CN111138498B - Application of disubstituted aromatic acid modified Anderson polyacid as coxsackie virus inhibitor - Google Patents

Abstract

The invention discloses application of disubstituted aromatic acid modified Anderson polyacid as a coxsackie virus inhibitor. Through the research experiment on the anti-CVB 3 activity of a plurality of double-substituted aromatic acid modified Anderson polyacids, the double-substituted aromatic acid modified Anderson polyacids show certain inhibitory activity to CVB3 viruses, including inhibiting cytopathic effect (CPE) generated by the CVB3 on host cell Hep-2 and enhancing cell survival rate, and the double-substituted aromatic acid modified Anderson polyacids have inhibitory effect on the CVB3 viruses, which indicates that the double-substituted aromatic acid modified Anderson polyacids have potential application in preparing anti-CVB 3 virus drugs.

Description

Application of Disubstituted Aromatic Acids Modified Anderson's Polyacids as Coxsackie Virus Inhibitors

technical field

The invention relates to the technical field of antiviral drugs, in particular to the application of a disubstituted aromatic acid modified Anderson polyacid as a Coxsackie virus inhibitor.

Background technique

Coxsaekievirus (CV) is a member of the Enterovirus genus of Picornaviridae, and its infection can cause a variety of diseases, such as hand, foot and mouth disease, aseptic meningitis, encephalitis, myocarditis , epidemic myositis, herpetic angina, etc. A total of 29 serotypes of CV have been reported. According to their pathogenic characteristics and cell susceptibility to suckling mice, they can be divided into two groups, A and B, namely CVA (CVA1-22, 24) and CVB ( CVB1-6). The infection of CVBs is the most common, among which CVB3 is the most pathogenic type among the six serotypes of CVB, and is the main pathogenic cause of viral myocarditis. According to the US Centers for Disease Control and Prevention (CDC) statistics, CVB (types 1-6) can cause about 5 million people to suffer from intestinal diseases every year, of which 10%-20% are acute myocarditis caused by CVB3. In recent years, the trend of HFMD caused by CVB3 is also on the rise, and there are many reports of CVB3-induced disease epidemic in my country. At present, there is no effective drug for coxsackie virus infection, and there is no targeted clinical treatment. Many researchers have found many compounds that inhibit CVB3 activity in vitro and in vivo, but these are still in the initial stage of laboratory experiments and are still far from practical clinical application. Therefore, it is imperative to develop specific and effective anti-CVB3 drugs.

Iodoaromatic acids are a class of biologically active organic compounds. For example, p-iodobenzoic acid is a potent inhibitor of cinnamate-4-hydroxylase (CINNAMATE 4-HYDROXYLASE), which is a key enzyme in the synthesis of lignin building blocks in the phenylpropane-like pathway (Dorien Van de Wouwer, et.al.Plant Physiology, 2016, 172, 198-220); metal salts of meta-iodobenzoic acid showed good inhibitory activity against Saccharomyces cerevisiae, Hansenula anomalies, 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 (Yan Jiqiong, et.al. Plant Physiological Communications, 1958, 3, 27-30). 3,5-bis(acetamido)-2,4,6-triiodobenzoic acid, also known as diatrizoic acid, is an important contrast agent, formulated as sodium diatrizoate, meglumine diatrizoate Injection, can be used for urinary system, cardiovascular, cerebrovascular and peripheral angiography (I Charles, et.al. 1986, US4567034). However, the antiviral activity of iodocarboxylic acid, including the inhibitory activity against CVB3 virus, has not been reported so far.

Polyoxometalates are polyanion clusters with specific structure and composition formed by condensation of oxygen-containing metal salts of vanadium, molybdenum, tungsten, niobium, tantalum and other pre-transition metals under certain conditions. Acids (POMs). Due to the wide variety of polyacids, their composition is rich and changeable, and they have unique physical, chemical, physiological and pharmacological activities, as well as rich and changeable physical properties such as light, electricity, and magnetism. They are widely used in nanoscience, materials, catalysis and medicinal chemistry The field has broad application prospects. In 1971, Raynaud et al. reported the inhibitory activity of [SiW ₁₂ O ₄₀ ] ^4- on mouse leukemia sarcoma virus (MLSV) (M.Raynaud, et.al.CRAcad.Sci.Hebd.Seances Acad.Sci.D 1971 , 272, 347). In 1985, French scientists discovered that (NH ₄ ) ₁₇ Na[NaSb ₉ W ₂₁ O ₈₆ ] (HPA-23) had an inhibitory effect on HIV reverse transcriptase (Dormont D., et.al.Ann.Inst.Pastour/Virol, 1985, 136E, 75). In 1988, Professor YAMASE from Japan found that (i-PrNH ₃ ) ₆ [Mo ₇ O ₂₄ ]·3H ₂ O(PM-8) exhibited good antitumor activity (Toshihiro Yamase, Inorg.Chem.Acta.1988,151 , 15-18).

However, there is no polyoxometalate derivative with anti-CV activity in the prior art, therefore, it is necessary to provide a polyoxometalate derivative with anti-CV activity, especially anti-CVB3 activity.

SUMMARY OF THE INVENTION

The purpose of the present invention is to develop new specific and effective anti-CVB3 drugs for the deficiencies of the prior art. In the present invention, through numerous screening experiments and verification by a large number of biological experiments, it is found that the disubstituted aromatic acid modified Anderson polyacid can inhibit the cytopathic effect (CPE) produced by CVB3 in the host cell Hep-2, and enhance the cell survival rate. , showed an inhibitory effect on CVB3 and a high therapeutic index, suggesting that it has the potential to be further developed into an effective drug for the treatment of CVB3 infection. Based on the discovery, the present invention provides an application of a disubstituted aromatic acid for modifying Andersen's polyacid.

The present invention provides an application of a disubstituted aromatic acid modified Anderson polyacid as a coxsackie virus inhibitor. The disubstituted aromatic acid modified Anderson polyacid includes A ₄ and A ₅ , wherein the molecular formula of A ₄ is (TBA) ₃ [ MnMo ₆ O ₁₈ ((OCH ₂ ) ₃ CNHCOC ₆ H ₃ -3-I-4-NH ₂ ) ₂ ], the cation is TBA, and the structural formula of the anion is as follows:

The molecular formula of A ₅ is (TBA) ₃ [MnMo ₆ O ₁₈ ((OCH ₂ ) ₃ CNHCO C ₆ H ₄ -3,4-I ₂ ) ₂ ], the cation is TBA, and the structural formula of the anion is as follows:

In the molecular formula, TBA is [(N(C ₄ H ₉ ) ₄ )] ⁺ .

Preferably, the coxsackie virus is of the B3 subtype, ie CVB3 virus.

More preferably, the disubstituted aromatic acid-modified Anderson polyacid is A ₅ .

Further, the application of the disubstituted aromatic acid modified Anderson polyacid as a coxsackie virus inhibitor, including the disubstituted aromatic acid modified Anderson polyacid and/or its pharmaceutically acceptable salt in the preparation of a drug against coxsackie virus Applications.

Further, the application of the disubstituted aromatic acid-modified Anderson polyacid as a coxsackie virus inhibitor also includes the combined use of the disubstituted aromatic acid-modified Anderson polyacid and/or a pharmaceutically acceptable salt thereof and ribavirin.

Further, the application of the disubstituted aromatic acid _- modified Anderson polyacid as a coxsackie virus inhibitor also includes the combination of A4 and _A5 .

The present invention also provides an anti-Coxsackie virus drug, comprising one or both of A ₄ , A ₅ or a pharmaceutically acceptable salt thereof.

Preferably, the present invention also provides a drug against Coxsackie virus B3 subtype, which is characterized by comprising one or both of A ₄ , A ₅ or a pharmaceutically acceptable salt thereof.

Further, the medicine also includes pharmaceutically acceptable excipients and carriers.

Further, the pharmaceutical preparation is granules, tablets, pills, capsules, injections or dispersions.

The beneficial effects of the present invention are:

1. The modification of Anderson polyacids with disubstituted aromatic acids, especially A ₄ and A ₅ , can inhibit the cytopathic effect (CPE) produced by CVB3 in the host cell Hep-2, and enhance the cell survival rate.

2. Disubstituted aromatic acids modified Anderson polyacids, especially A ₄ and A ₅ have significant inhibitory effect on CVB3, and have better anti-CVB3 virus effect than ribavirin, but the chemical structure is completely different from ribavirin , likely to have a completely different mechanism of action from ribavirin.

3. The double-substituted aromatic acid modified Anderson polyacid is a non-nucleoside drug, which is easy to synthesize.

4. Andersen's polyacid modified with disubstituted aromatic acids has the potential to be further developed into an effective drug for the treatment of CVB3 infection.

Description of drawings

Fig. 1 is a graph showing the effect of different concentrations of disubstituted aromatic acids modified Anderson polyacids A ₄ and A ₅ on the survival rate of Hep-2 cells under the action of CVB3;

Figure 2 is a graph showing the inhibitory effects of A ₄ and A ₅ on CVB3-induced CPE in Hep-2 cells.

Figure 3 is a graph showing the results of the inhibitory effect of A5 _on the viral yield of CVB3 progeny.

Detailed ways

The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be construed as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

Example 1: Preparation of A ₄ and A ₅

The molecular formula of the disubstituted aromatic acid modified Anderson polyacid A ₄ is (TBA) ₃ [MnMo ₆ O ₁₈ ((OCH ₂ ) ₃ CNHCOC ₆ H ₃ -3-I-4-NH ₂ ) ₂ ], wherein TBA is [( N(C ₄ H ₉ ) ₄ )] ⁺ , the cation is TBA, and the structural formula of the anion is as follows:

Referring to the method of the document Inorg.Chem. (2016, 55, 9497–9500), the corresponding amide ligand is prepared by reacting trihydroxymethylaminomethane with 3-iodo-4-aminobenzoyl chloride, and then the prepared The amide ligand reacts with octamolybdic acid and trivalent manganese acetate under reflux, and the obtained filtrate is diffused with ether to obtain the Anderson polyacid derivative A ₄ modified by monoiodobenzoic acid.

The molecular formula of A ₅ is (TBA) ₃ [MnMo ₆ O ₁₈ ((OCH ₂ ) ₃ CNHCO C ₆ H ₄ -3,4-I ₂ ) ₂ ], wherein TBA is [(N(C ₄ H ₉ ) ₄ )] ⁺ , the cation is TBA, and the structural formula of the anion is as follows:

Refer to the method of literature Inorg.Chem. (2016, 55, 9497-9500), specifically react with tris(hydroxymethyl)aminomethane and 3,4-diiodobenzoyl chloride to prepare the corresponding amide ligand, and then combine the prepared amide The ligand reacts with octamolybdic acid and trivalent manganese acetate under reflux, and the obtained filtrate is diffused with ether to obtain Anderson polyacid derivative A ₅ modified by monoiodobenzoic acid.

Example 2: Toxicity of Andersen's Polyacids A ₄ and A ₅ Modified by Disubstituted Aromatic Acids to Host Hep-2 Cells

Hep-2 cells were plated in a 96-well plate, cultured in a 37°C, 5% CO ₂ incubator until the monolayer became full, the cell culture medium was discarded, and cell maintenance solutions containing different concentrations of A ₄ and A ₅ were added to continue the culture. After 48h, the cytotoxicity was recorded by microscope and the cell viability was measured by MTT method. The specific steps of the MTT method are as follows: add 30 μL (5 mg·mL ^-1 ) of MTT to each well, remove the supernatant after incubation for 3-4 h, and add 50 μL of DMSO to dissolve the precipitate. The corresponding absorbance (OD ₄₉₂ value) was read at 492 nm with a microplate reader.

The Median cyctoxic concentration (CC50) of the drug to cells was calculated by SPSS 11.5 software.

Cell viability=(average OD ₄₉₂ value of drug group/average OD ₄₉₂ value of cell control group)×100%

Example 3: Inhibitory activity of Andersen's polyacids A ₄ and A ₅ modified by disubstituted aromatic acids on CVB3

The Hep-2 cells were plated in a 96-well plate, cultured at 37°C in a 5% CO ₂ incubator, and the culture medium was discarded. The cells were infected with CVB3 virus solution of 100 TCID50 for 1 h, and different concentrations (2.5 μg/mL) were added to the cells. , 5 μg/mL, 10 μg/mL, 20 μg/mL, 40 μg/mL, 80 μg/mL) of compounds A ₄ , A ₅ (ribavirin as a positive control drug) were incubated with cells. After culturing for about 48 hours, when about 90% of CPE lesions appeared in the virus control wells, the cytopathic effect (CPE) was observed under a microscope. CPE observation and recording method: no cytopathic lesions are recorded as -, 25% or less cytopathic lesions are recorded as +, 25%-50% cytopathic lesions are recorded as ++, 50%-75% cytopathic lesions are recorded as +++, more than 75% cytopathic lesions are recorded as +++ Cytopathies were recorded as ++++.

After the observation of CPE, the inhibitory rate of drugs on CVB3 was detected by MTT method. The specific steps are as follows: add 50 μL (5 mg·mL ^-1 ) of MTT to each well, remove the supernatant after incubating for 3-4 h, and add an equal volume of DMSO to dissolve the precipitate. The corresponding absorbance (OD ₄₉₂ value) was read at 492 nm with a microplate reader.

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

The following formulas were used to calculate the inhibition rates of CVB3 by the modified Anderson polyacids A ₄ and A ₅ with disubstituted aromatic acids.

Therapeutic Index (TI) of Andersen's Polyacid A ₄ , A ₅ Modified by Disubstituted Aromatic Acids

TI=CC50/EC50. The higher the therapeutic index, the greater the antiviral potential.

Combined with cytopathic effect analysis and MTT assay for cell viability detection methods, the anti-CVB3 activity of Andersen's polyacids A ₄ and A ₅ modified by disubstituted aromatic acids were evaluated respectively. The results are shown in Table 1, Figures 1 and 2.

Table 1 shows the test results of the cytotoxicity and anti-CVB3 activity of Andersen's polyacids A ₄ and A ₅ modified with disubstituted aromatic acids.

Table 1 Cytotoxicity and anti-CVB3 activity of Andersen's polyacids A ₄ and A ₅ modified with disubstituted aromatic acids

The effect of concentration-dependent disubstituted aromatic acid modification of Andersen's polyacids A ₄ and A ₅ on the survival rate of Hep-2 cells under the action of CVB3 is shown in Figure 1 . The results showed that both A ₄ and A ₅ of Andersen's polyacid modified with disubstituted aromatic acids had high inhibitory activity on CVB3, and A ₄ and A ₅ had low toxicity, CC50 was greater than 200μg/mL, and had a high therapeutic index. Among them, the inhibitory activity of A ₅ on CVB3 is better than that of A ₄ , the inhibitory effect of A 5 is better than that of the positive control drug ribavirin, and it has a higher therapeutic index than that of ribavirin.

Figure 2 shows the inhibition of CVB3-induced CPE in Hep2 cells by modification of Andersen's polyacids A ₄ and A ₅ with disubstituted aromatic acids. CVB3-infected Hep-2 cells became round and detached from the cell plate wall, while CVB3-infected Hep-2 cells treated with 50 μg/mL A ₄ and A ₅ grew well and were close to the morphological characteristics of the control group without virus infection. It indicated that A ₄ and A ₅ had a good inhibitory effect on the cytopathic effects caused by CVB3 infection, and further indicated that A ₄ and A ₅ showed excellent anti-CVB3 activity.

Example ₄ : Inhibition of CVB3 progeny virus production by benzoic acid derivative A5

Hep-2 cells in the logarithmic growth phase were plated in 24-well plates, and the cells were infected with 100 TCID ₅₀ CVB3 after they became monolayers. After incubating at 37°C for 1.5 h, the virus solution was removed, washed three times with PBS, and maintained by adding cells containing ₅₀ μg/mL A5. liquid. Cells and supernatant culture medium were collected at 12h and 36h, respectively, after three freeze-thaw lysis at -20°C and 37°C, CVB3 virus titer was determined by TCID ₅₀ method.

The results are shown in Figure 3, the CVB3 virus control group has shown obvious virus titer at 12h of infection, and until 36h of infection, the virus titer rises rapidly, and the increase is about 3.0log. However, the virus titer of the 50 μg/mL A ₅ treatment group was lower than that of the virus control group under the same time conditions. During the time period from 12h to 36h of virus infection, the increase was smaller, and the strongest inhibition was shown at 36h. effect. It shows that these compounds can strongly inhibit the replication and proliferation of virus in cells.

To sum up, the disubstituted aromatic acids modified Anderson polyacids A ₄ and A ₅ have certain inhibitory activity against CVB3, and the therapeutic index is high, indicating that compounds A ₄ and A ₅ have potential applications in the preparation of anti-CVB3 virus drugs. Among them, compound A ₅ has the best inhibitory effect, including inhibiting the cytopathic effect of Hep-2 caused by CVB3 virus, enhancing cell survival rate, and inhibiting the replication and proliferation of virus in cells.

Although the embodiments of the present invention have been shown and described above, it should be understood that the above-mentioned embodiments are exemplary and should not be construed as limiting the present invention. Embodiments are subject to variations, modifications, substitutions and variations.

Claims (10)

1. The application of the disubstituted aromatic acid modified Anderson polyacid in preparing the coxsackie virus inhibitor is characterized in that the disubstituted aromatic acid modified Anderson polyacid comprises A ₄ 、A ₅ Wherein A is ₄ Has a molecular formula of (TBA) ₃ [MnMo ₆ O ₁₈ ((OCH ₂ ) ₃ CNHCOC ₆ H ₃ -3-I-4-NH ₂ ) ₂ ]The cation is TBA, and the anion has the following structural formula:

A ₅ has a molecular formula of (TBA) ₃ [MnMo ₆ O ₁₈ ((OCH ₂ ) ₃ CNHCOC ₆ H ₄ -3,4-I ₂ ) ₂ ]The cation is TBA, and the anion has the following structural formula:

in the molecular formula, TBA is [ (N (C) ₄ H ₉ ) ₄ )] ⁺ 。

2. The use of claim 1, wherein the coxsackievirus is subtype B3.

3. The use according to claim 1 or 2, wherein the disubstituted aromatic acid modified anderson polyacid is a ₅ 。

4. Use according to claim 1 or 2, comprising the use of a disubstituted aromatic acid modified anderson polyacid and/or a pharmaceutically acceptable salt thereof in the preparation of a medicament against a coxsackievirus.

5. Use according to claim 1 or 2, comprising the combination of disubstituted aromatic acid modified anderson's polyacid and/or pharmaceutically acceptable salts thereof and ribavirin.

6. Use according to claim 1 or 2, characterized in that it comprises A ₄ And A ₅ The combination of (1).

7. A medicament for producing a coxsackievirus inhibitor, comprising A as defined in claim 1 ₄ 、A ₅ Or one or two of pharmaceutically acceptable salts thereof.

8. A medicament for the preparation of an inhibitor of Coxsackie virus subtype B3 comprising the A of claim 1 ₄ 、A ₅ Or one or both of pharmaceutically acceptable salts thereof.

9. The medicament of claim 7 or 8, further comprising pharmaceutically acceptable excipients and carriers.

10. Pharmaceutical according to claim 7 or 8, characterized in that the pharmaceutical formulation is a granule, tablet, pill, capsule or injection.

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