WO2017000789A1 - Inhibiteur de n-peptide réticulé de manière covalente - Google Patents

Inhibiteur de n-peptide réticulé de manière covalente Download PDF

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WO2017000789A1
WO2017000789A1 PCT/CN2016/085968 CN2016085968W WO2017000789A1 WO 2017000789 A1 WO2017000789 A1 WO 2017000789A1 CN 2016085968 W CN2016085968 W CN 2016085968W WO 2017000789 A1 WO2017000789 A1 WO 2017000789A1
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formula
compound
seq
stereoisomer
derivative
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Chinese (zh)
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刘克良
王潮
来文庆
姜喜凤
许笑宇
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中国人民解放军军事医学科学院毒物药物研究所
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Publication of WO2017000789A1 publication Critical patent/WO2017000789A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus human T-cell leukaemia-lymphoma virus
    • C07K14/155—Lentiviridae, e.g. human immunodeficiency virus [HIV], visna-maedi virus or equine infectious anaemia virus
    • C07K14/16—HIV-1 ; HIV-2
    • C07K14/162—HIV-1 ; HIV-2 env, e.g. gp160, gp110/120, gp41, V3, peptid T, CD4-Binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus human T-cell leukaemia-lymphoma virus
    • C07K14/155—Lentiviridae, e.g. human immunodeficiency virus [HIV], visna-maedi virus or equine infectious anaemia virus
    • C07K14/16—HIV-1 ; HIV-2
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV

Definitions

  • the invention belongs to the field of biomedicine and relates to a polypeptide against human immunodeficiency virus (HIV) infection, a derivative thereof, a stereoisomer, or a salt which is not physiologically toxic.
  • the present invention also relates to a pharmaceutical composition comprising the above polypeptide, a derivative thereof, a stereoisomer, or a salt which is not physiologically toxic, and a preparation of the polypeptide, a derivative thereof, a stereoisomer, or a salt which is not physiologically toxic in preparation Use in the treatment and/or prevention and/or adjuvant treatment of diseases associated with HIV infection, especially in the acquisition of immunodeficiency syndrome (AIDS).
  • HIV immunodeficiency virus
  • HIV-1 human immunodeficiency virus type 1
  • HIV-1 human immunodeficiency virus type 1
  • Drug treatment is still the only effective method at present.
  • the clinical application of anti-HIV-1 drugs, supplemented by highly active antiretroviral therapy, can prolong the survival time and improve the quality of life of HIV-infected patients to some extent.
  • HIV fusion inhibitors are novel anti-HIV drugs that interfere with the entry of viruses into target cells. They cut off the spread of the virus at the initial stage of infection, which has special significance for the prevention and control of HIV-1 infection, and thus becomes a new mechanism. Hot spots in HIV drug research.
  • Gp41 is a specific protein that mediates the fusion of HIV-1 virus with target cell membranes and is a major target for fusion inhibitors.
  • HR1, NHR or N-peptide the C-terminal repeat
  • HR2, CHR or C peptide C-terminal repeat
  • HR2 in three gp41 molecules interacts with HR1 to form a hexagonal core structure (6-HB).
  • the 6-HB formed in the gp41 molecule is the core structure of the whole fusion, and its crystal structure is the basic and basic model for designing fusion inhibitors. Designing peptide fusion inhibitors
  • the action of the helical region sequence corresponding to the gp41 molecule blocks the formation of the endogenous 6HB, thereby blocking
  • Fusion inhibitors based on natural C-peptide sequences have high activity and IC 50 can reach nanomolar levels. Therefore, the fusion inhibitors that have entered clinical development are both C-peptides and their derivatives.
  • Typical C-peptide fusion inhibitors are T20 and C34. Both are derived from the polypeptide of the CHR native sequence of gp41, and the target of action of these C-peptide drugs are all NHR trimers.
  • the researchers designed a new generation of fusion inhibitors, most of which use C34 as a template. Compared with T20, the activity and stability of the new inhibitors have been greatly improved, among which T1144 and Sifuvirtide have achieved good clinical results.
  • N-peptide fusion inhibitors based on the viral gp41-based NHR design are another development direction. From the structure and mechanism of the above three gp41 molecules to form 6HB, CHR and NHR are ligands, and the two have specific interaction characteristics, forming 6HB and releasing energy, driving the fusion of virus and cells. To date, although the N-peptide fusion inhibitor has not yet entered the clinical study, the first reported fusion inhibitor DP107 is the N-peptide. This shows from another side that its research problems are more and more difficult. The activity of the N-peptide is generally at the micromolar level, about 1000 times lower than the corresponding C-peptide.
  • N-peptide fusion inhibitors inhibit the formation of 6HB in gp41 molecules in two ways, thereby blocking the fusion process between HIV and cells: 1) exogenous N-peptide trimer and CHR in gp41 Role, the formation of heterologous 6HB, so that gp41 can not be intramolecular folding, interrupt the fusion; 2) single-stranded N-peptide complexed in NHR, forming a heterotrimer, inhibiting the formation of 6HB in gp41 molecule.
  • the N-peptide inhibitors that act in the first mode are more active, and the activities of the two methods differ by one to three orders of magnitude.
  • the researchers used various methods to make the N-peptide fusion inhibitor inhibit the formation of 6HB in gp41 molecule mainly by the first method, but the problem is that the N-peptide fragment itself is difficult to form a stable conformer (N3 helix) of active conformation, and The surface contains more hydrophobic residues, which are easily aggregated and inactivated under physiological conditions. Therefore, the research of N-peptide fusion inhibitors is mainly How to stabilize its trimer and improve physical and chemical properties. From the perspective of R&D drugs, CHR and NHR can be mutually targets. N-peptide fusion inhibitors target CHR in gp41, which is completely different from T20 (enfuvirtide) and Maraviroc. Is one of the effective ways to avoid or slow the cross-resistance with existing drugs.
  • the general method is to conjugate a natural N-peptide fragment to a helper polypeptide capable of forming a stable triple helix structure, or to use a disulfide bond to link the above-designed conjugated N-peptide to form Irreversible trimeric N-peptides, which increase the activity of N-peptide inhibitors by 2-3 orders of magnitude.
  • the above method also has its own drawbacks: the single-stranded N-peptide has low activity and is easy to aggregate and precipitate; the conjugation of the helper polypeptide makes the N-peptide sequence conjugated to be verbose; the disulfide-crosslinking can increase the activity of the N-peptide, but Linkage reaction sites and reaction specificity are uncontrollable, and so on. All of the above drawbacks limit the development of N-peptide inhibitors, which are still a long way from the drug.
  • the invention de novo has designed an artificial auxiliary sequence which can stably form a triple helix structure, and designs a new covalent bond forming method, chemically modifying at a suitable site, and using a lactam bond to obtain a fixed-point covalent cross-linking.
  • a trimer-assisted sequence which is then conjugated to a portion of the native N-peptide, such that the conjugated N-peptide is capable of interacting with the CHR of the virus, resulting in a covalently cross-linked, newly conjugated N-peptide inhibitor, thereby completing this invention.
  • the invention is based on a novel design idea and a new covalent bond formation method, and designs a novel N-peptide inhibitor to improve the inhibitory activity of the N-peptide.
  • a first aspect of the invention relates to a compound of formula I or formula II, a derivative thereof, a stereoisomer or a salt of no physiological toxicity,
  • the compound of formula II is a compound of three formula I which is covalently crosslinked by two a trimer,
  • X 1 is deleted or selected from L-type natural amino acids
  • X 2 , X 3 , X 4 , X 6 , and X 7 are each independently selected from natural L-type natural amino acids
  • X 5 is selected from the group consisting of L-type natural amino acids and L-type unnatural amino acids.
  • X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 are each independently selected from L-type natural amino acid ,
  • X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 exist simultaneously or simultaneously,
  • X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 exist simultaneously or simultaneously,
  • Z represents an N-terminal group and may be -NH 2 , -NR 1 C(O)R 2 , C 1-6 alkyl, C 1-6 alkoxy or -LR 3 ,
  • R 1 and R 2 are independently selected from H and an alkyl group
  • L is a linking fragment selected from a dicarboxylic acid (eg, oxalic acid, malonic acid, succinic acid, etc.), derivatized with a carboxyl group at the N-terminus and esterified with R 3 having a hydroxyl group,
  • a dicarboxylic acid eg, oxalic acid, malonic acid, succinic acid, etc.
  • R 3 is a group which can react with a carboxyl group such as R′OH, PEG, cholesterol, etc., and does not affect the crosslinking reaction, wherein R′ is an alkyl group,
  • B represents a C-terminal group and may be -COOH or -C(O)NR 4 R 5 , wherein R 4 and R 5 are independently selected from H and an alkyl group, and preferably R 4 and R 5 are both H.
  • the compound of Formula I or Formula II, a derivative, a stereoisomer or a non-physiologically acceptable salt thereof, according to the first aspect of the invention wherein said covalent cross-linking Is a covalent bond formed between X 5 in one compound of formula I and another glutamic acid at position 5 after X 5 in another compound of formula I, or the covalent cross-linking is in a compound of formula I X 2 forms a covalent bond with another glutamic acid at the 5th position after X 2 in the compound of the formula I, and the covalent bond is preferably an amide bond (-NC(O)-).
  • the amino acid is selected from the group consisting of glycine (Gly), alanine (Ala), leucine (Leu), isoleucine (Ile), glutamic acid (Glu), glutamine (Gln), and aspartic acid (Asp).
  • the L-type unnatural amino acid is ornithine (Orn, O).
  • the compound of Formula I or Formula II of Claim 1 of the first aspect of the invention a derivative thereof, a stereoisomer or a salt of no physiological toxicity, wherein:
  • X 1 is tryptophan (Trp)
  • X 2 is selected from the group consisting of arginine (Arg), lysine (Lys) and ornithine (Orn).
  • X 3 is selected from the group consisting of glutamine (Gln) and histidine (His).
  • X 4 is selected from the group consisting of glutamine (Gln) and histidine (His).
  • X 5 is selected from the group consisting of arginine (Arg), lysine (Lys) and ornithine (Orn).
  • X 6 is selected from the group consisting of glutamine (Gln) and histidine (His).
  • X 7 is selected from the group consisting of glutamine (Gln) and histidine (His).
  • X 8 is arginine (Arg)
  • X 10 is glutamine (Gln)
  • X 11 is glutamine (Gln)
  • X 12 is isoleucine (Ile),
  • X 13 is glutamic acid (Glu)
  • X 14 is glutamine (Gln)
  • X 15 is arginine (Arg),
  • X 16 is isoleucine (Ile),
  • X 17 is glutamic acid (Glu)
  • X 18 is alanine (Ala),
  • X 19 is glutamine (Gln)
  • X 20 is glutamine (Gln)
  • X 21 is histidine (His).
  • the compound of Formula I or Formula II, a derivative, a stereoisomer or a non-physiologically acceptable salt thereof, according to the first aspect of the invention wherein said covalent cross-linking Is the amide bond (-NC(O)-) formed by the lysine or ornithine represented by X 5 in the compound of the formula I and the glutamic acid at the 5th position after the X 5 in the compound of the formula I,
  • the covalent cross-linking is the formation of an amide bond between lysine or ornithine represented by X 2 in a compound of formula I and another glutamic acid at position 5 after X 2 in another compound of formula I ( -NC(O)-).
  • the compound of Formula I or Formula II, a derivative, a stereoisomer or a non-physiologically acceptable salt thereof, according to the first aspect of the invention is selected from the group consisting of:
  • a second aspect of the invention relates to a nucleic acid molecule encoding a compound of the above formula I or formula II according to any one of the first aspects of the invention, a derivative thereof, a stereoisomer or a salt of no physiological toxicity. .
  • a third aspect of the invention relates to a recombinant vector comprising the nucleic acid molecule of the second aspect of the invention.
  • the vector may be a prokaryotic expression vector or a eukaryotic expression vector.
  • a fourth aspect of the invention relates to a host cell comprising the nucleic acid molecule of the second aspect of the invention and/or the recombinant vector of the third aspect of the invention.
  • the cells are, for example, prokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., yeast cells, insect cells, mammalian cells).
  • a fifth aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one compound of the above formula I or formula II according to any one of the first aspects of the invention, a derivative thereof, a stereoisomer or none A physiologically toxic salt, or it comprises at least one nucleic acid molecule of the second aspect of the invention, optionally further comprising a pharmaceutically acceptable carrier or excipient.
  • a sixth aspect of the invention relates to the compound of the formula I or the formula II, the derivative, the stereoisomer or the non-physiologically-soluble salt thereof according to any one of the first aspects of the invention, or the second aspect
  • a seventh aspect of the invention relates to the above formula I according to any of the first aspects of the invention or A compound of the formula II, a derivative thereof, a stereoisomer or a non-physiologically toxic salt or the nucleic acid molecule of the second aspect or the pharmaceutical composition of the fifth aspect of the invention is prepared for inhibiting HIV (for example) Use of HIV-1) in cells fused to cells.
  • An eighth aspect of the invention relates to a method of inhibiting HIV (e.g., HIV-1)-cell fusion in vivo or in vitro, the method comprising using an effective amount of at least one of the aforementioned first aspects of the first aspect of the invention A step of the compound of Formula I or Formula II, a derivative, a stereoisomer or a non-physiologically acceptable salt thereof, or the nucleic acid molecule of the second aspect or the pharmaceutical composition of the fifth aspect of the invention.
  • HIV e.g., HIV-1
  • a ninth aspect of the invention relates to a method of treating and/or preventing and/or adjuvant treatment of a disease associated with HIV infection, in particular AIDS, comprising administering to a patient in need of such treatment a therapeutically and/or prophylactically effective amount At least one compound of the above formula I or formula II, a derivative, a stereoisomer or a non-physiologically acceptable salt thereof, or a nucleic acid molecule according to the second aspect or the present invention.
  • the step of the pharmaceutical composition of the fifth aspect of the invention is provided.
  • the present invention also relates to the compound of the above formula I or formula II, a derivative thereof, a stereoisomer or a non-physiologically toxic salt according to any one of the first aspects of the invention, wherein the formula I or formula II is a compound, a derivative thereof, a stereoisomer or a non-physiologically toxic salt for inhibiting HIV (e.g., HIV-1) and cell fusion, or for treating and/or preventing and/or adjuvant treatment of HIV infection-related diseases (especially It is AIDS).
  • HIV e.g., HIV-1
  • cell fusion e.g., HIV-1
  • HIV infection-related diseases especially It is AIDS
  • HIV Human Immunodeficiency Virus
  • HIV-1 virus HIV-1 virus
  • HIV-2 virus HIV-1 virus
  • the AIDS is an acquired immunodeficiency syndrome (AIDS).
  • AIDS acquired immunodeficiency syndrome
  • the extracellular domain of Gp41 of HIV-1 virus has two helical structural domains closely related to membrane fusion, namely the N-terminal repeat (HR1) and the C-terminal repeat (HR2).
  • HR1 N-terminal repeat
  • HR2 C-terminal repeat
  • HR2 in three gp41 molecules interacts with HR1 to form a hexagonal core structure (6-HB).
  • 6-HB core structure three N-peptides (ie, the 36-peptide of the N-terminal repeat, N36) form a centrally located trimer complex helix core, also called It is an N-helix trimer, or a trimer for short.
  • the compound of the formula I of the present invention is an N-peptide derivative, and therefore the compound of the formula I can also be referred to as an N-peptide in the present invention.
  • the compounds of formula II of the present invention form covalent cross-linking between the three polypeptides of the compound of formula I, thereby forming a more stable and more active trimer compound.
  • covalent crosslinking is lysine or ornithine represented by X 5 in one compound of formula I and the fifth position after X 5 in another compound of formula I (ie An amide bond (-NC(O)-) is formed between the glutamic acid at the X 5 +5 position, or the covalent cross-linking is a lysine or avian ammonia represented by X 2 in a compound of the formula I formation of an amide bond (-NC (O) -) between an acid of formula I and another compound of 5 X 2 behind (i.e. position X 2 +5) glutamic acid.
  • an amide bond may be formed between the two compounds of the formula I, so that among the trimers formed, three compounds of the formula I may be covalently linked to each other to form a bond. Three amide bonds, thereby achieving the purpose of stabilizing the trimer.
  • the compound of the formula I of the present invention can be synthesized by a standard Fmoc solid phase method, and a Rink Amide resin is selected, and the peptide chain is extended from the C terminal to the N terminal.
  • the condensing agent can be HBTU/HoBt/DIEA.
  • the deprotecting agent can be a piperidine/DMF solution.
  • the peptide sequence can be synthesized using a CS Bio peptide synthesizer, and finally the N-terminus of the polypeptide is blocked with acetic anhydride reagent acetate.
  • the cleavage agent may be trifluoroacetic acid/ethanedithiol/m-cresol (TFA/EDT/m-cresol), and the crude peptide is dissolved in water and stored by lyophilization. It can be separated and purified by medium pressure liquid chromatography or high pressure liquid chromatography (HPLC). The pure peptide content is >95%.
  • the molecular weight of the peptide sequence is determined by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS).
  • the compound of the formula II of the present invention can be synthesized by the following method:
  • the compounds of formula I X 2 rear section 5 i.e. 2 X +5 bits
  • glutamic acid sulfatide modified then dissolved in the reaction solution, it can form a trimeric structure, this time designed lysyl
  • the acid reacts with the sulphur-modified glutamic acid to form an amide bond between the N-peptides, i.e., the lysine represented by X 5 in a compound of formula I in the trimer structure formed by the thioester-modified compound of formula I is formed between an acid or ornithine with a compound of formula I in another back 5 of 5 X (i.e.
  • the distance is more suitable, which is conducive to the formation of covalent bonds, and the amino acids in the remaining positions are not suitable for the formation of covalent bonds due to the inappropriate spatial distance and spatial orientation, that is, the amino acids in the remaining positions are due to the spatial distance and The spatial orientation is not suitable and does not affect the formation of covalent bonds.
  • natural amino acid is an amino acid selected from the following (a commonly used three-letter symbol and one-letter symbol in parentheses): glycine (Gly, G), proline (Pro, P), alanine (Ala) , A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), methionine (Met, M), cysteine (Cys, C ), phenylalanine (Phe, F), tyrosine (Tyr, Y), tryptophan (Trp, W), histidine (His, H), lysine (Lys, K), refined ammonia Acid (Arg, R), glutamine (Gln, Q), asparagine (Asn, N), glutamic acid (Glu, E), aspartic acid (Asp, D), serine (Ser, S) And threonine (Thr, T). If there is a deviation from the commonly used symbol due to a typing
  • alkyl refers to a saturated straight or branched chain monovalent hydrocarbon group which may be substituted (single or multiple) or unsubstituted.
  • the alkyl group is a C 1-20 alkyl group, more preferably C 1-15 , more preferably a C 1-10 alkyl group, more preferably a C 1-8 alkyl group, more preferably a C 1-6 alkyl group, more A C 1-4 alkyl group is preferred.
  • alkyl groups include, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, tert-amyl, neopentyl, hexyl Base, n-heptyl and n-octyl groups, etc.
  • Suitable substituents include, for example, hydroxy, alkyl, halo, alkoxy, haloalkyl, haloalkoxy, amino, aminoalkyl or cycloalkyl.
  • the compounds of the invention may be used either as such or in the form of their derivatives, stereoisomers or salts which are not physiologically toxic.
  • the non-physiologically acceptable salt of a compound of Formula I or Formula II includes a pharmaceutically acceptable inorganic or organic acid, or a pharmaceutically acceptable inorganic or organic base Into the salt.
  • suitable acid addition salts include with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, perchloric acid, fumaric acid, acetic acid, propionic acid, succinic acid, glycolic acid, formic acid, lactic acid, maleic acid, tartaric acid.
  • citric acid pamoic acid
  • malonic acid hydroxymaleic acid
  • phenylacetic acid glutamic acid
  • benzoic acid salicylic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, benzene a salt formed from a sulfonic acid, a hydroxynaphthoic acid, hydroiodic acid, malic acid, citric acid or the like.
  • suitable base addition salts include sodium, lithium, potassium, magnesium, aluminum, calcium, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, B.
  • a salt formed by a diamine, N-methylglucamine, procaine or the like When a compound of the invention is referred to herein, it includes a compound of formula I or formula II and derivatives thereof, stereoisomers or salts which are not physiologically toxic.
  • the pharmaceutical composition comprises a compound of formula I or formula II of the invention together with a conventional pharmaceutical carrier or excipient.
  • the pharmaceutical compositions of the present invention can be prepared in a variety of dosage forms, including, but not limited to, tablets, capsules, solutions, suspensions, granules or injections, and the like, by conventional methods in the art.
  • the pharmaceutical composition can be administered by any of the following methods: oral, spray inhalation, rectal administration, nasal administration, buccal administration, vaginal administration, topical administration, parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal. Intraventricular, intrasternal, and intracranial injection or input, or by means of an explant reservoir. Among them, oral administration, intraperitoneal or intravenous administration and topical administration are preferred.
  • terapéuticaally and/or prophylactically effective amount refers to a sufficient amount of a compound to be a reasonable effect for any medical treatment and/or prophylaxis. It will be appreciated, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the particular therapeutically effective dosage level for any particular patient will depend on a number of factors, including the condition being treated and the severity of the disease; the activity of the particular compound employed; the particular composition employed; The age, weight, general health, sex and diet of the patient; the time of administration, the route of administration and the rate of excretion of the particular compound employed; the duration of treatment; the drug used in combination or concurrent with the particular compound employed; Similar factors are known in the medical field. For example, it is the practice in the art that the dosage of the compound be started from a level lower than that required to achieve the desired therapeutic effect, and the dosage is gradually increased until the desired effect is obtained.
  • the dosage for mammals may range from 0.001 to 1000 mg/kg body weight per day, such as from 0.01 to 100 mg/kg body weight per day, such as from 0.01 to 10 mg per kg body weight per day.
  • the present invention devises a novel covalent bond formation method (chemical modification at a suitable site, using a lactam bond) to obtain a site-covalent cross-linked trimer-assisted sequence, which is then combined with a portion of the native N-peptide. Conjugation allows the conjugated N-peptide to interact with the CHR of the virus, resulting in a covalently cross-linked, newly conjugated N-peptide inhibitor that significantly enhances the inhibitory activity of the N-peptide by covalent cross-linking.
  • the N-peptide inhibitor of the present invention has a different mechanism of action, mode of action and target of action than the currently used drugs, and is of great significance for finding a novel HIV-1 fusion inhibitor drug.
  • Figure 1 is a schematic cross-sectional view of a trimeric helical structure (compound of formula II) formed by three compounds of formula I, wherein g represents lysine and e represents glutamic acid, and an amide bond is formed between each single peptide.
  • Figure 2 is a sample preparation diagram of Example 8.
  • AIDS (Acquired Immune Deficiency Syndrome) AIDS, Acquired Immune Deficiency Syndrome
  • Env envelope glycoprotein
  • HIV Human Immunodeficiency Virus
  • HIV-1 human immunodeficiency virus type I HIV-1 human immunodeficiency virus type I
  • PBS Phosphate Buffered Saline
  • the solid phase synthesis carrier Rink amide resin used in the examples of the present invention is Tianjin Nankai Synthetic Co., Ltd.; HBTU, HOBt, DIEA, EDC hydrochloride and Fmoc protected natural amino acid are Shanghai Jill Biochemical Co., Ltd. and Chengdu Chengnuo New Technology Co., Ltd. company's product.
  • Trifluoroacetic acid (TFA) is a product of Beijing Bomaijie Technology Co., Ltd.; DMF and DCM are products of Beijing Bomaijie Technology Co., Ltd.; chromatographic pure acetonitrile is Fisher's product. Other reagents are domestically produced pure products if they are not described.
  • the unit "M" used in the examples of the present invention means mol/L.
  • Example 1 Preparation of monomeric polypeptide represented by SEQ ID NO: 5
  • Peptide synthesis uses the standard Fmoc solid phase method.
  • Rink Amide resin was selected and the peptide chain was extended from the C-terminus to the N-terminus.
  • the condensing agent is HBTU/HOBt/DIEA.
  • the deprotecting agent is a piperidine/DMF solution.
  • the peptide sequence was synthesized using a CS Bio peptide synthesizer, and finally the N-terminus of the polypeptide was blocked with acetic anhydride reagent acetate.
  • the cleavage agent is trifluoroacetic acid/ethylenedithiol/m-cresol (TFA/EDT/m-cresol), and the crude peptide is dissolved in water and stored by lyophilization.
  • Deprotecting agent 20v/v% piperidine in DMF solution
  • Blocking reagent 20 v/v% acetic anhydride in DMF solution.
  • Rink Amide resin 0.53g (0.23mmol) was weighed into the CS Bio automatic peptide synthesizer reactor, and then the protected amino acid, activator, activated base, deprotecting reagent, and blocking reagent were configured at the above concentrations, and then CS Bio was used. Automated peptide synthesizer for synthesis. After completion, the peptide resin was washed 3 times with DMF, then shrunk with anhydrous methanol, and dried under vacuum at room temperature to obtain about 2.02 g of a peptide resin.
  • the resulting crude peptide was purified by medium pressure or high pressure chromatography.
  • the column was a C8 column and the eluent was acetonitrile, water and a small amount of acetic acid.
  • the column was previously equilibrated with 200 ml of a mixed solution of acetonitrile solution and water containing 0.1 v/v% acetic acid (wherein the volume percentage of acetonitrile solution and water were 15% and 85%, respectively).
  • the mixture was washed with 200 ml of a mixed solution of acetonitrile solution and water containing 0.1 v/v% acetic acid (15% and 85% by volume of acetonitrile solution and water, respectively), and the fraction of the eluate was detected by high performance liquid chromatography. According to the liquid phase test results, the acetonitrile content is gradually increased until the purified polypeptide is The peak is eluted. The eluates were combined, and most of the solvent was removed by rotary evaporation, and the pure N peptide was freeze-dried, and the content of HPLC detection was more than 80%.
  • the N-peptide is purified by reverse phase preparative liquid phase.
  • the specific method is as follows: the intermediate-purified N-peptide is dissolved in 2 ml of acetonitrile and 8 ml of pure water, filtered through a 0.25 ⁇ m pore size filter, and then subjected to gradient elution. .
  • the phase A of the eluent is 0.1v/v% aqueous solution of trifluoroacetic acid; the phase B is a mixed solution of 0.1v/v% trifluoroacetic acid in acetonitrile solution and water (wherein the volume percentage of acetonitrile solution and water is 70% and 30%).
  • the reverse phase preparation liquid phase was first equilibrated with a mixed solution of 20 v/v% B phase and 80 v/v% A phase for 5 min. After loading, the elution gradient was adjusted as needed, and the content of phase B was gradually increased until the purified polypeptide was obtained. The main peak is eluted. The eluates detected by HPLC were greater than 95%, and most of the solvent was removed by rotary evaporation, and the pure N peptide was lyophilized.
  • Example 3 Preparation of a covalently crosslinked polypeptide represented by (SEQ ID NO: 1) 3
  • the monomer polypeptide sequence was synthesized as in the monomeric polypeptide of SEQ ID NO: 5 in Example 1, but when the resin was attached to the amino acid, the site-requiring E was replaced by E(OAll). After the sequence was synthesized on the resin, it was not cleaved and the following further chemical modification was performed.
  • the polypeptide resin after removing the side chain protecting group was added, and 275 ⁇ l of benzyl mercaptan, 315 mg of HOBt, and 450 mg of EDC hydrochloride were dissolved in a mixed solvent of 5 ml of DMF and 5 ml of DCM, and then added to the reactor for 6 hours. After that, the reaction solvent was drained, and 275 ⁇ l of benzyl mercaptan, 315 mg of HOBt, and 450 mg of EDC hydrochloride were again dissolved in a mixed solvent of 5 ml of DMF and 5 ml of DCM, and added to the reactor. After 12 hours of reaction, finally, DMF, DCM, and MeOH were respectively used. Wash, wash each solution twice and drain.
  • the purified thioester-modified N-peptide is dissolved in a reaction solvent (30% PBS/70% H 2 O) at a concentration of about 1 mg/ml, and reacted at 37 ° C for 40 hours.
  • the reaction is detected by HPLC to complete, and then The target N peptide was purified under reverse purification conditions for preparative high-performance liquid phase, and the purity was greater than 95%.
  • the prepared N-peptide solution is added to the cuvette, and the helical absorption value (without the blank control absorption value) is measured in a circular dichroism spectroscopy instrument, and converted into helicity according to the following formula:
  • concentration (c) refers to the concentration value of the N-peptide solution
  • path (L) refers to the reference cell length
  • number of residues (N) refers to the number of amide bonds of the N-peptide.
  • Example 8 Compound inhibition of HIV-1-mediated cell - cell fusion activity evaluation (IC 50)
  • the cell cryotube was taken out from the liquid nitrogen, and the temperature was rapidly raised in a 37 ° C water bath.
  • the cell cryopreservation solution (1 ml) was taken out, added to a 15 ml centrifuge tube, and 1 ml of the medium (purchased from Shanghai Lifei Biotechnology Co., Ltd.) was added and centrifuged ( 800 rpm, 10 min), the medium was removed, 1 ml of fresh medium was added again, and the cells were uniformly suspended by light blowing, and the cell suspension was completely transferred to a 75 cm 2 culture flask containing 15 ml of medium at 37 ° C, 5% CO 2 . Under cultivation.
  • the TZM-bl cells (supplied by the NIH AIDS Research and Reference Reagent Program) were diluted to 500,000/ml, and placed in a 96-well cell culture plate at 50 ⁇ l/well for 24 hours.
  • the test compound is diluted four times in the (1-10) column of the 96-well plate, 11 columns and 12 columns are blank solvents (blank solvent contains only medium, no sample to be tested, 11 of which are positive controls, TZM-bl cells and HL2/3 cells mixed at a concentration of 1:3 without sample inhibitors) ; 12 is a negative control, is the chemiluminescence signal of a single TZM-bl cell); DMSO content ⁇ 6%.
  • Sample preparation instructions (see Figure 2): Four samples were prepared for each 96-well sample plate (12 holes per row, 8 rows; Costar 3799, Corning Incorporation, USA), and each sample was repeated once, as shown in Figure 2.
  • the sample of the selected concentration was placed in the S1 well, and the sequence was diluted 4 times (ie, the sample concentration of the latter well was 1/4 of the previous well), and 10 concentration gradients were diluted accordingly.
  • the last two wells contained control medium only as the control, in which the 11th well contained the target cells and the effector cells were 100% fusion control (positive control), and the 12th well contained only the target cells as the unfused background control (negative control).
  • step D Immediately take the 20 ⁇ l/well sample from step B and add to the cell plate for 6 hours.
  • LA buffer (Luciferase Assay Buffer, Promega Cooperation, USA) was added to the LA substrate (Luciferase Assay Substrate, Promega Cooperation, USA) and mixed, and 40 ⁇ l/well was added to the 96-well phosphor plate.
  • the activity measurement results are shown in Table 2 below.
  • the data in Table 2 shows that the activity of the N-peptide after covalent cross-linking is significantly improved, and the best activity reaches a low nanomolar level.
  • T20, C34 is a C-peptide fusion inhibitor as an experimental control for cell fusion activity.
  • T20 is a marketed drug
  • C34 is a well-stabilized and stable fusion inhibitor in the laboratory.

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Abstract

L'invention concerne un composé représenté dans la Formule I ou la Formule II, ou un de ses sels physiologiquement non toxiques, une composition pharmaceutique contenant le composé, et une utilisation du composé dans la préparation de médicaments destinés au traitement et/ou à la prévention et/ou à l'aide au traitement de maladies provoquées par une infection par le VIH, en particulier le SIDA. Z-X1X2IX3X4IEQX5IX6X7IEQRIQQIEQX8X9X10X11X12X13X14X15X16X17X18X19X20X21LLQLTVWGIKQLQARIL-B(I) (Z-X1X2IX3X4IEQX5IX6X7IEQRIQQIEQX8X9X10X11X12X13X14X15X16X17X18X19X20X21LLQLTVWGIKQLQARIL-B)3(II)
PCT/CN2016/085968 2015-07-02 2016-06-16 Inhibiteur de n-peptide réticulé de manière covalente WO2017000789A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102307588A (zh) * 2009-02-09 2012-01-04 纽约血库公司 用于治疗或预防hiv感染的三聚体hiv融合抑制剂
CN103122024A (zh) * 2011-11-21 2013-05-29 中国人民解放军军事医学科学院毒物药物研究所 人工设计的抗hiv感染多肽、组合物以及用途
CN104203276A (zh) * 2012-03-20 2014-12-10 默沙东公司 Hiv-1 gp41前发夹中间物的稳定肽模拟物
CN104277113A (zh) * 2013-07-08 2015-01-14 中国人民解放军军事医学科学院毒物药物研究所 抑制hiv感染的二价多肽

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004072099A2 (fr) * 2003-02-11 2004-08-26 The United States Of America As Represented By The Secretary Of Health And Human Services, Nih Nouvel inhibiteur peptidique de fusion du vih faisant subir une disruption a la superhelice interne trimere de gp41

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102307588A (zh) * 2009-02-09 2012-01-04 纽约血库公司 用于治疗或预防hiv感染的三聚体hiv融合抑制剂
CN103122024A (zh) * 2011-11-21 2013-05-29 中国人民解放军军事医学科学院毒物药物研究所 人工设计的抗hiv感染多肽、组合物以及用途
CN104203276A (zh) * 2012-03-20 2014-12-10 默沙东公司 Hiv-1 gp41前发夹中间物的稳定肽模拟物
CN104277113A (zh) * 2013-07-08 2015-01-14 中国人民解放军军事医学科学院毒物药物研究所 抑制hiv感染的二价多肽

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