CN110898046A - Application of monoiodo aromatic acid as CVB3 virus inhibitor - Google Patents

Abstract

The invention discloses application of monoiodo aromatic acid as a CVB3 virus inhibitor. Through the research and experiment on the anti-CVB 3 activity of several mono-iodo aromatic acids, the mono-iodo aromatic acids show certain inhibitory activity on CVB3 viruses, including inhibition of cytopathic effect (CPE) generated by the CVB3 on host cell Hep-2 and enhancement of cell survival rate, and the mono-iodo aromatic acids have inhibitory effect on the CVB3 viruses, so that the application of the mono-iodo aromatic acids in preparation of anti-CVB 3 virus medicines is shown.

Description

Application of monoiodo aromatic acid as CVB3 virus inhibitor

Technical Field

The invention relates to the technical field of antiviral drugs, in particular to application of mono-iodo aromatic acid as a coxsackie virus inhibitor.

Background

Coxsackievirus (coxsaekkievirus, CV for short) is a member of Enterovirus (Enterovirus) of picornavirus (Picornaviridae), and infection of the coxsackievirus can cause various diseases, such as hand-foot-and-mouth disease, aseptic meningitis, encephalitis, myocarditis, epidemic myositis, herpangina and the like. CV has been reported to have 29 serotypes, and can be divided into two groups A and B, namely CVA (CVA1-22,24) and CVB (CVB1-6) according to the pathogenic characteristics and the sensitivity to cells of suckling mice. Infection with CVBs is most common, with CVB3 being the most pathogenic of the six serotypes of CVB and the most prominent causative cause of viral myocarditis. As shown by the american centers for disease prevention and control (CDC) statistics, CVB (types 1-6) can cause about 500 million people to develop bowel system disease each year, with 10% -20% of these cases being acute myocarditis caused by CVB 3. In recent years, the trend of the CVB3 to cause the hand-foot-and-mouth disease is also rising, and a plurality of reports of disease epidemics caused by the CVB3 exist in China. At present, no specific medicine is available for coxsackie virus infection, and no specific treatment means is available in clinic. Many researchers have found numerous compounds that inhibit CVB3 activity in vitro and in vivo, but these are still essentially in the first stages of laboratory testing and are far from practical clinical use. Therefore, the development of specific and effective anti-CVB 3 medicaments is imperative.

Iodo-aromatic acids are a class of organic compounds with biological activity. For example, p-iodobenzoic acid is a potent inhibitor of cinnamate-4-HYDROXYLASE (CINNAMATE 4-HYDROXYLASE), a key enzyme in the phenylpropanoid pathway for the synthesis of lignin building blocks (Dorien Van de Wouwer, et al. plant Physiology,2016,172, 198-220); the metal salt of m-iodobenzoic acid shows better inhibitory activity to Saccharomyces cerevisiae, Hansenula anomala, Escherichia coli and Bacillus subtilis (P.Koczon, et.al.J.Agric.food chem.2001,49, 2982-2986). For example, 2,3, 5-triiodobenzoic acid (TIBA) is an excellent plant growth regulator (asherman, et al. plant physiological communications, 1958,3, 27-30). 3, 5-bis (acetamido) -2,4, 6-triiodobenzoic acid, also known as diatrizoic acid (diatrizoic acid), is an important contrast agent, and is prepared into diatrizoate sodium and diatrizoate meglumine injection, which can be used for imaging urinary system, cardiovascular system, cerebrovascular system and peripheral blood vessels (I Charles, et al, 1986, US 4567034). However, the antiviral activity of iodine-containing carboxylic acids, including inhibitory activity against CVB3 virus, has not been reported so far.

Therefore, a new direction of research is being sought for iodine-containing carboxylic acids with anti-CV activity, in particular anti-CVB 3 activity, as CV virus inhibitors.

Disclosure of Invention

The invention aims to solve the defects of the prior art and develop a novel specific and effective anti-CVB 3 medicament. According to the invention, through numerous screening experiments and a large number of biological experiments, the monoiodo aromatic acid is found to be capable of inhibiting cytopathic effect (CPE) generated by CVB3 in host cell Hep-2, enhancing the cell survival rate, showing an inhibiting effect on CVB3 and having a high treatment index, and prompting that the monoiodo aromatic acid has potential to be further developed into a medicine for effectively treating CVB3 infection. Based on this discovery, the present invention provides an application of monoiodo aromatic acid.

The invention provides an application of monoiodo aromatic acid as CV virus inhibitor, wherein the monoiodo aromatic acid comprises L₁、L₂、L₃Wherein L is₁Is 2-iodobenzoic acid, L₁The structural formula of (A) is as follows:

L₂is 3-iodobenzoic acid, L₂The structural formula of (A) is as follows:

L₃is 4-iodobenzoic acid, L₃The structural formula of (A) is as follows:

preferably, the CV virus is a subtype B3, i.e. CVB3 virus.

Preferably, the monoiodo aromatic acid is L₂Or L₃I.e., 3-iodobenzoic acid or 4-iodobenzoic acid, more preferably, the monoiodo aromatic acid is L₃I.e. 4-iodobenzoic acid.

Further, the application of the monoiodo aromatic acid as the CVB3 virus inhibitor comprises the application of the monoiodo aromatic acid, namely 2-iodobenzoic acid, 3-iodobenzoic acid and 4-iodobenzoic acid, in the preparation of the medicine for resisting the CVB3 virus.

Further, the application of the monoiodo aromatic acid as the CVB3 virus inhibitor also comprises the combination of any one, any two or three of 2-iodobenzoic acid, 3-iodobenzoic acid and 4-iodobenzoic acid with ribavirin.

Further, the use of monoiodo aromatic acids as inhibitors of CVB3 virus also includes L₁、L₂、L₃Namely the combination of any two or three of 2-iodobenzoic acid, 3-iodobenzoic acid and 4-iodobenzoic acid.

The invention also provides a medicine for resisting CVB3 virus, which comprises L₁、L₂、L₃Namely one, two or three of 2-iodobenzoic acid, 3-iodobenzoic acid and 4-iodobenzoic acid.

Preferably, the invention also provides an anti-CVB 3 medicament, comprising L₁、L₂、L₃Namely one, two or three of 2-iodobenzoic acid, 3-iodobenzoic acid and 4-iodobenzoic acid.

Furthermore, the medicine also comprises pharmaceutically acceptable auxiliary materials and carriers.

Furthermore, the medicinal preparation is granules, tablets, pills, capsules, injections or dispersing agents.

The invention has the beneficial effects that:

1. mono-iodo aromatic acids, i.e. L₁、L₂、L₃Can inhibit cytopathic effect (CPE) generated by CVB3 in host cell Hep-2, enhance cell survival rate, greatly reduce progeny virus yield, and has high therapeutic index.

2. Mono-iodo aromatic acids, i.e. L₁、L₂、L₃The compound has a remarkable inhibition effect on CVB3, has a better anti-CVB 3 virus effect than ribavirin, is completely different from ribavirin in chemical structure, and probably has a completely different action mechanism.

3. The monoiodo aromatic acid is a non-nucleoside drug, has a simple structure and is easy to synthesize.

4. Monoiodo aromatic acids have the potential to be further developed into drugs effective in the treatment of CVB3 infection.

Drawings

FIG. 1 shows monoiodo aromatic acids L at various concentrations₁、L₂、L₃Graph of the results of the Hep-2 cell survival effect on CVB3 effect;

FIG. 2 is L₃Inhibition effect pattern on CPE of Hep-2 cells caused by CVB 3.

FIG. 3 is L₃Results for inhibition of virus production by CVB3 progeny.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

L₁、L₂、L₃Are all obtained commercially.

Example 1: monoiodo aromatic acid L₁、L₂、L₃Toxicity to host Hep-2 cells

Hep-2 cells were plated in 96-well plates at 37 ℃ with 5% CO₂Culturing in incubator until the culture medium grows to a monolayer, discarding cell culture solution, and adding L with different concentrations₁、L₂、L₃The cell maintenance solution is continuously cultured, after 48 hours, the cytotoxicity is visually observed and respectively recorded by a microscope, and the cell survival rate is measured by an MTT method. The MTT method comprises the following specific steps: MTT 30. mu.L (5 mg. multidot.mL) was added to each well^-1) After incubation for 3-4h, the supernatant was removed and 50. mu.L of DMSO was added to dissolve the pellet. The absorbance (OD) at 492nm was read with a microplate reader₄₉₂Value).

The Median cytotoxic concentration of the drug on the cells (CC 50) was calculated using SPSS 11.5 software.

Cell survival rate ═ (mean OD of drug groups)₄₉₂Value/cell control mean OD₄₉₂Value) × 100%

Example 2: monoiodo aromatic acid L₁、L₂、L₃Inhibitory Activity against CVB3

Hep-2 cells were plated in 96-well plates at 37 ℃ with 5% CO₂After the culture was completed in the incubator to a full monolayer, the culture medium was discarded, cells were infected with CVB3 virus solution of 100TCID50 for 1 hour, and Compound L was added at different concentrations (2.5. mu.g/mL, 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 40. mu.g/mL, 80. mu.g/mL)₁、L₂、L₃(ribavirin as a positive control drug) cells were incubated. After the culture is continued for about 48 hours, the cytopathic effect (CPE) is observed under a microscope when about 90% of CPE lesions appear in the virus control wells. Observation and recording method of CPE: no cytopathic effect is recorded as-below 25% cytopathic effect, 25% -50% cytopathic effect is recorded as +++, 50% -75% cytopathic effect is recorded as +++, and more than 75% cytopathic effect is recorded as ++++.

After the CPE is observed, the inhibition rate of the drug on the CVB3 is detected by using an MTT method. The method comprises the following specific steps: MTT 50. mu.L (5 mg. multidot.mL) was added to each well^-1) And after incubation for 3-4h, removing supernatant, and adding DMSO with the same volume to dissolve the precipitate. The absorbance (OD) at 492nm was read with a microplate reader₄₉₂Value).

The half effective Concentration of the drug (Concentration for 50% of maximum effect, EC50) was calculated using SPSS 11.5 software.

The monoiodo aromatic acid L was calculated using the following formula₁、L₂、L₃Inhibition of CVB 3.

Monoiodo aromatic acid L₁、L₂、L₃Therapeutic Index (TI)

TI CC50/EC 50. A higher therapeutic index indicates greater antiviral potential.

The monoiodo aromatic acid L is respectively subjected to cell survival rate detection by combining cytopathic effect analysis and MTT (methyl thiazolyl tetrazolium) determination method₁、L₂、L₃The anti-CVB 3 activity was evaluated and the results are shown in table 1, figures 1 and 2.

Monoiodo aromatic acid L₁、L₂、L₃The results of the cytotoxicity and anti-CVB 3 activity test are shown in table 1.

TABLE 1 Monoiodo aromatic acid L₁、L₂、L₃Cytotoxicity and anti-CVB 3 Activity

Concentration dependent monoiodo aromatic acid L₁、L₂、L₃The effect of Hep-2 cell viability on the effect of CVB3 is shown in FIG. 1. The results show that the invention detects the aromatic acid L except monoiodo₁Outer, L₂、L₃Has certain inhibitory activity on CVB3, wherein L₃The compound shows the best inhibition effect, is superior to a positive control compound ribavirin, and has higher therapeutic index than ribavirin.

Monoiodo aromatic acid L₃The effect of inhibiting cpp 2 cell CPE by CVB3 is shown in fig. 2. The CVB 3-infected Hep-2 cells rounded off from the cell plate wall, while the 50. mu.g/mL L3-treated CVB 3-infected Hep-2 cells grew well, approaching the morphological characteristics of the virus-free infected cell control group. The L3 has good inhibition effect on cytopathy caused by CVB3 infection, and L₃Shows excellent anti-CVB 3 activity.

Example 3: monoiodo aromatic acid L₃Inhibition of viral production in progeny of CVB3

Hep-2 cells in logarithmic growth phase are plated on 24-well plates and 100TCID after growing in monolayer₅₀CVB3 infected cells, incubated at 37 ℃ for 1.5h and transferredThe virus solution was removed, washed three times with PBS, and cell maintenance solutions containing 50. mu.g/mL L3 were added. Collecting cells and supernatant culture fluid at 12h and 36h respectively, freeze thawing at-20 deg.C and 37 deg.C for three times, and lysing to obtain TCID₅₀Methods CVB3 virus titers were determined.

The results are shown in fig. 3, the CVB3 virus control group already showed significant virus titer at 12h infection, and the virus titer rapidly increased by about 3.0log until 36h infection. And 50. mu.g/mL L₃The virus titer of the treated group is lower than that of the virus control group under the same time condition, the increase range is small in the period from 12h to 36h of virus infection, and the strongest inhibition effect is shown at 36 h. The compounds can strongly inhibit the replication and proliferation of viruses in cells.

As mentioned above, benzoic acid organic derivatives L₂、L₃Has stronger inhibition activity of CVB3, including inhibition of Hep-2 cytopathic effect caused by CVB3 virus, greatly reduces the yield of progeny virus, has higher therapeutic index, and is superior to a control medicament ribavirin. The two compounds are proved to have potential application in preparing the medicine for effectively resisting CVB3 infection clinically.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims (9)

1. The application of monoiodo aromatic acid as a CVB3 virus inhibitor is characterized in that the monoiodo aromatic acid comprises 2-iodobenzoic acid, 3-iodobenzoic acid and 4-iodobenzoic acid.

2. Use according to claim 1, wherein the monoiodo aromatic acid is 3-iodobenzoic acid or 4-iodobenzoic acid.

3. The use of claim 2, wherein the monoiodo aromatic acid is 4-iodobenzoic acid.

4. Use according to claim 1, comprising the use of a monoiodo aromatic acid for the preparation of a medicament against CVB3 virus.

5. The use of claim 1, comprising the combination of ribavirin with any one, any two or three of 2-iodobenzoic acid, 3-iodobenzoic acid, and 4-iodobenzoic acid.

6. The use of claim 1, comprising a combination of any two or three of 2-iodobenzoic acid, 3-iodobenzoic acid, and 4-iodobenzoic acid.

7. An anti-CVB 3 drug is characterized by comprising one, two or three of 2-iodobenzoic acid, 3-iodobenzoic acid and 4-iodobenzoic acid.

8. The medicament of claim 7, wherein: also comprises pharmaceutically acceptable auxiliary materials and carriers.

9. The pharmaceutical according to claim 7, wherein the pharmaceutical formulation is a granule, tablet, pill, capsule, injection or dispersion.

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