WO2016173429A1 - Inhibiteur du vih-1 d'un peptide n-terminal d'origine naturelle réticulé de manière covalente spécifique du site - Google Patents

Inhibiteur du vih-1 d'un peptide n-terminal d'origine naturelle réticulé de manière covalente spécifique du site Download PDF

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WO2016173429A1
WO2016173429A1 PCT/CN2016/079696 CN2016079696W WO2016173429A1 WO 2016173429 A1 WO2016173429 A1 WO 2016173429A1 CN 2016079696 W CN2016079696 W CN 2016079696W WO 2016173429 A1 WO2016173429 A1 WO 2016173429A1
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formula
compound
group
derivative
stereoisomer
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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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    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

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  • 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 N-terminal repeat
  • HR2 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.
  • 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 all C-peptides and their derivatives.
  • Typical C-peptide fusion inhibitors have T20, C34, both derived from the polypeptide of the CHR native sequence of gp41, 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 natural NHR sequences 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. On the other hand, it shows that the research problem is 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 former should be its main mode of action, and the activity is higher, the difference between the two is 1 to 3 orders of magnitude.
  • N-peptide fusion inhibitors are mainly how to stabilize their trimers and improve physical and chemical properties. From the perspective of R&D drugs, CHR and NHR can target each other. N-peptide fusion inhibitors target CHR in gp41, which is completely different from the first two fusion inhibitors. It avoids or slows the cross-resistance with existing drugs. A realistic approach to medicinal properties, although there are also induced drug resistance studies.
  • 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 an irreversible three.
  • the polymer N peptide can increase the activity of the N-peptide inhibitor, but the above method also has its own defects: the single-stranded N-peptide has low activity, is easy to aggregate and precipitate; and the auxiliary polypeptide is conjugated, so that the N-peptide sequence after conjugation is lengthy; Although disulfide cross-linking can increase the activity of N-peptide, the cross-linking reaction site and reaction specificity are uncontrollable, which limits the development of N-peptide inhibitors.
  • the invention uses a natural N-peptide (NHR) sequence as a template to design and synthesize a series of N-peptide fusion inhibitors with novel structures, and determines the aggregation state and stability of the designed N-peptide in solution.
  • NHR natural N-peptide
  • An N-peptide capable of forming a relatively stable triple helix structure is selected, and an appropriate site is selected, chemically modified, a functional group of an acyl transfer reaction is introduced, and finally, chemical crosslinking occurs under appropriate reaction conditions to obtain a fixed-point covalent cross-linking.
  • the N-peptide trimer is linked, thereby completing the present invention.
  • 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,
  • X 1 and X 2 are each independently selected from an L-form amino acid or a deletion
  • X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 and X 13 are each independently selected from L-form amino acids,
  • the compound represented by the formula II is a trimer formed by the three compounds of the formula I by covalent crosslinking.
  • X 1 and X 2 are co-present or co-deleted.
  • covalent crosslinking refers to a lysine between X 5 and X 6 in one compound of formula I and between X 8 and X 9 in another compound of formula I
  • covalent cross-linking of glutamic acid preferably, said covalent cross-linking refers to the formation of an amide bond; that is, when the compound of formula I is a 36-peptide (or 38-peptide), the sixth position of a compound of formula I (or the 8th position) lysine is covalently cross-linked with the 11th (or 13th) glutamic acid in another compound of formula I, such that the two molecules of the compound of formula I are cross-linked in pairs Forming a covalent crosslink, for example forming an amide bond.
  • X 1 is selected from the group consisting of leucine (Leu) and lysine (Lys), or X 1 is deleted.
  • X 2 is selected from the group consisting of isoleucine (Ile), alanine (Ala) and leucine (Leu), or X 2 is deleted.
  • X 3 is selected from serine (Ser), glutamine (Gln) and glutamate (Glu).
  • X 4 is selected from glycine (Gly), glutamic acid (Glu), and lysine (Lys).
  • X 5 is selected from a glutamine (Gln), lysine (Lys), glutamate (Glu) and arginine (Arg).
  • X 6 is selected from the group consisting of isoleucine (Ile) and glutamine (Gln).
  • X 7 is selected from histidine (His) and asparagine (Asn).
  • X 8 is selected from the group consisting of histidine (His) and asparagine (Asn).
  • X 9 is selected from alanine (Ala) and glutamic acid (Glu).
  • X 10 is selected from alanine (Ala), lysine (Lys) and glutamic acid (Glu).
  • X 11 is selected from isoleucine (Ile) and glutamine (Gln).
  • X 12 is selected from the group consisting of histidine (His) and lysine (Lys).
  • X 13 is selected from the group consisting of glutamine (Gln) and glutamic acid (Glu).
  • N-terminus of the compound of Formula I or Formula II is further attached to a C 1-6 alkyl acyl group (eg, acetyl), an oligopeptide sequence, or a lipophilic group, or
  • a C 1-6 alkyl acyl group eg, acetyl
  • an oligopeptide sequence e.g. a lipophilic group
  • the tether is linked to other small molecules (for example, the amino group at the N-terminus of the compound polypeptide can be reacted with succinic acid such that the terminal is a carboxyl group, which in turn can be attached to other groups reactive with the carboxyl group),
  • a C-terminal end with a carboxyl derivative eg, an amide group
  • a oligopeptide sequence e.g., a lipophilic group, or cholesterol
  • the compound of formula I or formula II is acetylated at the N-terminus and/or amidated at the C-terminus.
  • the oligopeptide sequence refers to an oligopeptide sequence containing 1-10 amino acid residues, such as EEE, KKK, GQAV (SEQ ID NO: 29), GEEE (SEQ ID NO: 30), and the like.
  • the lipophilic group means a fatty acid chain having 3 to 20 carbon atoms, preferably a fatty acid chain having 8 to 16 carbon atoms.
  • the compound of formula I or formula II, a derivative thereof, a stereoisomer or a salt of no physiological toxicity is selected from the group consisting of:
  • a second aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one compound of the 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
  • it also contains a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition is for use in the prevention and/or treatment of a disease associated with HIV infection, particularly AIDS.
  • the invention further relates to a compound of formula I or formula II, a derivative, a stereoisomer or a non-physiologically acceptable salt thereof, or a pharmaceutical composition according to any one of the second aspects, according to any one of the first aspects of the invention, is prepared Use in medicines for the prevention and/or treatment and/or adjuvant treatment of diseases associated with HIV infection, in particular AIDS.
  • HIV is an HIV-1 type virus.
  • the invention also relates to a method of preventing and/or treating and/or adjunctively treating a disease associated with HIV infection, in particular AIDS, comprising administering to a subject in need thereof an effective amount of the first aspect of the invention A step of any one of the compounds of Formula I or Formula II, a derivative, a stereoisomer or a non-physiologically acceptable salt thereof, or a pharmaceutical composition according to any one of the preceding aspects.
  • the invention further relates to a compound of formula I or formula II, a derivative, a stereoisomer or a non-physiologically acceptable salt thereof, or a pharmaceutical composition according to any one of the second aspects, according to any one of the first aspects of the invention, is prepared Use in drugs for inhibiting the fusion of HIV and cells.
  • HIV is an HIV-1 type virus.
  • the invention also relates to a method of inhibiting fusion of HIV and cells in vivo or in vitro, the method comprising the use of an effective amount of a compound of formula I or formula II of any of the first aspects of the invention, a derivative thereof, a stereo
  • the step of the isomer or the non-physiologically toxic salt or the pharmaceutical composition of any of the second aspects comprising the use of an effective amount of a compound of formula I or formula II of any of the first aspects of the invention, a derivative thereof, a stereo.
  • HIV is an HIV-1 type virus.
  • the invention further relates to a nucleic acid molecule encoding a compound of formula I, a derivative, a stereoisomer or a non-physiologically acceptable salt thereof, according to any one of the first aspects of the invention.
  • the invention also relates to a recombinant vector comprising the nucleic acid molecule of any of the invention.
  • the vector is, for example, a prokaryotic expression vector or a eukaryotic expression vector.
  • the invention also relates to a recombinant cell comprising the recombinant vector of any 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).
  • prokaryotic cells e.g., E. coli
  • eukaryotic cells e.g., yeast cells, insect cells, mammalian cells.
  • 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 known as an N-helix trimer, or a trimer for short.
  • the compounds of formula I of the present invention are N-peptide derivatives and are therefore also referred to as N-peptides in some places in the present invention.
  • the compound of the formula I of the present invention can naturally aggregate to form a trimer in solution.
  • the present invention further increases covalent cross-linking between the three polypeptides to form a more stable and A more active trimer compound, a compound of formula II.
  • covalent crosslinking refers to a lysine between X 5 and X 6 in one compound of formula I and between X 8 and X 9 in another compound of formula I
  • covalent cross-linking of glutamic acid preferably, said covalent cross-linking refers to the formation of an amide bond; that is, when the compound of formula I is a 36-peptide (or 38-peptide), the sixth position of a compound of formula I (or position 8) lysine is covalently cross-linked with the 11th (or 13th) glutamic acid in another compound of formula I, for example to form an amide bond.
  • an amide bond can be formed between the polypeptides of the two compounds of formula I, such that in the trimer formed, three amide linkages can be formed between the polypeptides of the three compounds of formula I, thereby Achieve the goal of stabilizing trimers.
  • the 11th (36 peptide) or 13th (38 peptide) glutamic acid of the compound of the formula I When the 11th (36 peptide) or 13th (38 peptide) glutamic acid of the compound of the formula I is subjected to thioester modification, and then dissolved in the reaction solution, it can form a trimer structure, which is designed at this time. Lysine and thioester-modified glutamate react to form an amide bond between the polypeptides, ie, in the trimeric structure formed by the thioester-modified formula I, the compound of formula I is a 36 peptide (or 38 peptide) In the case of a 6th (or 8th) lysine of a compound of formula I, an amide bond is formed between the 11th (or 13th) glutamic acid in another compound of formula I.
  • Z and B in Formula I or Formula II refer to the N-terminal group and the C-terminal group of the polypeptide, respectively; wherein Z may be NH 2 or a protecting group at the N-terminus of other polypeptides, such as C 1 -6 alkylamide group, such as AcNH, or bpy- ⁇ A, etc.; wherein B may be COOH, or a protective group at the C-terminus of other polypeptides, such as a carboxyl derivative such as CONH 2 or the like.
  • X 1 and X 2 may be present simultaneously or simultaneously; when X 1 and X 2 are present simultaneously, the compound of any of the first aspects of the invention is a 38 peptide, when X 1 and X 2 When simultaneously deleted, the compound of any one of the first aspects of the invention is a 36 peptide.
  • physiologically toxic salt means a salt of a compound of the present invention which is pharmaceutically acceptable and which has the desired pharmacological activity of the parent compound.
  • Such salts include: acid addition salts with inorganic acids or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; organic acids such as acetic acid, propionic acid, Caproic acid, cyclopentanoic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methylsulfonate Acid, ethanesulfonic acid, benzenesulfonic acid, naphthalene Acid, camphorsulfonic acid, glucoheptonic acid, gluconic acid, glutamic acid, hydroxynap
  • the term "effective amount” includes a dose that can achieve treatment, prevention, alleviation and/or alleviation of the disease or condition of the present invention in a subject.
  • the term "subject” may refer to a patient or other pharmaceutical composition according to any one of the present invention, which comprises the antimicrobial peptide, a derivative thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of the present invention.
  • An animal particularly a mammal, such as a human, a dog, a monkey, a cow, a horse, or the like, for treating, preventing, ameliorating, and/or ameliorating the disease or condition of the present invention.
  • disease and/or condition refers to a physical state of the subject that is associated with the disease and/or condition of the present invention.
  • C 1-6 alkylamido means C 1-6 alkyl-CO-NH
  • the C 1-6 alkyl group means a straight chain having 1 to 6 carbon atoms or
  • a branched monovalent saturated hydrocarbon group is, for example, a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, an isobutyl group, a t-butyl group, a pentyl group, a hexyl group or the like.
  • an amino acid means an L-form amino acid.
  • the N-terminus of the compound of Formula I or Formula II is NH 2 and the C-terminus is COOH; the N-terminus or C of the compound of Formula I or Formula II of the present invention
  • the terminal may also be bonded to other groups which do not affect the crosslinking reaction.
  • the N-terminal or C-terminal group when specifically indicated, it means a group obtained by modifying the group on the basis of NH 2 or COOH, for example, when the N-terminus is acetyl (Ac), the N-terminal group of the compound is AcNH, which is represented by Ac in the sequence, and when the C-terminus is an amide group, the C-terminal group of the compound is CONH 2 , which is represented in the sequence. For NH 2 .
  • the N-terminus and C-terminus of the compound of formula II are identical to the N-terminus and C-terminus of the compound of formula I in which the monopeptide is present.
  • the pharmaceutical composition usually contains 0.1 to 90% by weight of the compound according to any one of the present invention, a derivative thereof, a stereoisomer or a salt which is not physiologically toxic.
  • Pharmaceutical compositions can be prepared according to methods known in the art. When used for this purpose, the compounds of the invention, their derivatives, stereoisomers or non-physiologically toxic salts may be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants, if desired, It is prepared in a suitable form or dosage form for human use.
  • the compound of the present invention, a derivative thereof, a stereoisomer or a salt of no physiological toxicity or the pharmaceutical composition of the present invention can be administered in a unit dosage form, which can be enterally or parenterally, such as oral or muscle. , subcutaneous, nasal cavity, oral mucosa, skin, peritoneum or rectum.
  • Drug type For example, tablets, capsules, dropping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, lyophilized powders, and the like. It may be a general preparation, a sustained release preparation, a controlled release preparation, and various microparticle delivery systems.
  • carriers In order to form a unit dosage form into tablets, various carriers well known in the art can be widely used.
  • carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid.
  • wetting agent and binder such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, gum arabic, gelatin paste, sodium carboxymethyl cellulose , shellac, methyl cellulose, potassium phosphate, polyvinyl pyrrolidone, etc.
  • disintegrating agents such as dried starch, alginates, agar powder, brown algae starch, sodium bicarbonate and tannic acid, calcium carbonate, polyoxyethylene, Sorbitol fatty acid ester, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.
  • disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil, etc.
  • absorption promotion Agents such as quaternary ammonium salts, sodium lauryl sulfate, and the like
  • lubricants such as talc, silica,
  • Tablets may also be further formed into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer tablets and multilayer tablets.
  • various carriers known in the art can be widely used.
  • the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth, gelatin , ethanol, honey, liquid sugar, rice paste or batter; etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, and the like.
  • the polypeptide of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof may be formulated into a microcapsule, suspended in an aqueous medium to form a suspension, or may be enclosed in a hard capsule or used as an injection.
  • an injection preparation such as a solution, an emulsion, a lyophilized powder injection and a suspension
  • all diluents conventionally used in the art for example, water, ethanol, polyethylene glycol, 1, 3 may be used.
  • - propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, and the like in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin may be added to the preparation for injection, and a conventional solubilizer, a buffer, a pH adjuster or the like may be added.
  • coloring agents can also be added to pharmaceutical preparations.
  • coloring agents can also be added to pharmaceutical preparations.
  • the dose of the compound of the present invention, its derivative, stereoisomer or non-physiologically toxic salt or the pharmaceutical composition of the present invention depends on many factors such as the nature and severity of the disease to be prevented or treated, the patient or the animal. Gender, age, weight and individual response, the specific active ingredients used, the route of administration and the number of doses.
  • the above dosages may be administered in a single dosage form or divided into several, for example two, three or four dosage forms.
  • composition as used herein is intended to include a product comprising specified amounts of each of the specified ingredients, as well as any product that results, directly or indirectly, from the specified combination of the specified ingredients.
  • each active ingredient in the pharmaceutical compositions of the present invention can be varied so that the amount of active ingredient obtained is effective to the particular patient, and the compositions and modes of administration provide the desired therapeutic response.
  • the dosage level will be selected based on the activity of the particular active ingredient, the route of administration, the severity of the condition being treated, and the condition and past medical history of the patient to be treated. However, it is a practice in the art to dose the active ingredient from a level below that required to achieve the desired therapeutic effect, gradually increasing the dosage until the desired effect is achieved.
  • the therapeutically and/or prophylactically effective amount of a compound of the invention, a derivative, a stereoisomer or a non-physiologically acceptable salt thereof, when used in the above-mentioned therapeutic and/or prophylactic or adjunctive treatment, may be applied in pure form or in a pharmaceutically acceptable form.
  • the accepted ester or prodrug form (in the presence of these forms) is applied.
  • it may be administered as a pharmaceutical composition comprising a compound of the invention, a derivative thereof, a stereoisomer or a non-physiologically acceptable salt, and one or more pharmaceutically acceptable excipients.
  • the total daily usage of a compound of the invention, a derivative, a stereoisomer or a non-physiologically acceptable salt or a pharmaceutical composition of the invention will be determined by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend on a number of factors, including the disorder being treated and the severity of the disorder; the activity of the particular active ingredient 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 active ingredient employed; the duration of treatment; in combination with or in combination with the particular active ingredient employed Drugs; and similar factors well known in the medical field.
  • the dosage of the active ingredient be started from a level lower than that required to achieve the desired therapeutic effect, gradually increasing the dosage until the desired effect is achieved.
  • the compounds of the invention, their derivatives, stereoisomers or non-physiologically toxic salts may be used in mammals, especially humans, at doses ranging from 0.001 to 1000 mg/kg body weight per day, for example between 0.01 and 100 mg. /kg body weight / day, for example between 0.01-10 mg / kg body weight / day.
  • the invention designs a novel N-peptide sequence based on a novel design idea and research method, and Moreover, the inhibitory activity of the N peptide is remarkably enhanced by covalent crosslinking.
  • 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 Schematic cross-sectional view of a trimeric helical structure (compound of formula II) formed by three polypeptides of the compound of formula I, wherein each monopeptide forms a helical structure into seven repeats, forming two cycles per seven consecutive amino acid residues
  • the seven residues are represented by a, b, c, d, e, f, g, respectively, wherein g represents lysine, and e represents glutamic acid, and an amide bond is formed between each single peptide.
  • FIG. 1 Sample preparation of Example 8.
  • AIDS (Acquired Immure 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
  • Trp Tryptophan
  • the solid phase synthesis carrier Rink amide resin used in the examples is Tianjin Nankai Synthetic Co., Ltd.; HBTU, HOBT, DIEA, EDC hydrochloride and Fmoc protected natural amino acids are products of Shanghai Jill Biochemical Co., Ltd. and Chengdu Chengnuo New Technology Co., Ltd. .
  • 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.
  • Peptide 1 was synthesized using 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.
  • Blocking reagent 20% v/v acetic anhydride in DMF solution.
  • Lysis of peptide resin 1.95 g of peptide resin synthesized by CS Bio automatic peptide synthesizer was weighed, placed in a 500 ml eggplant-shaped flask, ice-cooled, and electromagnetically stirred. The lysate was prepared by adding 1 gram of the peptide resin to 10 ml. TFA needs to be cooled in the ice bath for 30 minutes in advance or stored in the refrigerator in advance; the prepared lysate is added to the peptide resin under ice bath conditions, electromagnetically stirred, the resin turns orange-red, and the reaction is carried out for 30 minutes under ice bath conditions, and then withdrawn. The reaction was stirred for 200 min at room temperature and the reaction was completed.
  • the sample was loaded, it was further washed with 200 ml of a 15% acetonitrile/water/0.1% glacial acetic acid solution, and the eluent component was detected in a high-performance liquid phase.
  • the acetonitrile content was gradually increased according to the liquid phase detection result until the main peak of the purified polypeptide was 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 was purified by reverse phase preparative liquid phase.
  • the specific method was as follows: the medium-purified N-peptide was dissolved in 2 ml of acetonitrile and 8 ml of pure water, filtered through a 0.25 ⁇ m pore size filter, and loaded.
  • the reverse phase preparative liquid phase was first equilibrated with 20% B phase for 5 min. After loading, the content of phase B was gradually increased according to the elution gradient until the main peak of the purified polypeptide was eluted. HPLC detection of more than 95% The eluates were combined, and most of the solvent was removed by rotary evaporation, and the pure N peptide was lyophilized.
  • the preparation method of the other uncrosslinked monomeric N-peptide is the same as that of the preparation of the polypeptide 1 in Example 1.
  • polypeptide sequence of polypeptide 6 is synthesized as the same as polypeptide 1, but when the resin is linked to an amino acid, the site-requiring E (from the N-terminal calculation of the 13th or 11th position) is replaced by E(OAll, O-allyl). . 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 ul of benzyl mercaptan, 315 mg of HOBt, 450 mg of EDC hydrochloride was dissolved in 5 ml of DMF/5 ml of DCM solvent, and then added to the reactor for 6 h and 12 h respectively. After the second, it was washed twice with DMF, DCM, MeOH and dried.
  • the method of purification and purification is carried out to obtain a pure peptide, and the purity of HPLC is more than 90%.
  • the purified thioester-modified N-peptide is dissolved in a reaction solvent (20% ACN/30% PBS/50% H 2 O at a concentration of about 1 mg/ml), and reacted at 37 ° C for 40 h, and the reaction is completely detected by HPLC. After purifying the high-performance liquid phase in reverse, the target N peptide is purified to a purity greater than 95%.
  • the single-stranded N-peptide in the present invention can form a trimer (N3 helix) structure, and in order to increase the stability of the trimer structure, we respectively assign a 13th or 11th glutamic acid of a single-stranded N-peptide to another
  • the 8th or 6th lysine of a single-stranded N-peptide is covalently cross-linked by an amide bond, thereby forming a stable triple helix structure.
  • Example 4 In addition to polypeptide 8, other cross-linked polypeptides (crosslinked polypeptides of 6, 10, 12, etc.) were prepared in the same manner as polypeptide 6 of Example 3.
  • 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.
  • 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 was added, and centrifuged (800 rpm, 10 min) to remove the medium and re- 1 ml of fresh medium was added, and the cells were uniformly suspended by light blowing, and the whole cell suspension was transferred to a 75 cm 2 culture flask containing 15 ml of the medium, and cultured at 37 ° C, 5% CO 2 .
  • 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, 50 ⁇ l/well, and cultured 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 inhibitor) ; 12 is a negative control, is the chemiluminescence signal of a single TZM-bl cell); DMSO content ⁇ 6%.
  • Sample preparation instructions ( 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.
  • Most The latter 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 h.
  • 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 helicity value is the measured value at 222 nm, Uni: deg * m 2 dmol -1 , and the value of 100% ⁇ helicity is -33,000.
  • 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.
  • the data in the table indicates that the activity of the N-peptide after cross-linking is significantly improved, and the best activity reaches a low nanomolar (nM) level.

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Abstract

La présente invention concerne un inhibiteur du virus de l'immunodéficience humaine de type 1. L'inhibiteur est un composé peptidique N-terminal réticulé de manière covalente spécifique à un site, son dérivé, son stéréoisomère ou sel présentant sa toxicité physiologique. Sont également prévues une composition pharmaceutique du composé et une utilisation du composé dans la préparation d'un médicament associé pour prévenir et/ou traiter une infection par le virus de l'immunodéficience humaine.
PCT/CN2016/079696 2015-04-28 2016-04-20 Inhibiteur du vih-1 d'un peptide n-terminal d'origine naturelle réticulé de manière covalente spécifique du site WO2016173429A1 (fr)

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US20120052090A1 (en) * 2009-05-18 2012-03-01 National University Corporation Tokyo Medical And Dental University Peptidic antigen that induces antibody recognizing three-dimensional structure of hiv and method for synthesizing same
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