CN1687033A - A kind of anti-respiratory virus medicine and application - Google Patents

Abstract

The invention discloses a compound of a general formula , wherein HO-represents hydroxyl, Br-represents bromine, CH₃-represents methyl, CH₂N(CH₃) ₂-represents a dimethylaminomethyl group, COOC₂H₅-ethyl hydroxyacid. With the compoundThe medicine composition with the active component has obvious antiviral effect on Coxsackie B3 virus, adenovirus Ad7, parainfluenza virus and rhinovirus, and the application of the medicine in preparing medicine for resisting Coxsackie virus, adenovirus Ad7, parainfluenza virus and rhinovirus.

Description

A kind of anti-respiratory virus medicine and application

technical field

The invention relates to a new antiviral drug Arbidol hydrochloride and the anti-multiple respiratory virus effects of the drug. Specifically, the present invention belongs to a new compound with good antiviral activity, and the application of the compound in the preparation of anti-coxsackie virus B3, adenovirus Ad7, parainfluenza virus and rhinovirus drugs.

Background technique

Influenza and acute respiratory virus infection are diseases with a high clinical incidence and a wide range of diseases. Due to the antigenic variability of influenza virus and its multi-drug resistance, a safe and effective drug for treatment and prevention is urgently needed. Chemical drugs play an extremely important role in the treatment of influenza and ARVI. So far, amantadine and amantadine derivative rimantadine have been widely used clinically as antiviral drugs, but their activity range is narrow (only effective against influenza A virus), and they have side effects. Its drug resistance also increases at the same time. Arbidol hydrochloride is a non-nucleoside antiviral drug, which was first developed by the Medicinal Chemistry Research Center of the former Soviet Union. It was first listed in Russia in 1993. It is a new antiviral drug and immunostimulant, used to prevent and treat influenza and For other acute respiratory virus infections, it has the most significant antiviral activity against influenza A and B viruses. The drug can inhibit the early stage of replication of influenza A and B viruses, and can inhibit the fusion of virus and cell membrane as well as virus and endocytosis Membrane fusion between vesicles (PH=5.0). At the same time, Arbidol hydrochloride can also induce the production of interferon in the body, which has a significant activation effect on the phagocytosis of macrophages and has an immune regulation effect. However, the effects of the drug on anti-Coxsackievirus B3, adenovirus Ad7, parainfluenza virus, and rhinovirus have not been reported or discovered yet.

Contents of the invention

The object of the present invention is to provide a compound with medicinal value-arbidol hydrochloride, which has obvious curative effect and prevention on influenza A and B, is convenient to take and well tolerated.

Another object of the present invention is to provide an application of Arbidol hydrochloride in the preparation of anti-coxsackie virus, adenovirus Ad7, parainfluenza virus and rhinovirus drugs.

A new antiviral drug--Arbidol Hydrochloride (Arbidol Hydrochloride), the chemical name is 6-bromo-4-(dimethylaminomethyl)-5-hydroxyl-1-methyl-2-(phenylthiomethyl) )-1H-indole-3-hydroxy acid ethyl ester hydrochloride-hydrate, white crystalline powder, molecular weight 531.89, does not contain additional compounds and other impurities affecting biological activity. Its chemical structural formula is as follows:

Where HO- means hydroxyl, Br- means bromine, CH ₃ - means methyl, CH ₂ N(CH ₃ ) ₂ - means dimethylaminomethyl, COOC ₂ H ₅ -hydroxy acid group, HCL means hydrochloric acid, -CH ₂ S- means thiomethyl, and H ₂ O means water.

Arbidol hydrochloride has pharmacodynamic effects against coxsackie virus B3, adenovirus Ad7, parainfluenza virus, and rhinovirus, and has obvious antiviral effect in antiviral biosynthesis.

Starting from 2 and 3, the target compound 1 was prepared through addition, cyclization, bromination, acylation, substitution and Mannich reaction. The total reaction yield is 26.6%. The synthetic technical route is as follows:

Arbidol hydrochloride is a water-soluble drug, but its solubility is general, and DMSO (dimethyl sulfoxide) can promote its dissolution. Factors such as whether it contains the solubilizing agent DMSO, the method of drug sterilization, and the time of drug action on cells affect the toxicity of Arbidol hydrochloride to HEp-2 cells (human throat cancer cells) and HEL cells (human embryonic lung cells). The toxic effects of drugs on cells are manifested as increased refraction, wall thickness, rounding, fragmentation, shedding, and a decrease in light absorption. The toxic effect of drugs on cells is within a certain range. With the increase of drug concentration, the cell survival rate decreases.

Arbidol hydrochloride has an anti-Coxsackie virus effect in vitro, and the anti-Coxsackie virus biosynthesis group of Arbidol hydrochloride can significantly reduce the CPE degree of CVB3-infected cells. With the increase of the drug dose, the cells shrink, become round, CPE characteristics such as shedding and fragmentation gradually weakened. Arbidol hydrochloride can reduce the virus titer in the supernatant, the virus inhibition rate is positively correlated with the drug concentration, and the virus titer is negatively correlated with the drug concentration. Arbidol hydrochloride anti-CVB3 index (TI) drug antiviral biosynthesis group > drug antiviral adsorption 8h group > drug direct inactivation group.

Arbidol hydrochloride can significantly inhibit the biosynthesis of adenovirus Ad7, which is manifested in: with the increase of drug concentration, typical CPE such as cell swelling and rounding, and enhanced refractive index gradually weakens, and the virus inhibition rate increases significantly. Within a certain range of drug concentration, with the increase of drug concentration, the inhibition rate of Ad7 increases, and shows a dose-effect relationship. Arbidol hydrochloride has neither blocking (preventing) effect nor direct killing effect on adenovirus invasion.

Arbidol hydrochloride has an anti-parainfluenza virus effect in vitro and can significantly interfere with virus biosynthesis. With the increase of drug concentration, typical CPE such as cell swelling, rounding, and syncytium formation gradually weaken, and the virus inhibition rate increases significantly. And within a certain concentration range, with the increase of drug concentration, the inhibition rate of RSV increases, and shows a dose-effect relationship. Arbidol hydrochloride can prevent the adsorption and penetration of RSV virus into cells, and the curative effect of pretreatment for 6 hours is obviously stronger than that of pretreatment for 2 hours. Arbidol hydrochloride has no direct killing effect on parainfluenza virus.

Arbidol hydrochloride has a significant inhibitory effect on the proliferation of rhinovirus in HEL cells. With the increase of drug concentration, the CPE characteristics such as cell shrinkage, rounding, shedding, and enhanced refraction gradually weaken, and the virus inhibition rate increases. , the antiviral activity of the drug was enhanced, and there was a positive correlation between the drug concentration and the virus inhibition rate.

Application of Arbidol hydrochloride in the preparation of anti-coxsackie virus, adenovirus Ad7, parainfluenza virus, rhinovirus and other acute respiratory virus infection drugs.

Arbidol hydrochloride can be made into film-coated tablets, capsules, granules, and dispersions for oral administration. In preparing the above formulations, conventional formulation techniques can be used. The drug component can be added to the drug for the treatment of acute respiratory virus infection and its complications or used in combination during the treatment. According to foreign research reports, Arbidol hydrochloride was used as an antiviral drug for clinical research during the influenza outbreak from 1983 to 1990, including a total of 9,500 patients. Take 0.1g of Arbidol coated tablets for adults as a medicine for treating and preventing influenza and other acute respiratory virus infections caused by A and B viruses. For the treatment of influenza, Arbidol recommends taking 0.2g orally, before meals, 4 times a day for 3 consecutive days. For people in contact with critically ill patients, it is recommended to take 0.2g orally before meals, once a day, for 5 to 10 consecutive days to prevent influenza. For the prevention of acute respiratory diseases during the flu period, Arbidol recommends taking 0.1g orally before meals, twice a week for 20 consecutive days. Arbidol has shown good drug resistance in clinical studies involving more than 9,000 patients; no side effects have been reported.

Compared with the prior art, the present invention has the following advantages and effects: 1. It is effective for influenza A and B viruses. 2. It has both therapeutic and preventive effects; 3. It has dual functions of directly inhibiting the virus and inducing endogenous interferon. 4. It can be administered orally and is convenient to use. 5. It is a non-nucleoside compound with low toxicity and good tolerance.

Detailed ways

Embodiment 1: Arbidol hydrochloride is to the cytotoxicity of HEp-2 cell and HEL cell

Use trypsin to disperse the well-growing HEp-2 cells and HEL cells into a single cell suspension, and divide them into 96-well plates at a concentration of 1×10 ⁵ /ml, 0.1ml per well, and culture at 37°C and 5% CO ₂ Cultivate in the box for 24h. After the cells grow into a single layer, the supernatant of the culture medium is discarded and replaced with a drug-containing maintenance solution of different concentrations. Four replicate wells are set for each concentration, and normal cells are set as a control. Continue culturing for 48 hours, discard the culture supernatant, add 50 μl of serum-free MEM medium containing 5 mg/ml MTT (tetramethylazozolium salt) to each well, place in a _CO2 incubator and continue culturing for 2 to 3 hours, then discard the MTT Wash the supernatant three times with PBS, add 100 μl of dissolving solution (DMSO:ethanol volume ratio: 1:1) to each well, shake for 5-10 minutes, read the OD value at the wavelength of 570 nm on a microplate reader, and calculate the cell density according to the following formula: For the survival rate, find out the half toxic concentration (TD50) and the maximum nontoxic concentration (TD ₀ ) of the drug on the cells.

Cell survival rate (%)=(OD value of experimental well/OD value of control group)×100%

The toxic effect of the drug on the two kinds of cells is manifested as an increase in refraction, wall thickness, rounding, fragmentation, shedding, and a decrease in light absorption. The toxic effect of drugs on cells is within a certain range. With the increase of drug concentration, the cell survival rate decreases. The results are shown in the table below

Table 1 Toxic effect of Arbidol hydrochloride on HEp-2 cells and HEL cells under different sterilization methods Experimental group Cell viability (%) at different drug concentrations (μg/mL) TC ⁵⁰ 10 20 40 80 100 160 250 320 500 640 1000 1 group 2 groups 3 groups 4 groups 117102.499.0589 6992.8084.3273 7080.6060.8080 63-44.1867 -81.07-- 26-41.8039 -70.4-- 32--40 -69.33-- 28--43 -51.2-- 199.2986.5485.39464.5

- Indicates that individual concentrations are not done

Group 1 Arbidol hydrochloride was made into an aqueous solution with dimethyl sulfoxide (DMSO) and double distilled water (V:V: 1:1), and its toxicity to HEp-2 cells after filter sterilization.

Two groups of Arbidol hydrochloride were dissolved in dimethyl sulfoxide (DMSO) and double distilled water (V:V 1:9), and the toxicity to HEp-2 cells after filter sterilization.

Three groups of Arbidol hydrochloride were dissolved in dimethyl sulfoxide (DMSO) and double distilled water (V:V: 1:9), and the toxicity to HEp-2 cells after 8 lbs 15 min high pressure.

The toxicity of 4 groups of Arbidol hydrochloride to HEL cells after making water with ddH2O and filter sterilization.

Embodiment 2: Arbidol hydrochloride anti-Coxsackie virus effect in vitro

It is proposed that the effect of Arbidol hydrochloride on CVB3 is divided into four groups. Group I (drug antiviral biosynthesis group): first add virus liquid to adsorb cells for 2 hours, then add drug-containing maintenance liquid. Group II (drug direct action group): the virus solution reacted with different concentrations of drugs for 2 hours, and then the mixed solution was incubated with the cells for 2 hours, and then the cell maintenance solution was replaced. Groups III and IV (drug antiviral adsorption 2h, 8h groups): After adding different concentrations of drugs into the cells for 2h and 8h respectively, the drug solution was discarded, and the virus solution was added, and the cell maintenance solution was replaced after 2 hours of adsorption.

Four replicate wells were set in each well of the above four experimental groups, and uninfected cell control, virus control and solvent control were set up at the same time. Culture at 35°C, 5% CO2, and observe cell changes under an inverted microscope every day. When the degree of cytopathic effect (CPE) of the virus control well is +++～++++, and the control cells are normal, the MTT method is used to stain the living cells, and the OD value is read at a wavelength of 570nm, and the different concentrations are judged according to the OD value The size of the drug's inhibitory effect on the virus, the formula is as follows:

Get the serial logarithmic dilution of the culture supernatant of experimental well, virus control well and cell control well, measure TCID50Probit regression method to calculate drug half poisoning concentration (TD50) and half effective concentration (ED50), therapeutic index (TI)=drug half poisoning Concentration (TD50)/half effective concentration (ED50), the TCID50 of the virus was calculated by the Reed-Muench method, and the pairwise comparison between different groups was carried out by the LSD method to determine whether the difference was significant, and the data was processed using SPSS 11.5 software Finish.

Experiments show that the Arbidol hydrochloride antiviral biosynthesis group can significantly reduce the degree of CPE of CVB3-infected cells. In the Arbidol antiviral biosynthesis group, with the increase of the drug dose, the cells shrink, become round, fall off, and fragment, etc. CPE features gradually weakened. Arbidol can reduce the virus titer in the supernatant, the virus inhibition rate is positively correlated with the drug concentration, and the virus titer is negatively correlated with the drug concentration. The DMSO control group showed that DMSO had no antiviral effect. Arbidol hydrochloride anti-CVB3 index (TI) drug antiviral biosynthesis group > drug antiviral adsorption 8h group > drug direct inactivation group, indicating that the drug mainly exerts antiviral effect by inhibiting virus biosynthesis.

Table 2 Comparison of virus inhibition rate of Arbidol hydrochloride in different modes of action against CVB3 Experimental group Drug Concentration (μg/mL) ^ED50 (μg/mL) Ti 5 10 20 40 80 160 Group I ① 0 44.63 46.83 73.28 75.48 66.67 46.48 (p<0.01) 19.29 ② ^10-11.3 10 ^-6.3 10 ^-5.8 10 ^-2.3 10 ^-2.3 10 ^-4.3 Group II ① 0 12.35 35.80 41.98 45.68 45.68 138.59 (p<0.01) 6.47 ② ^{10-11.3 10-11.3} 10 ^-8.8 10 ^-6.3 10 ^-6.3 10 ^-6.3 Group III ① 17.89 20.64 27.98 27.06 28.90 27.06 578.44 (p>0.05) 1.55 ② ^{10-11.3 10-11.3 10-11.3 10-11.3 10-11.3 10-11.3} Group IV ① 20.53 24.80 40.80 47.73 48.27 44.53 134.10 (p<0.01) 6.69 ② ^{10-11.3 10-11.3} 10 ^-5.8 10 ^-6.3 10 ^-5.8 10 ^-6.3

Group I: Drug antiviral biosynthesis group; Group II: Drug direct action group; Group III: Drug antiviral adsorption group for 2 hours; Group IV: Drug antiviral adsorption group for 8 hours; ①: Virus inhibition rate; ②: Supernatant virus drops Spend

Embodiment 3: Arbidol hydrochloride anti-adenovirus Ad7 effect in vitro

It is proposed that the effect of Arbidol hydrochloride on adenovirus Ad7 is divided into four groups. Group I (drug antiviral biosynthesis group): first add virus liquid to adsorb cells for 2 hours, then add drug-containing maintenance liquid. Group II (drug direct action group): the virus solution reacted with different concentrations of drugs for 2 hours, and then the mixed solution was incubated with the cells for 2 hours, and then the cell maintenance solution was replaced. Groups III, IV, and V (2h, 4h, and 8h groups of drug antiviral adsorption): add different concentrations of drugs into the cells for 2h, 4h, and 8h respectively, discard the drug solution, add virus solution, and absorb After 2 h, the cell maintenance solution was replaced.

Four replicate wells were set for each well of the above five groups of experimental groups, and uninfected cell control, virus control and solvent control were set up at the same time. Culture at 35°C, 5% CO2, and observe cell changes under an inverted microscope every day. When the degree of cytopathic effect (CPE) of the virus control well is +++～++++, and the control cells are normal, the MTT method is used to stain the living cells, and the OD value is read at a wavelength of 570nm, and the different concentrations are judged according to the OD value The size of the drug's inhibitory effect on the virus, the formula is as follows:

Probit regression method to calculate the half toxic concentration (TD50) and half effective concentration (ED50) of the drug, the therapeutic index (TI) = the half toxic concentration (TD50)/ half effective concentration (ED50) of the drug, and use LinearRegression to analyze the drug dose and its cellular effect (cell viability, inhibition rate) to conduct correlation analysis to determine whether there is a dose-effect relationship.

The experimental results show that Arbidol hydrochloride can significantly inhibit the biosynthesis of adenovirus Ad7, which is manifested in: with the increase of the drug concentration, the typical CPE such as cell swelling and rounding, and the refractive index enhancement gradually weakens, and the virus inhibition rate increases significantly. Within a certain range of drug concentration, with the increase of drug concentration, the inhibition rate of Ad7 increases, and shows a dose-effect relationship. Arbidol hydrochloride has neither blocking (preventing) effect nor direct killing effect on adenovirus invasion. The results are shown in the table below:

Table 3 Inhibitory effect of Arbidol hydrochloride on Ad7 biosynthesis Drug dosage (μg/mL) cytopathic effect Virus inhibition rate % _ED50 (μg/mL) Ti 51015202530 Cell Control Virus Control +++++++++++--++++ 26.1037.6150.3760.1565.5387.581000 15.39 4.26

++++: more than 75% cytopathic; +++: 75%~50% cytopathic; ++: 50%~25% cytopathic; +: less than 25% cytopathic; -: no cytopathic

Embodiment 4: Arbidol hydrochloride anti-parainfluenza virus effect in vitro

It is proposed that the effect of Arbidol hydrochloride on parainfluenza virus is divided into four groups. Group I (drug anti-low-titer virus biosynthesis group): add virus solution 10 ^-2 to adsorb cells for 2 hours, then add drug-containing maintenance solution. Group II (drug anti-viral biosynthesis group with high titer): add virus solution 10 ^-3 to adsorb cells for 2 hours, then add drug-containing maintenance solution. Groups III and IV (drug antiviral adsorption for 2h and 6h groups): After adding different concentrations of drugs into the cells for 2h and 6h respectively, the drug solution was discarded, and the virus solution was added, and the cell maintenance solution was replaced after 2h of adsorption.

Four replicate wells were set in each well of the above four experimental groups, and uninfected cell control, virus control and solvent control were set up at the same time. Culture at 35°C, 5% CO2, and observe cell changes under an inverted microscope every day. When the degree of cytopathic effect (CPE) of the virus control well is +++～++++, and the control cells are normal, the MTT method is used to stain the living cells, and the OD value is read at a wavelength of 570nm, and the different concentrations are judged according to the OD value The size of the drug's inhibitory effect on the virus, the formula is as follows:

Probit regression method to calculate the half toxic concentration (TD50) and half effective concentration (ED50) of the drug, the therapeutic index (TI) = the half toxic concentration (TD50)/ half effective concentration (ED50) of the drug, and use LinearRegression to analyze the drug dose and its cellular effect (cell viability, inhibition rate) to conduct correlation analysis to determine whether there is a dose-effect relationship.

The experimental results show that Arbidol hydrochloride has an anti-parainfluenza virus effect in vitro, and can significantly interfere with the biosynthesis of the virus. With the increase of the drug concentration, the typical CPE such as cell swelling, rounding, and syncytia formation gradually weakens, and the virus inhibition rate increases significantly. high. And within a certain concentration range, with the increase of drug concentration, the inhibition rate of RSV increases, and shows a dose-effect relationship. Arbidol hydrochloride can prevent the adsorption and penetration of RSV virus into cells, and the curative effect of pretreatment for 6 hours is obviously stronger than that of pretreatment for 2 hours. Arbidol hydrochloride has no direct killing effect on parainfluenza virus. The results are shown in the table below:

Table 4 Comparison of virus inhibition rate of Arbidol hydrochloride against parainfluenza virus in different modes of action Experimental group Drug Concentration (μg/mL) _ED50 (μg/mL) Ti 2 4 6 8 10 12.5 15 20 25 Group I Group II Group III Group IV Group 40.23--27.76 58.620-34.63 66.6710.67-46.30 70.1118.970- -20.55-52.14 -28.592.6878.60 81.6050.195.60- -50.5936.9877.56 --40.39- 2.4617.6425.188.00 34.714.843.3910.67

Group I: drug resistance to low titer virus biosynthesis group; Group II: drug resistance to high titer virus biosynthesis group; Group III: drug resistance to low titer virus adsorption group for 4 hours; Group IV drug resistance to low titer virus adsorption group for 6 hours

Embodiment 5: Inhibitory effect of Arbidol hydrochloride on rhinovirus proliferation in vitro

Infect the cells with 100TCID ₅₀ /0.1ml rhinovirus 0.1ml per well, absorb the virus for 2 hours, add different concentrations of drugs, set 4 replicate wells for each well dose, and set up uninfected cell control, virus control and Solvent control. Culture at 35°C, 5% CO2, and observe cell changes under an inverted microscope every day. When the degree of cytopathic effect (CPE) of the virus control well is +++～++++, and the control cells are normal, the MTT method is used to stain the living cells, read the OD value at a wavelength of 570nm, and judge the different concentrations according to the OD value The size of the drug's inhibitory effect on the virus, the formula is as follows:

Probit regression method to calculate the half toxic concentration (TD50) and half effective concentration (ED50) of the drug, the therapeutic index (TI) = the half toxic concentration (TD50)/ half effective concentration (ED50) of the drug, and use LinearRegression to analyze the drug dose and its cellular effect (cell viability, inhibition rate) to conduct correlation analysis to determine whether there is a dose-effect relationship.

Arbidol hydrochloride has a significant inhibitory effect on the proliferation of rhinovirus in HEL cells. With the increase of drug concentration, the CPE characteristics such as cell shrinkage, rounding, shedding, and enhanced refraction gradually weaken, and the virus inhibition rate increases. , the antiviral activity of the drug was enhanced, and there was a positive correlation between the drug concentration and the virus inhibition rate. The results are shown in the table below:

Table 5 Anti-rhinovirus effect of Arbidol hydrochloride Drug Concentration (μg/mL) ED50(μg/mL) 8 16 32 64 128 Virus suppression rate (%) 14 19 57 58 157 44.4

Claims (5)

1, following general formula (I) represents the compound of Arbidol hydrochloride: where HO- stands for hydroxyl Br- means bromine CH ₃ - means methyl CH ₂ N(CH ₃ ) ₂ - means dimethylaminomethyl -COOC ₂ H ₅ -Ethyl hydroxyate -CH ₂ S- means thiomethyl HCL means hydrochloric acid H ₂ O means water 2. The use of Arbidol hydrochloride in claim 1 in the preparation of anti-Coxsackievirus drugs. 3, the application of Arbidol hydrochloride in claim 1 in the preparation of anti-adenovirus Ad7 medicine 4. The application of Arbidol hydrochloride in claim 1 in the preparation of anti-parainfluenza virus medicine. 5. The application of Arbidol hydrochloride in claim 1 in the preparation of anti-rhinovirus drugs.