CN111728965A - Application of compounds in the preparation of antiviral drugs - Google Patents

Abstract

The present application discloses the application of compounds in the preparation of antiviral drugs. In an embodiment of the present application, the in vitro anti-herpes simplex virus type I activity of dihydromyricetin is verified by experiments, which can inhibit virus proliferation and related gene expression, and has low toxicity and can obtain a high therapeutic index. At the same time, it was confirmed that the mechanism of action of dihydromyricetin against herpes simplex virus is related to its inhibition of TLR9-dependent inflammatory response pathway. In an embodiment of the present application, it is disclosed that dihydromyricetin has potential value as a new type of anti-herpes simplex virus drug, and can be used in the preparation of anti-herpes simplex virus drugs.

Description

Application of compounds in the preparation of antiviral drugs

technical field

The invention belongs to the field of medicine, in particular to the application of compounds in the preparation of antiviral medicines.

Background technique

Herpes simplex virus belongs to the alphavirus subfamily of the family Herpesviridae, and is a DNA virus with a viral plasmid size of about 180 nanometers. The virus generally enters the body through the respiratory tract, genital mucosa and damaged skin, and resides in the normal human mucosa, blood, saliva and sensory ganglion cells. More than 90% of people in the population have been infected with herpes simplex virus. When the body's resistance decreases, the latent herpes simplex virus in the body is activated and the disease occurs. According to the difference in antigenicity, the virus is currently divided into type I and type II, and humans are its only host. Herpes simplex virus type I (HSV-1) mainly infects the skin and mucous membranes of the face, causing herpes labialis, herpetic keratitis, herpetic encephalopathy and other diseases; herpes simplex virus type II (HSV-2) is mostly transmitted through sexual contact. Invasion of the skin and mucous membrane junction, causing genital herpes. HSV-1 infection may be related to lip cancer, while HSV-2 infection may be related to cervical cancer. Therefore, countries around the world attach great importance to the research and control of HSV and its infection.

Epidemiological surveys show that viral keratitis is most commonly infected with herpes simplex virus, of which HSV-1 has a morbidity rate of up to 90%. Perforation, etc. and seriously endanger the patient's neurological function. However, there is still no effective preventive vaccine against HSV, and nucleoside analogs of lovir (such as acyclovir, ganciclovir) are generally used for treatment, and the replication of the simple disease virus is stopped in three ways: 1) Competitive inhibition of viral DNA polymerase; 2) entry and termination of extended viral DNA strands; 3) inactivation of viral DNA polymerase. Once the viral DNA polymerase breaks through, drug-resistant strains are prone to appear and cross-resistance occurs, especially in HIV-infected patients with HSV infection, the frequency of drug-resistant strains is higher. Therefore, it is of great significance to develop anti-HSV drugs with new targets.

SUMMARY OF THE INVENTION

One of the objects of the present invention is to provide the use of a compound or its derivatives, or its pharmaceutically acceptable salts or esters, solvates, tautomers and isomers in the preparation of antiviral drugs.

A further object of the present invention is to provide an antiviral drug that can treat or prevent viral infection.

A further object of the present invention is to provide an antiviral drug that can inhibit virus replication.

On the one hand, the present invention provides the compound shown in formula I or derivatives thereof, or pharmaceutically acceptable salts or esters, solvates, tautomers, isomers in the preparation of antiviral drugs Application, the virus is selected from at least one of herpes simplex virus, influenza A virus, coronavirus

wherein R ₁ -R ₆ are each independently hydrogen, hydroxy, methoxy or halogen.

In some embodiments, R ₁ is selected from hydrogen, and R ₂ -R ₆ are each independently hydrogen, hydroxy, or methoxy.

_In some embodiments, R1 is selected from hydrogen and at least _one of R2 _- R6 is hydroxy.

In some embodiments, R ₁ is selected from hydrogen, and at least one of R ₂ -R ₆ is methoxy.

In some embodiments, R ₁ is selected from hydroxy, and R ₂ -R ₆ are each independently hydrogen, hydroxy, or methoxy.

In some embodiments, R ₁ is selected from hydroxy, and at least one of R ₂ -R ₆ is hydroxy.

In some embodiments, R ₁ is selected from hydroxy, and at least one of R ₂ -R ₆ is methoxy.

In some embodiments, R ₁ is selected from methoxy, and R ₂ -R ₆ are each independently hydrogen, hydroxy, or methoxy.

In some embodiments, R ₁ is selected from methoxy, and at least one of R ₂ -R ₆ is hydroxy.

In some embodiments, R ₁ is selected from methoxy, and at least one of R ₂ -R ₆ is methoxy.

In some embodiments, R ₁ -R ₆ include 5 hydroxyl groups.

In some embodiments, R ₁ -R ₆ are all hydroxy.

In some embodiments, the compound is selected from dihydromyricetin.

In some embodiments, the dihydromyricetin has the structural formula shown in Formula II:

Dihydromyricelin (DHM), also known as ampelpsin (ampelopsin), dihydromyricetin, dihydromyricetin, etc., was first obtained by Kotake and Kubota in 1940 from Ampelopsis spp. Ampelopsis meliaefolia was isolated from the leaves of Ampelopsis meliaefolia. It is a dihydroflavonol flavonoid compound with a chemical structure of 3,5,7,3',4',5'-hexahydroxy-2,3-dihydroflavonol. Dihydromyricetin widely exists in the genus Rhododendron, and has important application value in many fields such as medicine, health care, food, animal husbandry, and feed.

Dihydromyricetin is the main pharmacodynamic material basis of the traditional Chinese medicine for clearing away heat and detoxification, and it reflects the effects of clearing heat and detoxification, reducing dampness and reducing swelling ("National Collection of Chinese Herbal Medicine", 3rd edition, Wang Guoqiang). Modern pharmacological studies have also proved that dimyricetin has various pharmacological activities such as antibacterial, anti-inflammatory, anti-oxidative, anti-tumor, antihypertensive, hypoglycemic, lipid-lowering and improving immunity, but its antiviral effects are rarely studied, only inventions Patents (application publication number CN 108354923A; CN 109512814A) disclose that dihydromyricetin can inhibit H1N1 influenza A virus; invention patent (application publication number CN 108524489A) discloses that dihydromyricetin can inhibit human immunodeficiency virus; invention patent (application Publication number CN 107311973A) discloses that dihydromyricetin derivatives have inhibitory activity against hepatitis B virus; invention patent (application publication number CN 1605335A) discloses that the composition containing dihydromyricetin and myricetin has anti-HBV activity in vitro and in ducks Reports of anti-duck hepatitis B virus activity. However, there is no relevant literature and patent report of dihydromyricetin against herpes simplex virus.

It is found through experiments in this application that dihydromyricetin has significant anti-HSV activity, can be used for preparing anti-herpes simplex virus drugs, and has good application prospects. Specifically, dihydromyricetin has a significant inhibitory effect on HSV in vitro, and its mechanism of action is related to the inhibition of Toll-like receptor 9 (TLR9)-dependent inflammatory response pathway.

On the one hand, the present application provides a compound represented by formula I or a derivative thereof, or a pharmaceutically acceptable salt or ester, solvate, tautomer, and isomer in the preparation of a virus inhibitor. Application, the virus is selected from at least one of herpes simplex virus, influenza A virus, and coronavirus.

On the one hand, the application provides a compound shown in formula I or a derivative thereof, or a pharmaceutically acceptable salt or ester, solvate, tautomer, and isomer in the preparation of treatment or alleviation of disease or Application in the medicine of the disorder: the disease or disorder is selected from the diseases or disorders caused by one or more infections in herpes simplex virus, influenza A virus, and coronavirus.

On the one hand, the application provides an antiviral pharmaceutical composition, comprising the compound shown in I as the first active ingredient or a derivative thereof, or a pharmaceutically acceptable salt or ester thereof, a solvate, a tautomer A isomer, an isomer and a second active ingredient, the second ingredient is selected from an antiviral substance that has no antagonistic effect with the first active ingredient.

In some embodiments, the compound is as previously described.

In some embodiments, the herpes simplex virus is selected from one or both of herpes simplex virus type I or type II.

In some embodiments, the influenza A virus is selected from one or both of the H1N1 or H3N2 types.

In some embodiments, the coronavirus is selected from the SARS-CoV-2 novel coronavirus.

In some embodiments, the antiviral is the treatment or prevention of viral infection.

In some embodiments, the antiviral is inhibition of viral replication.

On the one hand, the present application provides a compound represented by formula I or a derivative thereof, or a pharmaceutically acceptable salt or ester, solvate, tautomer, and isomer in the preparation of a gene inhibitor. Application, the gene is selected from at least one of ICP4, ICP22, ICP8, UL42, gB, and VP1/2.

In some embodiments, the dosage form of the medicament or pharmaceutical composition is selected from any pharmaceutically acceptable dosage form.

In some embodiments, the dosage form is selected from one or more of tablets, granules, capsules, pills, oral liquids, injections, and liposomes.

In some embodiments, the mode of administration of the drug/pharmaceutical composition is selected from oral administration, topical administration, intranasal administration, systemic administration, intravenous administration, subcutaneous administration, intramuscular administration, One or more of intraventricular, intrathecal, or transdermal administration.

In some embodiments, the present application confirms through experiments that dihydromyricetin has the effect of inhibiting HSV-1 virus proliferation and related gene expression, significantly inhibiting HSV-1 virus mRNA abundance, inhibiting HSV-1 virus proliferation; significantly inhibiting HSV-1 virus 1. mRNA expression of immediate early genes (ICP4, ICP22), early genes (ICP8, UL42) and late genes (gB, VP1/2) of the virus; can specifically inhibit Toll-like receptor 9 (TLR9) mRNA expression; can inhibit herpes simplex The role of the virus is less cytotoxic, and its mechanism of action is related to the inhibition of TLR9-dependent inflammatory response pathways.

Description of drawings

Fig. 1 is the cytotoxicity test result of different concentrations of dihydromyricetin;

Fig. 2 is the plaque assay chart of dihydromyricetin and acyclovir against HSV-1;

Figure 3 shows that dihydromyricetin can significantly inhibit the mRNA abundance of HSV-1 virus;

Figure 4 shows that dihydromyricetin can significantly inhibit the mRNA expression of immediate early genes (ICP4, ICP22), early genes (ICP8, UL42) and late genes (gB, VP1/2) of HSV-1 virus;

Figure 5 shows that dihydromyricetin specifically inhibits Toll-like receptor 9 (TLR9) mRNA expression without altering TLR2 and TLR3 mRNA levels.

Detailed ways

The following is a further description of the essential content of the present invention, the present invention is not limited by this, and all technologies implemented based on the above content of the present invention belong to the scope of the present invention.

Example 1: Preparation of dihydromyricetin

(1) get rattan tea (produced in Zhangping City, Fujian Province), pulverize, pass 50 mesh sieves, obtain rattan tea powder;

(2) get vine tea powder, add 10 times the amount of 75% ethanol, ultrasonicate for 30 minutes, filter, and filter to obtain dihydromyricetin extract;

(3) the above-mentioned dihydromyricetin extract was placed in a rotary evaporator, and the temperature was concentrated under reduced pressure in a 40°C water bath, and ethanol was recovered to no alcohol smell; 5 times of boiling water was added to dissolve, and it was placed in a low temperature environment of 4°C and allowed to cool. Crystallize, filter to obtain dihydromyricetin crude product;

(4) get the dihydromyricetin crude product and add 10 times the amount of boiling water to dissolve, add activated carbon (the amount is 0.5% of the crude product amount) and adsorb for 20 minutes, suction filtration while hot, and the filtrate is cooled and crystallized; repeat the operation 3 times, and separate out the crystallization for subsequent use ;

(5) take step (4) to separate out crystals, add 10 times of boiling water to dissolve, let cool to room temperature, add an equal volume of petroleum ether (boiling range is 30～60 ℃) for extraction, discard the petroleum ether layer, take the water layer and place it at 4 ℃ Under the low temperature environment, the crystals were filtered out and dried at 60° C. for 3 hours to obtain the refined dihydromyricetin product, which was detected by HPLC and the purity was 99.33% calculated by the area normalization method.

Example 2: Activity test of dihydromyricetin against herpes simplex virus HSV-1

1. Experimental materials

(1) Drugs: dihydromyricetin (purity of 99.5%; for example, can be prepared by the method of Example 1); Aciclovir (ACV) (purchased by Selleck Chemical Company, batch number: S180701)

(2) Cells: Vero cells

(3) Virus: HSV-1 (provided by Wuhan Institute of Virology, Chinese Academy of Sciences)

(4) Main reagents: DMEM (GiBCO), fetal bovine serum, trypsin (0.25% Trypsin-EDTA), double antibody, MTT reagent (Sigma-Aldrich), TRIzol reagent (Invitrogen), reverse transcription kit (ThermoScientific), RT-qPCR Amplification Kit (TaKaRa)

(5) Main instruments: ultra-clean workbench (ThermoScientific), microplate reader (Bio-Tek company), low temperature high-speed centrifuge (Eppendorf company), inverted microscope (Olympus company), autoclave (Shanghai Medical Equipment No. 5 Factory) ), pure water instrument (Millipore company), CO ₂ cell incubator (ThermoScientific), RT-qPCR detector (BioRad company)

2. MTT assay to detect the cytotoxicity of dihydromyricetin

100 μl of dihydromyricetin solutions diluted with DMEM to different concentrations (1, 2, 4, 8, 16, 32, 64, 128, 256 μM) were added to 96-well plates to grow monolayer Vero cells, and cultured for 24 h. The cells not treated with dihydromyricetin were the normal control group. The absorbance values of the normal control group and the drug group at a wavelength of 570 nm were detected by MTT method, and the cell viability was calculated (cell viability = average absorbance value of the drug group/average absorbance value of the normal control group × 100%), and the cells of dihydromyricetin were evaluated. toxic effects.

The results are shown in Figure 1. Dihydromyricetin is less toxic to Vero cells at low concentrations, and the survival rate of Vero cells is close to 100% at 8 μM, and the median lethal dose (CC ₅₀ ) of dihydromyricetin to Vero cells is calculated. As 41.04 μM, 16 μM and 32 μM of dihydromyricetin with a concentration less than CC ₅₀ were selected for subsequent antiviral studies.

3. In vitro anti-HSV-1 effect and mechanism of dihydromyricetin

After HSV-1 standard strain (MOI=0.1) infected Vero cells for 1 h, the cells were treated with dihydromyricetin solutions of different concentrations (1, 2, 4, 8, 16, 32 μM) for 24 h, and the cells were collected. Blank control. The plaque formation in the cell plate was detected by plaque assay, and the mRNA expression of HSV-1-related genes in cells was detected by real-time fluorescence quantitative PCR (RT-qPCR). Antiviral effects of HSV-1. Specific steps are as follows:

(1) Plaque assay to detect the anti-HSV-1 effect of dihydromyricetin

After HSV-1 strain infects Vero cells for 1 hour, the virus solution is discarded, and dihydromyricetin medium containing different concentrations is added, and acyclovir is selected as a positive control. After that, the unfixed components in the cell plate were washed away, and the plaque formation in the cell plate was observed and counted.

The results are shown in Figure 2. Acyclovir can significantly inhibit HSV-1 virus plaque formation at lower doses, and dihydromyricetin significantly inhibits HSV-1 replication and infection in Vero cells at concentrations of 16 and 32 μM. It can significantly inhibit the size of HSV-1 plaques in a dose-dependent manner.

(2) Detection of anti-HSV-1 effect and molecular mechanism of dihydromyricetin by real-time fluorescence quantitative PCR

Real-time quantitative PCR (RT-qPCR) was used to detect the expression of immediate early genes (ICP4, ICP22), early genes (ICP8, UL42) and late genes (gB, VP1/2) of HSV-1. GAPDH was used as an internal reference, and no dihydrogen was added. The myricetin-treated group served as the control group. The nucleotide sequences of the primers are as follows:

ICP4 gene upstream primer: 5'-CGACACGGATCCACGACCC-3'

ICP4 gene upstream primer: 5'-GATCCCCCTCCCGCGCTTCGTCCG-3'

ICP22 gene upstream primer: 5'-CGCCGCAGAAGACCGCAAGT-3'

ICP22 gene downstream primer: 5'-GTGCTGCACGGATAGGG-3'

ICP8 gene upstream primer: 5'-CGACAGTAACGCCAGAAG-3'

ICP8 gene downstream primer: 5'-GGAGACAAAGCCCAAGAC-3'

UL42 gene upstream primer: 5'-GCGGTATCGGCGGTATTT-3'

UL42 gene downstream primer: 5'-CCCGTCTTAGGTTTTCTTTAGGG-3'

gB gene upstream primer: 5'-AAACCGAAAAACCCACCGCC-3'

Downstream primer of gB gene: 5'-TGTTCTCCGCCTTGATGTCC-3'

VP1/2 gene upstream primer: 5'-CGGGTCAAAAAGGTATGCGG-3'

VP1/2 gene downstream primer: 5'-TGTCGTACACGCTCCTAACC-3'

TLR2 gene upstream primer: 5'-ATCCTCCAATCAGGCTTCTCT-3'

TLR2 gene downstream primer: 5'-GGACAGGTCAAGGCTTTTTACA-3'

TLR3 gene upstream primer: 5'-TTGCCTTGTATCTACTTTTGGGG-3'

TLR3 gene downstream primer: 5'-TCAACACTGTTATGTTTGTGGGT-3'

TLR9 gene upstream primer: 5'-CTGCCTTCCTACCCTGTGAG-3'

TLR9 gene downstream primer: 5'-GGATGCGGTTGGAGGACAA-3'

GAPDH gene upstream primer: 5'-GGTGGTCTCCTCTGACTTCAACA-3'

GAPDH gene downstream primer: 5'-GTTGCTGTAGCCAAATTCGTTGT-3'

Premix Ex Taq^TM检测试剂盒在荧光定量PCR仪上进行检测。反应程序为95℃预变性2min；95℃变性5s，60℃退火10s，72℃延伸15s，45个循环。扩增程序结束后，得到每个样本的CT值，通过2^-ΔΔCT计算每个样本中上述基因的mRNA相对表达含量。Take 1 μL of sample total RNA and reverse-transcribe it into cDNA: react at 37°C for 1 h, terminate the reaction at 95°C for 5 min, and store the product at 4°C. Using reverse transcribed cDNA as RT-qPCR reaction template, adding gene-specific primers, using

The Premix Ex Taq ^TM Assay Kit was assayed on a real-time PCR machine. The reaction program was pre-denaturation at 95 °C for 2 min; denaturation at 95 °C for 5 s, annealing at 60 °C for 10 s, and extension at 72 °C for 15 s, for 45 cycles. After the amplification procedure, the CT value of each sample was obtained, and the relative mRNA expression content of the above genes in each sample was calculated by 2 ^-ΔΔCT .

The test results show:

①Dihydromyricetin significantly inhibited the abundance of HSV-1 virus mRNA at concentrations of 16 and 32 μM (see Figure 3).

②Dihydromyricetin significantly inhibited the mRNA expression of immediate early genes (ICP4, ICP22), early genes (ICP8, UL42) and late genes (gB, VP1/2) of HSV-1 virus at concentrations of 16 and 32 μM (see Figure 4). ).

③Dihydromyricetin specifically inhibited the expression of Toll-like receptor 9 (TLR9) mRNA at the concentration of 16 and 32 μM, but did not change the mRNA levels of TLR2 and TLR3 (see Figure 5).

Dihydromyricetin has the effect of inhibiting HSV-1 virus proliferation and related gene expression, and can significantly inhibit HSV-1 replication and infection in Vero cells. Its antiviral mechanism may be related to its inhibition of TLR9-dependent inflammatory response pathway.

Other parts that are not described in detail are the prior art. Although the above embodiment has made a detailed description of the present invention, it is only a part of the embodiments of the present invention, rather than all the embodiments. People can also obtain other embodiments according to the present embodiment without creativity. These embodiments All belong to the protection scope of the present invention.

Claims (10)

1. the compound shown in formula I or its derivative or its pharmaceutically acceptable salt or ester, solvate, tautomer, the application of isomer in the preparation of antiviral medicine, the virus At least one selected from herpes simplex virus, influenza A virus, and coronavirus

wherein R ₁ -R ₆ are each independently hydrogen, hydroxy, methoxy or halogen. 2. The application according to claim 1, wherein R ₁ is selected from hydrogen, and R ₂ -R ₆ are independently hydrogen, hydroxyl or methoxy; Preferably, R ₁ is selected from hydrogen, and at least one of R ₂ -R ₆ is hydroxyl; Preferably, R ₁ is selected from hydrogen, and at least one of R ₂ -R ₆ is methoxy; Preferably, R ₁ is selected from hydroxyl, and R ₂ -R ₆ are each independently hydrogen, hydroxyl or methoxy; Preferably, R ₁ is selected from hydroxyl, and at least one of R ₂ -R ₆ is hydroxyl; Preferably, R ₁ is selected from hydroxyl, and at least one of R ₂ -R ₆ is methoxy; Preferably, R ₁ is selected from methoxy, and R ₂ -R ₆ are each independently hydrogen, hydroxyl or methoxy; Preferably, R ₁ is selected from methoxy, and at least one of R ₂ to R ₆ is hydroxyl; Preferably, R ₁ is selected from methoxy, and at least one of R ₂ -R ₆ is methoxy; Preferably, R ₁ -R ₆ include 5 hydroxyl groups; Preferably, R ₁ -R ₆ are all hydroxyl groups. 3. application according to claim 1, is characterized in that, described compound is selected from dihydromyricetin; Preferably, the dihydromyricetin has the structural formula shown in formula II:

4. the application of the compound shown in formula I or its derivative or its pharmaceutically acceptable salt or ester, solvate, tautomer, isomer in the preparation of virus inhibitor, the virus At least one selected from the group consisting of herpes simplex virus, influenza A virus, and coronavirus. 5. the compound shown in formula I or its derivative or its pharmaceutically acceptable salt or ester, solvate, tautomer, isomer in the preparation of the medicine for treatment or alleviation of disease or illness Application: The disease or condition is selected from diseases or conditions caused by one or more infections in herpes simplex virus, influenza A virus and coronavirus. 6. an antiviral pharmaceutical composition is characterized in that, comprises the compound shown in I as the first active ingredient or its derivative or its pharmaceutically acceptable salt or ester, solvate, tautomer isomers, isomers and a second active ingredient, the second ingredient is selected from an antiviral substance that has no antagonistic effect with the first active ingredient. 7. according to the application described in any one of claim 4-6, it is characterized in that, described compound is as shown in any one of claim 1-3; Described herpes simplex virus is selected from herpes simplex virus type I or type II one or both; Preferably, the influenza A virus is selected from one or both of H1N1 or H3N2 types; Preferably, the coronavirus is selected from SARS-CoV-2 novel coronavirus. 8. the application of the compound shown in formula I or its derivative or its pharmaceutically acceptable salt or ester, solvate, tautomer, isomer in the preparation of gene inhibitor, said gene At least one selected from ICP4, ICP22, ICP8, UL42, gB, and VP1/2. 9. The use according to claim 8, wherein the compound is as shown in any one of claims 1-3. 10. The application or pharmaceutical composition according to any one of claims 1-3, 5 and 6, wherein the dosage form of the medicine or the pharmaceutical composition is selected from any pharmaceutically acceptable dosage form; Preferably, the dosage form is selected from one or more of tablets, granules, capsules, pills, oral liquids, injections, and liposomes; Preferably, the mode of administration of the drug or the pharmaceutical composition is selected from oral administration, topical administration, intranasal administration, systemic administration, intravenous administration, subcutaneous administration, intramuscular administration, and intracerebroventricular administration One or more of drug, intrathecal or transdermal administration.

Images