CN114377015A - Application of naftopidil in preparation of anti-influenza virus medicine - Google Patents

Abstract

The invention belongs to the technical field of medicines, discloses application of naftopidil in preparation of anti-influenza virus medicines, and particularly discloses application of naftopidil shown in a structural formula (I) and pharmaceutically acceptable salts thereof in preparation of medicines for preventing or treating influenza virus infection. And the combined application of naftopidil and other antiviral drugs.

Description

Application of nafidil in the preparation of anti-influenza virus drugs

technical field

The invention belongs to the technical field of medicine, and particularly relates to the application of naftopidil (Naftopidil, CAS: 57149-07-2) in preparing a medicine for preventing or treating influenza virus infection. The present invention comprises the single or combined use of nafidil in the prevention or treatment of influenza virus infection.

Background technique

Influenza virus is an important threat to global public health security. According to the statistics of the World Health Organization (WHO), about 5-10% of adults and 20-30% of children are infected with influenza virus every year, of which 3-5 million are Severely ill patients, the number of deaths is about 250,000 to 500,000 [Ziegler T, Mamahit A, Cox NJ: 65 years of influenzasurveillance by a world health organization-coordinat ed global network. Influenza and other respiratory viruses (2018) 12(5):558-565 .]. Influenza viruses belong to the family Orthomyxoviridae, and are divided into four types: A, B, C, and D. Influenza viruses A, B, and C can infect humans and cause respiratory diseases. Influenza A virus is the most widespread and most harmful. Among them, the Spanish flu epidemic of 1918 killed 50 million people; and the 2009 swine flu epidemic also killed about 570,000 people worldwide [Firstglobal estimates of 2009h1n1pandemic mortality released by cdc-ledcollaboration:(2012).https://www. cdc.go v/flu/spotlights/pandemic-global-estimates.htm].

Influenza is spherical in shape, about 80-120 nm in diameter, and is an enveloped virus whose genome is a single-stranded segmented RNA. Type A has a total of 8 RNAs encoding at least 10 viral proteins [David M Knipe P, Peter M Howley, MD: Fieldsvirology, 6th edition. 2. Lippincott Williams & Wilkins (L WW), (2013)]. The influenza virus envelope is derived from the host cell membrane, on which three viral proteins are embedded: Hemagglutinin (HA), Neuraminidase (NA) and M2 ion channel. According to the serological classification of HA and NA, influenza A virus has 18 kinds of HA and 10 kinds of NA, so there are 180 subtypes in theory. Epidemiological data show that the H1N1 subtype has caused a large-scale lethal influenza epidemic. (1918 Spanish flu and 2009 swine flu), H2N2 subtype (1950 Asian flu), H3N2 subtype (1960 Hong Kong flu).

In addition to influenza pandemics, seasonal influenza occurs every year, mainly in winter, and is most commonly caused by influenza A or B viruses, of which H1N1 and H3N2 are more common. Symptoms of seasonal flu include sudden fever, cough (usually dry), headache, muscle and joint pain, sore throat and runny nose. The cough can be severe and last for 2 weeks or more. Most people recover from fever and other symptoms within a week without medical treatment. However, influenza can cause severe illness or death in high-risk groups [How can I avoid getting the flu? World HealthOrganization website: https://www.w ho.int/news-room/q-a-detail/how-can-i-avoid-getting-the-flu].

There are currently 7 anti-influenza drugs in three categories [Amarelle L, Lecuona E, Sznajder JI: Anti-influenza treatment: Drugs currently used and under development. Archiv osde bronconeumologia (2017) 53(1):19-26.] : M2 ion channel inhibitors amantadine and rimantadine; neuraminidase inhibitors oseltamivir, zanamivir, peramivir and lanamivir; CAP-dependent endonuclease inhibitor Barlow baloxavir marboxil. The above seven anti-influenza drugs have one thing in common: they are all drugs that target influenza virus proteins, so when the drug target mutates, the affinity of the drug will decrease, and the influenza virus will escape and become a drug-resistant virus. For example, after long-term use of influenza virus M2 ion channel inhibitors amantadine and rimantadine, the virus has developed stable drug resistance mutations. WHO has not recommended amantadine and rimantadine for the treatment of influenza A virus [Summary of influenza virus] enza antiviral susceptibility surveillance findings, September 2010-march 2011:(2011). https://www.who.int/influenza/gisrs_laboratory/updates/antiviral_susceptibi lity/en/].

Although there are 7 anti-influenza drugs in use or in use, 500 million to 1.5 billion people are infected with influenza virus and become sick each year. According to statistics from the US Centers for Disease Control and Prevention, from 2010 to 2018, the number of patients infected with influenza A virus in the United States was 9.3-49 million each year, and 12-79,000 died [Influenza(flu):(2020).https://www.cdc.gov /flu/about/burden/index.html]. That is, after the supply of anti-influenza virus drugs and the injection of influenza virus vaccine, 5%-20% of the entire population is still infected and sickened. Recombination (Reassortment) caused. For example, neuraminidase inhibitors are the most commonly used anti-influenza A virus drugs in clinical practice, of which oseltamivir (Tamiflu) is the most widely used. Good efficacy [Summary of influenza antiviral susceptibilitysurveillance findings, September 2010-March 2011:(2011).https://www.who.int/influenza/gisrs_lab oratory/updates/antiviral_susceptibility/en/].

Nafipidil is an oral α1 adrenergic receptor antagonist (α1 receptor blocker) first synthesized by Boehringer Mannheim Company of Germany and developed by Asahi Kasei Corporation of Japan. It is used for the treatment of benign prostatic hyperplasia (benign prostatic hyperplasia, Lower urinary tract symptoms (LUTS) due to BPH [Homma Y, Araki I, Igawa Y, et al. Japanese Society of NeurogenicBladder. Clinical guideline for male lower urinary tract symptoms. Int JUrol. 2009; 16(10 ):775–790.], listed in Japan in 1999, and currently used in Japan and Korea [Takei R-I, Ikegaki I, Shibata K, Tsujimoto G, Asano T. Naftopidil, a novel α1-adrenoceptorantagonist, displays selective inhibition of canine prostatic pressure and high affinity binding to cloned human α1-adrenoceptors. Jpn J Pharmacol. 1999;79(4):447–454.]. BPH is an adenoma formed by a mixture of increased numbers of epithelial and/or stromal cells and is a common histological disorder in older men. Benign prostate enlargement (BPE) caused by BPH can cause bladder outlet obstruction (BOO) and lead to lower urinary tract symptoms [McConnell J, Abrams P, Denis L, Khoury S, Roehrborn C, editors. Male LowerUrinary Tract Dysfunction Evaluation and Management. Ed 21. Paris: HealthPublications; 2006. pp. 69–142.]. There are two classes of drugs used to treat lower urinary tract symptoms due to BPH: 5α-reductase inhibitors (5ARI) and α1 adrenergic receptor antagonists, nafidil being the latter. Nafidil reduces prostate smooth muscle tone by blocking α1 receptors and improves bladder outlet obstruction, thereby reducing lower urinary tract symptoms [McConnell J, Abrams P, Denis L, Khoury S, Roehrborn C, editors. Male LowerUrinary Tract Dysfunction Evaluation and Management. Ed 21. Paris: Health Publications; 2006. pp. 143–194.]. Studies have shown that the expression levels of α1A and α1D subtype adrenergic receptors in BPH tissues are increased by 9 times and 3 times, respectively, and nafidil can bind to these two subtype receptors, among which α1D subtype adrenergic receptors. The affinity of the receptor (Ki: 1.2 nM) is 3 times that of the α1A subtype receptor (Ki: 3.7 nM) [Shibata K, Foglar R, HorieK, et al. KMD-3213, a novel, potent, alpha 1a-adrenoceptor -selective antagonist: characterization using recombinant human alpha 1-adrenoceptors and native tissues. Mol Pharmacol. 1995;48(2):250–258.]. Nafidil has a good safety profile. The study showed that 100 BPH patients took 75 mg orally daily for 6 consecutive weeks, and after 1 week of drug withdrawal, oral administration of 50 mg daily for 6 consecutive weeks showed no obvious adverse reactions [Oh-oka] H. Usefulness of naftopidil for dysuria in benign prostatic hyperplasia and its optimal dose: comparison between 75 and 50 mg. Urol Int. 2009;82(2):136–142.]. After literature search, there is no report on the anti-influenza virus activity or any antiviral activity of nafidil.

The present invention uses the influenza virus infection model to evaluate the antiviral activity of known compounds/marketed drugs, and finds that nafidil has broad-spectrum anti-influenza virus activity and strong inhibitory activity against influenza A and B influenza virus infection. The data show that the anti-influenza virus activity of nafidil is comparable to that of the first-line antiviral drug ribavirin, and the anti-influenza A virus activity is stronger than that of ribavirin, and the safety of nafidil is good. We believe that the new use of nafidil in anti-influenza virus is of high value and has application prospects. The present invention is an invention patent for a new use of a known compound.

SUMMARY OF THE INVENTION

The technical problem solved by the present invention is to provide the application of nafidil and its pharmaceutically acceptable salts in the preparation of medicines for preventing or treating influenza virus infection.

Specifically, in order to solve the technical problem of the present invention, the following technical solutions are adopted:

The first aspect of the technical solution of the present invention is to provide the application of napipidil and pharmaceutically acceptable salts thereof in the preparation of medicines for preventing or treating influenza virus as shown in structural formula (I)

The pharmaceutically acceptable salts of nafidil include pharmaceutically acceptable organic salts or inorganic salts, wherein the organic salts include sulfonates, carboxylates, amino acid salts and fatty acid salts, Inorganic salts include hydrochloride, bromate, iodate, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate and nitrate. Preferred are bisulfate, sulfate, hydrochloride and iodate.

The sulfonates include alkylsulfonates containing 1-15 carbon atoms and benzenesulfonates, p-toluenesulfonates, o-toluenesulfonates, and m-toluenesulfonates; carboxylates include tartrates , maleate, fumarate, citrate, malate, cinnamate, benzoate, malonate, succinate, glutarate, adipate, bisulfate Xonaphate and lactate; amino acid salts include glutamate, aspartate; fatty acid salts include long-chain fatty acid salts containing 2-18 carbon atoms.

Wherein, the influenza virus includes influenza A virus, influenza B virus, influenza C virus and influenza D virus.

Described influenza A virus comprises H1N1 subtype, H1N2 subtype, H2N2 subtype, H2N3 subtype, H3N1 subtype, H3N2 subtype, H3N8 subtype, H5N1 subtype, H5N2 subtype, H5N3 subtype, H5N6 subtype type, H5N8 subtype, H5N9 subtype, H6N1 subtype, H6N2 subtype, H7N1 subtype, H7N2 subtype, H7N3 subtype, H7N4 subtype, H7N7 subtype, H7N9 subtype, H9N2 subtype, H10N7 subtype, H10N8 subtype, H11N2 subtype, H11N9 subtype, H17N10 subtype, H18N11 subtype. Among them, influenza A H1N1 viruses include A/PurtoRico/8/1934, A/WSN/33, A/Hubei Hongshan/52/2005, A/Jingfang/262/1995, A/Guangdong Luohu/219/2006 and A/ FM/1/47 strains; Influenza A/H3N2 viruses include A/Jiangxi Donghu/312/2006, A/Jifang/15/90, A/Yuefang/243/1972, A/Hanfang/359/1995, A /New York/238/2015, A/Brisbane/10/07, A/Perth/16/09 and A/Udorn/307/72 strains. Influenza B viruses include B/Jiangxi Xinjian/BV/39/2008, B/Jifang/13/1997, B/Shenzhen/155/2005, B/Sichuan/63/2001, B/Zhejiang/2/2001, B /Shandong/7/97, B/Durban/39/98, B/Shandong Taian Taishan/1219/2009, B/Sichuan/34/2001B/Yamagata/16/88, B/Victoria/2/87, B/Johannesburg /1/99 and B/Maputo/1/99 strains.

The second aspect of the technical solution of the present invention provides the application of a pharmaceutical composition in the preparation of an anti-influenza virus drug, characterized in that the pharmaceutical composition comprises the nafidil shown in structural formula (I) and its pharmacy acceptable salts and pharmaceutically acceptable carriers or excipients; the pharmaceutical composition may also contain other antiviral drugs

Wherein, the influenza virus includes influenza A virus, influenza B virus, influenza C virus and influenza D virus.

Described influenza A virus comprises H1N1 subtype, H1N2 subtype, H2N2 subtype, H2N3 subtype, H3N1 subtype, H3N2 subtype, H3N8 subtype, H5N1 subtype, H5N2 subtype, H5N3 subtype, H5N6 subtype type, H5N8 subtype, H5N9 subtype, H6N1 subtype, H6N2 subtype, H7N1 subtype, H7N2 subtype, H7N3 subtype, H7N4 subtype, H7N7 subtype, H7N9 subtype, H9N2 subtype, H10N7 subtype, H10N8 subtype, H11N2 subtype, H11N9 subtype, H17N10 subtype, H18N11 subtype. Among them, influenza A H1N1 viruses include A/PurtoRico/8/1934, A/WSN/33, A/Hubei Hongshan/52/2005, A/Jingfang/262/1995, A/Guangdong Luohu/219/2006 and A/ FM/1/47 strains; Influenza A/H3N2 viruses include A/Jiangxi Donghu/312/2006, A/Jifang/15/90, A/Yuefang/243/1972, A/Hanfang/359/1995, A /New York/238/2015, A/Brisbane/10/07, A/Perth/16/09 and A/Udorn/307/72 strains. Influenza B viruses include B/Jiangxi Xinjian/BV/39/2008, B/Jifang/13/1997, B/Shenzhen/155/2005, B/Sichuan/63/2001, B/Zhejiang/2/2001, B /Shandong/7/97, B/Durban/39/98, B/Shandong Taian Taishan/1219/2009, B/Sichuan/34/2001B/Yamagata/16/88, B/Victoria/2/87, B/Johannesburg /1/99 and B/Maputo/1/99 strains.

The pharmaceutical composition can be prepared according to methods known in the art. Any dosage form suitable for human or animal use can be prepared by combining the compounds of the present invention with one or more pharmaceutically acceptable solid or liquid excipients and/or adjuvants.

The compound of the present invention or the pharmaceutical composition containing it can be administered in unit dosage form, and the route of administration can be enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung and Respiratory tract, skin, vagina, rectum, etc.

The dosage form for administration can be a liquid dosage form, a solid dosage form or a semi-solid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o/w, w/o and double emulsion), suspensions, injections (including water injection, powder injection and infusion), eye drops solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules ( Including hard capsules, soft capsules, enteric-coated capsules), granules, powders, pellets, drop pills, suppositories, films, patches, gas (powder) aerosols, sprays, etc.; semi-solid dosage forms can be ointments, Gels, pastes, etc.

The compounds of the present invention can be prepared into common preparations, as well as sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.

In order to formulate the compounds of the present invention into tablets, various excipients well known in the art can be widely used, including diluents, binders, wetting agents, disintegrating agents, lubricants, glidants. The diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; Propanol, etc.; the binder can be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia mucilage, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methylcellulose Base cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polymer Vinylpyrrolidone, croscarmellose sodium, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfonate, etc.; lubricants and flow aids The agent may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol and the like.

The tablets can also be further prepared as coated tablets, such as sugar-coated, film-coated, enteric-coated, or bilayer and multi-layer tablets.

In order to make the dosage unit into capsules, the active ingredients of the compounds of the present invention can be mixed with diluents and glidants, and the mixture can be directly placed in hard capsules or soft capsules. The compound of the present invention can also be made into granules or pellets with diluents, binders and disintegrating agents, and then placed in hard capsules or soft capsules. The various diluents, binders, wetting agents, disintegrants, and glidants used to prepare tablets of the compounds of the present invention can also be used to prepare capsules of the compounds of the present invention.

To prepare the compounds of the present invention into injections, water, ethanol, isopropanol, propylene glycol or their mixtures can be used as solvents and appropriate amount of solubilizers, cosolvents, pH adjusters and osmotic pressure adjusters commonly used in the art can be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjuster can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure adjuster can be It is sodium chloride, mannitol, glucose, phosphate, acetate, etc. For example, in the preparation of freeze-dried powder injection, mannitol, glucose, etc. can also be added as proppant.

In addition, colorants, preservatives, fragrances, flavors, or other additives can also be added to the pharmaceutical preparations, if desired.

The inventors of the present invention found that nafidil can block the infection of host cells by influenza virus. It can also be used in combination with other antiviral drugs.

In order to achieve the purpose of medication and enhance the therapeutic effect, the medicament or pharmaceutical composition of the present invention can be administered by any known administration method.

The dosage of the pharmaceutical composition of the compound of the present invention may vary widely according to the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the route of administration and the dosage form.

The compounds or compositions of the present invention may be administered alone or in combination with other therapeutic or symptomatic drugs. When the compound of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

beneficial technical effect

By evaluating the anti-influenza virus infection activity of 300 known compounds/marketed drugs, the inventors of the present invention found that nafidil has strong inhibitory activity against influenza A and B influenza virus infection. The influenza virus activity is comparable to that of the first-line antiviral drug ribavirin, and its anti-influenza A virus activity is stronger than that of ribavirin. The results of the clinical study on middle-aged and elderly male patients with LUTS/BPH showed that the patient tolerated nafidil well, and the patient had no obvious adverse reactions or side effects by oral administration of 75 mg/day for 12 consecutive weeks. Since the course of influenza virus infection is about 1 week, and the side effects of nafidil are very mild, we believe that the new use of nafidil in anti-influenza virus is of high value and has application prospects.

Description of drawings

Figure 1. The results of evaluating the activity of nafidil in blocking A549 cells infected with A/Puerto Rico/8/1934 (H1N1).

Figure 2. The results of the evaluation of the activity of nafidil in blocking A549 cells infected with A/Jiangxi Donghu/312/2006 (H3N2).

Figure 3. The results of evaluating the activity of nafidil in blocking the infection of MDCK cells with beta-B/Jiangxi Xinjian/BV/39/2008.

Figure 4. The effect of nafidil on the viability of A549 cells

Figure 5. The effect of nafidil on the viability of MDCK cells

Detailed ways

Example 1. Principle of detecting influenza virus infection model

Type A/Puerto Rico/8/1934(H1N1), Type A/Jiangxi Donghu/312/2006(H3N2) and Type B/Jiangxi Xinjian/BV/39/2008 are the classic seasonal influenza strains. Lung tissue is the main organ of influenza virus infection. This detection model mainly detects the inhibitory effect of compounds on influenza A virus (A/Puerto Rico/8/1934 and A/Jiangxi Donghu/312/2006) infecting lung cancer A549 cells, as well as the compounds on type B/Jiangxi Xinjian/BV/ 39/2008 Inhibitory effect of infection of MDCK cells.

In this detection model, the compound was pre-incubated with cells for 20 hours before infection, and then the cells were infected with virus, and the viability of A549 cells was detected 48 hours after infection. Inhibition rates of compounds against viral infection.

Example 2. Principle of cell viability detection model

ATP plays an important role in various physiological processes of cells and directly provides energy for the body. It is an important indicator of cell viability and is positively correlated with the number of living cells. Therefore, the number of viable cells in the test sample can be reflected by quantitative detection of ATP levels in cell lysates.

Luminescent Cell Viability Assay发光法细胞活力检测试剂盒(Promega公司)定量检测ATP水平，定量评价化合物对A549细胞和MDCK细胞活力的影响。This model uses

The Luminescent Cell Viability Assay (Promega) quantitatively detected the level of ATP, and quantitatively evaluated the effect of compounds on the viability of A549 cells and MDCK cells.

Example 3. Experimental methods and results of A549 cell model infected with Type A/Puerto Rico/8/1934 (H1N1)

发光法细胞活力检测试剂盒(Promega公司)检测细胞活力，即测定细胞裂解液中的相对荧光素酶活性(relative luminescence units,RLUs)。按公式(1)和(2)计算各个实验组的细胞病变率和病毒抑制率。采用Graph Pad Prism软件分析实验数据,以浓度-抑制率作散点图并用非线性拟合得到量效曲线，计算待测化合物的半数有效浓度EC₅₀。A549 cells were seeded in a 96-well plate at 4×10 ⁴ cells per well, and nafidil was added after 4 hours at a final concentration of 30 μM, 10 μM, 3 μM and 1 μM, respectively. The normal cell control group did not add any compound, and the solvent control The group added an equal volume of DMSO and continued to culture for 20 hours. Aspirate the medium in the culture plate, rinse the cells once with PBS, add A/PuertoRico/8/1934 virus infection (MOI=0.01), and incubate at 37°C for 1 hour. The medium was aspirated, washed once with PBS, and the medium containing the test compound was added, the normal cell control group was added with the medium, and the solvent control group was added with the medium containing the same amount of DMSO. Use after 48 hours

Cell viability was detected by luminescence cell viability assay kit (Promega company), that is, relative luciferase activity (relative luminescence units, RLUs) in cell lysate was determined. The cytopathic rate and virus inhibition rate of each experimental group were calculated according to formulas (1) and (2). Graph Pad Prism software was used to analyze the experimental data, the concentration-inhibition rate was used as a scatter plot and a dose-response curve was obtained by nonlinear fitting, and the half-effective concentration EC ₅₀ of the tested compound was calculated.

(1) Cytopathic rate %=(100-RLUs _{administration group} (or RLUs _{solvent control group} )/RLUs _{normal cell control group} )×100%

(2) Virus inhibition rate % = (cytopathic rate of solvent control group - cytopathic rate of administration group)/cytopathic rate of solvent control group × 100%

The results show that nafidil can block A549 cells infected by A/Puerto Rico/8/1934 (H1N1), and its antiviral activity is better than that of the first-line antiviral drug ribavirin (see Table 1 for the results, and see the dose-response curve). Figure 1).

Table 1 Activity evaluation results of compounds on A549 cells infected with influenza A virus A/Puerto Rico/8/1934 (H1N1)

Example 4. Experimental method and results of A549 cell model infected with Type A/Jiangxi Donghu/312/2006 (H3N2)

发光法细胞活力检测试剂盒(Promega公司)检测细胞活力，即测定细胞裂解液中的相对荧光素酶活性(relative luminescence units,RLUs)。按公式(1)和(2)计算各个实验组的细胞病变率和病毒抑制率。采用Graph Pad Prism软件分析实验数据,以浓度-抑制率作散点图并用非线性拟合得到量效曲线，计算待测化合物的半数有效浓度EC₅₀。A549 cells were seeded in a 96-well plate at 4×10 ⁴ cells per well, and nafidil was added after 4 hours at a final concentration of 30 μM, 10 μM, 3 μM and 1 μM, respectively. The normal cell control group did not add any compound, and the solvent control The group added an equal volume of DMSO and continued to culture for 20 hours. Aspirate the medium in the culture plate, rinse the cells once with PBS, add A/Jiangxi Donghu/312/2006 virus infection (MOI=0.02), and incubate at 37°C for 1 hour. The medium was aspirated and discarded, rinsed once with PBS, and the medium containing the test compound was added, the normal cell control group was added with the medium, and the solvent control group was added with the medium containing the same amount of DMSO. Use after 48 hours

Cell viability was detected by luminescence cell viability detection kit (Promega company), that is, relative luciferase activity (relative luminescence units, RLUs) in cell lysate was determined. The cytopathic rate and virus inhibition rate of each experimental group were calculated according to formulas (1) and (2). Graph Pad Prism software was used to analyze the experimental data, the concentration-inhibition rate was used as a scatter plot and the dose-response curve was obtained by nonlinear fitting, and the half-effective concentration EC ₅₀ of the test compound was calculated.

(1) Cytopathic rate %=(100-RLUs _{administration group} (or RLUs _{solvent control group} )/RLUs _{normal cell control group} )×100%

(2) Virus inhibition rate % = (cytopathic rate of solvent control group - cytopathic rate of administration group)/cytopathic rate of solvent control group × 100%

The results show that nafidil can block A549 cells infected with A/Jiangxi Donghu/312/2006 (H3N2), and its antiviral activity is better than that of the first-line antiviral drug ribavirin (see Table 2 for the results, and see the dose-response curve). Figure 2).

Table 2 Activity evaluation results of compounds on A549 cells infected with influenza A virus A/Jiangxi Donghu/312/2006 (H3N2)

Example 5. Experimental method and results of B-type B/Jiangxi Xinjian/BV/39/2008 infection of MDCK cell model

发光法细胞活力检测试剂盒(Promega公司)检测细胞活力，即测定细胞裂解液中的相对荧光素酶活性(relative luminescence units,RLUs)。按公式(1)和(2)计算各个实验组的细胞病变率和病毒抑制率。采用Grap hPad Prism软件分析实验数据,以浓度-抑制率作散点图并用非线性拟合得到量效曲线，计算待测化合物的半数有效浓度EC₅₀。MDCK cells were seeded in a 96-well plate at 4 × 10 ⁴ cells per well, and nafidil was added after 4 hours at a final concentration of 30 μM, 10 μM, 3 μM and 1 μM, respectively. The normal cell control group did not add any compound, and the solvent control The group added an equal volume of DMSO and continued to culture for 20 hours. Aspirate and discard the medium in the culture plate, rinse the cells once with PBS, add type B/Jiangxi Xinjian/BV/39/2008 virus infection (100*TCID ₅₀ ), and incubate at 37°C for 1 hour. The medium was aspirated and discarded, rinsed once with PBS, and the medium containing the test compound was added, the normal cell control group was added with the medium, and the solvent control group was added with the medium containing the same amount of DMSO. Use after 48 hours

Cell viability was detected by luminescence cell viability detection kit (Promega company), that is, relative luciferase activity (relative luminescence units, RLUs) in cell lysate was determined. The cytopathic rate and virus inhibition rate of each experimental group were calculated according to formulas (1) and (2). Graph hPad Prism software was used to analyze the experimental data, and the concentration-inhibition rate was used as a scatter plot and nonlinear fitting was used to obtain a dose-response curve, and the half-effective concentration EC ₅₀ of the tested compound was calculated.

(1) Cytopathic rate %=(100-RLUs _{administration group} (or RLUs _{solvent control group} )/RLUs _{normal cell control group} )×100%

(2) Virus inhibition rate % = (cytopathic rate of solvent control group - cytopathic rate of administration group)/cytopathic rate of solvent control group × 100%

The results show that nafidil can block B/Jiangxi Xinjian/BV/39/2008 infection of MDCK cells, and the inhibitory activity is comparable to the first-line antiviral drug ribavirin (the results are shown in Table 3, and the dose-response curve is shown in the accompanying drawings. 3).

Table 3 The results of the evaluation of MDCK activity of compounds against influenza B virus B/Jiangxi Xinjian/BV/39/2008 infection

Example 6. Effects of test compounds on cell viability

A549 cells or MDCK cells were seeded into a 96-well plate at a density of 8000 cells/well, 100 μL of cell fluid per well, and cultured at 37°C in 5% CO ₂ for 24 h. The next day, various concentrations of test compounds were added to the cells, and an equal amount of DMSO (0.1% v/v) was used as a solvent control. After culturing for 48h, add 100μL of CellTiter-Glo reagent to each well, shake and mix for 2min, incubate at room temperature for 10min, and measure the RLUs in each well [Tang K, He S, Zhang X, et al. Tangeretin, an extract from Citruspeels, blocks cellular entry of arenaviruses that cause viral hemorrhagicfever. Antiviral Res. 2018, 160: 87-93.]. Taking the RLUs value of the DMSO solvent well as 100%, the cell viability of the drug-added wells was calculated.

Cell survival %=fluorescence intensity _{administration group} /fluorescence intensity _{solvent control group} ×100%.

The experimental results showed that nafidil had no effect on the viability of A549 cells and MDCK cells at its half effective concentration (see Table 4 and Table 5 for the results, and Figure 4 and Figure 5 for the dose-response curve).

The effect of table 4 napipidil on the viability of A549 cells

Table 5 The effect of napipidil on the viability of MDCK cells

Claims (7)

1. the application of nafidil as shown in structural formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicine for preventing or treating influenza virus infection;

2. The use according to claim 1, wherein the pharmaceutically acceptable salts include pharmaceutically acceptable organic salts or inorganic salts. 3. application according to claim 2, is characterized in that, described organic salt comprises sulfonate, carboxylate, amino acid salt or fatty acid salt, and inorganic salt comprises hydrochloride, bromate, iodate, sulfuric acid salt, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate or nitrate. 4. according to the application of claim 3, it is characterized in that, described sulfonate comprises alkyl sulfonate, benzene sulfonate, p-toluene sulfonate, o-toluene sulfonate containing 1-15 carbon atoms , m-toluenesulfonate; carboxylates include tartrate, maleate, fumarate, citrate, malate, cinnamate, benzoate, malonate, succinic acid salts, glutamate, adipate, pamoate, and lactate; amino acid salts include glutamate, aspartate; fatty acid salts include long chains containing 2-18 carbon atoms fatty acid salts. 5. the application of a pharmaceutical composition in the preparation of the medicine for preventing or treating influenza virus infection, it is characterized in that, described pharmaceutical composition comprises nafidil shown in structural formula (I) and its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or excipient,

6. The application according to claim 5, wherein the pharmaceutical composition also contains other antiviral drugs. 7. The application according to any one of claims 1-5, wherein the influenza virus comprises influenza A virus, influenza B virus, influenza C virus or influenza D virus.

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