CN111000842A - Medicine for treating influenza virus infection - Google Patents
Medicine for treating influenza virus infection Download PDFInfo
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- CN111000842A CN111000842A CN201911319461.4A CN201911319461A CN111000842A CN 111000842 A CN111000842 A CN 111000842A CN 201911319461 A CN201911319461 A CN 201911319461A CN 111000842 A CN111000842 A CN 111000842A
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- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
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Abstract
The invention relates to a medicament for treating influenza virus infection. Specifically, the invention provides a compound, or an isomer thereof, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure shown in the formula I. The compound, or the isomer thereof, or the pharmaceutically acceptable salt thereof has a remarkable inhibitory effect on influenza virus, and is used for preventing and/or treating influenza virus infection.
Description
Technical Field
The invention relates to the field of medicaments, and particularly provides a medicament for treating influenza virus infection.
Background
The influenza virus belongs to the genus of influenza virus of the family Orthomyxoviridae (Orthomyxoviridae). Influenza viruses are classified into A, B, C types, depending on the antigenic and genetic properties of the virion Nucleoprotein (NP) and matrix protein (M). The influenza a virus genome consists of 8 single negative stranded RNAs of different sizes, designated segment 1 to segment 8, respectively. Influenza a viruses can be further divided into 17H (H1-H17) and 10N (N1-N10) subtypes, depending on the surface glycoproteins Hemagglutinin (HA) and Neuraminidase (NA) of the virion. Human influenza viruses are predominantly of the H1, H2 and H3 subtypes. Most of the current highly pathogenic avian influenza with serious harm are H5, H7 and H9 subtypes, wherein the lethality rate is highest by using the H5N1 subtype.
The entire life cycle of influenza virus needs to be completed in the cytoplasm and nucleus. The infection is initiated by the recognition and binding of the spike HA on the surface of the virion to a sialic acid receptor on the surface of the host cell, which binds the virion to the host cell as an endocytosed form. Under the acidic pH condition of endocytosis, the conformation of virus HA protein is changed, the fusion peptide at the N end of the light chain is exposed, and the virus envelope is fused with the cell membrane. The low pH environment also results in large amounts of H+Access to the interior of the virion via the M2 ion channel results in dissociation of the M1 protein from vRNP. The combined result of both is the release of the vRNP of the virion into the cytoplasm of the infected cell. The vRNP is then transferred into the nucleus for genome replication and transcription, and during replication the virus first synthesizes complementary RNA (cRNA) using self RNA as a template, and then synthesizes vRNA using cRNA as a template. The mRNA produced by transcription is transferred from the nucleus to the cytoplasm and is translated into the structural and non-structural proteins of the virus. Part of the synthesized protein (such as NP) needs to be transferred into nucleus again to form vRNP with newly generated vRNA, vRNP begins to assemble into new virion with other virus protein after nucleus emergence, and newly generated progeny virus hydrolyzes glycoprotein on the cell surface through Neuraminidase (NA) to release N-acetylneuraminic acid, so that the virion is promoted to be released from the budding site.
The basic means for preventing and treating influenza are divided into vaccine injection and drug therapy. The effectiveness of the vaccine establishes the similarity between the strain for preparing the vaccine and the influenza virus strain existing in the environment or about to cause epidemic, but because the influenza virus is easy to mutate, the prediction accuracy is difficult, and the prevention and treatment effect of the vaccine is greatly influenced. In the case that the effectiveness of the vaccine is difficult to grasp, the research of the anti-influenza virus medicament is particularly important. While the current FDA approved marketed anti-influenza drugs are only four: amantadine, rimantadine, oseltamivir, zanamivir. The first two are M2 ion channel inhibitors, which inhibit viral replication by inhibiting viral RNA release into the cytoplasm. The latter two belong to inhibitors of NA activity, which inhibit viral replication by inhibiting the release and spread of viral particles. However, the development of new anti-influenza virus drugs is imminent due to problems such as development of viral resistance to these drugs and side effects caused by these drugs.
Therefore, there is a need in the art to develop a novel, highly effective, powerful medicament for the prevention and/or treatment of influenza virus infection.
Disclosure of Invention
The present invention is directed to a drug which is effective in preventing and/or treating influenza virus infection.
In a first aspect of the present invention, there is provided a use of a compound, or an isomer thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention and/or treatment of influenza virus infection:
wherein the compound has the structure of formula I:
in formula I:
r1, R2 and R3 are each independently substituted or unsubstituted C6-C20Aryl, or substituted or unsubstituted 5-20 membered heteroaryl;
r4 and R5 are each independently hydrogen, substituted or unsubstituted C1-C4Alkyl, substituted or unsubstituted C3-C6A cycloalkyl group;
wherein any "substitution" means that one or more (preferably 1, 2, 3 or 4) hydrogen atoms on the group are substituted with a substituent selected from the group consisting of: halogen, C1-C4Alkyl radical, C1-C4Haloalkyl, C3-C6Cycloalkyl radical, C3-C6Halocycloalkyl, hydroxy, mercapto, amino, nitro, C6-C12Aryl, or 5-8 membered heteroaryl;
the heteroaryl group has 1 to 3 (preferably 1, 2 or 3) heteroatoms selected from N, O and S.
In another preferred embodiment, the heteroaryl group has 1N heteroatom.
In another preferred embodiment, R1, R2 and R3 are each independently substituted or unsubstituted C6-C16Aryl, or substituted or unsubstituted 5-12 membered heteroaryl.
In another preferred embodiment, R1, R2 and R3 are each independently substituted or unsubstituted C6-C12An aryl group, a heteroaryl group,
in another preferred embodiment, R1, R2, and R3 are each independently halogen substituted phenyl.
In another preferred embodiment, R4 and R5 are each independently hydrogen.
In another preferred embodiment, the halogen is Cl.
In another preferred embodiment, the compound has the structure of formula Ia:
in the formula:
r4 and R5 are as defined above;
r6, R7 and R8 are each independently halogen, C1-C4Alkyl radical, C1-C4Haloalkyl, C3-C6Cycloalkyl radical, C3-C6A halocycloalkyl group;
a is 1, 2, 3, 4 or 5;
b is 1, 2, 3, 4 or 5;
c is 1, 2, 3, 4 or 5.
In another preferred embodiment, a is 1.
In another preferred embodiment, b is 1.
In another preferred embodiment, c is 1.
In another preferred embodiment, R6 is halogen.
In another preferred embodiment, R7 is halogen.
In another preferred embodiment, R8 is halogen.
In another preferred embodiment, the compound has the structure of formula Ib:
in the formula:
r6, R7 and R8 are each independently halogen, C1-C4Alkyl radical, C1-C4Haloalkyl, C3-C6Cycloalkyl radical, C3-C6A halocycloalkyl group.
In another preferred embodiment, R6 is chloro.
In another preferred embodiment, R7 is chloro.
In another preferred embodiment, R8 is chloro.
In another preferred embodiment, the compound is
In another preferred embodiment, the influenza virus is an influenza a virus.
In another preferred embodiment, the influenza virus is an RNA virus or a DNA virus.
In another preferred embodiment, the influenza virus is an RNA virus.
In another preferred embodiment, the influenza a virus is an influenza H subtype and/or an influenza N subtype, and preferably, the influenza a virus is an influenza H1N1 subtype.
In another preferred embodiment, the influenza virus subtype H is an influenza virus subtype H1, H2, H3, H5, H7 or H9.
In another preferred embodiment, the influenza virus subtype N is influenza virus subtype N1.
In another preferred embodiment, said preventing and/or treating influenza virus infection means:
preventing and/or treating influenza virus infection by inhibiting the binding of influenza virus to host cell membrane.
In another preferred embodiment, the dosage form of the drug is a solid preparation, a liquid preparation or a semisolid preparation.
In another preferred embodiment, the medicament is in the form of tablets, powder, pills, injections, capsules, films, suppositories, paste, granules, injections, infusion solutions and powder injections.
In a second aspect of the present invention, there is provided a pharmaceutical composition for the prophylaxis and/or treatment of influenza virus infection, said pharmaceutical composition comprising a compound according to the first aspect of the present invention, or an isomer thereof, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.
In a third aspect of the invention, there is provided an in vitro non-therapeutic and non-diagnostic method of inhibiting influenza virus, said method comprising the steps of: contacting an influenza virus or an influenza virus infected cell with a compound according to the first aspect of the invention, or an isomer thereof, or a pharmaceutically acceptable salt thereof, thereby inhibiting the influenza virus.
In a fourth aspect of the present invention, there is provided a method for the prophylaxis and/or treatment of an influenza virus infection by administering a compound according to the first aspect of the present invention, or an isomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the second aspect of the present invention to a subject in need of prophylaxis and/or treatment of an influenza virus infection.
In another preferred embodiment, the subject includes human and non-human mammals (rodents, rabbits, monkeys, domestic animals, dogs, cats, etc.).
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 is a graph showing the inhibitory effect of NSC13294, ribavirin and DMSO, the compound of example 2, on H1N1 subtype influenza virus.
FIG. 2 shows the results of the inhibition of HA pseudovirus activity by NSC13294 and DMSO, the compounds of example 3.
Detailed Description
The present inventors have surprisingly found, for the first time, a compound of formula I, or an isomer thereof, or a pharmaceutically acceptable salt thereof, through extensive and intensive studies. Experiments show that the compound has obvious inhibitory effect on influenza virus. The compounds of the present invention are effective in preventing and/or treating influenza virus infection. On the basis of this, the present invention has been completed.
Term(s) for
As used herein, the terms "comprises," "comprising," "includes," "including," and "including" are used interchangeably and include not only closed-form definitions, but also semi-closed and open-form definitions. In other words, the term includes "consisting of … …", "consisting essentially of … …".
As used herein, "R1", "R1" and "R1"has the same meaning as" and can be substituted for "another, and other similar definitions have the same meaning.
The term "alkyl" refers to a straight-chain (i.e., unbranched) or branched-chain saturated hydrocarbon group containing only carbon atoms, or a combination of straight-chain and branched-chain groups. When the alkyl group is preceded by a carbon atom number limitation (e.g. C)1-C4Alkyl) means that the alkyl group contains 1 to 4 carbon atoms, e.g. C1-C4Alkyl refers to an alkyl group containing 1 to 4 carbon atoms, and representative examples include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.
The term "cycloalkyl" refers to a cyclic, bicyclic or polycyclic (fused, bridged or spiro) ring system group having a saturated or partially saturated unit ring. When a cycloalkyl group is preceded by a carbon atom number limitation (e.g. C)3-C6) When used, means that the cycloalkyl group has 3 to 6 carbon atoms. In some preferred embodiments, the term "C3-C6Cycloalkyl "refers to a saturated or partially saturated monocyclic or bicyclic alkyl group having 3 to 6 carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, or the like.
The term "aryl" refers to an all-carbon monocyclic or fused polycyclic ring (i.e., sharing adjacent carbon atoms) having a conjugated pi-electron systemPara-ring), when the aryl group is preceded by a carbon atom number limitation, such as C6-C20Aryl means that the aryl group has 6 to 20 carbon atoms, such as phenyl and naphthyl. The aryl ring may be fused to other cyclic groups (including saturated or unsaturated rings) but must not contain heteroatoms such as nitrogen, oxygen, or sulfur, while the point of attachment to the parent must be at a carbon atom on the ring with the conjugated pi-electron system. Representative examples of aryl groups include, but are not limited to: phenyl, naphthyl, or the like.
The term "heteroaryl" refers to an aromatic heterocyclic system having one to more (preferably 1, 2, 3 or 4) heteroatoms, which may be monocyclic (monocyclic) or polycyclic (bicyclic, tricyclic or polycyclic) fused together or covalently linked, where the heteroatoms referred to herein include oxygen, sulfur and nitrogen. When heteroaryl is pre-defined, examples of, for example, 5-membered heteroaryl include (but are not limited to): examples of pyrrole, furan, thiophene, imidazole, oxazole, thiazole, 6-membered heteroaryl include, but are not limited to, pyridine, pyrazine, pyridazine, pyrimidine. The heteroaryl ring may be fused to an aryl, heterocycloalkyl, or cycloalkyl ring, wherein the ring joined together with the parent structure is a heteroaryl ring.
The term "halogen" refers to F, Cl, Br and I.
The term "halo" means that one or more hydrogens (preferably 1, 2, 3 or 4) of the group are replaced with a halogen, for example "haloalkyl" means that one or more hydrogens (preferably 1, 2, 3 or 4) on the alkyl group are replaced with a halogen.
The term "hydroxy" denotes-OH.
The term "mercapto" denotes-SH.
The term "amino" denotes-NH3。
The term "nitro" denotes-NO2。
Active ingredient
As used herein, "compounds of the invention" or "compounds of formula I" are used interchangeably and refer to compounds having the structure of formula I, or isomers thereof, or pharmaceutically acceptable salts thereof. It is to be understood that the term also includes mixtures of the above components, where in the compound, if a chiral carbon atom is present, the chiral carbon atom may be in the R configuration, also in the S configuration, or a mixture of both (e.g., a racemate).
The compound of the invention is as described in the first aspect of the invention.
The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention with an acid or base that is suitable for use as a pharmaceutical. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed with acids of the compounds of the present invention, and suitable acids for forming salts include (but are not limited to): inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, etc., organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, phenylmethanesulfonic acid, benzenesulfonic acid, etc.; and acidic amino acids such as aspartic acid and glutamic acid. One preferred class of salts are metal salts of the compounds of the present invention formed with bases, suitable bases for forming the salts include (but are not limited to): inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogencarbonate and sodium phosphate, and organic bases such as ammonia, triethylamine and diethylamine.
The compound of formula I of the present invention can be converted into a pharmaceutically acceptable salt thereof by a conventional method, for example, a solution of the corresponding acid can be added to a solution of the above compound, and the corresponding salt of the compound of the present invention can be obtained by removing the solvent after salt formation is completed.
Preferably, the compound is:
compound NSC 13294: from the NCI Diversity Set II library, and CAS: 5450-03-3.
Use of
The present invention provides a method for preventing and/or treating influenza virus infection. The compounds of the present invention can be used for the prevention and/or treatment of influenza virus infections. The compound can be prepared into a medicament for preventing and/or treating influenza virus infection, and the dosage form of the medicament can be solid preparation, liquid preparation or semisolid preparation. Typically, the dosage form of the medicine is tablets, powder, pills, injections, capsules, films, suppositories, paste, granules, injections, infusion solutions and powder injections.
The term "influenza virus" as used herein is synonymous with the meaning commonly understood by those skilled in the art, consisting of a nucleic acid molecule (DNA or RNA) and a protein or consisting of a protein only (e.g., a prion). The virus is small and simple in structure. Influenza viruses have no cellular structure and cannot replicate themselves because there is no fundamental system necessary to achieve metabolism. However, when it comes into contact with the host cell, its nucleic acid substance invades into the host cell, and a new virus is replicated by the latter replication system according to the instruction of the viral gene.
The influenza virus described herein is preferably an RNA virus (RNA virus). RNA viruses are a type of biological virus whose genetic material consists of ribonucleic acid (RNA ribonuclear acid), usually the nucleic acid is single-stranded (ssrnaingle-stranded RNA) and also double-stranded (dsRNA-stranded RNA).
In a specific embodiment, the influenza virus of the present invention is an influenza a virus. In a preferred embodiment, said influenza a virus is an influenza H subtype and/or an influenza N subtype virus. Typically, the influenza a virus is an influenza virus subtype H1N 1.
In another preferred embodiment, the influenza virus subtype H is an influenza virus subtype H1, H2, H3, H5, H7 and H9.
In another preferred embodiment, the influenza virus subtype N is influenza virus subtype N1.
In a preferred embodiment of the present invention, said preventing and/or treating influenza virus infection means:
preventing and/or treating influenza virus by inhibiting the binding of influenza virus to host cell membrane.
The present invention also provides an in vitro non-therapeutic and non-diagnostic method of inhibiting influenza virus, said method comprising the steps of: influenza virus or an influenza virus-infected cell is contacted with a compound of the present invention, or an isomer thereof, or a pharmaceutically acceptable salt thereof, thereby inhibiting influenza virus.
Compositions and methods of administration
The invention provides a composition for preventing and/or treating influenza virus infection. The composition includes (but is not limited to): pharmaceutical compositions, food compositions, dietary supplements, beverage compositions, and the like.
Typically, the composition is a pharmaceutical composition comprising a compound of the invention, or an isomer thereof, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.
In the present invention, the dosage form of the pharmaceutical composition includes (but is not limited to) oral preparations, injections, and external preparations.
Representative include (but are not limited to): tablet, injection, infusion solution, paste, gel, solution, microsphere, and pellicle.
The term "pharmaceutically acceptable carrier" refers to: one or more compatible solid, semi-solid, liquid or gel fillers which are suitable for human or animal use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant that the components of the pharmaceutical composition and the active ingredient of the drug are blended with each other and not significantly detract from the efficacy of the drug.
It is to be understood that, in the present invention, the carrier is not particularly limited and may be selected from materials commonly used in the art, or prepared by a conventional method, or commercially available. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., tween), wetting agents (e.g., sodium lauryl sulfate), buffers, chelating agents, thickeners, pH adjusters, transdermal enhancers, colorants, flavors, stabilizers, antioxidants, preservatives, bacteriostats, pyrogen-free water, etc.
Typically, liquid dosage forms may contain, in addition to the active pharmaceutical ingredient, inert diluents commonly employed in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, especially cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like. In addition to these inert diluents, the compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents and the like
The pharmaceutical preparation should be compatible with the mode of administration. The agents of the invention may also be used with (including before, during or after) other co-therapeutic agents. In using the pharmaceutical composition or formulation, a safe and effective amount of the drug, typically at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably from about 10 micrograms/kg body weight to about 1 mg/kg body weight, is administered to a subject in need thereof (e.g., a human or non-human mammal). Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The main advantages of the invention include:
the invention discovers a series of compounds with broad-spectrum and excellent antiviral activity for the first time, the compounds have high-efficiency inhibitory action on influenza viruses, thereby preventing and/or treating influenza virus infection, and meanwhile, the compounds lay a material foundation for researching and developing new generation antiviral drugs, thereby having important academic value and practical significance.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
Examples
The following are reagents, cells, kits, materials, influenza virus strains, vectors, and the like used in the examples:
PBS buffer (pH7.0-7.2): NaCl 8g, KCl 0.2g, Na2HPO4·12H2O 3.58g、KH2PO40.27g of ultrapure water was added thereto, and the volume was adjusted to 1L.
293T cells (human renal epithelial cells): ATCC, accession number CRL-11268.
DMEM Medium (Dulbecco's Modified Eagle's Medium): gibco Corp.
Transfection reagent PEI (operating as described in the instructions): polyscience, Inc.
Luciferase activity measurement kit: promega corporation.
Ribavirin (ribavirin): sigma Co.
Protease inhibitors: purchased from Roche corporation.
A/WSN/33 influenza virus strain (A/WSN/33 strain for short): is an Influenza A virus subtype H1N1 strain described in "Neumann G1, Watanabe T, Ito H, et al.Generation of Influenza A virus from cloned cDNAs. PNAS,1999(16):9345-9350," entitled "Influenza viruses Ayny 33 (WSH 1N 1)" publicly available from Li-Tech Biotech, Inc., Zhejiang.
Plasmids expressing the PA gene of influenza virus, Plasmids expressing the PB1 gene of influenza virus, Plasmids expressing the PB2 gene of influenza virus, and Plasmids expressing the NP gene of influenza virus (i.e., "Plasmids expressing PA, PB1, PB2, and NP genes", "Plasmids for expressing non-tagged PB1, PB2, PAand NP" in the literature): generally described in "Zhang J, LiuT, Tong X, et al identification of novel vitamins inhibitors by antifluence A viral fibrous reporter cell based screening. additive Ras,2012(1): 48-54", publicly available from Li-En Biotech, Zhejiang.
pRL-TK plasmid: promega corporation.
The pREP4 vector is described in "Liyunfang, Zhang Haoyi, Hourong et al plasmid transfection effects on expression of the native β 2-adrenoceptor in HEK293 and DDT 1-MF 2 cells" journal of Beijing university, medical edition, 5 th 2001, "publicly available from Li En Biotech, Inc., Zhejiang.
pHH21 vector: described in "Neumann G1, Watanabe T, Ito H, et al, Generation of nonfluenza A viruses from bound cDNAs, PNAS,1999(16): 9345-.
pNLLucE-R-HIV-Luc plasmid, pEWSN-HA plasmid and pCAGGS-NA plasmid: reference documents: junjie zhang, Ting Liu, Xiamei Tong, Jinghua Yan, Xin Ye, et al.identification of novelvus inhibitors by antifluence A virus specific reporter cell searching.2011; 93:48-54, publicly available from rien biotechnology limited, zhejiang.
The NSC13294 compound is:
compound NSC 13294: from the NCI Diversity Set II library, and CAS: 5450-03-3.
Example 1 preparation of 293T-IAV-Luc cells
1. Construction of pREP4-IAV-Luc plasmid
1.1, artificially synthesizing a DNA fragment shown as a sequence 1SEQ ID NO. 1 in a sequence table (see a patent CN 106562957A). The sequence 1 consists of 1748 nucleotides, the 14 th to 58 th sites are marked as segment 1, the 59 th to 1711 th sites are firefly luciferase coding gene (reporter gene), the 1712 th and 1734 th sites are marked as segment 2, and the two ends are recognition site sequences of BsmB I. Wherein, the segment 1 and the segment 2 are promoters of NP protein of influenza virus, and under the condition of existence of influenza virus, the promoter positioned on the fusion plasmid can be started, and the firefly luciferase can be expressed.
1.2, cutting a DNA fragment shown in a sequence 1SEQ ID NO. 1 in a sequence table by using a restriction enzyme BsmBI, recovering the cut fragment, positively connecting the cut fragment with a large skeleton fragment of a pHH21 vector cut by the same enzyme, and naming the intermediate plasmid with correct sequencing verification as a pHH21-IAV-Luc plasmid. The pHH21-IAV-Luc plasmid was digested with NheI and PciI endonucleases (Takara), the digested fragments were recovered, the ends were filled with Klenow enzyme (purchased from Takara), and the fragments were ligated into pREP4 vector digested with PvuII endonucleases, and the recombinant plasmid whose sequencing was confirmed was named pREP 4-IAV-Luc.
2. Preparation of 293T-IAV-Luc cell
The recombinant plasmid pREP4-IAV-Luc was introduced into 293T cells to obtain recombinant cells, which were designated as 293T-IAV-Luc cells.
Example 2 Compound NSC13294 inhibits replication of influenza virus subtype H1N1
1. The A/WSN/33 strain, DMEM medium and the compound NSC13294 are mixed to obtain a mixed solution. The mixture contained 0.5MOI virus and 50. mu. mol/L of compound NSC 13294.
2. 293T-IAV-Luc cells prepared in example 1 were uniformly plated on a 96-well plate (20000 cells per well), incubated at 37 ℃ for 12 hours, the supernatant was discarded, and the cells in the wells were washed with PBS buffer.
3. After completion of step 2, the 96-well plate was taken, the mixture obtained in step 1 (MOI ═ 0.5) was added thereto, and the mixture was incubated at 37 ℃ for 1 hour with standing, and the supernatant was discarded.
4. After completion of step 3, the 96-well plate was taken, DMEM medium containing 10% (by volume) fetal bovine serum and 50. mu. mol/L of compound NSC13294 was added thereto, the mixture was left to stand at 37 ℃ for 12 hours, the supernatant was discarded, and the cells in the well were washed with PBS buffer.
5. And (4) after the step 4 is completed, adding the lysis solution in the luciferase activity measurement kit into the 96-well plate, standing and incubating for 30 minutes at 37 ℃, and taking the supernatant.
6. And (5) taking the supernatant obtained in the step (5), and detecting the expression level of the luciferase reporter gene by adopting a luciferase activity measurement kit.
Meanwhile, DMSO and ribavirin are adopted to replace a compound NSC13294 to serve as a negative control (DMSO group) and a positive control (ribavirin group) in the experimental process respectively, the experimental group and the control group are subjected to three times of repeated tests, and the results are averaged.
Results
The inhibitory effect of compounds NSC13294, ribavirin and DMSO on influenza virus subtype H1N1 is shown in table 1 and figure 1:
table 1 inhibitory effect (fluorescein gene expression level) of compounds NSC13294, ribavirin and DMSO on H1N1 subtype influenza virus (N ═ 3)
| Experimental group | | Ribavirin | NSC | 13294 |
| Parallel group 1 | 31384 | 938 | 6554 | |
| Parallel group 2 | 31508 | 906 | 6290 | |
| Parallel group 3 | 30665 | 719 | 6358 |
As can be seen from table 1 and fig. 1, compound NSC13294 is able to significantly inhibit the activity of influenza virus subtype H1N 1.
Example 3 inhibition of HA pseudovirus Activity by Compound NSC13294
3.1 preparation of HA pseudovirus
The 293T cells are inoculated to a cell culture dish, after the density reaches 80 percent, 6 mu g of pNLLucE-R-HIV-Luc plasmid, 6 mu g of pEWSN-HA plasmid and 6 mu g of pCAGGS-NA plasmid are co-transfected by virtue of a transfection reagent PEI, after 6 hours, the plasmids are replaced by a DMEM complete culture medium containing 10 percent (volume fraction) fetal calf serum, after 48 hours, the whole culture system is transferred into a 15ml centrifuge tube, cells are blown away, after one freeze-thaw operation, the cells are filtered by a 0.22 mu m filter membrane, and filtrate is collected, namely virus liquid of HA pseudovirus, and the virus liquid is stored at the temperature of minus 80 ℃.
3.2 Compound NSC13294 inhibits the Activity of HA pseudovirus
1. 293T cells were plated uniformly on a 24-well plate (16 ten thousand cells per well), incubated at 37 ℃ for 15 hours, and the supernatant was discarded.
2. After completion of step 1, the 24-well plate was taken, 100. mu.L of the virus solution of HA pseudovirus prepared in step one and 400. mu.L of DMEM medium containing compound NSC13294 (so that the concentration of compound NSC13294 in the system was 50. mu.M) were added to each well, incubated at 37 ℃ for 18 hours with standing, and the cells in the wells were washed with PBS buffer.
3. And (3) after the step 2 is completed, adding the lysis solution in the luciferase activity measurement kit into the 24-well plate, standing and incubating for 30 minutes at 37 ℃, and taking the supernatant.
4. And (3) taking the supernatant obtained in the step (3), and detecting the expression level of the luciferase reporter gene by adopting a luciferase activity measurement kit.
A negative control was set up using DMSO instead of compound NSC 13294.
Meanwhile, DMSO was used as a negative control instead of compound NSC13294 during the experiment. The experimental group and the control group were subjected to three replicates and the results were averaged.
Results
The results of inhibition of HA pseudovirus activity by compound NSC13294 and DMSO are shown in table 2 and fig. 2:
table 2 inhibition results (fluorescein gene expression level) of HA pseudovirus activity by NSC13294 and DMSO compound (n ═ 3)
As can be seen from table 2 and fig. 2, compound NSC13294 significantly inhibits the activity of HA pseudovirus, indicating that compound NSC13294 can significantly inhibit the binding of influenza virus to host cell membrane, thereby preventing and/or treating influenza virus infection.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Zhejiang Lien Biotechnology Ltd
<120> a medicament for treating influenza virus infection
<130>P2018-1876
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<170>SIPOSequenceListing 1.0
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cgctggagag caactgcata aggctatgaa gagatacgcc ctggttcctg gaacaattgc 180
ttttacagat gcacatatcg aggtggacat cacttacgct gagtacttcg aaatgtccgt 240
tcggttggca gaagctatga aacgatatgg gctgaataca aatcacagaa tcgtcgtatg 300
cagtgaaaac tctcttcaat tctttatgcc ggtgttgggc gcgttattta tcggagttgc 360
agttgcgccc gcgaacgaca tttataatga acgtgaattg ctcaacagta tgggcatttc 420
gcagcctacc gtggtgttcg tttccaaaaa ggggttgcaa aaaattttga acgtgcaaaa 480
aaagctccca atcatccaaa aaattattat catggattct aaaacggatt accagggatt 540
tcagtcgatg tacacgttcg tcacatctca tctacctccc ggttttaatg aatacgattt 600
tgtgccagag tccttcgata gggacaagac aattgcactg atcatgaact cctctggatc 660
tactggtctg cctaaaggtg tcgctctgcc tcatagaact gcctgcgtga gattctcgca 720
tgccagagat cctatttttg gcaatcaaat cattccggat actgcgattt taagtgttgt 780
tccattccat cacggttttg gaatgtttac tacactcgga tatttgatat gtggatttcg 840
agtcgtctta atgtatagat ttgaagaaga gctgtttctg aggagccttc aggattacaa 900
gattcaaagt gcgctgctgg tgccaaccct attctccttc ttcgccaaaa gcactctgat 960
tgacaaatac gatttatcta atttacacga aattgcttct ggtggcgctc ccctctctaa 1020
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cgccgccgtt gttgttttgg agcacggaaa gacgatgacg gaaaaagaga tcgtggatta 1560
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agacgtat 1748
Claims (10)
1. Use of a compound, or an isomer thereof, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the prevention and/or treatment of an influenza virus infection:
wherein the compound has the structure of formula I:
in formula I:
r1, R2 and R3 are each independently substituted or unsubstituted C6-C20Aryl, or substituted or unsubstituted 5-20 membered heteroaryl;
r4 and R5 are each independently hydrogen, substituted or unsubstituted C1-C4Alkyl, substituted or unsubstituted C3-C6A cycloalkyl group;
wherein any "substitution" means that one or more (preferably 1, 2, 3 or 4) hydrogen atoms on the group are substituted with a substituent selected from the group consisting of: halogen, C1-C4Alkyl radical, C1-C4Haloalkyl, C3-C6Cycloalkyl radical, C3-C6Halocycloalkyl, hydroxy, mercapto, amino, nitro, C6-C12Aryl, or 5-8 membered heteroaryl;
the heteroaryl group has 1 to 3 (preferably 1, 2 or 3) heteroatoms selected from N, O and S.
2. The use of claim 1, wherein the compound has the structure of formula Ia:
in the formula:
r4 and R5 are as described in claim 1;
r6, R7 and R8 are each independently halogen, C1-C4Alkyl radical, C1-C4Haloalkyl, C3-C6Cycloalkyl radical, C3-C6A halocycloalkyl group;
a is 1, 2, 3, 4 or 5;
b is 1, 2, 3, 4 or 5;
c is 1, 2, 3, 4 or 5.
3. The use of claim 1, wherein said compound has the structure of formula Ib:
in the formula:
r6, R7 and R8 are each independently halogen, C1-C4Alkyl radical, C1-C4Haloalkyl, C3-C6Cycloalkyl radical, C3-C6A halocycloalkyl group.
4. The use according to claim 1, wherein the compound is
5. The use according to claim 1, wherein the influenza virus is an influenza a virus.
6. The use according to claim 1, wherein the influenza a virus is an influenza H subtype and/or an influenza N subtype, preferably the influenza a virus is an influenza H1N1 subtype.
7. The use according to claim 1, wherein the prevention and/or treatment of influenza virus infection is:
preventing and/or treating influenza virus infection by inhibiting the binding of influenza virus to host cell membrane.
8. The use of claim 1, wherein the medicament is in the form of a solid, liquid or semi-solid formulation.
9. A pharmaceutical composition for preventing and/or treating influenza virus infection, comprising the compound of claim 1, or an isomer thereof, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.
10. An in vitro non-therapeutic and non-diagnostic method of inhibiting influenza virus comprising the steps of: contacting an influenza virus or an influenza virus-infected cell with the compound of claim 1, or an isomer thereof, or a pharmaceutically acceptable salt thereof, thereby inhibiting the influenza virus.
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| CN108653279A (en) * | 2018-06-26 | 2018-10-16 | 南方医科大学 | The pyrrolin analog derivative of application and a kind of resisiting influenza virus of the pyrrolin analog derivative on preparing influenza virus inhibitor |
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| CN108653279A (en) * | 2018-06-26 | 2018-10-16 | 南方医科大学 | The pyrrolin analog derivative of application and a kind of resisiting influenza virus of the pyrrolin analog derivative on preparing influenza virus inhibitor |
Non-Patent Citations (4)
| Title |
|---|
| BY: LUTZ等: "Antimalarials. α-Alkyl and dialkylaminomethyl-2-phenyl-4-quinolinemethanols", 《JOURNAL OF THE AMERICAN CHEMICAL SOCIETY》 * |
| KHARE G等: "Identification of inhibitors against Mycobacterium tuberculosis thiamin phosphate synthase, an important target for the development of anti-TB drugs", 《PLOS ONE》 * |
| 汪世雄等: "蛋白激酶抑制剂Flavopiridol 对流感病毒复制的体外抑制作用", 《微生物学报》 * |
| 赖文彬等: "Colletodiol 降低聚合酶活性抑制流感病毒( WSN/H1N1) 复制", 《微生物学报》 * |
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Denomination of invention: A drug to treat influenza virus infection Effective date of registration: 20220921 Granted publication date: 20220624 Pledgee: Pinghu Rural Commercial Bank of Zhejiang, Limited by Share Ltd. Pledgor: ZHEJIANG GLLION BIOSCIENCE Co.,Ltd. Registration number: Y2022990000662 |