WO2017000789A1 - Covalently crosslinked n-peptide inhibitor - Google Patents

Abstract

Provided are a compound represented in Formula I or Formula II, or a physiologically non-toxic salt thereof, a pharmaceutical composition containing the compound, and a use of the compound in preparing drugs for treating and/or preventing and/or assisting in the treatment of diseases caused by HIV infection, AIDS in particular. Z-X₁X₂IX₃X₄IEQX₅IX₆X₇IEQRIQQIEQX₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁LLQLTVWGIKQLQARIL-B(I) (Z-X₁X₂IX₃X₄IEQX₅IX₆X₇IEQRIQQIEQX₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁LLQLTVWGIKQLQARIL-B)₃(II)

Description

Covalently crosslinked N-peptide inhibitor Technical field

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).

Background technique

AIDS is mainly a highly lethal infectious disease caused by human immunodeficiency virus type 1 (HIV-1) infection, which is widespread and spreads rapidly. At present, the disease is still unable to cure and lacks an effective vaccine. 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. However, due to the slow progress of HIV vaccine research and the increasing awareness of drug resistance, it is still a top priority to develop new anti-HIV drugs with new mechanisms of action and new targets.

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. There are two helical structural domains closely related to membrane fusion in the extracellular domain of Gp41, namely the N-terminal repeat (abbreviated as HR1, NHR or N-peptide) and the C-terminal repeat (abbreviated as HR2, CHR or C peptide). . During membrane fusion, 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 the fusion of the virus and the cell.

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. By optimizing the native sequence of gp41, 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. Although newly developed fusion inhibitors can efficiently inhibit existing T20 resistant strains, cross-resistance to T20 is often present. Because of the same target, these cross-resistances are expected to become more pronounced after these new drugs enter clinical use. Therefore, the development of fusion inhibitors with novel structures and mechanisms of action for different target sequences should be the focus of future drug development.

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. Based on the structure and mechanism of action of 6HB, 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. In contrast, 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. Therefore, 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.

At present, there are also related studies for improving the inhibitory activity of N-peptide. 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. However, 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.

Summary of the invention

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,

ZX ₁ X ₂ IX ₃ X ₄ IEQX ₅ IX ₆ X ₇ IEQRIQQIEQX 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 LLQLTVWGIKQLQARIL-B(I)

(ZX ₁ X ₂ IX ₃ X ₄ IEQX ₅ IX ₆ X ₇ IEQRIQQIEQX 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 LLQLTVWGIKQLQARIL-B) ₃ (II )

among them:

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 ,

Wherein 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,

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.

In one embodiment of the invention, 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)-).

In one embodiment of the invention, the compound of Formula I or Formula II, a derivative, a stereoisomer or a non-physiologically toxic salt thereof, according to the first aspect of the invention, wherein said natural L-form natural 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). ), asparagine (Asn), valine (Val), lysine (Lys), serine (Ser), threonine (Thr), arginine (Arg), histidine (His), color Amino acid (Trp), tyrosine (Tyr),

The L-type unnatural amino acid is ornithine (Orn, O).

In one embodiment of the invention, 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).

And X ₂ and X _{5 are} not simultaneously arginine (Arg),

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 ₉ is isoleucine (Ile),

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).

In one embodiment of the invention, 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, Or 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)-).

In one embodiment of the invention, 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. In the present 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. In the present 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 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 Use of a nucleic acid molecule or a pharmaceutical composition according to the fifth aspect of the invention for the manufacture of a medicament for the treatment and/or prevention and/or adjuvant treatment of a disease associated with HIV infection, in particular AIDS.

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.

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.

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).

In an embodiment of the invention, wherein said HIV refers to Human Immunodeficiency Virus (HIV), including HIV-1 virus and HIV-2 virus, preferably HIV-1 virus.

In an embodiment of the invention, the AIDS is an acquired immunodeficiency syndrome (AIDS).

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). During membrane fusion, HR2 in three gp41 molecules interacts with HR1 to form a hexagonal core structure (6-HB). In the 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.

In an embodiment of the invention, wherein said 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. In a specific embodiment of the present invention, 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. X ₅ bits +5) glutamic acid amide bond (-NC (O) -), or a compound of formula I formation of an amide bond (-NC (O) between the X ₂ represents lysine or ornithine with a compound of formula I, X ₂ another after the first five (i.e. position X ₂ +5) glutamic acid - ). As shown in Figure 1, in the compound of formula II, three single-chained I molecules self-polymerize into a triple helix structure in which one lysine represented by X ₅ (or X ₂ ) on the molecule of formula I and another formula The glutamic acid at the X ₅ +5 (or X ₂ +5) position in the I molecule is located at the position of g and e' of the polypeptide (e' indicates a peptide molecule different from the g position), in the spatial direction and space. 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.

The term "natural amino acid" as used herein 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 error, the commonly used symbol will prevail.

The term "alkyl" as used herein refers to a saturated straight or branched chain monovalent hydrocarbon group which may be substituted (single or multiple) or unsubstituted. Preferably, 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. Particularly 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. Examples of 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. Examples of 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.

According to the invention, 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.

The term "therapeutically and/or prophylactically effective amount" of a compound of the invention herein 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. In general, the compounds of formula I of the invention The dosage for mammals, particularly humans, 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.

DRAWINGS

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.

detailed description

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, however, the following examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Those who do not specify the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.

The abbreviations used in the present invention have the following meanings:

AIDS (Acquired Immune Deficiency Syndrome) AIDS, Acquired Immune Deficiency Syndrome

DCM (Dichloromethane) dichloromethane

DMF (N, N-Dimethyl malonate) dimethylformamide

Env (Envelope glycoprotein) envelope glycoprotein

Fmoc(Fluorenylmethoxycarbonyl)芴methoxycarbonyl

HBTU 2-(1H-1-hydroxybenzotriazole)-1,1,3,3-tetramethylurea hexafluorophosphate

HOBt(1-Hydroxylbenzotriazole anhydrous) 1-hydroxybenzotriazole

6-HB (six-helix bundle)

HR1 (N-terminal heptad repeat, NHR) N-terminal repeat

HR2 (C-terminal heptad repeat, CHR) C-terminal repeat

HIV (Human Immunodeficiency Virus)) human immunodeficiency virus

HIV-1 human immunodeficiency virus type I

HPLC (High performance liquid chromatography) high performance liquid chromatography

OAll allyl

TFA (trifluoroacetic acid) trifluoroacetic acid

DIEAN, N-diisopropylethylamine

EDC hydrochloride 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

PBS (Phosphate Buffered Saline) phosphate buffer (pH=7.2)

MALDI-TOF-MS matrix-assisted laser desorption time-of-flight mass spectrometry

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. Separation and purification were carried out by medium pressure liquid chromatography or high pressure liquid chromatography (HPLC) with a pure peptide content of >95%, and the molecular weight of the peptide sequence was determined by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS).

The synthesis conditions are as follows:

Protected amino acid: 0.25M amino acid in DMF solution,

Activator: 0.2M HBTU/0.2M HOBt in DMF solution,

Activated base: 0.4M DIEA in DMF solution,

Deprotecting agent: 20v/v% piperidine in DMF solution,

Blocking reagent: 20 v/v% acetic anhydride in DMF solution.

0.53g (0.23mmol) of Rink Amide resin 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.

Lysate (% by volume): trifluoroacetic acid: ethanedithiol: m-cresol: water = 87.5..5..5..2.5.

Pyrolysis of peptide resin: 2.02 g of a 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. The 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 reacted in an ice bath for 30 minutes, then The ice bath was removed, and the reaction was further stirred at room temperature for 200 min, and the reaction was completed. 500 ml of cold diethyl ether was added to the reactor under vigorous stirring, and a white precipitate was precipitated, and stirring was continued for 60 min; the precipitate was filtered off with a G3 sand core, and washed repeatedly with cold ether for 3 times and dried. 150 ml of a 10% (v/v) aqueous acetic acid solution and 5 ml of acetonitrile were added to sufficiently dissolve the solid, and the filtrate was lyophilized to 927 mg of a crude peptide.

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. Specific procedure: Weigh 867mg of crude peptide, add 20ml of water, 5ml of acetonitrile to completely dissolve the solid, filter through 0.25μm pore size filter and load. 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). After the application, 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 μm 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 2: Preparation of other monomeric polypeptides

Other uncrosslinked monomeric N peptides (SEQ ID NO: 1 to SEQ ID NO: 4, and monomeric polypeptides represented by SEQ ID NO: 6 to SEQ ID NO: 11) are prepared in the same manner as in Example 1 The preparation method of the monomeric polypeptide represented by ID NO: 5 is the same.

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.

On the resin, the side chain protecting group OAll (O-allyl) of E(OAll) in the polypeptide sequence is removed.

The synthesized peptide resin was transferred into a 50 ml eggplant-shaped flask, and 0.26 g of tetrakis(triphenylphosphine)palladium (Pd(PPh ₃ ) ₄ ) was weighed, and 0.31 g of 5,5-dimethylcyclohexanedione was dissolved. 8 ml of a mixture solvent of anhydrous tetrahydrofuran and dichloromethane (v/v = 1/1) was placed in an eggplant-shaped flask and allowed to react in the dark for 6 hours. In the peptide reactor, wash 3 times with DCM, then 3 times with DMF, then 5 times with 50 ml of 0.5 v / v% DIEA in DMF solution, 50 ml of 0.5% (mass / volume) copper reagent DMF solution wash 5 After that, it was washed with DMF, DCM, and MeOH, respectively, and each solution was washed twice and drained.

On the resin, the side chain of the polypeptide after the side chain protecting group is removed is thiolated.

In the polypeptide reactor, the polypeptide resin after removing the side chain protecting group was added, and 275 μl 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 μl 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.

Then, according to the cleavage method and purification method of the monomeric polypeptide shown by SEQ ID NO: 5 in Example 1, a pure peptide was obtained, and the purity was more than 90% by HPLC.

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%.

Example 4: Preparation of other covalently crosslinked N-peptides

Preparation of other covalently cross-linked N-peptides (e.g., covalently cross-linked polypeptides represented by (SEQ ID NO: 2) ₃ to (SEQ ID NO: 11) ₃ ) and in Example 3 (SEQ ID NO: 1) _The crosslinked polypeptides shown in _{3 are} identical.

The molecular weights of the above respective monomeric polypeptides and covalently crosslinked polypeptides are shown in Table 1.

Table 1 molecular weight of the polypeptide

Example 5: Characterization of alpha helicity of N-peptide

1.N peptide solution configuration

About 1 mg of the pure peptide was weighed, dissolved in 700 μld of H ₂ O, shaken, centrifuged, and the supernatant was taken, and the concentration of the N-peptide solution was calibrated on a nanodrop 2000 apparatus. The buffer solution PBS (pH = 7.4) was used as a diluent, and an N peptide solution of 10 μM and 500 μl was placed.

2. Determination of the helicity of the N-peptide solution

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:

Wherein the concentration (c) refers to the concentration value of the N-peptide solution, the path (L) refers to the reference cell length, and the number of residues (N) refers to the number of amide bonds of the N-peptide.

The helicity values of the respective N peptides are shown in Table 2.

Example 8: Compound inhibition of HIV-1-mediated cell - cell fusion activity evaluation (IC ₅₀₎

Cell-cell fusion assay:

1. Resuscitation/freezing of TZM-bl cells and HL2/3 cells

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.

After digesting the cells and counting, centrifuge, discard the supernatant, add the frozen solution and gently blow the cells to suspend the cells evenly (1 million/ml), and dispense them into a cryotube (1 ml/tube), and place them at 4 ° C (30 min). Store at -20 ° C (2 hours), -80 ° C (12 hours), and -196 ° C.

2. Subculture

Remove the cell culture flask, pour off the medium, add 2 ml of digestive juice (purchased from gibico), gently spread it on the cell surface, pour the digestive juice, re-add 2 ml of the digestive juice, spread, and digest at 37 ° C for 2 min. The digestion was terminated by adding 4 ml of the medium, all the liquid was taken out, centrifuged, the supernatant was discarded, 4 ml of the medium was added, and the cells were uniformly suspended by light blowing, and 10 μl of the cells were counted, and 40-500,000 cells were placed in a 75 cm ² culture flask for subculture.

3. Fusion experiment

A. 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 μl/well for 24 hours.

B. Matching the sample: Take the compound to be tested, first estimate the IC ₅₀ value of the compound, based on this estimated value, multiply by two 4, and multiply by 6 to obtain the formulated concentration of the test compound, for example: IC of estimated sample ₅₀ is 10nM, the sample is prepared at a concentration of 10*4*4*6=960nM. Based on the concentration, 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. In the first behavior example, 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).

C. Take HL2/3 cells (supplied by NIH AIDS Research and Reference Reagent Program) and dilute to 1 million/ml, add (1-11)×(AH), 50 μl/well, 12×( AH) supplemented with 50 μl/well of medium.

D. Immediately take the 20 μl/well sample from step B and add to the cell plate for 6 hours.

E. The medium (120 μl/well) in each well of the cell plate was removed and washed twice with PBS, 150 μl/time.

Add the diluted lysate (1×), 50 μl/well, and lyse for 5 min; Solution (1×) The lysate (5×) in the Luciferase Kit (Promega, USA) was diluted with water and freshly prepared according to the amount.

F. Take 20 μl/well of cell lysate onto a 96-well phosphor plate.

G. The melted LA buffer (Luciferase Assay Buffer, Promega Cooperation, USA) was added to the LA substrate (Luciferase Assay Substrate, Promega Cooperation, USA) and mixed, and 40 μl/well was added to the 96-well phosphor plate.

H. Immediately detect luminescence on a microplate reader. The negative control of the experiment is the chemiluminescence signal of single TZM-bl cells, which is represented by Min; the positive control is TZM-bl cells and HL2/3 cells mixed at a concentration of 1:3 without sample inhibitor, expressed by Max; The value is the signal value of a certain sample at a certain concentration, indicated by X; the cell fusion rate = (X-Min) / (Max-Min) * 100%.

According to the above method, 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.

Table 2

Note: 1.a) Uni: deg*m ² dmol ^-1 , the absorption at 222 nm and normalized, the value of 100% alpha helicity is -33000; b) the data in the table indicates that after cross-linking The activity of the N peptide was significantly improved, and the best activity reached a low nanomolar level.

2. T20, C34 is a C-peptide fusion inhibitor as an experimental control for cell fusion activity. Among them, T20 is a marketed drug, and C34 is a well-stabilized and stable fusion inhibitor in the laboratory.

Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and alterations of the details are possible in light of the teachings of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (16)

a compound of formula I or formula II, a derivative thereof, a stereoisomer or a salt of no physiological toxicity, ZX ₁ X ₂ IX ₃ X ₄ IEQX ₅ IX ₆ X ₇ IEQRIQQIEQX 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 LLQLTVWGIKQLQARIL-B (I) (ZX ₁ X ₂ IX ₃ X ₄ IEQX ₅ IX ₆ X ₇ IEQRIQQIEQX 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 LLQLTVWGIKQLQARIL-B) ₃ (II ) among them: The compound of the formula II is a trimer formed by the covalent crosslinking of three compounds represented by the formula I, 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 , Wherein 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, R ₃ is a group which can react with a carboxyl group such as R′OH, PEG, cholesterol, etc., 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. A compound of formula I or formula II, a derivative, a stereoisomer or a non-physiologically acceptable salt thereof, wherein said covalent crosslinking is X ₅ in another compound of formula I with another compounds of formula I form a covalent bond between the X at position 5 in the back ₅ of glutamate, or the covalent crosslinking in a compound of formula ₂ with another compound I of formula I in X X ₂ after the first A covalent bond is formed between the glutamic acid at the 5-position, and the covalent bond is preferably an amide bond (-NC(O)-). A compound of formula I or formula II, a derivative, a stereoisomer or a non-physiologically acceptable salt thereof, wherein said natural L-type natural amino acid is selected from the group consisting of glycine (Gly) and alanine (Ala) ), leucine (Leu), isoleucine (Ile), glutamic acid (Glu), glutamine (Gln), aspartic acid (Asp), asparagine (Asn), proline ( Val), Lys, Lys, Ser, Thr, Arginine, Histidine The L-type unnatural amino acid is ornithine (Orn, O). A compound of formula I or formula II, a derivative, a stereoisomer or a non-physiologically acceptable salt thereof, 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). And X ₂ and X _{5 are} not simultaneously arginine (Arg), 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 ₉ is isoleucine (Ile), 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). A compound of formula I or formula II, a derivative, a stereoisomer or a non-physiologically acceptable salt thereof, wherein said covalent crosslinking is represented by X ₅ in a compound of formula I An amide bond (-NC(O)-) is formed between lysine or ornithine and another glutamic acid at the 5th position after X _{5 in the} compound of formula I, or the covalent cross-linking is in the formula I The lysine or ornithine represented by X ₂ in the compound forms an amide bond (-NC(O)-) with another glutamic acid at the 5th position after X _{2 in the} compound of the formula I. A compound of the formula I or formula II according to claim 4, a derivative thereof, a stereoisomer or a non-physiologically toxic salt selected from the group consisting of: Ac-WRIQQIEQKIHHIEQRIQQIEQLLQLTVWGIKQLQARIL-NH ₂ (SEQ ID NO: 1) (Ac-WRIQQIEQKIHHIEQRIQQIEQLLQLTVWGIKQLQARIL-NH ₂ ) ₃ (SEQ ID NO:1) ₃ Ac-WRIQQIEQKIHHIEQRIQQIEQRIQQIEQLLQLTVWGIKQLQARIL-NH ₂ (SEQ ID NO: 2) (Ac-WRIQQIEQKIHHIEQRIQQIEQRIQQIEQLLQLTVWGIKQLQARIL-NH ₂ ) ₃ (SEQ ID NO: 2) ₃ Ac-WRIQQIEQKIHHIEQRIQQIEQRIEAQQHLLQLTVWGIKQLQARIL-NH ₂ (SEQ ID NO: 3) (Ac-WRIQQIEQKIHHIEQRIQQIEQRIEAQQHLLQLTVWGIKQLQARIL-NH ₂ ) ₃ (SEQ ID NO: 3) ₃ Ac-WRIQQIEQKIHHIEQRIQQIEQRIQQIEQRIEAQQHLLQLTVWGIKQLQARIL-NH ₂ (SEQ ID NO: 4) (Ac-WRIQQIEQKIHHIEQRIQQIEQRIQQIEQRIEAQQHLLQLTVWGIKQLQARIL-NH ₂ ) ₃ (SEQ ID NO: 4) ₃ Ac-RIQQIEQKIHHIEQRIQQIEQLLQLTVWGIKQLQARIL-NH ₂ (SEQ ID NO: 5) (Ac-RIQQIEQKIHHIEQRIQQIEQLLQLTVWGIKQLQARIL-NH ₂ ) ₃ (SEQ ID NO: 5) ₃ Ac-KIHHIEQRIQQIEQRIQQIEQLLQLTVWGIKQLQARIL-NH ₂ (SEQ ID NO: 6) (Ac-KIHHIEQRIQQIEQRIQQIEQLLQLTVWGIKQLQARIL-NH ₂ ) ₃ (SEQ ID NO: 6) ₃ Ac-RIQQIEQOIHHIEQRIQQIEQLLQLTVWGIKQLQARIL-NH ₂ (SEQ ID NO: 7) (Ac-RIQQIEQOIHHIEQRIQQIEQLLQLTVWGIKQLQARIL-NH ₂ ) ₃ (SEQ ID NO: 7) ₃ Ac-RIQQIEQKIHHIEQRIQQIEQRIQQIEQLLQLTVWGIKQLQARIL-NH ₂ (SEQ ID NO: 8) (Ac-RIQQIEQKIHHIEQRIQQIEQRIQQIEQLLQLTVWGIKQLQARIL-NH ₂ ) ₃ (SEQ ID NO: 8) ₃ Ac-RIQQIEQKIHHIEQRIQQIEQRIEAQQHLLQLTVWGIKQLQARIL-NH ₂ (SEQ ID NO: 9) (Ac-RIQQIEQKIHHIEQRIQQIEQRIEAQQHLLQLTVWGIKQLQARIL-NH ₂ ) ₃ (SEQ ID NO: 9) ₃ Ac-RIQQIEQKIHHIEQRIQQIEQRISGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL-NH ₂ (SEQ ID NO: 10) (Ac–RIQQIEQKIHHIEQRIQQIEQRISGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL-NH ₂ ) ₃ (SEQ ID NO:10) ₃ Ac-LLQLTVWGIKQLQARILARIQQIEQKIHHIEQRIQQIEQ-NH ₂ (SEQ ID NO: 11) (Ac-LLQLTVWGIKQLQARILARIQQIEQKIHHIEQRIQQIEQ-NH ₂ ) ₃ (SEQ ID NO: 11) ₃ A nucleic acid molecule encoding a compound of Formula I or Formula II of any of claims 1-6, a derivative thereof, a stereoisomer or a salt of no physiological toxicity. A recombinant vector comprising the nucleic acid molecule of claim 8. A host cell comprising the nucleic acid molecule of claim 8 and/or the recombinant vector of claim 9. A pharmaceutical composition comprising at least one compound of Formula I or Formula II of any one of claims 1-6, a derivative, a stereoisomer or a non-physiologically acceptable salt thereof, or which contains at least one of the rights Requires a nucleic acid molecule of 7, Optionally, it also contains a pharmaceutically acceptable carrier or excipient. The compound of the formula I or formula II according to any one of claims 1 to 6, a derivative thereof, a stereoisomer or a salt of no physiological toxicity or the nucleic acid molecule of claim 7 or the pharmaceutical composition of claim 10 is prepared Use in a medicament for the treatment and/or prevention and/or adjuvant treatment of diseases associated with HIV infection, in particular AIDS. The compound of the formula I or formula II according to any one of claims 1 to 6, a derivative thereof, a stereoisomer or a salt of no physiological toxicity or the nucleic acid molecule of claim 7 or the pharmaceutical composition of claim 10 is prepared Use in a medicament for inhibiting the fusion of HIV (e.g., HIV-1) with cells. A method of inhibiting HIV (e.g., HIV-1) and cell fusion in vivo or in vitro, the method comprising using an effective amount of at least one compound of Formula I or Formula II of any one of claims 1 to 6, A step of a derivative, a stereoisomer or a non-physiologically acceptable salt or the nucleic acid molecule of claim 7 or the pharmaceutical composition of claim 10. A method of treating and/or preventing and/or adjuvant treatment of a disease associated with HIV infection, particularly AIDS, comprising administering to a patient in need of such treatment a therapeutically and/or prophylactically effective amount of at least one of claims 1 to 6 A step of a compound of Formula I or Formula II, a derivative, a stereoisomer or a non-physiologically acceptable salt thereof, or the nucleic acid molecule of Claim 7 or the pharmaceutical composition of Claim 10. A compound of the formula I or formula II according to any one of claims 1 to 6, a derivative thereof, a stereoisomer or a salt of no physiological toxicity or a nucleic acid molecule or claim of claim 7. The pharmaceutical composition of claim 10 for use in the treatment and/or prevention and/or adjuvant treatment of diseases associated with HIV infection, particularly AIDS. A compound of the formula I or formula II, a derivative, a stereoisomer or a non-physiologically acceptable salt thereof, or a nucleic acid molecule according to claim 7 or a pharmaceutical composition according to claim 10, according to any one of claims 1 to 6 To inhibit the fusion of HIV (such as HIV-1) with cells.

Images