CN102731626B - Cyclopeptide and its medicinal application - Google Patents

Cyclopeptide and its medicinal application Download PDF

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Publication number
CN102731626B
CN102731626B CN201110091273.8A CN201110091273A CN102731626B CN 102731626 B CN102731626 B CN 102731626B CN 201110091273 A CN201110091273 A CN 201110091273A CN 102731626 B CN102731626 B CN 102731626B
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Prior art keywords
hiv
phe
derivatives
pro
peptide
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Expired - Fee Related
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CN201110091273.8A
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CN102731626A (en
Inventor
刘克良
贾启燕
姜喜凤
刘叔文
李琳
程卯生
蔡利锋
郑保华
王昆
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Institute of Pharmacology and Toxicology of AMMS
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Institute of Pharmacology and Toxicology of AMMS
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Priority to CN201110091273.8A priority Critical patent/CN102731626B/en
Priority to PCT/CN2012/073988 priority patent/WO2012139519A1/en
Publication of CN102731626A publication Critical patent/CN102731626A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • AIDS & HIV (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention relates to a novel cyclopeptide that has HIV (human immunodeficiency virus) infection inhibiting activity and is as shown in formula (I), its derivatives, a preparation method thereof, medicinal compositions containing the cyclopeptide and its derivatives, as well as application of the medicinal components in treating and preventing related diseases (such as AIDS (acquired immune-deficiency syndrome)) resulted from HIV infection. The formula (I) is shown as: cyclo(Cys-Pro-AA1-Val-AA2-Leu-AA3).

Description

Cyclic peptide and medicinal use thereof
Technical field
The present invention relates to there is the cyclic peptide that suppresses HIV target cell infection activity, its preparation method, containing they pharmaceutical composition and they are at treatment or prevention acquired immune deficiency syndrome (AIDS) (acquired immune-deficiency syndrome, AIDS, also referred to as acquired immune deficiency syndrome (AIDS)) and the purposes that infects the relative disease cause due to HIV.
Background technology
The process of human immunodeficiency virus (human immunodeficiency virus, HIV) target cell infection, is first the fusion of viral coated film and target cell membrane, and then the genetic material RNA of virus enters in target cell, copies the also virus of release new.It is to be mediated by the acceptor on viral envelope glycoprotein mixture (gp120/gp41) and target cell (CD4 acceptor and Chemokine Receptors CCR-5 or CXCR-4) that HIV enters target cell process.First the CD4 receptors bind on HIV envelope glycoprotein surface subunit gp120 and target cell, is attached on cell; Subsequently again with accessory receptor (chemokine receptor CCR 5 or the CXCR4 etc.) combination of target cell; Cause the conformation of cross-film subunit gp41 to change, the fusogenic peptide of its N end is inserted in host cell membrane, start the fusion of peplos and target cell membrane, complete cell entry host cell course of infection (N Engl J Med 2003,348:2228-33).Inhibition to above-mentioned any one link, all can suppress HIV and enter target cell, thus the infection of prevention and treatment HIV.
In recent years, along with deepening continuously of HIV film fusion process Mechanism Study, the Research progress of drugs that inhibition HIV is entered to target cell is very fast.This class medicine is called HIV entry inhibitor.The keying action being risen in film fusion process due to gp41, thereby become the novel targets of inverase research and development.This class be take the medicine that gp41 is action target and is called HIV fusion inhibitor.
Owens etc. have reported that gp41 fusogenic peptide N-terminal six peptide sequence AVGIGA act on HIV-1 gp41 fusogenic peptide, have the cytogamy activity that suppresses HIV-1 induction, and its IC50 value is about 1mM.Mar í a J.G ó mara etc. are by the screening active ingredients to synthetic compound library, finding the full D type six peptide GQIDEV of synthetic and GQIDQV to take fusogenic peptide, as target spot has, to suppress the cytogamy of HIV-1 virus induction active, IC50 value is about 50 μ M, and in 2006, has applied for European patent.The application such as Eckert mirror image phage display method, screening obtains a peptide storehouse, a series of D type ring 18 peptides with medium inhibition activity have wherein been found, IC50 value is about 10 μ M, they can specific binding N-trimer hydrophobic pocket district, thereby show that simple pocket region also can be used as small molecules fusion inhibitor action target.By same method, Welch etc. have reported one group of active D type ring 15 peptide (KGACDYKEWQWLCAA) preferably, and IC50 value is about 11～270 μ M.
The present invention seeks to searching and there is lower molecular weight, compared with the material of the anti-HIV of high biological activity.
Summary of the invention
Contriver is discoverable type (I) polypeptide or derivatives thereof, its steric isomer or its pharmaceutical salts after deliberation, has good inhibition HIV target cell infection active.Therefore, formula (I) polypeptide or derivatives thereof, its steric isomer or its salt without physiology toxicity can be used as the infection that medicine is used for the treatment of or prevents HIV.
One aspect of the present invention relates to the cyclic peptide or derivatives thereof of formula I, its steric isomer or its pharmaceutical salts:
Cyclo(Cys-Pro-AA 1-Val-AA 2-Leu-AA 3)
Formula (I)
Wherein
AA 1can be the natural or alpha-non-natural amino acid of D type or L-type;
AA 2can be the natural of D type or L-type or non-natural fragrant amino acid;
AA 3can be the natural or alpha-non-natural amino acid of D type or L-type.
In the present invention, particularly,
AA1 is preferably phenylalanine, 4-amino-benzene L-Ala, 4-chlorophenylalanine, L-glutamic acid, glutamine, aspartic acid or l-asparagine.
AA2 is preferably the phenylalanine, 4-amino-benzene L-Ala, 4-chlorophenylalanine, naphthylalanine, β-piperonyl L-Ala, morpholine methyl phenylalanine of L or D type or to urea groups phenylalanine.
AA3 is preferably L-glutamic acid, glutamine, aspartic acid, l-asparagine, methionine(Met) or arginine.
According to the preferred embodiments of the invention, cyclic peptide of the present invention is selected from peptide or its steric isomer or its pharmaceutical salts below:
(1)cyclo(Cys-Pro-Phe-Val-Phe-Leu-Glu)；
(2)cyclo(Cys-Pro-Phe-Val-Phe-Leu-Arg)；
(3)cyclo(Cys-Pro-Phe-Val-Phe-Leu-Met)；
(4)cyclo(Cys-Pro-Glu-Val-Phe-Leu-Met)；
(5)cyclo(Cys-Pro-Glu-Val-Phe-Leu-Arg)。
The preferred cyclic peptide of the present invention is the polypeptide of numbering (1), (2).
Further aspect of the present invention relates to the pharmaceutical composition that comprises at least one formula (I) polypeptide or its steric isomer or its pharmaceutical salts and pharmaceutically acceptable carrier or vehicle.
The invention still further relates to formula (I) polypeptide or its steric isomer or its pharmaceutical salts purposes in the medicine of preparation treatment or pre-preventing HIV infection relative disease (as acquired immune deficiency syndrome (AIDS)) or symptom.
The invention still further relates to the preparation method of formula (I) polypeptide or its steric isomer or its pharmaceutical salts.
The preparation of the compounds of this invention adopts solid phase synthesis process; take mbha resin as carrier; Boc-protects strategy; HBTU is condensation reagent; DIEA is activation alkali, and TFA is deprotecting regent, according to the aminoacid sequence of prepared compound; from C, hold N end, by the Boc solid-phase peptide synthesis synthetic peptide resin of standard.
In the present invention, cyclo refers to cyclic peptide.
Term of the present invention " derivative " comprises cysteine sulfydryl modification in formula (I).
Term used herein " formula (I) polypeptide steric isomer " refers to its corresponding D-or L-steric configuration.
Term used herein " pharmacologically acceptable salt " or " without the salt of physiology toxicity " refer to that can retain parent compound expects physiologically active and can not produce the salt of any unexpected toxic side effect or their composition.For example: hydrochloride, hydrobromate, vitriol, phosphoric acid salt, nitrate, and acetate, oxalate, tartrate, succinate, malate, benzoate, embonate, alginates, mesylate, naphthalenesulfonate etc.According to the positively charged ion containing in salt, can be again: sylvite, lithium salts, zinc salt, mantoquita, barium salt, bismuth salt, the inorganic salt such as calcium salt, also can be such as organic salts such as trialkyl ammonium salts.
According to the present invention, pharmaceutical composition of the present invention can be by the preparation of this area ordinary method, for example, by formula (I) polypeptide and pharmaceutical carrier or mixed with excipients.In the present invention, pharmaceutical carrier used or vehicle are generally pharmaceutical carrier or the vehicle that pharmacy field is conventional.Here " conventional drug excipient " comprises any or all solvents, dispersion medium, dressing, antiseptic-germicide or anti-mycotic agent, Deng oozing and slowly-releasing reagent, and similar physiology compatibility agent, to be applicable to intravenous injection, intramuscular injection, subcutaneous injection, or other administering mode, as oral administration.According to the mode of administration, active compound dressing can be avoided to acid or the impact of other natural condition and inactivation with protection compound.About the example of carrier, as saline based and various aqueous buffer solution, ethanol or other polyvalent alcohol, liposome, poly(lactic acid), vinyl-acetic ester, polyanhydride, polyglycolic acid, collagen, poe etc.
Formula of the present invention (I) polypeptide can be used alone or use with pharmaceutical compositions.Using method can be oral or parenteral route.Oral administration preparation can be tablet, capsule or granule etc.Non-intestinal drug delivery agent can be injection, sprays, patch etc.The present composition also can be made into sustained release preparation, as microballoon etc.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example is only for the present invention is described, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, carries out according to the condition of normal condition or manufacturers's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
The abbreviation of using in the present invention has implication below:
AIDS (Acquired Immune Deficiency Syndrome) acquired immune deficiency syndrome (AIDS),
Acquired immune deficiency syndrome (AIDS)
Ala (Alanine, A) L-Ala
Arg (Arginine, R) arginine
Boc (t-Butoxycarbonyl) tertbutyloxycarbonyl
Cys (Cysteine, C) halfcystine
DIEA (N, N-Diisopropylethylamine) DIPEA
Glu (Glutamic acid, E) L-glutamic acid
HBTU 2-(1H-1-hydroxybenzotriazole)-1,1,3,3-tetramethyl-urea phosphofluoric acid
HoBt (1-Hydroxylbenzotriazole anhydrous) 1-hydroxy benzo triazole
HIV (Human Immunodeficiency Virus)) human immunodeficiency virus
HIV-1 human immunodeficiency virus I type
HIV-2 human immunodeficiency virus II type
HPLC (High performance liquid chromatography) high performance liquid chromatography
Leu (Leucine, L) leucine
MBHA phenylamino methyl resin
MPA (3-Mercaptopropionic) thiohydracrylic acid
Phe (Phenylalanine, F) phenylalanine
Pro (Proline) proline(Pro)
TFA (trifluoroacetic acid) trifluoroacetic acid
Trt (Trityl) trityl
Embodiment
Example below and biological activity test are used for further illustrating the present invention, but this and do not mean that any limitation of the invention.
Embodiment solid-phase synthesized carrier mbha resin used is the synthetic responsibility of Tianjin Nankai company limited product; The natural amino acid of HBTU, DIEA and Boc protection or the alpha-non-natural amino acid of D type are gill biochemical corp, Shanghai and Chengdu Cheng Nuo new technology limited liability company product.
Embodiment 1: compound (1) synthetic
Synthesizing of linear peptides thioesters: the 0.93g mbha resin (1mmol) of take is solid phase carrier, HBTU/DIEA is condensing agent, first Trt-MPA is connected with resin, obtain Trt-MPA-MBHA-Resin, again according to the aminoacid sequence of the formula I polypeptide that will obtain, from C, hold N end, by the Boc solid-phase peptide synthesis (reference: Huang Weide of standard, Chen Changqing work, polypeptide is synthetic, Science Press, 1985) operation, by L-glutamic acid, leucine, phenylalanine, α-amino-isovaleric acid, phenylalanine, proline(Pro), halfcystine is condensation successively, after above-mentioned peptide resin is dry, put into the reactor of HF cutting device, add 1.0mL methyl-phenoxide, 1ml dithioglycol, use cooled with liquid nitrogen reactor, proceed to the liquid HF of about 20mL, in 0 ℃ of reaction 60 minutes.With oil pump, take HF away, take off reactor, add cold anhydrous diethyl ether to be settled out solid, then suspension is transferred in sand core funnel.With a small amount of cooling anhydrous diethyl ether washing three times, with the aqueous solution, rinse to resin and no longer mutually adheres to, collect washings, must white dry powder after lyophilize.Through RP-HPLC purifying, obtain the peptide thioesters sterling of CPFVFLE.MS:940.7 (theoretical value: 941.2).
Synthesizing of cyclic peptide (1): the linear peptides thioesters after freeze-drying is added to 0.1M Na 2hPO 4and containing in 50% (v/v) acetonitrile/water solution of 3eq TCEP (three propyloic phosphines), make peptide concentration be about 2mg/mL and pH approximately 7.0, and reaction is monitored by HPLC, and product, through RP-HPLC separation and purification, obtains the sterling of compound (1) after freeze-drying.MS:835.6 (theoretical value: 836.0).
Embodiment 2
According to the same procedure of embodiment 1, use the amino acid in corresponding compound number to synthesize the cyclic peptide in table 1:
Table 1
Compound number MW (mass spectrum result) MW (theoretical value)
(2) 862.9 863.1
(3) 837.8 838.1
(4) 819.7 820.0
(5) 844.6 845.0
Embodiment 3: the active detection of cytogamy of suppressing HIV-1 mediation.
Non-infectious cytogamy test:
On 96 well culture plates, the target cell MT-2 cell (1 * 10 that adds 50 μ l to contain CD4 acceptor and CXCR4 accessory receptor 6/ ml) and certain density testing compound, it is peak concentration that testing compound be take 25 μ M, and 2 times of dilutions are 6 times step by step, and each concentration repeats to do three times.Then the effector cell CHO-WT cell (5 * 10 that adds 100 μ l stably express HIV-1 envelope protein gp160 5/ ml) (CHO-WT and MT-2 derive from America NI H AIDS Research and Reference Reagent Program), under 37 ℃ of conditions, cultivate 48 hours, at the plasmodial formational situation of inverted micro-Microscopic observation, suppress the IC50 value Calcusyn computed in software of cytogamy.
According to the method described above, determination of activity the results are shown in Table 2.
Table 2
Wherein compound (1), (2) are corresponding with the compound number in the embodiment of the present invention, and T20 is control drug.
Experimental result shows, compound of the present invention can effectively suppress the cytogamy of HIV-1 mediation.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.

Claims (4)

1. following cyclic peptide or derivatives thereof or its steric isomer or its pharmaceutical salts, it is selected from:
(1) cyclo (Cys-Pro-Phe-Val-Phe-Leu-Glu); With
(2)cyclo(Cys-Pro-Phe-Val-Phe-Leu-Arg)。
2. pharmaceutical composition, the cyclic peptide or derivatives thereof that it contains at least one claim 1 or its steric isomer or its pharmaceutical salts, and pharmaceutically acceptable carrier or vehicle.
3. the cyclic peptide or derivatives thereof of claim 1 or its steric isomer or its pharmaceutical salts purposes in the medicine for the preparation for the treatment of or pre-preventing HIV infection relative disease.
4. the purposes of claim 3, it is acquired immune deficiency syndrome (AIDS) that wherein said HIV infects relative disease.
CN201110091273.8A 2011-04-13 2011-04-13 Cyclopeptide and its medicinal application Expired - Fee Related CN102731626B (en)

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PCT/CN2012/073988 WO2012139519A1 (en) 2011-04-13 2012-04-13 Cyclic peptide and medical use thereof

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CN107236021B (en) * 2017-06-12 2020-12-01 湖北泓肽生物科技有限公司 Synthetic method of polypeptide derivative

Citations (1)

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Publication number Priority date Publication date Assignee Title
CN101845079A (en) * 2009-03-27 2010-09-29 中国人民解放军军事医学科学院毒物药物研究所 Hexapeptide or derivative thereof and medical application thereof

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WO2011127624A1 (en) * 2010-04-13 2011-10-20 中国人民解放军军事医学科学院毒物药物研究所 Anti-hiv peptide

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101845079A (en) * 2009-03-27 2010-09-29 中国人民解放军军事医学科学院毒物药物研究所 Hexapeptide or derivative thereof and medical application thereof

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