WO2020241737A1 - Anti-hiv pharmaceutical composition - Google Patents

Anti-hiv pharmaceutical composition Download PDF

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Publication number
WO2020241737A1
WO2020241737A1 PCT/JP2020/021080 JP2020021080W WO2020241737A1 WO 2020241737 A1 WO2020241737 A1 WO 2020241737A1 JP 2020021080 W JP2020021080 W JP 2020021080W WO 2020241737 A1 WO2020241737 A1 WO 2020241737A1
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group
hiv
pharmaceutical composition
capsid
composition according
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PCT/JP2020/021080
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French (fr)
Japanese (ja)
Inventor
天野　将之
朋文 中村
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国立大学法人熊本大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/502—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with carbocyclic ring systems, e.g. cinnoline, phthalazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a novel pharmaceutical composition for treating or preventing HIV, which comprises a compound having anti-HIV activity, and a method for treating HIV infection.
  • the HIV particles are spherical with a diameter of about 120 nm, and are an envelope consisting of a lipid bilayer membrane derived from a host cell in which the viral glycoprotein Env (gp120, gp41) is planted.
  • a matrix that is one of the viral protein group Gag that lines the envelope. (MA) inside it is a cone-shaped core made of capsid (CA) protein, which is also one of the hosts.
  • CA capsid protein
  • HIV Autoimmune disease
  • HIV-1 is a common type that is distributed worldwide, while HIV-2 causes AIDS primarily in West Africa.
  • HIV is a retrovirus, and like many viruses, HIV is infected by forming virus particles (virions) during the infection cycle.
  • the HIV-1 billion is spherical and contains a high electron density pyramidal core, which is enveloped in a lipid envelope derived from the host cell membrane.
  • the viral cores are (1) major capsid protein p24 (CA), (2) nucleocapsid protein p7 / p9 (NC), (3) two copies of genomic RNA, and (4) three viral enzymes (protease (PR)). , Reverse Transcriptase (RT), and Integrase).
  • the viral core is wrapped in a matrix protein, the so-called p17, which is located below the viral envelope.
  • the viral envelope has two viral glycoproteins, gp120 and gp41.
  • the HIV provirus genome contains gag, pol, and env genes, which encode various viral proteins.
  • the products of the gag and pol genes are first translated into a large precursor protein, which is cleaved by viral proteases to produce mature proteins.
  • CA is first synthesized as a region within the 55 kDa Gag precursor polyprotein. Approximately 4,000 copies of Gag assemble on the plasma membrane and germinate to form immature viral particles. After budding, when Gag is cleaved by proteolysis, CA is released, which triggers a change in the three-dimensional structure, and this change promotes the construction of capsid particles. Two copies of the viral genome and enzymes essential for infectivity are encapsulated within the central pyramidal capsid of the mature virion.
  • HIV-1 Gag protein inhibitor has not yet reached clinical application.
  • Non-Patent Document 1 V. Novitsky, et al., J Virol, 2002, 76: 5435-51, HIV Sequence Compendium 2014, Los Alamos National Laboratory, USA. Therefore, CA is attracting attention as a promising antiviral target.
  • Several reports have been made as HIV-1 CA inhibitors.
  • Non-Patent Document 2 F. Li, et al. PNAS vol. 100, no. 23, pp.13555-13560, 2003.
  • the capsid assembly inhibitor (CAI) is a 12-mer peptide that binds to the C-terminal side of CA and exhibits an action of suppressing virus budding from infected cells (Non-Patent Document 3: Jana Stickt, et. Al). ., Nat Struct Mol Biol, pp. 671-677, 2005).
  • Non-Patent Document 4 Chun Tang, et al., J Mol Biol, vol. 327, pp. 1013-1020, 2003. It has been reported that PF-3450074 over-promotes CA multimeric stabilization and inhibits normal CA function (Non-Patent Document 5: Wada S. Blair, PLoS Pathog, vol., 6, Issue 12, e1001220). , 2010).
  • An object of the present invention is to provide a molecule having novel anti-HIV activity.
  • the present invention also aims to provide novel anti-HIV agents targeting the HIV-1 capsid.
  • the present inventors are new in that they can suppress the growth of HIV-1 by specifically binding to CA, which is a structural protein forming HIV-1 particles, and further inducing excessive stabilization of CA.
  • CA which is a structural protein forming HIV-1 particles
  • the HIV-1 infection process goes through unshelling, which is the process in which the CA shell breaks after infecting the target cells and the viral genes inside are transferred to the cytoplasm or nucleus.
  • unshelling is the process in which the CA shell breaks after infecting the target cells and the viral genes inside are transferred to the cytoplasm or nucleus.
  • E45A and N74D mutations in amino acids in the CA region of HIV-1 promote the stability of HIV-1 CA and impair CA shell shedding compared to wild-type CA.
  • a compound that strongly binds to the region containing these amino acids on the surface of the CA protein can induce unshelling disorder of the CA shell and thus inhibit the infection / proliferation of HIV-1.
  • a compound that excessively stabilizes CA was found, and the present invention was completed.
  • the present invention includes the following aspects.
  • X and Y each independently represent C or N
  • Z represents C or bond
  • Ra and Rb each independently represent a hydrogen or optionally substituted phenyl group. Together with each other, they form a 6-membered carbocycle that may be substituted with X and Y, where R 1 contains C1-C5 alkyl or alkenyl groups, phenyl groups, benzyl groups, or 2-morpholinoethyl groups. Represent.
  • a pharmaceutical composition for treating or preventing HIV which comprises a pharmaceutically acceptable salt thereof as an active ingredient.
  • R 2 represents an alkyl or alkenyl group, a benzyl group, or a 2-morpholinoethyl group of C1 to C5.
  • R 2 represents an alkyl or alkenyl group, a benzyl group, or a 2-morpholinoethyl group of C1 to C5.
  • R 2 is an alkyl or an alkenyl group of C1 to C3 in the above formula (II).
  • R 2 is selected from the group consisting of a methyl group, an ethyl group, and an allyl group in the above formula (II).
  • R 2 is 2-morpholinoethyl in the above formula (II).
  • R 3 represents an alkyl group, an alkenyl group, or a phenyl group of C1 to C5.
  • the pharmaceutical composition according to the above [1] which comprises the compound represented by [1] or a pharmaceutically acceptable salt thereof.
  • R 3 is selected from the group consisting of a methyl group, an ethyl group, and an allyl group.
  • R 3 is a phenyl group in the above formula (III).
  • composition according to the above [2] which comprises, as an active ingredient, a compound selected from the group consisting of or a pharmaceutically acceptable salt thereof.
  • composition according to the above [2] which comprises, as an active ingredient, a compound selected from the group consisting of or a pharmaceutically acceptable salt thereof.
  • the other anti-HIV agent is a chemotherapeutic agent, an antiretroviral inhibitor, a cytokine, a hydroxyurea, a monoclonal antibody that binds to a Gag protein, or an inhibitor of other retrovirus replication.
  • the pharmaceutical composition according to. [15] For patients in need of treatment or prevention of HIV, the following formula (I):
  • X and Y independently represent C or N
  • Z represents C or a single bond
  • Ra and Rb represent phenyl groups which may be independently hydrogenated or substituted, respectively. Together they form a 6-membered carbocycle that may be substituted with X and Y, where R 1 contains C1-C5 alkyl or alkenyl groups, phenyl groups, benzyl groups, or 2-morpholinoethyl groups.
  • Represent. Or a method for treating or preventing HIV, which comprises administering to a patient a pharmaceutically acceptable salt thereof.
  • the compound or a pharmacologically acceptable salt thereof is the following formula (II):
  • R 2 represents an alkyl or alkenyl group, a benzyl group, or a 2-morpholinoethyl group of C1 to C5. ]
  • the method according to the above [15] which is a pharmaceutically acceptable salt thereof.
  • the compound or a pharmacologically acceptable salt thereof is described in Formula (III) :.
  • R 3 represents an alkyl group, an alkenyl group, or a phenyl group of C1 to C5.
  • a method for screening a substance having anti-HIV activity wherein the following steps: (A) A step of incubating a candidate substance in a buffer at a temperature of 37 ⁇ 2 ° C. with a capsid prepared from cells expressing the wild-type capsid of HIV-1, wherein the cell is of HIV-1.
  • the present invention provides a novel anti-HIV-1 drug based on a completely new mechanism of action and target of action that induces stabilization of the capsid of HIV-1.
  • X to Y when used, they are used to mean that X is included as the lower limit and Y is included as the upper limit, or that X is used as the upper limit and Y is included as the lower limit.
  • the HIV-1 virus repeats proliferation through a process in which the capsid (CA) shell is broken and the internal viral gene is transferred into the cytoplasm or cell nucleus (called "unshelling").
  • CA capsid
  • the E45A and N74D mutations promote the stability of HIV-1 CA and impair the unshelling of the CA shell compared to wild-type CA.
  • a compound that strongly binds to the region containing the same amino acid on the surface of the CA protein can induce unshelling disorder of the CA shell and, in turn, inhibit the infection and proliferation of HIV-1.
  • the present inventors performed a detailed crystal structure analysis of the capsid (CA) of HIV-1.
  • CA capsid
  • hydrophobic pockets of a size capable of binding the compound were identified around amino acids E45 and N74.
  • the binding score of each compound to the target pocket on CA was calculated by the method of in silico docking simulation targeting the same pocket, and the compound with a good score was actually measured.
  • the anti-HIV-1 activity of the compound was evaluated by the MTT assay method (evaluating the ability to prevent cell death due to HIV-1 infection) using the wild strains HIV-1 LAI and MT2 cells. As a result, dozens of compounds having anti-HIV-1 activity were identified.
  • the effect on the stability of HIV-1 CA was evaluated. Specifically, a plasmid that expresses HIV-1CA alone is prepared, CA is expressed in cells, a cell lysate is prepared, and a CA antibody is used to change the CA over time after standing at 37 ° C. It was analyzed by the ELISA method and the like.
  • the anti-HIV-1 compound of the present invention is a compound represented by the following structural formula (I) or a pharmaceutically acceptable salt thereof.
  • X and Y independently represent C or N
  • Z represents C or bond (bond means that Y and N are directly bonded)
  • Ra and Rb are.
  • R 1 is an alkyl group or alkenyl of C1-C5. It represents a group, a phenyl group, a benzyl group, or a 2-morpholinoethyl group, preferably an alkyl group or an alkenyl group of C1 to C3.
  • R 1 is preferably an alkyl or alkenyl group of C1 to C5, more preferably an alkyl or alkenyl group of C1 to C3, and even more preferably a methyl, ethyl or allyl group.
  • One group of preferred anti-HIV compounds of the present invention is the compound represented by the following structural formula (II) or a pharmaceutically acceptable salt thereof.
  • R 2 represents an alkyl or alkenyl group of C1 to C5, a benzyl group, or a 2-morpholinoethyl group, preferably an alkyl or alkenyl group of C1 to C3, more preferably a methyl group. , Ethyl group or allyl group.
  • a more preferable compound or salt thereof in the compound represented by the above formula (II) or a salt thereof is at least one compound selected from the compounds represented by the following formula or a salt thereof.
  • At least one compound selected from the compounds represented by the following chemical formulas or a salt thereof is at least one compound selected from the compounds represented by the following chemical formulas or a salt thereof.
  • Another preferred group of anti-HIV compounds of the present invention is the compound represented by the following structural formula (III) or a pharmaceutically acceptable salt thereof.
  • R 3 represents an alkyl or alkenyl group of C1 to C5, or a phenyl group, preferably an alkyl or alkenyl group of C1 to C3, or a phenyl group, more preferably an ethyl or phenyl group.
  • a more preferable compound or salt thereof in the compound represented by the above formula (III) or a salt thereof is a compound selected from the compounds represented by the following formula or a salt thereof.
  • the compounds that can be used in the present invention can be synthesized by referring to known methods used in the technical field of compound synthesis of low molecules, but some of them can also be purchased. Techniques known in the art of compound synthesis at the time of this application can be used without limitation in the present invention.
  • composition for the treatment or prevention of HIV infection, which comprises a therapeutically effective amount of a compound represented by any of the above formulas (I) to (III) or a pharmaceutically acceptable salt thereof according to the present invention.
  • the composition is provided.
  • “Pharmaceutically acceptable salt thereof” refers to any non-toxic salt formed from a compound represented by any of the above formulas (I)-(III). Suitable salts include, for example, but not limited to, hydrochlorides, hydrobromates, hydroiodates, phosphates, hydrogen phosphates, inorganic acid salts such as sulfates, acetates, birds.
  • Salts of acidic groups such as salts, lower alkylamines such as methylamine, ethylamine, cyclohexylamine, substitutions with organic bases such as diethanolamine and triethanolamine, glycine salts, lysine salts, arginine salts, ornithine.
  • Examples include salts, amino acid salts such as glutamate and asparaginate.
  • the present invention also includes pharmaceutical compositions containing solvates and hydrates formed from compounds represented by any of the above formulas (I)-(III).
  • the compounds used in the present invention may be obtained in the form of their hydrates or may contain other solvents used for their crystallization.
  • the compounds used in the present invention may, essentially or by design, form a solvate with a pharmaceutically acceptable solvent (including water). Therefore, the compounds used in the present invention include both solvated and non-solvated forms.
  • solvate refers to a molecular complex of one or more solvent molecules and the above compounds, including pharmaceutically acceptable salts thereof. Such solvent molecules are commonly used in the field of pharmaceutical technology and are known to be harmless to humans.
  • hydrate refers to a complex in which the solvent molecule is water.
  • the compounds used in the present invention including their salts, hydrates and solvates, can form polymorphs. Solvates or hydrates may be useful in producing crystalline forms of the compounds represented by any of the formulas (I)-(III).
  • the pharmaceutical composition of the present invention comprises one or more pharmaceutically acceptable carriers and, as appropriate, other HIV antiviral agents.
  • Antiviral agents and immunomodulators can be included.
  • the term "pharmaceutically acceptable carrier” is used in any and all solvents, dispersion media, coatings, antioxidants, chelating agents, as is known to those skilled in the art. Preservatives (eg, antibacterial agents, antifungal agents), surfactants, buffers, osmotic pressure regulators, absorption retarders, salts, drug stabilizers, excipients, diluents, binders, disintegrants, sweeteners , Fragrances, abundants, dyes, etc., and combinations thereof. Unless any carrier is incompatible with the active ingredient of the invention, it can be used in the therapeutic or pharmaceutical compositions of the invention.
  • the term "therapeutically effective amount” refers to an amount of the above formulas (I)-(III) sufficient to produce a therapeutic effect when administered to a mammal in need of treatment. A compound represented by either.
  • the therapeutically effective amount depends on the subject and the disease symptoms to be treated, the weight and age of the subject, the severity of the disease symptoms, the administration method, and the like, and can be easily determined by those skilled in the art.
  • object refers to animals.
  • the animal is a mammal.
  • the subject also refers to, for example, primates (eg, humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds and the like.
  • the subject is a primate, preferably a human.
  • compositions containing the compound represented by any of the above formulas (I) to (III) of the present invention can be formulated according to a known method for prescribing a pharmaceutical composition.
  • Representative pharmaceutical compositions may include the pharmaceutically acceptable carriers described above. The use of these carriers is well known in the art. Further, a method for preparing a pharmaceutical composition containing an active ingredient is well known in the art.
  • the pharmaceutical composition of the present invention can be formulated to suit a specific route of administration according to the purpose of use. Routes of administration include, but are not limited to, oral, parenteral, intravenous, intradermal, subcutaneous, transdermal, inhalation, topical, transmucosal, or rectal administration.
  • the pharmaceutical composition of the present invention may be formulated in solid or liquid form. Fixed forms include, but are not limited to, tablets, capsules, pills, granules, powders, or suppositories. Liquid forms include, but are not limited to, solutions, suspensions, or emulsions.
  • the pharmaceutical composition of the present invention is preferably administered orally.
  • the dose of the compound of the present invention is appropriately selected depending on the type of disease, the symptom of the administration target, the age, the administration method, and the like.
  • the dose of the compound of the present invention is appropriately selected depending on the type of disease, the symptom of the administration target, the age, the administration method, and the like.
  • usually 0.1 to 5000 mg, preferably 1 to 2000 mg, more preferably 5 to 100 mg per day may be administered in one or several divided doses. ..
  • the toxicity and therapeutic effects of the compounds of the invention are, for example, according to standard methods using cell cultures or laboratory animals representing the viral activity of HIV-1, eg LD50 (lethal dose to 50% of the population) and ED50 (lethal dose to 50% of the population) and ED50 ( It can be estimated by determining the therapeutically effective dose in 50% of the population and determining the therapeutic index (LD50 / ED50 ratio).
  • LD50 lethal dose to 50% of the population
  • ED50 lethal dose to 50% of the population
  • ED50 ED50
  • a compound showing a large therapeutic index is selected.
  • Compounds with addictive side effects can also be used, but in such cases it is possible to design a delivery system that directs the compound to the site of infected tissue in order to minimize damage to non-infected cells and reduce side effects. is there.
  • the dose of the compound is preferably in the range of circulating concentrations containing ED50, which has little or no toxicity.
  • the dosage can vary within this range depending on the route of administration used and the formulation used.
  • therapeutically effective doses can be estimated first from cell culture assays. Dosages are examined in animal models to achieve a circulating plasma concentration range containing IC50s (ie, concentrations of test compounds that achieve half-maximal inhibition) as measured in cell culture. ⁇ Can be decided. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured by methods known in the art, such as high performance liquid chromatography.
  • the present invention also includes a method for treating HIV infection, which comprises administering to a patient a therapeutically effective amount of a compound represented by any of the above formulas (I) to (III).
  • the present invention also includes methods for the treatment of HIV infection (combination) and combinations of two or more active pharmaceutical ingredients (combination), including administration to a patient in combination with other known anti-HIV agents. ..
  • Other anti-HIV agents include, but are not limited to, chemotherapeutic agents, antiretroviral inhibitors, cytokines, hydroxyurea, monoclonal antibodies that bind to the Gag protein, or other retrovirus replication inhibitors. be able to.
  • anti-retrovirus inhibitors include reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors (raltegravir, etc.), CCR5 inhibitors (maraviroc), and fusion inhibitors.
  • the present invention also provides a method of inhibiting viral replication of HIV-1, which comprises the step of administering to a subject a therapeutically effective amount of the above pharmaceutical composition.
  • the method is further selected from the group consisting of antiretroviral inhibitors, cytokines, hydroxyureas, monoclonal antibodies that bind to Gag proteins, or other inhibitors of retroviral replication. Including being used in combination with the treatment of.
  • antiretroviral inhibitors include reverse transcriptase inhibitors, protease inhibitors, and fusion inhibitors.
  • Reverse-transcriptase inhibitors include nucleoside / nucleotide reverse-transcriptase inhibitors (NRTI) (collectively referred to as nucleoside reverse-transcriptase inhibitors) and non-nucleoside / nucleotide reverse-transcriptase inhibitors (NNRTI) (hereinafter collectively referred to as nucleoside reverse-transcriptase inhibitors). Collectively, non-nucleoside reverse transcriptase inhibitors) are included.
  • NRTI nucleoside / nucleotide reverse-transcriptase inhibitors
  • NRTI non-nucleoside / nucleotide reverse-transcriptase inhibitors
  • Nucleoside reverse transcriptase inhibitors include, but are not limited to, zidovudine (ZDV, formerly known as azidothymidine (AZT)), didanosine (dideoxyinosine (ddI)), zalcitabine (dideoxycitidine (dideoxycitidine)).
  • ZDV zidovudine
  • ddI didanosine
  • ddI didanosine
  • zalcitabine dideoxycitidine
  • dideoxycitidine dideoxycitidine
  • ddC) Lamibudine
  • d4T Stavudine
  • Abacavir Abacavir
  • TDF Tenohovir
  • non-nucleoside reverse transcriptase inhibitors of HIV include, but are not limited to, nevirapine, delavirdine mesylate, and efavirenz.
  • HIV protease inhibitors include, but are not limited to, amprenavir, saquinavir mesylate, ritonavir, indinavir sulfate, nelfinavir mesylate, lopinavir and ritonavir, atazanavir, fosamprenavir. You can raise the building. HIV fusion inhibitors include, but are not limited to, enfuvirtide.
  • the present invention also includes methods for screening substances with anti-HIV activity.
  • the method of the present invention is characterized in that the capsid of HIV-1 itself is used to screen for a substance that induces stabilization of the capsid.
  • the capsid of HIV-1 the capsid expressed by introducing a plasmid containing the gene of the HIV-1 capsid into cultured cells may be used, or the capsid isolated or purified from the HIV-1 virus may be used. However, preferably, the HIV-1 capsid expressed in cells is used.
  • the means for expressing the HIV-1 capsid in cells is not limited to this, but can be carried out, for example, as follows.
  • Preparation of expression plasmid For example, but not limited to this, an expression plasmid is prepared as follows. A wild-type CA single expression plasmid is prepared using a pCMV-Myc vector (# 631604, Takara Bio Inc.). The region containing the Myc epitope is deleted with a restriction enzyme to linearize the vector. The gene region encoding the capsid is amplified by PCR using HIV-1 NL4-3 as a template, and then introduced into a linearization vector. Expression of CA protein The prepared capsid expression plasmid is transfected into cells to express the capsid protein in the cells.
  • the cells are not particularly limited, but for example, COS-7 cells can be used.
  • the expressed capsid protein can be used in any of a cell lysate state, a crudely purified state, and a purified state.
  • the crude purification and purification of the expressed capsid can be carried out with reference to a known method.
  • a compound that induces stabilization of the capsid can be screened using the wild-type capsid of HIV-1 prepared as described above. Not limited to this, for example, using a cell lysate of a cell expressing a capsid, the lysate together with the target compound at an arbitrary temperature (for example, 37 ° C.) for a predetermined time (for example, several hours to several days). ) Incubate and confirm capsid disintegration. Disintegration of the capsid is not limited to this, but can be confirmed, for example, by measuring the amount of the capsid antigen by ELISA using an antibody against the capsid.
  • Example 1 Hydrophobic cavities on the surface that contribute to capsid stabilization
  • E45A and N74D mutations have been reported to delay capsid shell shedding compared to wild-type capsids.
  • N74 the 45th amino acid
  • Example 2 Identification of compounds that bind to capsid cavities Structural data of more than about 8 million compounds that can be purchased are obtained from academic and commercial compound databases, and when they are actually administered to a living body, the raw material of each compound is obtained. Taking into consideration the factors that affect ADME in the body, about 7 million compounds having molecular properties that can be used as drugs were extracted. About 7 million selected compounds, MMFF94 (Merck Molecular Force Field 94) was used to perform three-dimensional structural conversion and energy minimization calculation to optimize the structure of each compound in the solvent, and then virtual docking. The binding score of each compound to the target cavity on the capsid was calculated by a simulation (in silico docking simulation) method, and thousands of compounds having a good binding score were selected.
  • MMFF94 Merck Molecular Force Field 94
  • Example 3 Evaluation of anti-HIV-1 activity
  • the compounds selected in Example 2 were evaluated for anti-HIV-1 activity by the MTT assay method as follows.
  • an MTT assay using MT-2 cells and the laboratory wild strain HIV-1 LAI was used.
  • Each compound was serially diluted on a 96-well plate, and a mixed solution of virus and MT-2 cells having a concentration of 100 TCID 50 was added to the well to prepare a well containing only MT-2 cells as a control. After culturing for one day, MTT reagent was added to each well and a color reaction was carried out in the culture chamber.
  • the absorbance of each well is measured and compared with the absorbance of the well in which only the cells are cultured to anti-HIV-1. If there is activity, it can be calculated as an EC 50 value, which is a concentration that inhibits cell damage due to HIV-1 infection by 50%.
  • This system was used to evaluate the anti-HIV-1 activity of the compounds selected in Example 2. As a result, more than 40 kinds of compounds were selected.
  • Example 4 Evaluation of Capsid Stabilization Inducing Activity
  • the compound selected in Example 3 was evaluated for the stabilization inducing activity of the capsid of HIV-1.
  • (1) Preparation of wild-type capsid expression plasmid A wild-type capsid expression plasmid was prepared as follows. A wild-type CA single expression plasmid was prepared using a pCMV-Myc vector (# 631604, Takara Bio Inc.). The region containing the Myc epitope was deleted with a restriction enzyme to linearize the vector, and the gene region encoding the capsid was amplified by PCR using HIV-1 NL4-3 as a template, and then introduced into the above linearized vector.
  • the prepared cell lysates were dispensed into four tubes in equal amounts, and allowed to stand at a constant temperature at 37 ° C. for different times (0 to 72 hours).
  • the amount of capsid antigen in each cell lysate was measured by ELISA using a monoclonal antibody against capsid (Lumipulse p24 cartridge Fujirebio), and the stable ratio of capsid was calculated from the following formula. (P24 antigen amount of each sample / p24 antigen amount of unincubated sample) ⁇ 100 The one to which only DMSO was added was used as a control.
  • the following compound (PF3450074) which has been reported to over-promote the stabilization of the multimer of CA, was used as a comparative example.
  • Compound B The proportion of stable CA after culturing for 72 hours was 79.4% for DMSO (control) and 99.1% for compound A. Compound B was 95.7% and PF3450074 was 75.7%. Compounds A and B induced significant capsid stabilization, whereas PF3450074 was not different from control (DMSO).
  • Example 5 Evaluation of anti-HIV-1 activity of other similar compounds Obtained the compounds listed in the table below as compounds having similar structures to each of the two compounds (Compounds A and B) identified in Example 4. Then, their anti-HIV-1 activity was evaluated in the same manner as in Example 3. The compound can also be synthesized by appropriately referring to a known synthesis method. The results are shown in the table below.
  • Example 6 using a plasmid expressing the confirmation of inhibitory activity HIV wild-type strain (pHIV-1 NL4-3) to viral synthesis and budding, the influence of the compounds on viral synthesis and sprouting after transfection (Compound A and Compound B) was confirmed as follows.
  • the day before transfection (TF) COS-7 cells were seeded on a 6-well plate at 1.5 ⁇ 10 5 cells / well, and after confirming that the cells were 70% or more confluent on the day, lipofectamine LTX (Invitrogen) was used.
  • compounds A and B were added to the TF medium at concentrations of 1, 10 and 100 ⁇ M, respectively.
  • the expression level of HIV-1 p24 in the culture supernatant after 72 hours of TF was measured by ELISA (Lumipulse f, Fujirebio), and the effect of the compound on virus budding was measured. As a result, as shown in FIG. 2, it was found that compounds A and B did not affect virus synthesis and budding after HIV-1 NL4-3 expression plasmid TF.
  • Example 7 Confirmation of cytotoxicity The MTT assay method was used to confirm the cytotoxicity of the compound.
  • COS-7 cells or MT-4 cells were added to a 96-well plate, and a compound diluted in 10 steps was added at the same time to prepare a well containing only COS-7 cells or MT-4 cells as a control, and cultured for 7 days.
  • MTT reagent was added to each well and a color reaction was carried out in a culture chamber.
  • MTT solubilized solution was added to each well to dissolve formazan crystals (synthesized in living cells)
  • the absorbance of each well is measured and compared with the absorbance of the well in which only cells are cultured, resulting in 50% cytotoxicity.
  • the concentration (CC 50 ) was calculated.
  • both compounds A and B were CC 50 :> 100 ⁇ M for COS-7 cells and 87 and 39 ⁇ M for MT-4 cells, respectively, and compound A and compound B were almost cytotoxic. It was confirmed not to show
  • Example 8 Confirmation of Cytotoxicity to Human-Derived Cells Similar to Example 7, for Compound A and Compound B, using the MTT assay method, human hepatobiliary system-derived cells (Li-7, HLE and YSCCC cells) and Cytotoxicity to kidney line-derived cells (HEK293 cells) was confirmed. The results are shown in the table below. Compound A showed no cytotoxicity to human-derived cells. In addition, Compound B showed no cytotoxicity to human hepatobiliary cells and showed almost no cytotoxicity to renal cells.
  • Example 9 Evaluation of entry into target cells by MAGI assay
  • the HIV Tat protein is a transcriptional activator of the HIV gene and is believed to be co-localized with the HIV gene in the virion. Then, when HIV infects the target cell, shedding occurs, and the Tat protein is also sent from the cytoplasm of the target cell into the nucleus together with the HIV gene (HIV genomic RNA).
  • the Maggi assay using U373-Magi CD4 + CXCR4 + cells incorporating a fusion gene of the HIV-1 LTR and the Escherichia coli lacZ gene shows that when the Magi cells are infected with HIV, the HIV-1 LTR pre-integrated in the infected cells becomes HIV.
  • Tat which is a transcriptional regulator of -1
  • ⁇ -gal activity is increased by the lacZ gene downstream of it, so that the number of blue-stained infected cells after the X-gal reaction can be measured and infectivity can be evaluated.
  • the outline of the assay system is shown in FIG. The virus was forcibly expressed in COS-7 cells, and the virus supernatant of HIV-1 WT was infected with U373-Magi CD4 + CXCR4 + cells so as to contain the same amount of p24 antigen, and the number of infected cells was microscopically measured. The infectivity of the virus was evaluated by measuring below.
  • Virus supernatants transfect only pHIV-1 NL4-3, Compound A (100 [mu] M), with a viral supernatant obtained by culturing 3 days after transfection with the conditions of compound B (100 [mu] M).
  • a viral supernatant obtained by culturing 3 days after transfection with the conditions of compound B (100 [mu] M).
  • 6 ⁇ 10 4 cells per well were seeded and cultured for 1 day.
  • the above virus supernatant was diluted to contain an equal amount of p24 (10 ng / ml), added to cells together with DEAE dextran, infected with the virus, and then cultured for 2 hours (adsorption reaction). Then, a medium was further added and the cells were cultured for 48 hours.
  • Example 10 Suggestion of increased thermal stability of capsid Using scanning fluorescence measurement, the effect of the compound on the thermal stability of HIV-1 capsid was confirmed as follows.
  • Suggestion Scanning fluorescence measurement is a method in which the three-dimensional structure of the target protein is denatured by raising the temperature of the environment in which the target protein is present, and the exposure of the hydrophobic region is increased, so that the binding of the added fluorescent dye is increased.
  • This is a method for measuring the denaturation temperature (Tm) of a protein by analyzing the change in fluorescence intensity.
  • Example 11 Concentration of triplicate drug resistance induced test MT-4 cells (3 ⁇ 10 5 / well) with HIV-1 NL4-3 at a concentration of 500TCID 50 in EC 50 near concentration (at resistance induced initiation) After confirming the growth of the virus by culturing in the presence of the above compound and measuring the amount of p24 antigen in the supernatant on day 7, the virus propagated and only the virus supernatant containing the highest concentration of the compound was collected. , Infect the cells with the virus by adding the new MT-4 cells to the collected virus supernatant and culturing for 5 hours or more, and after washing the infected cells, three compounds with different concentrations (one is the latest one).
  • the compound of the present invention exhibits anti-HIV activity and is therefore useful as an antiviral inhibitor.

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Abstract

The purpose of the present invention is to provide a novel small molecule having anti-HIV activity. The purpose of the present invention is also to provide a novel anti-HIV drug that targets an HIV-1 capsid. The present invention provides a novel compound having anti-HIV activity and represented by formula(I): [In formula (I), X and Y each independently represent C or N, Z represents C or a bond, Ra and Rb each independently represent hydrogen or an optionally substituted phenyl group, or together form an optionally substituted six-membered carbon ring together with X and Y, R1 represents a C1-C5 alkyl group or alkenyl group, a phenyl group, a benzyl group, or a 2-morpholinethyl group].

Description

抗ＨＩＶ医薬組成物Anti-HIV pharmaceutical composition
　本発明は、抗ＨＩＶ活性を有する化合物を含む新規なＨＩＶの治療又は予防のための医薬組成物、並びに、ＨＩＶ感染の治療方法に関する。 The present invention relates to a novel pharmaceutical composition for treating or preventing HIV, which comprises a compound having anti-HIV activity, and a method for treating HIV infection.
　ＨＩＶの粒子は直径約１２０ｎｍの球状で、ウイルス糖タンパク質Ｅｎｖ（ｇｐ１２０，ｇｐ４１）が植え付けられた宿主細胞由来の脂質二重膜からなるエンベロープ、エンベロープを裏打ちするウイルスタンパク群Ｇａｇの一つであるマトリックス（ＭＡ）、その内側にやはりＧａｇの一つであるキャプシド（ＣＡ）タンパクからなる円錐台状をしたコアが存在する。コアの内側にヌクレオキャプシドタンパク質（ＮＣ）に取り巻かれて二重体化しているポジティブ一本鎖ＲＮＡのウイルスゲノムが存在する。 The HIV particles are spherical with a diameter of about 120 nm, and are an envelope consisting of a lipid bilayer membrane derived from a host cell in which the viral glycoprotein Env (gp120, gp41) is planted. A matrix that is one of the viral protein group Gag that lines the envelope. (MA), inside it is a cone-shaped core made of capsid (CA) protein, which is also one of the hosts. Inside the core is the viral genome of positive single-strand RNA that is doubled around the nucleocapsid protein (NC).
　自己免疫疾患（ＡＩＤＳ）はＨＩＶによって生じる。遺伝学的に異なる２つの型のＨＩＶ、いわゆるＨＩＶ－１及びＨＩＶ－２がＡＩＤＳ患者から単離されている。ＨＩＶ－１は、世界的に分布している一般的なタイプであり、一方、ＨＩＶ－２は主に西アフリカにおいてＡＩＤＳを引き起こしている。 Autoimmune disease (AIDS) is caused by HIV. Two genetically distinct types of HIV, the so-called HIV-1 and HIV-2, have been isolated from AIDS patients. HIV-1 is a common type that is distributed worldwide, while HIV-2 causes AIDS primarily in West Africa.
　ＨＩＶはレトロウイルスであり、多くのウイルスと同様、ＨＩＶも感染サイクルの中でウイルス粒子（ビリオン）の形になることで感染する。ＨＩＶ－１ビリオンは球状であって、高電子密度の錐体形コアを含んでおり、それは宿主細胞膜由来の脂質エンベロープに包まれている。ウイルスコアは（１）主要なキャプシドタンパク質ｐ２４（ＣＡ）、（２）ヌクレオキャプシドタンパク質ｐ７／ｐ９（ＮＣ）、（３）２コピーのゲノムＲＮＡ、及び（４）３つのウイルス酵素（プロテアーゼ（ＰＲ）、逆転写酵素（ＲＴ）、及びインテグラーゼ）を含有する。ウイルスコアはマトリクスタンパク質、いわゆるｐ１７に包まれ、これはウイルスエンベロープの下側に位置している。ウイルスエンベロープには２つのウイルス糖タンパク質、ｇｐ１２０及びｇｐ４１が付いている。 HIV is a retrovirus, and like many viruses, HIV is infected by forming virus particles (virions) during the infection cycle. The HIV-1 billion is spherical and contains a high electron density pyramidal core, which is enveloped in a lipid envelope derived from the host cell membrane. The viral cores are (1) major capsid protein p24 (CA), (2) nucleocapsid protein p7 / p9 (NC), (3) two copies of genomic RNA, and (4) three viral enzymes (protease (PR)). , Reverse Transcriptase (RT), and Integrase). The viral core is wrapped in a matrix protein, the so-called p17, which is located below the viral envelope. The viral envelope has two viral glycoproteins, gp120 and gp41.
　ＨＩＶプロウイルスのゲノムはｇａｇ、ｐｏｌ、及びｅｎｖ遺伝子を含有し、これらは種々のウイルスタンパク質をコードする。ｇａｇ及びｐｏｌ遺伝子の産物はまず、大きな前駆体タンパク質に翻訳されるが、このタンパク質は、ウイルスのプロテアーゼによって切断され、成熟タンパク質を生成する。 The HIV provirus genome contains gag, pol, and env genes, which encode various viral proteins. The products of the gag and pol genes are first translated into a large precursor protein, which is cleaved by viral proteases to produce mature proteins.
　ＣＡはまず、５５ｋＤａのＧａｇ前駆体ポリタンパク質内の一領域として合成される。約４，０００コピーのＧａｇが原形質膜で集合し、出芽して未成熟なウイルス粒子を形成する。出芽後、タンパク質分解によってＧａｇが切断されるとＣＡが遊離し、それが引き金となって立体構造が変化し、この変化によりキャプシド粒子の構築が促進される。２コピーのウイルスゲノム及び感染性に必須の酵素は、成熟ビリオンの中心の錐体形キャプシド内に内包される。 CA is first synthesized as a region within the 55 kDa Gag precursor polyprotein. Approximately 4,000 copies of Gag assemble on the plasma membrane and germinate to form immature viral particles. After budding, when Gag is cleaved by proteolysis, CA is released, which triggers a change in the three-dimensional structure, and this change promotes the construction of capsid particles. Two copies of the viral genome and enzymes essential for infectivity are encapsulated within the central pyramidal capsid of the mature virion.
　既存の抗ＨＩＶ－１剤は、主にウイルス側の酵素である逆転写酵素、プロテアーゼ、インテグラーゼを標的として開発されている。しかし、ＨＩＶ－１の逆転写酵素に起因する高い突然変異発生率及びＨＩＶ－１の酵素群が持つ遺伝子変異への高い許容性により、薬剤耐性株出現の問題が惹起されている。そこで、現行の治療薬とは全く異なる機序でＨＩＶ－１の増殖を阻止する新たな治療法の開発が急務であり、ＨＩＶ－１の骨格形成因子であるＧａｇタンパク群（ＨＩＶ－１最外層の膜を形成するマトリクスタンパク質、ウイルス内でＨＩＶ－１遺伝子を包むキャプシドタンパク質、ウイルス遺伝子のシャペロンとして働くヌクレオキャプシドタンパク質等）の阻害は、ウイルス学・創薬学的に魅力的なターゲットであるが、ＨＩＶ－１ Ｇａｇタンパク阻害剤に関しては未だ臨床応用に至っていない。 Existing anti-HIV-1 agents have been developed mainly targeting viral side enzymes such as reverse transcriptase, protease, and integrase. However, the high mutation rate caused by the reverse transcriptase of HIV-1 and the high tolerance for gene mutations of the HIV-1 enzyme group have caused the problem of the emergence of drug-resistant strains. Therefore, there is an urgent need to develop a new treatment method that blocks the growth of HIV-1 by a mechanism completely different from the current therapeutic agents, and the Gag protein group (the outermost layer of HIV-1), which is a skeletal formation factor of HIV-1. Inhibition of the matrix protein that forms the membrane of HIV-1, the capsid protein that wraps the HIV-1 gene in the virus, the nucleocapsid protein that acts as a chapellon of the viral gene, etc.) is an attractive target in terms of virology and drug discovery. The HIV-1 Gag protein inhibitor has not yet reached clinical application.
　最近のいくつかの研究では、適正なＣＡの構築がウイルスの感染性に極めて重要であることが示されている。ＣＡの構築を阻害するＣＡの変異は致死であり、ＣＡの安定性を変化させる変異は複製を著しく減弱させる。また、ＣＡは非常に保存された領域である（非特許文献１：V. Novitsky, et al., J Virol, 2002, 76:5435-51, HIV Sequence Compendium 2014, Los Alamos National Laboratory, USA）。よって、ＣＡは有望な抗ウイルス標的として関心を集めている。ＨＩＶ－１ ＣＡ阻害剤として、いくつか報告がなされている。Ｂｅｖｉｒｉｍａｔ（ＰＡ－４５７）は、ＣＡとｐ２間でのプロテアーゼによる最終的な開裂を阻害するＣＡ成熟阻害剤として報告されている（非特許文献２：F. Li, et al. PNAS vol. 100, no. 23, pp.13555-13560, 2003）。キャプシドアセンブリー阻害剤（ＣＡＩ）は、ＣＡのＣ末端側に結合する１２－ｍｅｒのペプチドであり、感染細胞からウイルスの出芽を抑制する作用を示す（非特許文献３：Jana Sticht, et. al., Nat Struct Mol Biol, pp. 671-677, 2005）。N-(3-chloro-4-methylphenyl)-N0-{2-[({5-[(dimethylamino)-methyl]-2-furyl}-methyl)-sulfanyl]ethyl}urea)（ＣＡＰ－１）は、ＣＡのＮ末端に結合する化合物である（非特許文献４：Chun Tang, et al., J Mol Biol, vol. 327, pp. 1013-1020, 2003）。ＰＦ－３４５００７４は、ＣＡ多量体安定化を過剰促進させ、正常なＣＡの機能を阻害すると報告されている（非特許文献５：Wada S. Blair, PLoS Pathog, vol., 6, Issue 12, e1001220, 2010）。 Several recent studies have shown that building a proper CA is extremely important for virus infectivity. Mutations in CA that inhibit the construction of CA are lethal, and mutations that alter the stability of CA significantly attenuate replication. In addition, CA is a very conserved area (Non-Patent Document 1: V. Novitsky, et al., J Virol, 2002, 76: 5435-51, HIV Sequence Compendium 2014, Los Alamos National Laboratory, USA). Therefore, CA is attracting attention as a promising antiviral target. Several reports have been made as HIV-1 CA inhibitors. Bevirimat (PA-457) has been reported as a CA maturation inhibitor that inhibits the final cleavage by protease between CA and p2 (Non-Patent Document 2: F. Li, et al. PNAS vol. 100, no. 23, pp.13555-13560, 2003). The capsid assembly inhibitor (CAI) is a 12-mer peptide that binds to the C-terminal side of CA and exhibits an action of suppressing virus budding from infected cells (Non-Patent Document 3: Jana Stickt, et. Al). ., Nat Struct Mol Biol, pp. 671-677, 2005). N- (3-chloro-4-methylphenyl) -N0- {2-[({5-[(dimethylamino) -methyl] -2-furyl} -methyl) -sulfanyl] ethyl} urea) (CAP-1) , A compound that binds to the N-terminus of CA (Non-Patent Document 4: Chun Tang, et al., J Mol Biol, vol. 327, pp. 1013-1020, 2003). It has been reported that PF-3450074 over-promotes CA multimeric stabilization and inhibits normal CA function (Non-Patent Document 5: Wada S. Blair, PLoS Pathog, vol., 6, Issue 12, e1001220). , 2010).
　本発明は、新規な抗ＨＩＶ活性を有する分子を提供することを目的とする。本発明はまた、ＨＩＶ－１キャプシドをターゲットとした新規な抗ＨＩＶ薬を提供することを目的とする。 An object of the present invention is to provide a molecule having novel anti-HIV activity. The present invention also aims to provide novel anti-HIV agents targeting the HIV-1 capsid.
　本発明者らは、ＨＩＶ－１粒子を形成する構造蛋白であるＣＡに対して特異的に結合し、さらにＣＡの過剰な安定化を誘導することで、ＨＩＶ－１の増殖を抑制し得る新しい機序を有する抗ＨＩＶ－１剤の開発を目指して鋭意検討した結果、ＨＩＶ－１のＣＡの過剰安定性を誘導する化合物を見いだし、本発明を完成させた。 The present inventors are new in that they can suppress the growth of HIV-1 by specifically binding to CA, which is a structural protein forming HIV-1 particles, and further inducing excessive stabilization of CA. As a result of diligent studies aimed at the development of an anti-HIV-1 agent having a mechanism, a compound that induces hyperstability of CA of HIV-1 was found, and the present invention was completed.
　ＨＩＶ－１の感染工程は、標的細胞に感染した後にＣＡ殻が壊れ内部のウイルス遺伝子が細胞質や核内に移行する過程である脱殻を経る。本発明者らは、ＨＩＶ－１のＣＡ領域のアミノ酸におけるＥ４５Ａ変異やＮ７４Ｄ変異は、ＨＩＶ－１のＣＡの安定性を促進し、野生型ＣＡと比較してＣＡ殻の脱殻が障害されるとの知見を基に、ＣＡ蛋白表面のこれらのアミノ酸を含む領域に強固に結合する化合物は、ＣＡ殻の脱殻障害を誘発し、ひいてはＨＩＶ－１の感染・増殖を阻害し得ると考え、化合物の検索、開発、検討を行った結果、ＣＡを過剰に安定化する化合物を見いだし、本発明を完成させた。本発明は以下の態様を含むものである。 The HIV-1 infection process goes through unshelling, which is the process in which the CA shell breaks after infecting the target cells and the viral genes inside are transferred to the cytoplasm or nucleus. We found that E45A and N74D mutations in amino acids in the CA region of HIV-1 promote the stability of HIV-1 CA and impair CA shell shedding compared to wild-type CA. Based on the findings of the above, it is considered that a compound that strongly binds to the region containing these amino acids on the surface of the CA protein can induce unshelling disorder of the CA shell and thus inhibit the infection / proliferation of HIV-1. As a result of searching, developing, and studying, a compound that excessively stabilizes CA was found, and the present invention was completed. The present invention includes the following aspects.
［１］下記式（Ｉ）： [1] The following formula (I):
Figure JPOXMLDOC01-appb-C000007
 ［式（Ｉ）中、Ｘ及びＹは、それぞれ独立にＣ又はＮを表し、Ｚは、Ｃ又は結合を表し、Ｒａ及びＲｂは、それぞれ独立に、水素又は置換されてもよいフェニル基を表すか互いに一緒になってＸとＹとともに置換されてもよい６員炭素環を形成し、Ｒ1は、Ｃ１～Ｃ５のアルキル基又はアルケニル基、フェニル基、ベンジル基、あるいは２－モルホリノエチル基を表す。］で表される化合物、又はその薬学的に許容される塩を有効成分として含むＨＩＶの治療又は予防のための医薬組成物。
［２］下記式（ＩＩ）：
Figure JPOXMLDOC01-appb-C000007
 [In formula (I), X and Y each independently represent C or N, Z represents C or bond, and Ra and Rb each independently represent a hydrogen or optionally substituted phenyl group. Together with each other, they form a 6-membered carbocycle that may be substituted with X and Y, where R 1 contains C1-C5 alkyl or alkenyl groups, phenyl groups, benzyl groups, or 2-morpholinoethyl groups. Represent. ], Or a pharmaceutical composition for treating or preventing HIV, which comprises a pharmaceutically acceptable salt thereof as an active ingredient.
[2] The following formula (II):
Figure JPOXMLDOC01-appb-C000008
 ［式（ＩＩ）中、Ｒ2は、Ｃ１～Ｃ５のアルキル基又はアルケニル基、ベンジル基、あるいは２－モルホリノエチル基を表す。］で表される化合物、又はその薬学的に許容される塩を有効成分として含む、上記［１］に記載の医薬組成物。
［３］上記式（ＩＩ）において、Ｒ2は、Ｃ１～Ｃ３のアルキル基又はアルケニル基である、上記［２］に記載の医薬組成物。
［４］上記式（ＩＩ）において、Ｒ2は、メチル基、エチル基、及びアリル基からなる群より選ばれる上記［２］に記載の医薬組成物。
［５］上記式（ＩＩ）において、Ｒ2は、２－モルホリノエチルである、上記［２］に記載の医薬組成物。
［６］下記式（ＩＩＩ）：
Figure JPOXMLDOC01-appb-C000008
 [In formula (II), R 2 represents an alkyl or alkenyl group, a benzyl group, or a 2-morpholinoethyl group of C1 to C5. ] As an active ingredient, the pharmaceutical composition according to the above [1].
[3] The pharmaceutical composition according to the above [2], wherein R 2 is an alkyl group or an alkenyl group of C1 to C3 in the above formula (II).
[4] The pharmaceutical composition according to the above [2], wherein R 2 is selected from the group consisting of a methyl group, an ethyl group, and an allyl group in the above formula (II).
[5] The pharmaceutical composition according to the above [2], wherein R 2 is 2-morpholinoethyl in the above formula (II).
[6] The following formula (III):
Figure JPOXMLDOC01-appb-C000009
 ［式（ＩＩＩ）中、Ｒ3は、Ｃ１～Ｃ５のアルキル基又はアルケニル基、あるいはフェニル基を表す。］で表される化合物、又はその薬学的に許容される塩を有効成分として含む、上記［１］に記載の医薬組成物。
［７］上記式（ＩＩＩ）において、Ｒ3は、Ｃ１～Ｃ３のアルキル基又はアルケニル基である、上記［６］に記載の医薬組成物。
［８］上記式（ＩＩＩ）において、Ｒ3は、メチル基、エチル基、及びアリル基からなる群より選ばれる上記［６］に記載の医薬組成物。
［９］上記式（ＩＩＩ）において、Ｒ3は、フェニル基である上記［６］に記載の医薬組成物。
［１０］以下の化合物：
Figure JPOXMLDOC01-appb-C000009
 [In formula (III), R 3 represents an alkyl group, an alkenyl group, or a phenyl group of C1 to C5. ] As an active ingredient, the pharmaceutical composition according to the above [1], which comprises the compound represented by [1] or a pharmaceutically acceptable salt thereof.
[7] The pharmaceutical composition according to the above [6], wherein in the above formula (III), R 3 is an alkyl group or an alkenyl group of C1 to C3.
[8] The pharmaceutical composition according to the above [6], wherein in the above formula (III), R 3 is selected from the group consisting of a methyl group, an ethyl group, and an allyl group.
[9] The pharmaceutical composition according to the above [6], wherein R 3 is a phenyl group in the above formula (III).
[10] The following compounds:
Figure JPOXMLDOC01-appb-C000010
からなる群から選ばれる化合物又はその薬学的に許容される塩を有効成分として含む、上記［２］に記載の医薬組成物。
［１１］以下の化合物：
Figure JPOXMLDOC01-appb-C000010
The pharmaceutical composition according to the above [2], which comprises, as an active ingredient, a compound selected from the group consisting of or a pharmaceutically acceptable salt thereof.
[11] The following compounds:
Figure JPOXMLDOC01-appb-C000011
 からなる群から選ばれる化合物又はその薬学的に許容される塩を有効成分として含む、上記［２］に記載の医薬組成物。
［１２］以下の化合物：
Figure JPOXMLDOC01-appb-C000011
 The pharmaceutical composition according to the above [2], which comprises, as an active ingredient, a compound selected from the group consisting of or a pharmaceutically acceptable salt thereof.
[12] The following compounds:
Figure JPOXMLDOC01-appb-C000012
 からなる群から選ばれる化合物又はその薬学的に許容される塩を有効成分として含む、上記［６］に記載の医薬組成物。
［１３］他の抗ＨＩＶ薬と併用されることを特徴とする上記［１］から［１２］のいずれか一つに記載の医薬組成物。
［１４］前記他の抗ＨＩＶ薬が、化学療法剤、抗レトロウイルス阻害剤、サイトカイン、ヒドロキシウレア、Ｇａｇタンパク質に結合するモノクローナル抗体、又は他のレトロウイルス複製の阻害剤である、上記［１３］に記載の医薬組成物。
［１５］ＨＩＶの治療又は予防が必要とされている患者に、治療有効量の下記式（Ｉ）：
Figure JPOXMLDOC01-appb-C000012
 The pharmaceutical composition according to the above [6], which comprises, as an active ingredient, a compound selected from the group consisting of or a pharmaceutically acceptable salt thereof.
[13] The pharmaceutical composition according to any one of the above [1] to [12], which is characterized in that it is used in combination with another anti-HIV drug.
[14] The other anti-HIV agent is a chemotherapeutic agent, an antiretroviral inhibitor, a cytokine, a hydroxyurea, a monoclonal antibody that binds to a Gag protein, or an inhibitor of other retrovirus replication. The pharmaceutical composition according to.
[15] For patients in need of treatment or prevention of HIV, the following formula (I):
Figure JPOXMLDOC01-appb-C000013
 ［式（Ｉ）中、Ｘ及びＹは、それぞれ独立にＣ又はＮを表し、Ｚは、Ｃ又は単結合を表し、Ｒａ及びＲｂは、それぞれ独立に水素又は置換されてもよいフェニル基を表すか互いに一緒になってＸとＹとともに置換されてもよい６員炭素環を形成し、Ｒ1は、Ｃ１～Ｃ５のアルキル基又はアルケニル基、フェニル基、ベンジル基、あるいは２－モルホリノエチル基を表す。］で表される化合物、又はその薬学的に許容される塩を患者に投与することを特徴とするＨＩＶの治療又は予防方法。
［１６］前記化合物又はその薬理学的に許容される塩が、下記式（ＩＩ）：
Figure JPOXMLDOC01-appb-C000013
 [In formula (I), X and Y independently represent C or N, Z represents C or a single bond, and Ra and Rb represent phenyl groups which may be independently hydrogenated or substituted, respectively. Together they form a 6-membered carbocycle that may be substituted with X and Y, where R 1 contains C1-C5 alkyl or alkenyl groups, phenyl groups, benzyl groups, or 2-morpholinoethyl groups. Represent. ], Or a method for treating or preventing HIV, which comprises administering to a patient a pharmaceutically acceptable salt thereof.
[16] The compound or a pharmacologically acceptable salt thereof is the following formula (II):
Figure JPOXMLDOC01-appb-C000014
 ［式（ＩＩ）中、Ｒ2は、Ｃ１～Ｃ５のアルキル基又はアルケニル基、ベンジル基、あるいは２－モルホリノエチル基を表す。］で表される化合物、又はその薬学的に許容される塩である、上記［１５］に記載の方法。
［１７］前記化合物又はその薬理学的に許容される塩が、記式（ＩＩＩ）：
Figure JPOXMLDOC01-appb-C000014
 [In formula (II), R 2 represents an alkyl or alkenyl group, a benzyl group, or a 2-morpholinoethyl group of C1 to C5. ], The method according to the above [15], which is a pharmaceutically acceptable salt thereof.
[17] The compound or a pharmacologically acceptable salt thereof is described in Formula (III) :.
Figure JPOXMLDOC01-appb-C000015
 ［式（ＩＩＩ）中、Ｒ3は、Ｃ１～Ｃ５のアルキル基又はアルケニル基、あるいはフェニル基を表す。］で表される化合物、又はその薬学的に許容される塩である、上記［１５］に記載の方法。
［１８］さらに他の抗ＨＩＶ薬を投与することを特徴とする上記［１５］から［１７］のいずれか一つに記載の方法。
［１９］抗ＨＩＶ活性を有する物質をスクリーニングする方法であって、以下の工程：
（ａ）ＨＩＶ－１の野生型キャプシドを発現する細胞からの調製したキャプシドとともに候補物質を緩衝液中で３７±２℃の温度にてインキュベートする工程、ここで、該細胞は、ＨＩＶ－１の野生型キャプシドの発現プラスミドを用いて形質転換した細胞であり、キャプシド以外のＨＩＶ－１由来の成分を発現しない細胞である、及び
（ｂ）キャプシドの崩壊を検出し、対照に対し、キャプシドの崩壊が抑制された物質を選択する工程、
を含むスクリーニング方法。
［２０］前記工程（ｂ）は、無傷（intact）のキャプシドの抗原量を、抗キャプシド抗体を用いて測定する工程である、上記［１９］に記載のスクリーニング方法。
Figure JPOXMLDOC01-appb-C000015
 [In formula (III), R 3 represents an alkyl group, an alkenyl group, or a phenyl group of C1 to C5. ], The method according to the above [15], which is a pharmaceutically acceptable salt thereof.
[18] The method according to any one of the above [15] to [17], which comprises administering yet another anti-HIV drug.
[19] A method for screening a substance having anti-HIV activity, wherein the following steps:
(A) A step of incubating a candidate substance in a buffer at a temperature of 37 ± 2 ° C. with a capsid prepared from cells expressing the wild-type capsid of HIV-1, wherein the cell is of HIV-1. Cells transformed with a wild-type capsid expression plasmid and do not express components derived from HIV-1 other than capsid, and (b) capsid disruption was detected and capsid disruption was detected as opposed to control. The process of selecting substances in which capsids are suppressed,
Screening method including.
[20] The screening method according to the above [19], wherein the step (b) is a step of measuring the antigen amount of an intact capsid using an anti-capsid antibody.
　本発明は、ＨＩＶ－１のキャプシドの安定化を誘導するという全く新しい作用機序・作用標的に基づく新規な抗ＨＩＶ－１薬を提供するものである。 The present invention provides a novel anti-HIV-1 drug based on a completely new mechanism of action and target of action that induces stabilization of the capsid of HIV-1.
同定したcavityのキャプシド表面上における位置を示す。四角で囲まれた部分がcavityである。疎水性の表面領域を、グレーで示している。The position of the identified cavity on the capsid surface is shown. The part surrounded by a square is the cavity. Hydrophobic surface areas are shown in gray. ウイルス合成及び出芽に対する影響を確認した結果である。This is the result of confirming the effect on virus synthesis and budding. Ｍａｇｉアッセイの評価系を説明する図である。It is a figure explaining the evaluation system of the Magi assay. 本発明の化合物で処理されたウイルス上清を用いてＭａｇｉアッセイを行った結果である。This is the result of performing a Magi assay using a virus supernatant treated with the compound of the present invention. ＨＩＶ－１キャプシドの熱安定性に対する本発明の化合物の効果を確認した結果である。This is the result of confirming the effect of the compound of the present invention on the thermal stability of the HIV-1 capsid. 本発明の化合物に対する薬剤耐性誘導を確認した結果である。This is the result of confirming the induction of drug resistance to the compound of the present invention.
　以下、本発明を、例示的な実施態様を例として、本発明の実施において使用することができる好ましい方法及び材料とともに説明する。なお、文中で特に断らない限り、本明細書で用いるすべての技術用語及び科学用語は、本発明が属する技術分野の当業者に一般に理解されるのと同じ意味をもつ。また、本明細書に記載されたものと同等または同様の任意の材料及び方法は、本発明の実施において同様に使用することができる。また、本明細書に記載された発明に関連して本明細書中で引用されるすべての刊行物及び特許は、例えば、本発明で使用できる方法や材料その他を示すものとして、本明細書の一部を構成するものである。 Hereinafter, the present invention will be described together with preferred methods and materials that can be used in the practice of the present invention, using exemplary embodiments as examples. Unless otherwise specified in the text, all technical terms and scientific terms used in the present specification have the same meanings as generally understood by those skilled in the art to which the present invention belongs. Also, any material and method equivalent to or similar to that described herein can be used similarly in the practice of the present invention. In addition, all publications and patents cited herein in connection with the invention described herein are described herein, for example, as indicating methods, materials, etc. that can be used in the present invention. It constitutes a part.
　本明細書中で、「Ｘ～Ｙ」という表現を用いた場合は、下限としてＸを上限としてＹを含む意味、或いは、上限としてＸを下限としてＹを含む意味で用いる。 In the present specification, when the expressions "X to Y" are used, they are used to mean that X is included as the lower limit and Y is included as the upper limit, or that X is used as the upper limit and Y is included as the lower limit.
　ＨＩＶ－１ウイルスは、ウイルスが標的細胞に感染した後に、キャプシド（ＣＡ）殻が壊れ、内部のウイルス遺伝子が細胞質や細胞核内に移行する過程（「脱殻」と呼ばれる）を経て増殖を繰り返す。
　本発明者らは、ＨＩＶ－１のキャプシド（ＣＡ）領域のアミノ酸において、Ｅ４５Ａ変異やＮ７４Ｄ変異は、ＨＩＶ－１ ＣＡの安定性を促進し、野生型ＣＡと比較してＣＡ殻の脱殻が障害されるといった知見をもとに、ＣＡタンパク質表面の同アミノ酸を含む領域へ強固に結合する化合物は、ＣＡ殻の脱殻障害を誘発し、延いてはＨＩＶ－１の感染・増殖を阻害し得るとの仮説を立て、新規メカニズムに基づく新たなＨＩＶ治療法につながる化合物を探索・検討を行った。その結果、本発明を完成した。 After the virus infects the target cell, the HIV-1 virus repeats proliferation through a process in which the capsid (CA) shell is broken and the internal viral gene is transferred into the cytoplasm or cell nucleus (called "unshelling").
In the amino acids in the capsid (CA) region of HIV-1, the E45A and N74D mutations promote the stability of HIV-1 CA and impair the unshelling of the CA shell compared to wild-type CA. Based on the finding that, a compound that strongly binds to the region containing the same amino acid on the surface of the CA protein can induce unshelling disorder of the CA shell and, in turn, inhibit the infection and proliferation of HIV-1. We searched for and investigated compounds that lead to new HIV treatment methods based on a new mechanism. As a result, the present invention was completed.
　本発明者らは、ＨＩＶ－１のキャプシド（ＣＡ）の詳細な結晶構造解析を行った。結晶構造解析により得られたＨＩＶ－１ ＣＡ単量体の表面構造上、Ｅ４５番アミノ酸及びＮ７４番アミノ酸の周囲に化合物が結合可能なサイズの疎水性ポケットを同定した。
　次いで、数百万の化合物構造データをもとに、同ポケットを標的としたin silico ドッキングシミュレーションの手法により各化合物のＣＡ上の標的ポケットとの結合スコアを計算し、スコアの良い化合物に関して実際に購入し、野生株であるＨＩＶ－１LAI及びＭＴ２ 細胞を用いたＭＴＴ ａｓｓａｙ法（ＨＩＶ－１感染による細胞死に対する阻止能を評価）により化合物の抗ＨＩＶ－１活性を評価した。その結果、数十種の抗ＨＩＶ－１活性を有する化合物を同定した。 The present inventors performed a detailed crystal structure analysis of the capsid (CA) of HIV-1. On the surface structure of the HIV-1 CA monomer obtained by crystal structure analysis, hydrophobic pockets of a size capable of binding the compound were identified around amino acids E45 and N74.
Next, based on millions of compound structure data, the binding score of each compound to the target pocket on CA was calculated by the method of in silico docking simulation targeting the same pocket, and the compound with a good score was actually measured. The anti-HIV-1 activity of the compound was evaluated by the MTT assay method (evaluating the ability to prevent cell death due to HIV-1 infection) using the wild strains HIV-1 LAI and MT2 cells. As a result, dozens of compounds having anti-HIV-1 activity were identified.
　さらに、抗ＨＩＶ－１活性を有する化合物に関し、ＨＩＶ－１ ＣＡの安定性に与える影響を評価した。具体的には、ＨＩＶ－１ ＣＡを単独で発現するプラスミドを作成し、細胞にＣＡを発現させ、細胞溶解液を作成し、３７℃で静置後のＣＡの経時変化を、ＣＡ抗体を用いたＥＬＩＳＡ法等により解析した。 Furthermore, regarding the compound having anti-HIV-1 activity, the effect on the stability of HIV-1 CA was evaluated. Specifically, a plasmid that expresses HIV-1CA alone is prepared, CA is expressed in cells, a cell lysate is prepared, and a CA antibody is used to change the CA over time after standing at 37 ° C. It was analyzed by the ELISA method and the like.
抗HＩＶ化合物
　本発明の抗ＨＩＶ－１化合物は、下記の構造式（Ｉ）により表される化合物又はその薬学的に許容される塩である。 Anti-HIV Compound The anti-HIV-1 compound of the present invention is a compound represented by the following structural formula (I) or a pharmaceutically acceptable salt thereof.
Figure JPOXMLDOC01-appb-C000016
 　式（Ｉ）中、Ｘ及びＹは、それぞれ独立にＣ又はＮを表し、Ｚは、Ｃ又は結合（結合とは、ＹとＮが直接結合することを意味する）を表し、Ｒａ及びＲｂは、それぞれ独立に水素又は置換されてもよいフェニル基を表すか互いに一緒になってＸとＹとともに置換されてもよい６員炭素環を形成し、Ｒ1は、Ｃ１～Ｃ５のアルキル基又はアルケニル基、フェニル基、ベンジル基、或いは２－モルホリノエチル基を表し、好ましくは、Ｃ１～Ｃ３のアルキル基又はアルケニル基を表す。
　Ｘ、Ｙ及びＺがＣである場合は、好ましくは、Ｒａ及びＲｂは互いに一緒になって６員炭素環となり、フタラジン環を形成する。Ｘ及びＹがＮである場合は、好ましくは、Ｚは単なる結合であって、テトラゾール環を形成する。
　Ｒ1は、好ましくはＣ１～Ｃ５のアルキル基又はアルケニル基、より好ましくはＣ１～Ｃ３のアルキル基又はアルケニル基、さらに好ましくは、メチル基、エチル基又はアリル基である。
Figure JPOXMLDOC01-appb-C000016
 In formula (I), X and Y independently represent C or N, Z represents C or bond (bond means that Y and N are directly bonded), and Ra and Rb are. , Representing hydrogen or optionally substituted phenyl groups, respectively, or together to form a 6-membered carbon ring which may be substituted with X and Y, where R 1 is an alkyl group or alkenyl of C1-C5. It represents a group, a phenyl group, a benzyl group, or a 2-morpholinoethyl group, preferably an alkyl group or an alkenyl group of C1 to C3.
When X, Y and Z are C, preferably Ra and Rb together form a 6-membered carbon ring to form a phthalazine ring. When X and Y are N, preferably Z is just a bond and forms a tetrazole ring.
R 1 is preferably an alkyl or alkenyl group of C1 to C5, more preferably an alkyl or alkenyl group of C1 to C3, and even more preferably a methyl, ethyl or allyl group.
　好ましい本発明の抗ＨＩＶ化合物の一つのグループは、下記の構造式（ＩＩ）により表される化合物又はその薬学的に許容される塩である。 One group of preferred anti-HIV compounds of the present invention is the compound represented by the following structural formula (II) or a pharmaceutically acceptable salt thereof.
Figure JPOXMLDOC01-appb-C000017
 　式（ＩＩ）中、Ｒ2は、Ｃ１～Ｃ５のアルキル基又はアルケニル基、ベンジル基、あるいは２－モルホリノエチル基を表し、好ましくはＣ１～Ｃ３のアルキル基又はアルケニル基、より好ましくは、メチル基、エチル基又はアリル基を表す。
　上記式（ＩＩ）で表される化合物又はその塩においてより好ましい化合物又はその塩は、以下の式で表される化合物から選ばれる少なくとも一つの化合物又はその塩である。
Figure JPOXMLDOC01-appb-C000017
 In formula (II), R 2 represents an alkyl or alkenyl group of C1 to C5, a benzyl group, or a 2-morpholinoethyl group, preferably an alkyl or alkenyl group of C1 to C3, more preferably a methyl group. , Ethyl group or allyl group.
A more preferable compound or salt thereof in the compound represented by the above formula (II) or a salt thereof is at least one compound selected from the compounds represented by the following formula or a salt thereof.
Figure JPOXMLDOC01-appb-C000018
Figure JPOXMLDOC01-appb-C000018
　よりさらに好ましいのは、以下の化学式で表される化合物から選ばれる少なくとも一つの化合物又はその塩である。 Even more preferable is at least one compound selected from the compounds represented by the following chemical formulas or a salt thereof.
Figure JPOXMLDOC01-appb-C000019
Figure JPOXMLDOC01-appb-C000019
　好ましい本発明の抗ＨＩＶ化合物の別の一つのグループは、下記の構造式（ＩＩＩ）により表される化合物又は薬学的に許容されるその塩である。 Another preferred group of anti-HIV compounds of the present invention is the compound represented by the following structural formula (III) or a pharmaceutically acceptable salt thereof.
Figure JPOXMLDOC01-appb-C000020
 式（ＩＩＩ）中、Ｒ3は、Ｃ１～Ｃ５のアルキル基又はアルケニル基、あるいはフェニル基を表し、好ましくはＣ１～Ｃ３のアルキル基又はアルケニル基、あるいはフェニル基、より好ましくはエチル基又はフェニル基を表す。
　上記式（ＩＩＩ）で表される化合物又はその塩においてより好ましい化合物又はその塩は、以下の式で表される化合物から選ばれる化合物又はその塩である。
Figure JPOXMLDOC01-appb-C000020
 In formula (III), R 3 represents an alkyl or alkenyl group of C1 to C5, or a phenyl group, preferably an alkyl or alkenyl group of C1 to C3, or a phenyl group, more preferably an ethyl or phenyl group. Represents.
A more preferable compound or salt thereof in the compound represented by the above formula (III) or a salt thereof is a compound selected from the compounds represented by the following formula or a salt thereof.
Figure JPOXMLDOC01-appb-C000021
Figure JPOXMLDOC01-appb-C000021
　よりさらに好ましいのは、以下の化学式で表される化合物又はその塩である。 Even more preferable is a compound represented by the following chemical formula or a salt thereof.
Figure JPOXMLDOC01-appb-C000022
Figure JPOXMLDOC01-appb-C000022
　本発明で用いることができる化合物は、低分子の化合物合成の技術分野において用いられている公知の方法を参照して合成することができるが、その一部は購入することも可能である。本出願時において化合物合成の技術分野において公知の技術は、本発明において制限なく用いることができる。 The compounds that can be used in the present invention can be synthesized by referring to known methods used in the technical field of compound synthesis of low molecules, but some of them can also be purchased. Techniques known in the art of compound synthesis at the time of this application can be used without limitation in the present invention.
医薬組成物
　本発明に従って、治療上有効量の上記式（Ｉ）～（ＩＩＩ）のいずれかで表される化合物又は薬学的に許容されるその塩を含むＨＩＶ感染の治療又は予防のための医薬組成物が提供される。 Pharmaceutical Composition A medicament for the treatment or prevention of HIV infection, which comprises a therapeutically effective amount of a compound represented by any of the above formulas (I) to (III) or a pharmaceutically acceptable salt thereof according to the present invention. The composition is provided.
「薬学的に許容されるその塩」とは、上記式（Ｉ）～（ＩＩＩ）のいずれかで表される化合物から形成されるあらゆる非毒性の塩を示す。適切な塩としては、例えば、これに限定しないが、塩酸塩、臭化水素酸塩、ヨウ化水素酸塩、リン酸塩、リン酸水素塩、硫酸塩などの無機酸塩、酢酸塩、トリフルオロ酢酸塩、リンゴ酸塩、コハク酸塩、酒石酸塩、乳酸塩、クエン酸塩、マレイン酸塩、フマル酸塩、ソルビン酸塩、アスコルビン酸塩、サリチル酸塩、フタル酸塩、メタンスルホン酸塩、トリフルオロメチルスルホン酸塩、ベンゼンスルホン酸塩などの有機酸塩、アンモニウム塩などの無機塩、ナトリウム塩、カリウム塩などのアルカリ金属塩、カルシウム塩、マグネシウム塩などのアルカリ土類金属塩、カルボン酸塩などの酸性基の塩、メチルアミン、エチルアミン、シクロヘキシルアミンなどの低級アルキルアミン、ジエタノールアミン、トリエタノールアミンなどの置換低級アルキルアミンなどの有機塩基との塩、グリシン塩、リジン塩、アルギニン塩、オルニチン塩、グルタミン酸塩、アスパラギン酸塩などのアミノ酸塩などをあげることができる。 "Pharmaceutically acceptable salt thereof" refers to any non-toxic salt formed from a compound represented by any of the above formulas (I)-(III). Suitable salts include, for example, but not limited to, hydrochlorides, hydrobromates, hydroiodates, phosphates, hydrogen phosphates, inorganic acid salts such as sulfates, acetates, birds. Fluoroacetate, malate, succinate, tartrate, lactate, citrate, maleate, fumarate, sorbate, ascorbate, salicylate, phthalate, methanesulfonate, Organic acid salts such as trifluoromethyl sulfonate and benzene sulfonate, inorganic salts such as ammonium salt, alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, and carboxylic acids. Salts of acidic groups such as salts, lower alkylamines such as methylamine, ethylamine, cyclohexylamine, substitutions with organic bases such as diethanolamine and triethanolamine, glycine salts, lysine salts, arginine salts, ornithine. Examples include salts, amino acid salts such as glutamate and asparaginate.
　本発明はまた、上記式（Ｉ）～（ＩＩＩ）のいずれかで表される化合物から形成される溶媒和物及び水和物を含む医薬組成物を包含する。本発明で用いる化合物（それらの塩を含む）は、それらの水和物の形態で得られるか、またはそれらの結晶化のために用いられる他の溶媒を含み得る。本発明で用いる化合物は、本質的にまたは設計によって、薬学的に許容される溶媒（水を含む）との溶媒和物を形成してもよい。したがって、本発明で用いる化合物は、溶媒和及び非溶媒和形態の両方を包含する。「溶媒和物」という用語は、１種以上の溶媒分子と上記の化合物（その薬学的に許容される塩を含む）の分子複合体を指す。このような溶媒分子は、医薬技術分野で一般的に用いられるものであり、ヒトに無害であると知られている。例えば、これに限定されないが、水、エタノールなどである。「水和物」という用語は、溶媒分子が水である複合体を指す。本発明で用いる化合物は、その塩、水和物及び溶媒和物を含め、多形を形成し得る。溶媒和物または水和物は、前記式（Ｉ）～（ＩＩＩ）のいずれかで表される化合物の結晶形態の生成において有用となることがある。 The present invention also includes pharmaceutical compositions containing solvates and hydrates formed from compounds represented by any of the above formulas (I)-(III). The compounds used in the present invention (including salts thereof) may be obtained in the form of their hydrates or may contain other solvents used for their crystallization. The compounds used in the present invention may, essentially or by design, form a solvate with a pharmaceutically acceptable solvent (including water). Therefore, the compounds used in the present invention include both solvated and non-solvated forms. The term "solvate" refers to a molecular complex of one or more solvent molecules and the above compounds, including pharmaceutically acceptable salts thereof. Such solvent molecules are commonly used in the field of pharmaceutical technology and are known to be harmless to humans. For example, but not limited to, water, ethanol, and the like. The term "hydrate" refers to a complex in which the solvent molecule is water. The compounds used in the present invention, including their salts, hydrates and solvates, can form polymorphs. Solvates or hydrates may be useful in producing crystalline forms of the compounds represented by any of the formulas (I)-(III).
　本発明の医薬組成物は、上記式（Ｉ）～（ＩＩＩ）のいずれかで表される化合物に加え、１以上の薬学的に許容される担体、及び、適宜、他のＨＩＶ抗ウイルス薬、抗感染症薬、及び免疫調整薬を含むことができる。 In addition to the compounds represented by any of the above formulas (I) to (III), the pharmaceutical composition of the present invention comprises one or more pharmaceutically acceptable carriers and, as appropriate, other HIV antiviral agents. Antiviral agents and immunomodulators can be included.
　本明細書で使用される「薬学的に許容される担体」という用語は、当業者に知られているように、任意及びすべての、溶媒、分散媒体、コーティング剤、酸化防止剤、キレート剤、保存剤（例えば、抗菌剤、抗真菌剤）、界面活性剤、緩衝剤、浸透圧調節剤、吸収遅延剤、塩、薬物安定剤、賦形剤、希釈剤、結合剤、崩壊剤、甘味剤、芳香剤、潤沢剤、染料など、及びそれらの組合せを含む。何れかの担体が本発明の活性成分と不適合である場合以外は、本発明の治療または医薬組成物において使用することができる。 As used herein, the term "pharmaceutically acceptable carrier" is used in any and all solvents, dispersion media, coatings, antioxidants, chelating agents, as is known to those skilled in the art. Preservatives (eg, antibacterial agents, antifungal agents), surfactants, buffers, osmotic pressure regulators, absorption retarders, salts, drug stabilizers, excipients, diluents, binders, disintegrants, sweeteners , Fragrances, abundants, dyes, etc., and combinations thereof. Unless any carrier is incompatible with the active ingredient of the invention, it can be used in the therapeutic or pharmaceutical compositions of the invention.
　本明細書で使用される「治療上有効量」という用語は、治療を必要とするほ乳類に投与される場合、治療効果を生じさせるのに十分な量の上記式（Ｉ）～（ＩＩＩ）のいずれかで表される化合物をいう。治療上有効量は、対象及び治療する疾患症状、対象の体重及び年齢、疾患症状の重症度、投与方法などに依存して異なり、当該技術分野における当業者により容易に決定されることができる。 As used herein, the term "therapeutically effective amount" refers to an amount of the above formulas (I)-(III) sufficient to produce a therapeutic effect when administered to a mammal in need of treatment. A compound represented by either. The therapeutically effective amount depends on the subject and the disease symptoms to be treated, the weight and age of the subject, the severity of the disease symptoms, the administration method, and the like, and can be easily determined by those skilled in the art.
　本明細書で使用される「対象」という用語は、動物を指す。典型的には、動物は、哺乳動物である。対象は、例えば、霊長類（例えば、ヒト）、ウシ、ヒツジ、ヤギ、ウマ、イヌ、ネコ、ウサギ、ラット、マウス、魚類、鳥類なども指す。ある種の実施形態において、対象は霊長類、好ましくはヒトである。 The term "object" as used herein refers to animals. Typically, the animal is a mammal. The subject also refers to, for example, primates (eg, humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds and the like. In certain embodiments, the subject is a primate, preferably a human.
　本発明の上記式（Ｉ）～（ＩＩＩ）のいずれかで表される化合物を含む医薬組成物は、医薬組成物の処方のための既知の方法に準じて処方することができる。代表的な医薬組成物には、上記した薬学的に許容される担体が含まれうる。これらの担体の使用は、当該技術分野においてよく知られている。また、活性成分を含む医薬組成物を調製するための方法は、当該技術分野においてよく知られている。 The pharmaceutical composition containing the compound represented by any of the above formulas (I) to (III) of the present invention can be formulated according to a known method for prescribing a pharmaceutical composition. Representative pharmaceutical compositions may include the pharmaceutically acceptable carriers described above. The use of these carriers is well known in the art. Further, a method for preparing a pharmaceutical composition containing an active ingredient is well known in the art.
　本発明の医薬組成物は、使用目的に応じた特定の投与経路に適合するように製剤化されうる。投与経路は、これに限定されないが、例えば、経口、非経口、静脈内、皮内、皮下、経皮、吸入、局所、経粘膜的、又は直腸投与がある。本発明の医薬組成物は、固体形態又は液体形態で製剤化されうる。固定形態は、これに限定されないが、例えば、錠剤、カプセル剤、丸剤、顆粒剤、散剤、又は坐剤を含む。液体形態は、これに限定されないが、例えば、溶液剤、懸濁剤、又は乳濁剤を含む。本発明の医薬組成物は、好ましくは経口により投与される。 The pharmaceutical composition of the present invention can be formulated to suit a specific route of administration according to the purpose of use. Routes of administration include, but are not limited to, oral, parenteral, intravenous, intradermal, subcutaneous, transdermal, inhalation, topical, transmucosal, or rectal administration. The pharmaceutical composition of the present invention may be formulated in solid or liquid form. Fixed forms include, but are not limited to, tablets, capsules, pills, granules, powders, or suppositories. Liquid forms include, but are not limited to, solutions, suspensions, or emulsions. The pharmaceutical composition of the present invention is preferably administered orally.
　本発明の化合物の投与量は、疾患の種類、投与対象の症状、年齢、投与方法等により適宜選択される。例えば、これに限定されないが、経口剤であれば、通常、１日当たり０．１～５０００ｍｇ、好ましくは１～２０００ｍｇ、より好ましくは５～１００ｍｇを、１回又は数回に分けて投与すればよい。 The dose of the compound of the present invention is appropriately selected depending on the type of disease, the symptom of the administration target, the age, the administration method, and the like. For example, but not limited to this, in the case of oral preparations, usually 0.1 to 5000 mg, preferably 1 to 2000 mg, more preferably 5 to 100 mg per day may be administered in one or several divided doses. ..
　本発明の化合物の毒性及び治療効果は、例えば、ＨＩＶ－１のウイルス活性を表す細胞培養又は実験動物を用いた標準的な方法に従って、例えば、ＬＤ５０（集団の５０％に対する致死量）及びＥＤ５０（集団の５０％において治療的に有効な用量）を決定し、治療係数（ＬＤ５０／ＥＤ５０の比）を求めることにより推測することができる。好ましくは、大きい治療係数を示す化合物が選択される。中毒性副作用を示す化合物も使用されうるが、係る場合は、非感染細胞へのダメージを最小限にして、副作用を減少させるため、該化合物を感染組織の部位に導く送達システムの設計も可能である。 The toxicity and therapeutic effects of the compounds of the invention are, for example, according to standard methods using cell cultures or laboratory animals representing the viral activity of HIV-1, eg LD50 (lethal dose to 50% of the population) and ED50 (lethal dose to 50% of the population) and ED50 ( It can be estimated by determining the therapeutically effective dose in 50% of the population and determining the therapeutic index (LD50 / ED50 ratio). Preferably, a compound showing a large therapeutic index is selected. Compounds with addictive side effects can also be used, but in such cases it is possible to design a delivery system that directs the compound to the site of infected tissue in order to minimize damage to non-infected cells and reduce side effects. is there.
　細胞培養アッセイ及び動物試験から得られたデータを、ヒトでの使用の投与量の範囲を決定する際に用いることができる。多くの場合、化合物の投与量は、ほとんど毒性を有さないかまたは全く毒性がないＥＤ５０を含む循環濃度の範囲内が好ましい。投与量は、利用される投与経路及び利用される製剤形に依存してこの範囲内で変動しうる。本発明の化合物について、治療的に有効な用量が、細胞培養アッセイより最初に見積もられ得る。細胞培養中で測定されたようなＩＣ５０（すなわち、症状の半分最大阻害（half-maximal inhibition）を達成する試験化合物の濃度）を含む循環血漿濃度範囲を達成するために、用量は動物モデルで検討・決定されうる。このような情報はヒトにおいて有用な用量をより正確に決定するために使用することができる。血漿レベルは、当該分野における公知の方法、例えば、高速液体クロマトグラフィーによって測定され得る。 Data obtained from cell culture assays and animal studies can be used to determine the range of doses for use in humans. In many cases, the dose of the compound is preferably in the range of circulating concentrations containing ED50, which has little or no toxicity. The dosage can vary within this range depending on the route of administration used and the formulation used. For the compounds of the invention, therapeutically effective doses can be estimated first from cell culture assays. Dosages are examined in animal models to achieve a circulating plasma concentration range containing IC50s (ie, concentrations of test compounds that achieve half-maximal inhibition) as measured in cell culture.・ Can be decided. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured by methods known in the art, such as high performance liquid chromatography.
　本発明はまた、治療有効量の上記式（Ｉ）から（ＩＩＩ）のいずれかで表される化合物を、患者に投与することを含む、ＨＩＶ感染の治療のための方法を含む。本発明はまた、他の既知の抗ＨＩＶ薬と組み合わせて患者に投与することを含む、ＨＩＶ感染の治療のための方法（併用）及び２つ以上の医薬有効成分の組合せ（配合剤）を含む。他の抗ＨＩＶ薬としては、これに限定されないが、例えば、化学療法剤、抗レトロウイルス阻害剤、サイトカイン、ヒドロキシウレア、Ｇａｇタンパク質に結合するモノクローナル抗体、又は他のレトロウイルス複製の阻害剤をあげることができる。抗レトロウイルス阻害剤の例としては、逆転写酵素阻害剤、プロテアーゼ阻害剤、インテグラーゼ阻害剤（ラルテグラビル等）、ＣＣＲ５阻害剤（マラビロク）、及び融合阻害剤をあげることができる。 The present invention also includes a method for treating HIV infection, which comprises administering to a patient a therapeutically effective amount of a compound represented by any of the above formulas (I) to (III). The present invention also includes methods for the treatment of HIV infection (combination) and combinations of two or more active pharmaceutical ingredients (combination), including administration to a patient in combination with other known anti-HIV agents. .. Other anti-HIV agents include, but are not limited to, chemotherapeutic agents, antiretroviral inhibitors, cytokines, hydroxyurea, monoclonal antibodies that bind to the Gag protein, or other retrovirus replication inhibitors. be able to. Examples of anti-retrovirus inhibitors include reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors (raltegravir, etc.), CCR5 inhibitors (maraviroc), and fusion inhibitors.
　本発明はまた、治療有効量の上記医薬組成物を対象に投与する工程を含む、ＨＩＶ－１のウイルス複製を阻害する方法を提供する。本発明の１つの態様において、該方法はさらに、抗レトロウイルス阻害剤、サイトカイン、ヒドロキシウレア、Ｇａｇタンパク質に結合するモノクローナル抗体、またはレトロウイルス複製の他の阻害剤からなる群から選択される抗ウイルスの治療と併用されることを含む。抗レトロウイルス阻害剤の例には、逆転写酵素阻害剤、プロテアーゼ阻害剤、及び融合阻害剤がある。 The present invention also provides a method of inhibiting viral replication of HIV-1, which comprises the step of administering to a subject a therapeutically effective amount of the above pharmaceutical composition. In one embodiment of the invention, the method is further selected from the group consisting of antiretroviral inhibitors, cytokines, hydroxyureas, monoclonal antibodies that bind to Gag proteins, or other inhibitors of retroviral replication. Including being used in combination with the treatment of. Examples of antiretroviral inhibitors include reverse transcriptase inhibitors, protease inhibitors, and fusion inhibitors.
　逆転写酵素阻害剤には、ヌクレオシド／ヌクレオチド系逆転写酵素阻害剤（ＮＲＴＩ）（以下まとめて、ヌクレオシド系逆転写酵素阻害剤という）及び非ヌクレオシド／ヌクレオチド系逆転写酵素阻害剤（ＮＮＲＴＩ）（以下まとめて、非ヌクレオシド系逆転写酵素阻害剤という）が含まれる。ヌクレオシド系逆転写酵素阻害剤として、これらに限定されるものではないが、例えば、ジドブジン（ＺＤＶ、以前はアジドチミジン（ＡＺＴ）として知られる）、ジダノシン（ジデオキシイノシン（ｄｄＩ））、ザルシタビン（ジデオキシシチジン（ｄｄＣ））、ラミブジン（３ＴＣ）、スタブジン（ｄ４Ｔ）、アバカビル（ＡＢＣ）、テノホビル（ＴＤＦ）をあげることができる。ＨＩＶの非ヌクレオシド系逆転写酵素阻害剤として、これらに限定されるものではないが、例えば、ネビラピン、メシル酸デラビルジン、エファビレンツをあげることができる。ＨＩＶのプロテアーゼ阻害剤（ＰＩ）としては、これらに限定されるものではないが、例えば、アンプレナビル、メシル酸サキナビル、リトナビル、インジナビル硫酸塩、メシル酸ネルフィナビル、ロピナビル及びリトナビル、アタザナビル、ホスアンプレナビルをあげることができる。ＨＩＶの融合阻害剤としては、これに限定されるものではないが、例えば、エンフビルチドをあげることができる。 Reverse-transcriptase inhibitors include nucleoside / nucleotide reverse-transcriptase inhibitors (NRTI) (collectively referred to as nucleoside reverse-transcriptase inhibitors) and non-nucleoside / nucleotide reverse-transcriptase inhibitors (NNRTI) (hereinafter collectively referred to as nucleoside reverse-transcriptase inhibitors). Collectively, non-nucleoside reverse transcriptase inhibitors) are included. Nucleoside reverse transcriptase inhibitors include, but are not limited to, zidovudine (ZDV, formerly known as azidothymidine (AZT)), didanosine (dideoxyinosine (ddI)), zalcitabine (dideoxycitidine (dideoxycitidine)). ddC)), Lamibudine (3TC), Stavudine (d4T), Abacavir (ABC), Tenohovir (TDF) can be mentioned. Examples of non-nucleoside reverse transcriptase inhibitors of HIV include, but are not limited to, nevirapine, delavirdine mesylate, and efavirenz. HIV protease inhibitors (PIs) include, but are not limited to, amprenavir, saquinavir mesylate, ritonavir, indinavir sulfate, nelfinavir mesylate, lopinavir and ritonavir, atazanavir, fosamprenavir. You can raise the building. HIV fusion inhibitors include, but are not limited to, enfuvirtide.
抗ＨＩＶ物質のスクリーニング
　本発明はまた、抗ＨＩＶ活性を有する物質をスクリーニングする方法を含む。
　本発明の方法においては、ＨＩＶ－１のキャプシドそのものを用いて、キャプシドの安定化を誘導する物質をスクリーニングすることを特徴とする。ＨＩＶ－１のキャプシドは、培養細胞にＨＩＶ－１キャプシドの遺伝子を含むプラスミドを導入して発現させたキャプシドを用いても、ＨＩＶ－１ウイルスからキャプシドを単離又は精製したものを用いてもよいが、好ましくは、細胞に発現させたＨＩＶ－１キャプシドが用いられる。 Screening for Anti-HIV Substances The present invention also includes methods for screening substances with anti-HIV activity.
The method of the present invention is characterized in that the capsid of HIV-1 itself is used to screen for a substance that induces stabilization of the capsid. As the capsid of HIV-1, the capsid expressed by introducing a plasmid containing the gene of the HIV-1 capsid into cultured cells may be used, or the capsid isolated or purified from the HIV-1 virus may be used. However, preferably, the HIV-1 capsid expressed in cells is used.
　ＨＩＶ－１キャプシドを細胞に発現させる手段は、これに限定されないが、例えば以下のようにして行うことができる。
発現プラスミドの作成
　これに限定されないが、例えば、以下のようにして発現プラスミドを作成する。
　ｐＣＭＶ－Ｍｙｃベクター（＃６３１６０４，タカラバイオ社）を用いて野生型ＣＡ単独発現ｐｌａｓｍｉｄを作成する。制限酵素によりＭｙｃエピトープを含む領域を削除してベクターを線状化する。ＨＩＶ－１NL4-3を鋳型としてＰＣＲでキャプシドをコードする遺伝子領域を増幅した後、線状化ベクターに導入する。
ＣＡタンパクの発現
　作成したキャプシド発現プラスミドを細胞にトランスフェクトし、細胞にキャプシドタンパク質を発現させる。細胞は、特に制限されないが、例えば、ＣＯＳ－７細胞を用いることができる。本発明のスクリーニング方法においては、発現させたキャプシドタンパクは、細胞溶解物の状態、粗精製の状態、精製した状態のいずれでも用いることができる。発現したキャプシドの粗精製、精製は、公知の方法を参考にして行うことができる。 The means for expressing the HIV-1 capsid in cells is not limited to this, but can be carried out, for example, as follows.
Preparation of expression plasmid For example, but not limited to this, an expression plasmid is prepared as follows.
A wild-type CA single expression plasmid is prepared using a pCMV-Myc vector (# 631604, Takara Bio Inc.). The region containing the Myc epitope is deleted with a restriction enzyme to linearize the vector. The gene region encoding the capsid is amplified by PCR using HIV-1 NL4-3 as a template, and then introduced into a linearization vector.
Expression of CA protein The prepared capsid expression plasmid is transfected into cells to express the capsid protein in the cells. The cells are not particularly limited, but for example, COS-7 cells can be used. In the screening method of the present invention, the expressed capsid protein can be used in any of a cell lysate state, a crudely purified state, and a purified state. The crude purification and purification of the expressed capsid can be carried out with reference to a known method.
　本発明のスクリーニング方法によれば、上記のようにして調製したＨＩＶ－１の野生型キャプシドを用いて、キャプシドの安定化を誘導する化合物をスクリーニングすることができる。これに限定されないが、例えば、キャプシドを発現した細胞の細胞溶解物を用い、該溶解物を目的の化合物とともに、任意の温度（例えば３７℃）にて所定の時間（例えば、数時間から数日）インキュベートし、キャプシド崩壊を確認する。キャプシドの崩壊は、これに限定されないが、例えば、キャプシドに対する抗体を用いて、キャプシドの抗原量をＥＬＩＳＡにて測定することにより確認できる。 According to the screening method of the present invention, a compound that induces stabilization of the capsid can be screened using the wild-type capsid of HIV-1 prepared as described above. Not limited to this, for example, using a cell lysate of a cell expressing a capsid, the lysate together with the target compound at an arbitrary temperature (for example, 37 ° C.) for a predetermined time (for example, several hours to several days). ) Incubate and confirm capsid disintegration. Disintegration of the capsid is not limited to this, but can be confirmed, for example, by measuring the amount of the capsid antigen by ELISA using an antibody against the capsid.
　以下、実施例により、本発明を具体的に説明するが、本発明は以下の実施例に限定されるものではない。
実施例１：キャプシドの安定化に寄与する表面上の疎水性キャビティ
　ＨＩＶ－１キャプシド領域のアミノ酸において、Ｅ４５Ａ変異やＮ７４Ｄ変異は野生型キャプシドと比較してキャプシド殻の脱殻を遅延させるとの報告がある（例えば、非特許文献６）。そこで、本発明者らは、４５番目のアミノ酸（Ｅ４５）、７４番目のアミノ酸（Ｎ７４）がＣＡの安定性に影響を及ぼすと考え、Ｅ４５、Ｎ７４の周囲に、化合物が結合可能な大きさを有する疎水性キャビティの検索を行った。その結果、Ｅ４５及びＮ７４の周囲に疎水性アミノ酸残基からなる表面領域が存在し、低分子化合物が結合し得る十分な空間が存在することが判った。キャビティの位置を図１に示す。 Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to the following Examples.
Example 1: Hydrophobic cavities on the surface that contribute to capsid stabilization In the amino acids in the HIV-1 capsid region, E45A and N74D mutations have been reported to delay capsid shell shedding compared to wild-type capsids. (For example, Non-Patent Document 6). Therefore, the present inventors consider that the 45th amino acid (E45) and the 74th amino acid (N74) affect the stability of CA, and determine the size at which the compound can be bound around E45 and N74. The hydrophobic cavity having was searched. As a result, it was found that a surface region composed of hydrophobic amino acid residues exists around E45 and N74, and there is a sufficient space to which the low molecular weight compound can bind. The position of the cavity is shown in FIG.
実施例２：キャプシドキャビティに結合する化合物の同定
　購入可能な約８００万個超の化合物の構造データを、学術・商用の化合物データベースより入手し、実際に生体に投与された場合に各化合物の生体内でのＡＤＭＥに影響を及ぼす要素を考慮した上で、薬剤となりうる分子特性を有する化合物約７００万個を抽出した。選択した約７００万個の化合物について、ＭＭＦＦ９４（Merck Molecular Force Field 94）を用いて、３次元構造変換及びエネルギー極小化計算を行い、溶媒中における各化合物の構造を最適化した上で、バーチャルドッキングシミュレーション（in silico ドッキングシミュレーション）の手法により各化合物のキャプシド上の標的キャビティ（ｃａｖｉｔｙ）との結合スコアを計算し、結合スコアのよい化合物数千個を選定した。 Example 2: Identification of compounds that bind to capsid cavities Structural data of more than about 8 million compounds that can be purchased are obtained from academic and commercial compound databases, and when they are actually administered to a living body, the raw material of each compound is obtained. Taking into consideration the factors that affect ADME in the body, about 7 million compounds having molecular properties that can be used as drugs were extracted. About 7 million selected compounds, MMFF94 (Merck Molecular Force Field 94) was used to perform three-dimensional structural conversion and energy minimization calculation to optimize the structure of each compound in the solvent, and then virtual docking. The binding score of each compound to the target cavity on the capsid was calculated by a simulation (in silico docking simulation) method, and thousands of compounds having a good binding score were selected.
実施例３：抗ＨＩＶ－１活性評価
　実施例２で選定した化合物について、以下のようにしてＭＴＴアッセイ法により抗ＨＩＶ－１活性を評価した。
　化合物の抗ＨＩＶ－１活性の評価として、ＭＴ－２細胞と実験室野生株であるＨＩＶ－１LAI によるＭＴＴアッセイを用いた。９６ ｗｅｌｌ ｐｌａｔｅ 上で各化合物を段階希釈し、１００ ＴＣＩＤ50 の濃度となるウイルスとＭＴ－２細胞の混和溶液をｗｅｌｌに加え、対照として、ＭＴ－２細胞のみを加えたｗｅｌｌを作成し、７日間培養後、各ｗｅｌｌにＭＴＴ試薬を加え培養庫で呈色反応を行った。各ｗｅｌｌにＭＴＴ可溶化溶液を加えホルマザン結晶（生細胞において合成される）を溶かした後、各ｗｅｌｌの吸光度を測定、細胞のみを培養したｗｅｌｌでの吸光度と比較することで、抗ＨＩＶ－１活性があった場合には、ＨＩＶ－１感染による細胞障害を５０％阻害する濃度であるＥＣ50 値として算定できる。この系を用いて、実施例２で選定した化合物の抗ＨＩＶ－１活性を評価した。その結果、４０種類以上の化合物を選定した。 Example 3: Evaluation of anti-HIV-1 activity The compounds selected in Example 2 were evaluated for anti-HIV-1 activity by the MTT assay method as follows.
To evaluate the anti-HIV-1 activity of the compound, an MTT assay using MT-2 cells and the laboratory wild strain HIV-1 LAI was used. Each compound was serially diluted on a 96-well plate, and a mixed solution of virus and MT-2 cells having a concentration of 100 TCID 50 was added to the well to prepare a well containing only MT-2 cells as a control. After culturing for one day, MTT reagent was added to each well and a color reaction was carried out in the culture chamber. After adding an MTT solubilizing solution to each well to dissolve formazan crystals (synthesized in living cells), the absorbance of each well is measured and compared with the absorbance of the well in which only the cells are cultured to anti-HIV-1. If there is activity, it can be calculated as an EC 50 value, which is a concentration that inhibits cell damage due to HIV-1 infection by 50%. This system was used to evaluate the anti-HIV-1 activity of the compounds selected in Example 2. As a result, more than 40 kinds of compounds were selected.
実施例４：キャプシド安定化誘導活性評価
　実施例３で選定した化合物について、ＨＩＶ－１のキャプシドの安定化誘導活性を評価した。
（１）野生型キャプシド発現プラスミドの作成
　野生型キャプシド発現プラスミドを以下のようにして作成した。
　ｐＣＭＶ－Ｍｙｃベクター（＃６３１６０４，タカラバイオ社）を用いて野生型ＣＡ単独発現ｐｌａｓｍｉｄを作成した。制限酵素によりＭｙｃエピトープを含む領域を削除してベクターを線状化し、ＨＩＶ－１NL4-3を鋳型としてＰＣＲでキャプシドをコードする遺伝子領域を増幅した後、上記の線状化ベクターに導入した。
（２）野生型キャプシド発現細胞を用いた安定化誘導活性測定
　上記のようにして作成した野生型キャプシド発現プラスミドを細胞（ＣＯＳ－７細胞）に、プラスミドを１ μｇ/well（12 well plate）の割合にて混合することによりトランスフェクトさせ、４８時間後に細胞を回収した。トランスフェクトの１日前の細胞数で細胞（5 × 104 cells/day-1）当たり、１００μMの濃度となるように評価化合物を溶解した溶解液（M-PER Mammalian Protein Extraction Reagent （#78501, Thermo Fisher Scientific社））２００μLを加えて、細胞を溶解して、細胞溶解物を作成した。
　作成した細胞溶解物を等量ずつ４本のチューブに分注し、３７℃で、異なった時間（０～７２時間）、定温静置した。
　各細胞溶解物中の、キャプシド抗原量を、キャプシドに対するモノクローナル抗体（ルミパルスp24カートリッジ・富士レビオ）を用いて、ＥＬＩＳＡにより測定し、安定したキャプシドの割合を以下の式から求めた。
　（各サンプルのp24抗原量／インキュベートしていないサンプルのp24抗原量）× 100
　ＤＭＳＯのみを添加したものを対照とした。また、比較例として、ＣＡの多量体の安定化を過剰促進すると報告されている以下の化合物（ＰＦ３４５００７４）を比較例として用いた。 Example 4: Evaluation of Capsid Stabilization Inducing Activity The compound selected in Example 3 was evaluated for the stabilization inducing activity of the capsid of HIV-1.
(1) Preparation of wild-type capsid expression plasmid A wild-type capsid expression plasmid was prepared as follows.
A wild-type CA single expression plasmid was prepared using a pCMV-Myc vector (# 631604, Takara Bio Inc.). The region containing the Myc epitope was deleted with a restriction enzyme to linearize the vector, and the gene region encoding the capsid was amplified by PCR using HIV-1 NL4-3 as a template, and then introduced into the above linearized vector.
(2) Measurement of stabilization-inducing activity using wild-type capsid-expressing cells The wild-type capsid expression plasmid prepared as described above was used as cells (COS-7 cells), and the plasmid was added to 1 μg / well (12 well plate). The cells were transfected by mixing in proportions and the cells were harvested after 48 hours. M-PER Mammalian Protein Extraction Reagent (# 78501, Thermo) in which the evaluation compound was lysed to a concentration of 100 μM per cell (5 × 10 4 cells / day-1) at the number of cells 1 day before transfection. Fisher Scientific)) 200 μL was added to lyse the cells to prepare a cell lysate.
The prepared cell lysates were dispensed into four tubes in equal amounts, and allowed to stand at a constant temperature at 37 ° C. for different times (0 to 72 hours).
The amount of capsid antigen in each cell lysate was measured by ELISA using a monoclonal antibody against capsid (Lumipulse p24 cartridge Fujirebio), and the stable ratio of capsid was calculated from the following formula.
(P24 antigen amount of each sample / p24 antigen amount of unincubated sample) × 100
The one to which only DMSO was added was used as a control. Further, as a comparative example, the following compound (PF3450074), which has been reported to over-promote the stabilization of the multimer of CA, was used as a comparative example.
Figure JPOXMLDOC01-appb-C000023
 化合物ＰＦ３４５００７４
Figure JPOXMLDOC01-appb-C000023
 Compound PF3450074
　その結果、ＣＡの安定化過剰促進能を有する化合物として、以下の２つの化合物を同定した。 As a result, the following two compounds were identified as compounds having the ability to promote overstabilization of CA.
Figure JPOXMLDOC01-appb-C000024
 化合物Ａ
Figure JPOXMLDOC01-appb-C000024
 Compound A
Figure JPOXMLDOC01-appb-C000025
 化合物Ｂ
　７２時間培養後の安定ＣＡの割合は、ＤＭＳＯ（対照）が７９．４％、化合物Ａが９９．１％。化合物Ｂが９５．７％、ＰＦ３４５００７４が７５．７％であった。化合物Ａ及びＢは、顕著なキャプシドの安定化を誘導したが、ＰＦ３４５００７４は、対照（ＤＭＳＯ）と差がなかった。
　また、同定した２つの化合物（化合物Ａ及びＢ）の抗ＨＩＶ－１活性（ＥＣ50）は、化合物Ａは０．３μＭ、化合物Ｂは０．６μＭであった。
Figure JPOXMLDOC01-appb-C000025
 Compound B
The proportion of stable CA after culturing for 72 hours was 79.4% for DMSO (control) and 99.1% for compound A. Compound B was 95.7% and PF3450074 was 75.7%. Compounds A and B induced significant capsid stabilization, whereas PF3450074 was not different from control (DMSO).
The anti-HIV-1 activity (EC 50 ) of the two identified compounds (Compounds A and B) was 0.3 μM for Compound A and 0.6 μM for Compound B.
実施例５：他の類似化合物の抗ＨＩＶ－１活性評価
　実施例４で同定した２つの化合物（化合物Ａ及びＢ）のそれぞれに類似の構造を有する化合物として、下記の表に記載の化合物を入手し、それらの抗ＨＩＶ－１活性を実施例３と同様にして評価した。化合物はまた、公知の合成方法を適宜参照して合成することもできる。結果を以下の表に示す。 Example 5: Evaluation of anti-HIV-1 activity of other similar compounds Obtained the compounds listed in the table below as compounds having similar structures to each of the two compounds (Compounds A and B) identified in Example 4. Then, their anti-HIV-1 activity was evaluated in the same manner as in Example 3. The compound can also be synthesized by appropriately referring to a known synthesis method. The results are shown in the table below.
Figure JPOXMLDOC01-appb-T000026
Figure JPOXMLDOC01-appb-T000026
Figure JPOXMLDOC01-appb-T000027
Figure JPOXMLDOC01-appb-T000027
実施例６：ウイルス合成・出芽に対する阻害活性の確認
　ＨＩＶ野生株（ｐＨＩＶ－１NL4-3）を発現するプラスミドを用い、トランスフェクト後のウイルス合成・出芽に対する化合物（化合物Ａ及び化合物Ｂ）の影響を以下のようにして確認した。
　トランスフェクション（ＴＦ）前日にＣＯＳ－７細胞を６ｗｅｌｌ ｐｌａｔｅに１．５×１０5 ｃｅｌｌｓ／ｗｅｌｌずつ播種し、当日に細胞が７０％以上コンフルエントである事を確認した上で、リポフェクタミンＬＴＸ（Ｉｎｖｉｔｒｏｇｅｎ）を用いてｐＨＩＶ－１NL4-3のＴＦを行った。この際、化合物Ａ、Ｂをそれぞれ１、１０、１００μＭの濃度でＴＦ培地に添加した。ＴＦ ７２時間後における培養上清中のＨＩＶ－１ ｐ２４発現量をＥＬＩＳＡ（ルミパルスｆ、富士レビオ）にて測定し、化合物のウイルス出芽への影響を測定した。その結果、図２に示すように、化合物Ａ、Ｂは、ＨＩＶ－１NL4-3発現プラスミドＴＦ後のウイルス合成・出芽に影響を及ぼさないことが判った。 Example 6: using a plasmid expressing the confirmation of inhibitory activity HIV wild-type strain (pHIV-1 NL4-3) to viral synthesis and budding, the influence of the compounds on viral synthesis and sprouting after transfection (Compound A and Compound B) Was confirmed as follows.
The day before transfection (TF), COS-7 cells were seeded on a 6-well plate at 1.5 × 10 5 cells / well, and after confirming that the cells were 70% or more confluent on the day, lipofectamine LTX (Invitrogen) was used. was TF of pHIV-1 NL4-3 using. At this time, compounds A and B were added to the TF medium at concentrations of 1, 10 and 100 μM, respectively. The expression level of HIV-1 p24 in the culture supernatant after 72 hours of TF was measured by ELISA (Lumipulse f, Fujirebio), and the effect of the compound on virus budding was measured. As a result, as shown in FIG. 2, it was found that compounds A and B did not affect virus synthesis and budding after HIV-1 NL4-3 expression plasmid TF.
実施例７：細胞毒性の確認
　化合物の細胞毒性の確認には、ＭＴＴアッセイ法を用いた。ＣＯＳ－７細胞もしくはＭＴ－４細胞を９６ｗｅｌｌ ｐｌａｔｅに加え、１０段階ずつ希釈した化合物を同時に添加し、対照として、ＣＯＳ－７細胞もしくはＭＴ－４細胞のみを加えたｗｅｌｌを作成し、７日間培養後、各ｗｅｌｌにＭＴＴ試薬を加え培養庫で呈色反応を行った。各ｗｅｌｌにＭＴＴ可溶化溶液を加えホルマザン結晶（生細胞において合成される）を溶かした後、各ｗｅｌｌの吸光度を測定、細胞のみを培養したｗｅｌｌでの吸光度と比較する事で、５０％細胞毒性濃度（ＣＣ50）を算定した。その結果、化合物Ａ、ＢはいずれもＣＯＳ－７細胞に対してＣＣ50： ＞ １００ μＭ、ＭＴ－４細胞に対してそれぞれ８７，３９ μＭであり、化合物Ａ及び化合物Ｂは、細胞毒性を殆ど示さないことが確認された。 Example 7: Confirmation of cytotoxicity The MTT assay method was used to confirm the cytotoxicity of the compound. COS-7 cells or MT-4 cells were added to a 96-well plate, and a compound diluted in 10 steps was added at the same time to prepare a well containing only COS-7 cells or MT-4 cells as a control, and cultured for 7 days. After that, MTT reagent was added to each well and a color reaction was carried out in a culture chamber. After adding MTT solubilized solution to each well to dissolve formazan crystals (synthesized in living cells), the absorbance of each well is measured and compared with the absorbance of the well in which only cells are cultured, resulting in 50% cytotoxicity. The concentration (CC 50 ) was calculated. As a result, both compounds A and B were CC 50 :> 100 μM for COS-7 cells and 87 and 39 μM for MT-4 cells, respectively, and compound A and compound B were almost cytotoxic. It was confirmed not to show.
実施例８：ヒト由来細胞に対する細胞毒性の確認
　実施例７と同様にして、化合物Ａ及び化合物Ｂについて、ＭＴＴアッセイ法を用い、ヒト肝胆道系由来細胞（Ｌｉ－７、ＨＬＥ及びＹＳＣＣＣ細胞）及び腎臓系由来細胞（ＨＥＫ２９３細胞）に対する細胞毒性を確認した。結果を以下の表に示す。化合物Ａは、ヒト由来細胞に細胞毒性を示さなかった。また化合物Ｂは、ヒト肝胆道系由来細胞には細胞毒性を示さず、腎臓系由来細胞には細胞毒性を殆ど示さなかった。 Example 8: Confirmation of Cytotoxicity to Human-Derived Cells Similar to Example 7, for Compound A and Compound B, using the MTT assay method, human hepatobiliary system-derived cells (Li-7, HLE and YSCCC cells) and Cytotoxicity to kidney line-derived cells (HEK293 cells) was confirmed. The results are shown in the table below. Compound A showed no cytotoxicity to human-derived cells. In addition, Compound B showed no cytotoxicity to human hepatobiliary cells and showed almost no cytotoxicity to renal cells.
Figure JPOXMLDOC01-appb-T000028
Figure JPOXMLDOC01-appb-T000028
実施例９：ＭＡＧＩアッセイによる標的細胞内進入の評価
　ＨＩＶのＴａｔタンパク質は、ＨＩＶ遺伝子の転写活性因子であり、ビリオン内でＨＩＶ遺伝子と共局在していると考えられている。そして、ＨＩＶが標的細胞に感染すると、脱殻が起こり、標的細胞の細胞質から核内へと、ＨＩＶ遺伝子（ＨＩＶゲノムＲＮＡ）とともにＴａｔタンパク質も送られる。ＨＩＶ－１ ＬＴＲ と大腸菌 ｌａｃＺ 遺伝子の融合遺伝子を組み込んだ Ｕ３７３－ＭａｇｉCD4+CXCR4+ 細胞を用いたＭａｇｇｉアッセイは、Ｍａｇｉ細胞にＨＩＶが感染すると、感染した細胞においてあらかじめ組み込まれたＨＩＶ－１ ＬＴＲがＨＩＶ－１の転写調節因子であるＴａｔにより促進され、その下流のｌａｃＺ遺伝子によりβ－ｇａｌ活性が上昇する事でＸ－ｇａｌ反応後の青染した感染細胞数を測定でき、感染性の評価が可能である。アッセイ系の概略を図３に示す。
　ＣＯＳ－７細胞にウイルスを強制発現させ、ＨＩＶ－１WTのウイルス上清を、同量のｐ２４抗原量を含むようにして、Ｕ３７３－ＭａｇｉCD4+CXCR4+ 細胞に感染させた上で、感染細胞数を顕微鏡下で測定しウイルスの感染性を評価した。トランスフェクトさせるウイルス上清は、ｐＨＩＶ－１NL4-3のみ、化合物Ａ（１００μＭ）、化合物Ｂ(１００μＭ)の条件でトランスフェクト後３日間培養して得られたウイルス培養上清を用いた。２４ウェルプレートに、ウェルあたり、６ × 104 の細胞を播き、１日間培養した。上記のウイルス上清を等量のｐ２４を含むように希釈し（１０ｎｇ／ｍｌ）ＤＥＡＥデキストランとともに細胞に添加し、ウイルスを感染させた後、２時間培養した（吸着反応）。次いで、さらに培地を加え４８時間培養した。細胞を洗浄し、１％ホルムアルデヒド及び０．２％グルタルアルデヒドで固定した（固定反応）。染色液（Ｘ－ｇａｌ、フェリシアン化カリウム、Ｍｇ2Ｃｌ含有）を用いて２時間染色した（染色反応）。その後、顕微鏡下で、染色された陽性細胞数をカウントした。結果を図４に示す。化合物Ａ及び化合物Ｂで前処理されたウイルス上清は、対照に比べ、陽性細胞数の減少を認め、脱殻が阻害されている事が示唆された。 Example 9: Evaluation of entry into target cells by MAGI assay The HIV Tat protein is a transcriptional activator of the HIV gene and is believed to be co-localized with the HIV gene in the virion. Then, when HIV infects the target cell, shedding occurs, and the Tat protein is also sent from the cytoplasm of the target cell into the nucleus together with the HIV gene (HIV genomic RNA). The Maggi assay using U373-Magi CD4 + CXCR4 + cells incorporating a fusion gene of the HIV-1 LTR and the Escherichia coli lacZ gene shows that when the Magi cells are infected with HIV, the HIV-1 LTR pre-integrated in the infected cells becomes HIV. It is promoted by Tat, which is a transcriptional regulator of -1, and β-gal activity is increased by the lacZ gene downstream of it, so that the number of blue-stained infected cells after the X-gal reaction can be measured and infectivity can be evaluated. Is. The outline of the assay system is shown in FIG.
The virus was forcibly expressed in COS-7 cells, and the virus supernatant of HIV-1 WT was infected with U373-Magi CD4 + CXCR4 + cells so as to contain the same amount of p24 antigen, and the number of infected cells was microscopically measured. The infectivity of the virus was evaluated by measuring below. Virus supernatants transfect only pHIV-1 NL4-3, Compound A (100 [mu] M), with a viral supernatant obtained by culturing 3 days after transfection with the conditions of compound B (100 [mu] M). In a 24-well plate, 6 × 10 4 cells per well were seeded and cultured for 1 day. The above virus supernatant was diluted to contain an equal amount of p24 (10 ng / ml), added to cells together with DEAE dextran, infected with the virus, and then cultured for 2 hours (adsorption reaction). Then, a medium was further added and the cells were cultured for 48 hours. Cells were washed and fixed with 1% formaldehyde and 0.2% glutaraldehyde (fixation reaction). Staining was performed for 2 hours using a staining solution (containing X-gal, potassium ferricyanide, and Mg 2 Cl) (staining reaction). Then, the number of stained positive cells was counted under a microscope. The results are shown in FIG. The virus supernatants pretreated with Compound A and Compound B showed a decrease in the number of positive cells as compared with the control, suggesting that shedding was inhibited.
実施例１０：キャプシドの熱安定性の上昇
　示唆走査蛍光測定法を用い、ＨＩＶ－１キャプシドの熱安定性に対する化合物の影響を以下のようにして確認した。
　示唆走査蛍光測定法とは、対象となる蛋白が存在する環境の温度を上昇させることにより対象蛋白の立体構造が変性し、疎水性領域の露出が増加することで添加した蛍光色素の結合が増加、蛍光強度の変化を解析する事により、蛋白の変性温度（Ｔｍ）を測定する手法である。大腸菌にＨＩＶ－１ ＣＡを単独で発現・抽出したサンプルに化合物Ａ、Ｂを添加し、更に蛍光色素ＳＹＰＲＯ－Ｏｒａｎｇｅを加え、温度を変化させる事によりＣＡの安定性に与える影響を評価した。対照として、ＤＭＳＯのみ、臨床的に使用されているＨＩＶ－１インテグラーゼ阻害剤であるラルテグラビル（ＲＡＬ）を用いた。結果を図５に示す。評価した化合物Ａ及び化合物Ｂは、ＣＡの熱安定性を上昇させていることがわかった。 Example 10: Suggestion of increased thermal stability of capsid Using scanning fluorescence measurement, the effect of the compound on the thermal stability of HIV-1 capsid was confirmed as follows.
Suggestion Scanning fluorescence measurement is a method in which the three-dimensional structure of the target protein is denatured by raising the temperature of the environment in which the target protein is present, and the exposure of the hydrophobic region is increased, so that the binding of the added fluorescent dye is increased. This is a method for measuring the denaturation temperature (Tm) of a protein by analyzing the change in fluorescence intensity. Compounds A and B were added to a sample in which HIV-1 CA was expressed and extracted independently in Escherichia coli, and a fluorescent dye SYPRO-Orange was further added to evaluate the effect on the stability of CA by changing the temperature. As a control, DMSO only, the clinically used HIV-1 integrase inhibitor raltegravir (RAL), was used. The results are shown in FIG. It was found that the evaluated compounds A and B increased the thermal stability of CA.
実施例１１：薬剤耐性誘導性の試験
　ＭＴ－４細胞（３×１０5 ／ｗｅｌｌ）を５００ＴＣＩＤ50の濃度のＨＩＶ－１NL4-3とともにＥＣ50濃度（耐性誘導開始時）付近における３通りの濃度の化合物存在下で培養し、ｄａｙ ７における上清中のｐ２４抗原量を測定することでウイルスの増殖を確認した後、ウイルスが増殖し・かつ最も高濃度の化合物を含むウイルス上清のみを回収、新たなＭＴ－４細胞を回収したウイルス上清に加え５時間以上培養することで細胞にウイルスを感染させ、感染した細胞を洗浄した後、３通りの濃度が異なる化合物（１つは直近の継代時にウイルス増殖を確認できたうち、最も高い濃度、他２つはそれより更に高い濃度）の存在下で培養・継代（ｐａｓｓａｇｅ）を継続した（ｃｅｌｌ ｆｒｅｅ ｍａｎｎｅｒ）。化合物Ａ、Ｂに対する耐性誘導において、それぞれ１５週（ｐａｓｓａｇｅ１５）、１９週（ｐａｓｓａｇｅ１９）における化合物濃度よりも高い濃度の化合物存在下ではウイルスの増殖が不能となり、また最終ｐａｓｓａｇｅで回収した感染細胞よりＤＮＡを抽出、組み込まれたプロウイルスＤＮＡの配列を解析した結果、ＣＡをコードする遺伝子領域に変異は認められず、これより化合物Ａ、Ｂの、ＨＩＶ－１の薬剤耐性獲得に対するｇｅｎｅｔｉｃ ｂａｒｒｉｅｒが高い（ＨＩＶ－１の薬剤耐性化が起こりにくい）ことが示唆された。結果を図６に示す。 Example 11: Concentration of triplicate drug resistance induced test MT-4 cells (3 × 10 5 / well) with HIV-1 NL4-3 at a concentration of 500TCID 50 in EC 50 near concentration (at resistance induced initiation) After confirming the growth of the virus by culturing in the presence of the above compound and measuring the amount of p24 antigen in the supernatant on day 7, the virus propagated and only the virus supernatant containing the highest concentration of the compound was collected. , Infect the cells with the virus by adding the new MT-4 cells to the collected virus supernatant and culturing for 5 hours or more, and after washing the infected cells, three compounds with different concentrations (one is the latest one). Among the virus growth confirmed at the time of passage, the culture and passage (passage) were continued in the presence of the highest concentration (the other two had higher concentrations) (cell free manager). In the induction of resistance to compounds A and B, virus growth became impossible in the presence of a compound having a concentration higher than the compound concentration at 15 weeks (passage 15) and 19 weeks (passage 19), respectively, and DNA from infected cells recovered in the final passage. As a result of analyzing the sequence of the provirus DNA in which the virus was extracted and integrated, no mutation was found in the gene region encoding CA, and the genetic barrier for the acquisition of drug resistance of HIV-1 of compounds A and B was higher than this ( It was suggested that drug resistance of HIV-1 is unlikely to occur). The results are shown in FIG.
　上記の詳細な記載は、本発明の目的及び対象を単に説明するものであり、添付の特許請求の範囲を限定するものではない。添付の特許請求の範囲から離れることなしに、記載された実施態様に対しての、種々の変更及び置換は、本明細書に記載された教示より当業者にとって明らかである。 The above detailed description merely explains the purpose and subject of the present invention, and does not limit the scope of the appended claims. Various changes and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings set forth herein, without departing from the appended claims.
　本発明の化合物は、抗ＨＩＶ活性を示すので抗ウイルス阻害剤として有用である。 The compound of the present invention exhibits anti-HIV activity and is therefore useful as an antiviral inhibitor.

Claims (16)

  1. 　下記式（Ｉ）：
    Figure JPOXMLDOC01-appb-C000001
     ［式（Ｉ）中、Ｘ及びＹは、それぞれ独立にＣ又はＮを表し、Ｚは、Ｃ又は結合を表し、Ｒａ及びＲｂは、それぞれ独立に、水素又は置換されてもよいフェニル基を表すか互いに一緒になってＸとＹとともに置換されてもよい６員炭素環を形成し、Ｒ1は、Ｃ１～Ｃ５のアルキル基又はアルケニル基、フェニル基、ベンジル基、あるいは２－モルホリノエチル基を表す。］で表される化合物、又はその薬学的に許容される塩を有効成分として含むＨＩＶの治療又は予防のための医薬組成物。     The following formula (I):
    Figure JPOXMLDOC01-appb-C000001
     [In formula (I), X and Y each independently represent C or N, Z represents C or bond, and Ra and Rb each independently represent a hydrogen or optionally substituted phenyl group. Together with each other, they form a 6-membered carbocycle that may be substituted with X and Y, where R 1 contains C1-C5 alkyl or alkenyl groups, phenyl groups, benzyl groups, or 2-morpholinoethyl groups. Represent. ], Or a pharmaceutical composition for treating or preventing HIV, which comprises a pharmaceutically acceptable salt thereof as an active ingredient.
  2. 　下記式（ＩＩ）：
    Figure JPOXMLDOC01-appb-C000002
     ［式（ＩＩ）中、Ｒ2は、Ｃ１～Ｃ５のアルキル基又はアルケニル基、ベンジル基、あるいは２－モルホリノエチル基を表す。］で表される化合物、又はその薬学的に許容される塩を有効成分として含む、請求項１に記載の医薬組成物。     The following formula (II):
    Figure JPOXMLDOC01-appb-C000002
     [In formula (II), R 2 represents an alkyl or alkenyl group, a benzyl group, or a 2-morpholinoethyl group of C1 to C5. ] As an active ingredient, the pharmaceutical composition according to claim 1.
  3. 　上記式（ＩＩ）において、Ｒ2は、Ｃ１～Ｃ３のアルキル基又はアルケニル基である、請求項２に記載の医薬組成物。 The pharmaceutical composition according to claim 2, wherein in the above formula (II), R 2 is an alkyl group or an alkenyl group of C1 to C3.
  4. 　上記式（ＩＩ）において、Ｒ2は、メチル基、エチル基、及びアリル基からなる群より選ばれる、請求項２に記載の医薬組成物。 The pharmaceutical composition according to claim 2, wherein in the above formula (II), R 2 is selected from the group consisting of a methyl group, an ethyl group, and an allyl group.
  5. 　上記式（ＩＩ）において、Ｒ2は、ベンジル基又は２－モルホリノエチルである、請求項２に記載の医薬組成物。 The pharmaceutical composition according to claim 2, wherein in the above formula (II), R 2 is a benzyl group or 2-morpholinoethyl.
  6. 　下記式（ＩＩＩ）：
    Figure JPOXMLDOC01-appb-C000003
     ［式（ＩＩＩ）中、Ｒ3は、Ｃ１～Ｃ５のアルキル基又はアルケニル基、あるいはフェニル基を表す。］で表される化合物、又はその薬学的に許容される塩を有効成分として含む、請求項１に記載の医薬組成物。     The following formula (III):
    Figure JPOXMLDOC01-appb-C000003
     [In formula (III), R 3 represents an alkyl group, an alkenyl group, or a phenyl group of C1 to C5. ] As an active ingredient, the pharmaceutical composition according to claim 1.
  7. 　上記式（ＩＩＩ）において、Ｒ3は、Ｃ１～Ｃ３のアルキル基又はアルケニル基である、請求項６に記載の医薬組成物。 The pharmaceutical composition according to claim 6, wherein in the above formula (III), R 3 is an alkyl group or an alkenyl group of C1 to C3.
  8. 　上記式（ＩＩＩ）において、Ｒ3は、メチル基、エチル基、及びアリル基からなる群より選ばれる、請求項６に記載の医薬組成物。 The pharmaceutical composition according to claim 6, wherein in the above formula (III), R 3 is selected from the group consisting of a methyl group, an ethyl group, and an allyl group.
  9. 　上記式（ＩＩＩ）において、Ｒ3は、フェニル基である請求項６に記載の医薬組成物。 The pharmaceutical composition according to claim 6, wherein R 3 is a phenyl group in the above formula (III).
  10. 　以下の化合物：
    Figure JPOXMLDOC01-appb-C000004
     からなる群から選ばれる化合物又はその薬学的に許容される塩を有効成分として含む、請求項２に記載の医薬組成物。     The following compounds:
    Figure JPOXMLDOC01-appb-C000004
     The pharmaceutical composition according to claim 2, which comprises, as an active ingredient, a compound selected from the group consisting of or a pharmaceutically acceptable salt thereof.
  11. 　以下の化合物：
    Figure JPOXMLDOC01-appb-C000005
     からなる群から選ばれる化合物又はその薬学的に許容される塩を有効成分として含む、請求項２に記載の医薬組成物。     The following compounds:
    Figure JPOXMLDOC01-appb-C000005
     The pharmaceutical composition according to claim 2, which comprises, as an active ingredient, a compound selected from the group consisting of or a pharmaceutically acceptable salt thereof.
  12. 　以下の化合物：
    Figure JPOXMLDOC01-appb-C000006
     からなる群から選ばれる化合物又はその薬学的に許容される塩を有効成分として含む、請求項６に記載の医薬組成物。     The following compounds:
    Figure JPOXMLDOC01-appb-C000006
     The pharmaceutical composition according to claim 6, which comprises, as an active ingredient, a compound selected from the group consisting of or a pharmaceutically acceptable salt thereof.
  13. 　他の抗ＨＩＶ薬と併用されることを特徴とする請求項１から１２のいずれか一つに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 12, characterized in that it is used in combination with another anti-HIV drug.
  14. 　前記他の抗ＨＩＶ薬が、化学療法剤、抗レトロウイルス阻害剤、サイトカイン、ヒドロキシウレア、Ｇａｇタンパク質に結合するモノクローナル抗体、又は他のレトロウイルス複製の阻害剤である、請求項１３に記載の医薬組成物。 13. The medicament according to claim 13, wherein the other anti-HIV agent is a chemotherapeutic agent, an anti-retrovirus inhibitor, a cytokine, a hydroxyurea, a monoclonal antibody that binds to a Gag protein, or another inhibitor of retrovirus replication. Composition.
  15. 　抗ＨＩＶ活性を有する物質をスクリーニングする方法であって、以下の工程：
    （ａ）ＨＩＶ－１の野生型キャプシドを発現する細胞から調製したキャプシドとともに候補物質を緩衝液中で３７±２℃の温度にてインキュベートする工程、ここで、該細胞は、ＨＩＶ－１の野生型キャプシドの発現プラスミドを用いて形質転換した細胞であり、キャプシド以外のＨＩＶ－１由来の成分を発現しない細胞である、及び
    （ｂ）キャプシドの崩壊を検出し、対照に対し、キャプシドの崩壊が抑制された物質を選択する工程、
    を含むスクリーニング方法。     A method of screening for substances with anti-HIV activity, the following steps:
    (A) A step of incubating a candidate substance in a buffer at a temperature of 37 ± 2 ° C. together with a capsid prepared from cells expressing the wild-type capsid of HIV-1, where the cells are wild in HIV-1. Cells transformed with a type capsid expression plasmid, which do not express components derived from HIV-1 other than capsid, and (b) capsid disruption was detected, and capsid disruption occurred as opposed to control. The process of selecting suppressed substances,
    Screening method including.
  16. 　前記工程（ｂ）は、無傷（intact）のキャプシドの抗原量を、抗キャプシド抗体を用いて測定する工程である、請求項１５に記載のスクリーニング方法。 The screening method according to claim 15, wherein the step (b) is a step of measuring the antigen amount of an intact capsid using an anti-capsid antibody.
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