylated ribavirin compound, its preparation method, and its application. Background Technology

[0002] Ribavirin, also known as Virazole, has the chemical name 1-β-D-ribofuranosyl-1H-1,2,4,-triazole-3-carboxamide and the chemical formula C8H10. 12 N4O5 is a purine nucleoside analog. Ribavirin has broad-spectrum antiviral activity, targeting both DNA and RNA viruses. Initial reports showed that ribavirin inhibited 16 DNA and RNA viruses in cell culture and in mice. Due to this significant antiviral activity, ribavirin is widely used clinically. Even before HCV was discovered in 1989, ribavirin was approved in the mid-1980s for the treatment of respiratory syncytial virus (RSV) infection. In vitro, it inhibits the growth of various viruses, including RSV, influenza virus, hepatitis A virus, and adenovirus.

[0003] Enzymes that catalyze glycosyltransferase 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. Urate-diphosphate-dependent glycosyltransferases (UGTs), which utilize UDP-activated sugar donors, constitute the largest class of enzymes in the glycosyltransferase family. Glycosylation is a key modification in biosynthesis, and the modified products exist as glycosides and exert biological activity.

[0004] Glycosides exhibit a wide variety of glycosyl substitutions, with varying numbers and positions of substitutions, resulting in an extremely rich diversity of structures. Small-molecule glycosylation modifications significantly impact their solubility, stability, and bioactivity. This process can also reduce or eliminate the toxicity of endogenous and exogenous substances, making glycosides attractive compounds for use as food additives, therapeutic agents, or health supplements.

[0005] Although ribavirin can be obtained through chemical synthesis, there are no reports on the biological or chemical synthesis pathways for glycosylated ribavirin. Therefore, it is necessary to develop a glycosylated ribavirin compound and its preparation method. Summary of the Invention

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

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

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

[0009]

[0010] In a second aspect of the invention, a method for producing 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 was synthesized under the catalysis of glycosyltransferase. The chemical reaction formula is as follows:

[0012]

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

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

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

[0016] Furthermore, the antiviral drugs include drugs against influenza viruses or drugs against enteroviruses.

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

[0018] Furthermore, the dosage form of the drug includes one of the following: 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 synthesizing 3′-O-β-glucosyl-ribavirin compound provided by the present invention uses uridine diphosphate-glucose and ribavirin as raw materials to synthesize 3′-O-β-glucosyl-ribavirin compound under the catalysis of glycosyltransferase. The reaction conditions are mild, the operation is simple, and the yield is high, making it suitable for industrial production.

[0021] 2. The 3′-O-β-glucosyl-ribavirin compound prepared in this invention has a significant inhibitory effect on influenza virus and enterovirus, and its antiviral effect is higher than that of the current clinical drug ribavirin, and it can be used as an antiviral drug candidate. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying 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 effort.

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

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

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

[0026] Figure 4 The 1H NMR spectrum is for 3′-O-β-glucosyl-ribavirin.

[0027] Figure 5 The two-dimensional spectrum of 3′-O-β-glucosyl-ribavirin. Detailed Implementation

[0028] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0029] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0030] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

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

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

[0033] The avpGT gene (see reference 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, which was then transformed into E. coli BL21(DE3). Heterologous overexpression and purification were performed in E. coli, and the relatively pure protein AvpGT was obtained by SDS-PAGE analysis. The specific operation was as follows: ① Positive single clones were picked and cultured overnight at 37°C in 5 mL LB medium, and then transferred at 1% to 500 mL LB medium and cultured at 37°C until the bacterial cell OD reached the target value. 600① Add IPTG (final concentration 0.1-0.2 mM) to the culture medium to 0.5-0.8, induce incubation at 18℃ for 20 h, and collect bacterial cells by centrifugation at 6000 rpm for 15 min; ② Add an appropriate amount (20 mL-30 mL) of lysis buffer to the collected bacterial cells, shake to mix the bacteria, and then use an ultrasonic homogenizer to sonicate and homogenize the E. coli cells. Centrifuge at 12000 rpm for 20 min at 4℃ to collect the supernatant. At 4℃, load the supernatant into a gravity column filled with nickel packing and elute with Tris buffer containing different concentrations (20 mM-300 mM) of imidazole. Perform SDS-PAGE analysis on the samples eluted at different concentrations and collect the purer protein samples. This invention uses ribavirin as a substrate and adds UDP-glucose and catalyst AvpGT (in other embodiments, the catalyst can also be commercially available) to carry out an in vitro biochemical reaction under the conditions of pH 8.0 Tris-HCl buffer. The molar ratio of ribavirin to UDP-glucose is 1:(5-20), and the reaction conditions include 0.5-8h at 30-60℃.

[0034] After the reaction was completed, analysis using liquid chromatography and mass spectrometry revealed the formation of 3′-O-β-glucosyl-ribavirin, such as... Figure 3 As shown.

[0035] The obtained product was characterized by 1H NMR and 1C NMR spectra. Figure 4 The results are 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 the compound

[0037] The ability of unmodified ribavirin and the glycosylated 3'-O-β-glucosyl-ribavirin compound of Example 1 of this invention to inhibit influenza virus PR8 strain and enterovirus EVA71, as well as the drug toxicity in 293T cells, were detected in 293T cells and RD cells, respectively. The results are shown in the table below.

[0038] Table 1

[0039]

[0040] As shown in the table above, compared with the unmodified ribavirin, the glycosylated 3'-O-β-glucosyl-ribavirin compound of the present invention has significantly improved antiviral activity and exhibits significant inhibitory effects against influenza virus and enterovirus, and can be used as a candidate antiviral drug.

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

[0042] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0043] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends 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: Formula I.

2. A method for producing a 3′-O-β-glucosyl-ribavirin compound, characterized in that, The method includes: Using uridine diphosphate-glucose and ribavirin as raw materials, 3′-O-β-glucosyl-ribavirin was synthesized under the catalysis of glycosyltransferase. The chemical reaction formula is as follows: 。 3. The method for the 3′-O-β-glucosyl-ribavirin compound according to claim 2, characterized in that, The molar ratio of ribavirin to UDP-glucose is 1:(5-20).

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

5. The use of the 3′-O-β-glucosyl-ribavirin compound of claim 1 in the preparation of antiviral drugs.

6. The application according to claim 5, characterized in that, The antiviral drug is selected from drugs that fight influenza viruses or drugs that fight enteroviruses.

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

8. The application according to claim 5, characterized in that, The dosage form of the drug is selected from one of the following: capsules, pills, powders, tablets, granules, oral liquids, and injections.

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