WO2020241737A1 - Composition pharmaceutique anti-vih - Google Patents

Composition pharmaceutique anti-vih 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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Japanese (ja)
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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

La présente invention a pour objet de fournir une nouvelle molécule de faible poids moléculaire ayant une activité anti-VIH. La présente invention a également pour objet de fournir un nouveau médicament anti-VIH ciblant la capside du VIH-1. L'invention concerne un nouveau composé ayant une activité anti-VIH et représenté par la formule (I) : [Dans la formule (I), X et Y représentent chacun indépendamment C ou N, Z représente C ou une liaison, Ra et Rb représentent chacun indépendamment un atome d'hydrogène ou un groupe phényle éventuellement substitué, ou forment ensemble un cycle carboné à six chaînons éventuellement substitué avec X et Y, R1 représente un groupe alkyle ou alcényle en C1-C5, un groupe phényle, un groupe benzyle ou un groupe 2-morpholinoéthyle].
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US20090088420A1 (en) * 2007-04-12 2009-04-02 University Of Southern California Compounds with hiv-1 integrase inhibitory activity and use thereof as anti-hiv/aids therapeutics

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Publication number Priority date Publication date Assignee Title
US20090088420A1 (en) * 2007-04-12 2009-04-02 University Of Southern California Compounds with hiv-1 integrase inhibitory activity and use thereof as anti-hiv/aids therapeutics

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