WO2019136824A1 - 抑制MERS-CoV感染的多肽 - Google Patents

抑制MERS-CoV感染的多肽 Download PDF

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WO2019136824A1
WO2019136824A1 PCT/CN2018/079474 CN2018079474W WO2019136824A1 WO 2019136824 A1 WO2019136824 A1 WO 2019136824A1 CN 2018079474 W CN2018079474 W CN 2018079474W WO 2019136824 A1 WO2019136824 A1 WO 2019136824A1
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group
substituted
mers
unsubstituted
polypeptide compound
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PCT/CN2018/079474
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English (en)
French (fr)
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王潮
刘克良
张培宇
孟广鹏
梁国栋
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中国人民解放军军事科学院军事医学研究院
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Publication of WO2019136824A1 publication Critical patent/WO2019136824A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the field of biomedicine, relates to a polypeptide compound against MERS-CoV infection, in particular to a polypeptide compound having at least 80% identity with the general formula (I):
  • a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof, and/or a functionally equivalent variant thereof also relates to a pharmaceutical composition comprising such a polypeptide compound, a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof and/or a functionally equivalent variant thereof, and a polypeptide compound thereof
  • a pharmaceutical composition of a construct, a mixture thereof, a pharmaceutically acceptable salt thereof, and/or a functionally equivalent variant thereof for the treatment or prevention of a disease associated with MERS-CoV infection, particularly Middle East respiratory syndrome.
  • MERS-CoV Middle East Respiratory Syndrome Coronavirus
  • the MERS-CoV fusion protein is mainly composed of the S1 subunit and the S2 subunit.
  • the S1 subunit is responsible for the specific recognition of CD26 receptor on the surface of the host cell;
  • the S2 subunit is responsible for mediating the fusion between the virus and the target cell membrane.
  • the S2 subunit can be further divided into a fusion peptide (FP), an N-terminal heptad repeat (NHR, also known as HR1), and a C-terminal heptad repeat. , CHR, also known as HR2) functional area.
  • FP fusion peptide
  • NHR N-terminal heptad repeat
  • HR2 C-terminal heptad repeat
  • the S2 subunit When the S1 subunit binds to the CD26 receptor, the S2 subunit undergoes a conformational change, and its FP is inserted into the host cell membrane, and the HR1 and HR2 regions are induced to interact to form a six-strand ⁇ -helix bundle (6HB) structure, thereby mediating the virus and Fusion of host cell membranes.
  • 6HB six-strand ⁇ -helix bundle
  • Peptide binding technology is one of the effective strategies for "helix stabilization.” Covalently bridged polypeptide binding techniques can more effectively "lock” the alpha helical conformation of a polypeptide in solution. Stapled peptides constructed using this technique can be used to selectively modulate protein-protein interactions.
  • the general idea of the peptide binding technology is to orthogonally react the side chain of the amino acid residue at the i ⁇ i+3 or i ⁇ i+4 or i ⁇ i+7 position of the polypeptide sequence on the side of the ⁇ -helix that does not interact with the target protein. Covalent bridging is performed to lock the alpha-helical conformation of the polypeptide.
  • the present inventors constructed a highly active, metabolically stable MERS-CoV fusion inhibitor based on the MERS-CoV HR2 alpha helix region polypeptide sequence, thereby providing a solution for treating and preventing MERS-CoV.
  • the invention relates to a polypeptide compound having at least 80% identity to the general formula (I):
  • X a is an amino acid residue capable of hydrophobic interaction with MERS-COV HR2, which is selected from the following D-type or L-type hydrophobic amino acids: alanine (Ala), valine (Val), leucine ( Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), tryptophan (Trp), norleucine (n-Leu) and methionine (Met);
  • X 1 , X 2 , X 3 and X 4 are each independently selected D-form or L-form amino acid;
  • U and W are absent or each are independently selected one or more additional D- or L-form amino acids
  • R 1 is selected from the group consisting of: H, a polymer derived from polyethylene glycol, an acyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyclic group, a substituted or unsubstituted a heterocyclic group, a substituted or unsubstituted heteroarylalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group and R 5 -CO-, wherein R 5 is selected from the group consisting of: H, acyclically substituted or unsubstituted aliphatic group, substituted or unsubstituted alicyclic group, substituted or unsubstituted aryl group, substituted or unsubstituted aralkyl group, substituted Or unsubstituted heterocyclic group and substituted or unsubstituted heteroarylalkyl;
  • R 2 is selected from the group consisting of: -NR 3 R 4 , -OR 3 and -SR 3 , wherein R 3 and R 4 are independently selected from: H, a polymer derived from polyethylene glycol, acyclic substituted or not Substituted aliphatic group, substituted or unsubstituted alicyclic group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted heteroarylalkyl group, substituted or unsubstituted aryl group a group, and a substituted or unsubstituted aralkyl group;
  • R 1 and R 2 are not D-form or L-form amino acids.
  • the invention relates to at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the formula (I) Or a 100% identity polypeptide compound or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof, and/or a functionally equivalent variant thereof.
  • X a is selected from the group consisting of the following D or L amino acids: methionine, leucine, isoleucine, valine and norleucine.
  • R 1 is selected from the group consisting of H, acetyl, lauroyl, myristoyl and palmitoyl. In a preferred embodiment, R 1 is selected from: H or R 5 -CO-; wherein R 5 is selected from: H or substituted or unsubstituted C 1 -C 24 alkyl.
  • R 2 is selected from the group consisting of: -NR 3 R 4 and -OR 3 , wherein R 3 and R 4 are independently selected from the group consisting of H, methyl, ethyl, hexyl, dodecyl and hexadecane base.
  • R 2 is selected from: -NR 3 R 4 or -OR 3 wherein R 3 and R 4 are independently selected from: H or substituted or unsubstituted C 1 -C 24 alkyl.
  • X 1 , X 2 , X 3 and X 4 in the general formula (I) are located on the side that does not interact with the target protein, but Exposure to the side that is in contact with the solvent.
  • the inventors have surprisingly found that the introduction of one or more alpha helix stable amino acids in X 1 , X 2 , X 3 and X 4 can help stabilize the polypeptide compounds of the invention in an alpha helix conformation, thereby enabling efficient It inhibits MERS-CoV fusion and is suitable for the treatment and prevention of MERS-CoV.
  • X 1, X 2, X 3 and X 4 is one or more substituents independently stabilizing amino acid is ⁇ helix.
  • two or more of X 1 , X 2 , X 3 and X 4 are independently alpha helix stable amino acids.
  • X 1 and X 2 , X 1 and X 3 , X 2 and X 3 and/or X 3 and X 4 are alpha helix stable amino acids.
  • X 1 , X 2 and X 3 are alpha helix stable amino acids.
  • X 1 , X 3 and X 4 are alpha helix stable amino acids.
  • X 2 , X 3 and X 4 are alpha helix stable amino acids.
  • X 2 , X 3 and X 4 are alpha helix stable amino acids.
  • X 1 , X 2 , X 3 and X 4 are alpha helix stable amino acids.
  • the amino acid residues that are not alpha helix stable amino acids in X 1 , X 2 , X 3 and X 4 may each independently be selected from any amino acid, preferably selected from the following D or L amino acids : glutamine, alanine and serine.
  • alpha helix stable amino acid means an amino acid that increases stability to the alpha helix conformation of a MERS-CoV fusion inhibitor peptide.
  • Alpha helix stable amino acids are well known in the art. Suitable alpha helical stable amino acids suitable for the purposes herein include, but are not limited to, alanine, norvaline, norleucine, alpha-aminobutyric acid, alpha-aminoisobutyric acid (AIB), leucine, Isoleucine, valine, and the like.
  • the alpha helix stable amino acid is any amino acid that is part of the alpha helix of a naturally occurring protein, such as Leu, Phe, Ala, Met, Gly, Ile, Ser, Asn, Glu, Asp, Lys, Arg.
  • the alpha helix stable amino acid is an alpha, alpha-disubstituted amino acid.
  • the alpha, alpha-disubstituted amino acid comprises a first substituent and a second substituent, each of the first substituent and the second substituent being bonded to an alpha carbon, wherein the first substituent and the second substituent
  • the groups are each independently selected from a C1-C4 alkyl group optionally substituted with a hydroxy, amide, fluorenyl, or halo group, or the first substituent and the second substituent form a ring with the alpha carbon to which they are attached (eg, C3- C8 ring).
  • the first substituent and the second substituent are each independently selected from the group consisting of: methyl, ethyl, propyl, and n-butyl, or the first substituent and the second substituent form a cyclooctane or Cycloheptane (e.g., 1-aminocyclooctane-1-carboxylic acid).
  • the first substituent is the same as the second substituent.
  • the first substituent is different from the second substituent.
  • the first substituent and the second substituent are each a C1-C4 alkyl group.
  • the first substituent and the second substituent are each a C1 or C2 alkyl group. In an exemplary embodiment, the first substituent and the second substituent are each a methyl group such that the ⁇ , ⁇ -disubstituted amino acid is ⁇ -aminoisobutyric acid (AIB). In other exemplary embodiments, the alpha, alpha-disubstituted amino acid is 1-aminocyclopropane-1-carboxylic acid (ACPC). In other exemplary embodiments, the alpha, alpha-disubstituted amino acid may also be an alpha, alpha-disubstituted amino acid having an olefinic side chain.
  • AIB ⁇ -aminoisobutyric acid
  • ACPC 1-aminocyclopropane-1-carboxylic acid
  • the alpha, alpha-disubstituted amino acid may also be an alpha, alpha-disubstituted amino acid having an olefinic side
  • Non-limiting examples of ⁇ , ⁇ -disubstituted amino acids having an olefinic side chain may include, for example, (R)-2-(4'-pentenyl)alanine (R 5 ), (S)-2- (4'-pentenyl) alanine (S 5 ), (S)-2-(4'-octenyl)alanine (S8), (R)-2-(4'-octenyl) Alanine (R8) and 2-amino-2-(4-pentenyl)-6-heptenoic acid (B 5 ).
  • the invention relates to having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% with any one of SEQ ID NO. 1-37 a 97%, 98%, 99% or 100% identity polypeptide compound or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof and/or a functionally equivalent variant thereof.
  • the inventors also utilize peptide binding techniques to crosslink two or more amino acid residues in a polypeptide compound of the invention to form a so-called binding peptide.
  • binding peptide refers to a peptide that is stabilized in a desired conformation by intramolecular crosslinks.
  • the binding peptide is a peptide that is stabilized in the alpha helical conformation by intramolecular crosslinks.
  • a binding peptide constructed based on the MERS-CoV HR2 alpha helix region polypeptide sequence comprising an intramolecular crosslinker at a specific amino acid site is a very highly active MERS-CoV fusion inhibitor, It is effective in the treatment and prevention of MERS-CoV infection and diseases associated with MERS-CoV infection.
  • a MERS-CoV fusion inhibitor that is effective in the treatment and prevention of MERS-CoV infection and diseases associated with MERS-CoV infection.
  • the invention relates to a polypeptide compound having at least 80% identity to the general formula (I):
  • X a is an amino acid residue capable of hydrophobic interaction with MERS-COV HR2, which is selected from the following D-type or L-type hydrophobic amino acids: alanine (Ala), valine (Val), leucine ( Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), tryptophan (Trp), norleucine (n-Leu) and methionine (Met);
  • X 1 , X 2 , X 3 and X 4 are each independently selected D-form or L-form amino acid, and form a crosslinked body between two or more of X 1 , X 2 , X 3 and X 4 ;
  • U and W are absent or each are independently selected one or more additional D- or L-form amino acids
  • R 1 is selected from the group consisting of: H, a polymer derived from polyethylene glycol, an acyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyclic group, a substituted or unsubstituted a heterocyclic group, a substituted or unsubstituted heteroarylalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group and R 5 -CO-, wherein R 5 is selected from the group consisting of: H, acyclically substituted or unsubstituted aliphatic group, substituted or unsubstituted alicyclic group, substituted or unsubstituted aryl group, substituted or unsubstituted aralkyl group, substituted Or unsubstituted heterocyclic group and substituted or unsubstituted heteroarylalkyl;
  • R 2 is selected from the group consisting of: -NR 3 R 4 , -OR 3 and -SR 3 , wherein R 3 and R 4 are independently selected from: H, a polymer derived from polyethylene glycol, acyclic substituted or not Substituted aliphatic group, substituted or unsubstituted alicyclic group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted heteroarylalkyl group, substituted or unsubstituted aryl group a group, and a substituted or unsubstituted aralkyl group;
  • R 1 and R 2 are not D-form or L-form amino acids.
  • the invention relates to at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the formula (I) Or a 100% identity polypeptide compound or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof, and/or a functionally equivalent variant thereof.
  • X a is selected from the group consisting of the following D or L amino acids: methionine, leucine, isoleucine, valine and norleucine.
  • R 1 is selected from the group consisting of H, acetyl, lauroyl, myristoyl and palmitoyl. In a preferred embodiment, R 1 is selected from: H or R 5 -CO-; wherein R 5 is selected from: H or substituted or unsubstituted C 1 -C 24 alkyl.
  • R 2 is selected from the group consisting of: -NR 3 R 4 and -OR 3 , wherein R 3 and R 4 are independently selected from the group consisting of H, methyl, ethyl, hexyl, dodecyl and hexadecane base.
  • R 2 is selected from: -NR 3 R 4 or -OR 3 wherein R 3 and R 4 are independently selected from: H or substituted or unsubstituted C 1 -C 24 alkyl.
  • a crosslinker is formed between X 1 and X 2 , X 1 and X 3 , X 2 and X 3 and/or X 3 and X 4 .
  • a crosslinked body is formed between X 1 , X 2 and X 3 .
  • a crosslinked body is formed between X 1 , X 3 and X 4 .
  • a crosslinked body is formed between X 2 , X 3 and X 4 .
  • a crosslinked body is formed between X 1 , X 2 , X 3 and X 4 .
  • the polypeptide compounds of the invention comprise one or more crosslinkers formed between X 1 and X 2 , X 1 and X 3 , X 2 and X 3 and X 3 and X 4 .
  • X 1 , X 2 , X 3 and X 4 in the general formula (I) are located on the side that does not interact with the target protein, but exposed surface in contact with the solvent, and by X 1, X 2, two of X 4 X 3 or more and crosslink, can help stabilize the polypeptide compounds of the invention in ⁇ -helical conformation.
  • X 1 , X 2 , X 3 and X 4 may be selected from any amino acid as long as it is capable of being linked to each other by a cross-linker as described herein.
  • amino acid residues that are not crosslinked in X 1 , X 2 , X 3 and X 4 may each independently be selected from any amino acid, preferably selected from the following D or L amino acids: Glutamine, alanine and serine.
  • crosslinker refers to an intramolecular linkage of two peptide domains (ie, for example, two loops of a helical peptide).
  • the cross-linker can be a macrocycle which is the core of the outer (part of the non-helical peptide structure) or an intrinsic (non-crosslinked) helical peptide structure.
  • the macrocycle may comprise a perhydrocarbon linking ring and comprises a side chain attached to the a-carbon of at least two amino acids of the peptide.
  • the crosslinked peptide has at least one crosslinked body.
  • the cross-linked peptide has 1, 2 or 3 or at least 1, 2 or 3 cross-links.
  • the peptides disclosed herein comprise only one crosslinker.
  • crosslinker known in the art may be used as long as it is capable of linking two or more amino acids of the X 1 , X 2 , X 3 and X 4 positions of the general formula (I).
  • the crosslinker can be a non-covalent or covalent crosslinker.
  • non-covalent crosslinker Any non-covalent crosslinker known in the art may be used as long as it is capable of linking two or more amino acids of the X 1 , X 2 , X 3 and X 4 positions of the formula (I).
  • the non-covalent crosslinker may be selected from, but not limited to, crosslinkers comprising hydrogen bonds, electrostatic interactions or metal chelation (see, for example, V. Azzarito, K. Long, NSMurphy and AJ Wilson, Nat. Chem., 2013, 5, 161 - 173; V. Haridas, Eur. J. Org. Chem., 2009, 5112 - 5128.; each of which is incorporated herein by reference in its entirety.
  • covalent crosslinker Any covalent crosslinker known in the art can also be used as long as it is capable of linking two or more amino acids of the X 1 , X 2 , X 3 and X 4 positions of the formula (I).
  • the covalent crosslinker may be selected from, but not limited to, an azide-alkyne cyclone comprising a hydrocarbon linkage, a molecular lactam linkage, a disulfide bond, a thioether bond, or a Cu(I)-catalyzed Crosslinking of the (CuAAC) reaction (see, for example, YH Lau et al., "Peptide stapling techniques based on different macrocyclisation chemistries", Cheminform, 2015, 46 (15); White CJ, Yudin AK, "Contemporary strategies for peptide macrocyclization” , Nature chemistry, 2011, 3(7): 509; all incorporated herein by reference.
  • a linker can also be used to indirectly link two or more amino acids of the X 1 , X 2 , X 3 and X 4 positions of the general formula (I) to form a crosslinked body comprising a linker.
  • Such joints suitable for the purposes herein are well known in the art and can be any suitable linker.
  • Examples of such linkers may include, for example, an ⁇ , ⁇ -diaminoalkane linker, a 1,4-diaminopropane linker or a 1,5-diaminopentane linker photoswitchable linker, a functionalized double click link (based on CuAAC) Reaction) and the like (see, for example, YH Lau et al. (supra); White CJ et al. (supra)).
  • the crosslinker comprising a hydrocarbon linkage is a perhydrocarbon crosslinker.
  • the all-carbon hydrogen-crosslinking amino acid residue formed in X 1 , X 2 , X 3 and X 4 is an ⁇ , ⁇ -disubstituted amino acid having an olefinic side chain. It should be understood that in the polypeptide compound of the present invention, X 1 , X 2 , X 3 and X 4 may be any ⁇ , ⁇ -disubstituted amino acid having an olefinic side chain as long as it can pass through the olefinic side with each other. The chain forms a full carbon hydrogen crosslinker.
  • Non-limiting examples of such an ⁇ , ⁇ -disubstituted amino acid having an olefinic side chain may include, for example, (R)-2-(4'-pentenyl)alanine (R 5 ), (S)- 2-(4'-pentenyl)alanine (S 5 ), (S)-2-(4'-octenyl)alanine (S8), (R)-2-(4'-octyl alkenyl yl) alanine (R8) and 2-amino-2- (4-pentenyl) -6-heptenoic acid (B 5).
  • R 5 2-(4'-pentenyl)alanine
  • S 5 2-(4'-pentenyl)alanine
  • S8 2-(4'-octenyl)alanine
  • R8 2-amino-2- (4-pentenyl) -6-heptenoic acid
  • the invention relates to having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% with any one of SEQ ID NO. 38-69 a 97%, 98%, 99% or 100% identity polypeptide compound or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof and/or a functionally equivalent variant thereof.
  • the present inventors have found that the polypeptide compound of the present invention is effective for inhibiting fusion of MERS-CoV with cells.
  • the polypeptide compounds of the invention are useful for inhibiting MERS-CoV fusion.
  • the polypeptide compounds of the invention are useful for treating or preventing a disease associated with MERS-CoV infection.
  • the MERS-CoV infection associated disease is Middle East Respiratory Syndrome.
  • the present invention is also a pharmaceutical composition
  • a pharmaceutical composition comprising at least one polypeptide compound of the present invention, or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof, and/or a functionally equivalent change thereof And at least one pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
  • the peptide itself may be present in the composition in any of a wide variety of forms. For example, two or more peptides may be only mixed together, or may be more closely associated by complexation, crystallization, or ionic or covalent bonding.
  • the invention relates to a MERS-CoV fusion inhibitor comprising at least one polypeptide compound of the invention, or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof, and/or a functionally equivalent change thereof body.
  • a peptide or a pharmaceutical composition thereof, or a MERS-CoV fusion inhibitor thereof, as described herein, can be administered in a number of ways, depending on whether local or systemic administration is desired and depends on the area to be treated.
  • the peptide or a pharmaceutical composition thereof, or a MERS-CoV fusion inhibitor thereof can be administered to a patient in the following manner: oral or rectal, or transmucosal, or enteral, or intramuscular, or subcutaneous, or Intramedullary, or intrathecal, or directly intraventricular, or intravenous, or intravitreal, or intraperitoneal, or intranasal, or intraocular.
  • Suitable pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, buffer, phosphate buffered saline, buffered sodium chloride, vegetable oil, minimal essential medium (MEM), MEM with HEPES, and the like.
  • the invention relates to the use of a polypeptide compound of the invention, or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof, and/or a functionally equivalent variant thereof, for inhibiting MERS-CoV fusion.
  • the invention relates to a polypeptide compound of the invention, or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof, and/or a functionally equivalent variant thereof, for use in the treatment or prevention of a disease associated with MERS-CoV infection the use of.
  • the MERS-CoV infection associated disease is Middle East Respiratory Syndrome.
  • the invention relates to the use of a polypeptide compound of the invention, or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof, and/or a functionally equivalent variant thereof, for the preparation of a MERS-CoV fusion inhibitor .
  • the invention relates to a polypeptide compound of the invention, or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof, and/or a functionally equivalent variant thereof, for use in the manufacture or treatment of a MERS-CoV infection Use in drugs for related diseases.
  • the MERS-CoV infection associated disease is Middle East Respiratory Syndrome.
  • the present invention relates to a method for preparing a polypeptide compound of the present invention, comprising:.. A synthesis of formula (I) having at least 80% identity to the polypeptide, and b X 1, X 2, X 3, and Two or more of X 4 are connected by crosslinking.
  • X 1 and X 2 , X 1 and X 3 , X 2 and X 3 and/or X 3 and X 4 are linked by crosslinking.
  • X 1 , X 2 and X 3 are linked by crosslinking.
  • X 1 , X 3 and X 4 are linked by crosslinking.
  • X 2 , X 3 and X 4 are linked by crosslinking.
  • X 1 , X 2 , X 3 and X 4 are linked by crosslinking.
  • X 1 , X 2 , X 3 and X 4 are linked by crosslinking.
  • X 1 , X 2 , X 3 and X 4 are
  • polypeptide compounds, their stereoisomers, mixtures thereof, their pharmaceutically acceptable salts and/or functionally equivalent variants thereof as described herein can be Any conventional method known in the art is carried out, for example, by solid phase peptide synthesis [Stewart JMy Young JD, "Solid Phase Peptide Synthesis, 2nd edition", (1984), Pierce Chemical Company, Rockford, Illinois; Bodanzsky My Bodanzsky A., "The practice of Peptide Synthesis", (1994), Springer Verlag, Berlin; Lloyd Williams P.
  • the compounds can also be obtained by fermentation of a genetically engineered or unmodified bacterial strain intended to produce the desired sequence, or by free passage of a peptide containing at least the desired sequence from an animal, fungus or preferably plant-derived protein. Controlled hydrolysis to obtain.
  • a nucleic acid sequence encoding an amino acid sequence of a polypeptide as described herein, and optionally an appropriate amino acid modification can be used to produce a compound of the invention.
  • methods of obtaining polypeptide compounds of the invention, their stereoisomers, and mixtures thereof may include the following stages:
  • two or more amino acids in the peptide sequence are cross-linked by a suitable cross-linker.
  • the C-terminus is bound to a solid support and the process is carried out in a solid phase, thus comprising amino acids that bind the N-terminally protected and C-terminal free amino acids to the N-terminal free and C-terminus to the polymeric carrier.
  • the functional groups of the amino acid side chains are conveniently protected with temporary or permanent protective groups and are capable of being deprotected simultaneously or orthogonally with the process of cleaving the peptide from the polymeric carrier.
  • solid phase synthesis can be carried out using a converging strategy: coupling the peptide to a polymeric carrier or to a peptide or amino acid previously bound to a polymeric carrier.
  • Polymerization strategies are widely known to those skilled in the art and are described in Lloyd-Williams P. et al., "Convergent Solid-Phase Peptide Synthesis", (1993), Tetrahedron, 49 (48), 11065-11133.
  • the process of the invention can include additional N-terminal and C-terminal deprotection and/or stages of cleavage of the peptide from the polymeric carrier in an indiscriminate sequence, using standard procedures and conditions known in the art; These terminal functional groups can then be modified.
  • Optional modifications of the N-terminus and C-terminus can be made when the peptide of formula (I) is immobilized to the polymeric carrier or once the peptide has been separated from the polymeric carrier.
  • R 1 can be introduced by reacting the N-terminus of the compound of the invention with a R 1 -X compound by nucleophilic substitution reaction in the presence of a suitable base and solvent, wherein R 1 has the aforementioned meaning and X is removed
  • the group is, for example and not limited to, a tosyl group, a methanesulfonyl group, a halogen group and the like; and a fragment having a functional group which is not involved in the formation of the NC bond is suitably protected with a temporary or permanent protective group.
  • the R 2 residue can be introduced in a suitable solvent and base such as N,N-diisopropylethylamine (DIEA) or triethylamine or an additive such as 1-hydroxybenzotriazole
  • a suitable solvent and base such as N,N-diisopropylethylamine (DIEA) or triethylamine or an additive such as 1-hydroxybenzotriazole
  • DIEA N,N-diisopropylethylamine
  • HOAt 1-hydroxyazabenzotriazole
  • a dehydrating agent such as a carbodiimide, a urea sulfonium salt, a phosphonium salt or a phosphonium salt 3 , -NR 3 R 4 or -SR 3 , reacting with a complementary fragment corresponding to a compound of formula (I), wherein R 2 is -OH, or by pre-forming an acid halide with, for example, thionyl chloride, and thereby obtaining
  • protecting group relates to a group that blocks an organic functional group and is capable of being removed under controlled conditions.
  • Protective groups, their relative reactivity and the conditions under which they remain inert are known to those skilled in the art.
  • amides such as amide acetate, amide benzoic acid, amide pivalate; carbamates such as benzyloxycarbonyl (Cbz or Z), 2-chlorobenzyl Base (CIZ), p-nitrobenzyloxycarbonyl (pNZ), tert-butoxycarbonyl (Boc), 2,2,2-trichloroethoxycarbonyl (Troc), 2-(trimethylsilyl) Ethyloxycarbonyl (Teoc), 9-fluorenylmethyloxycarbonyl (Fmoc) or allyloxycarbonyl (Alloc), trityl (Trt), methoxytrityl (Mtt , 2,4-dinitrophenyl (Dnp), N-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde), 1 -(4,4-dimethyl-2
  • Examples of representative protective groups for carboxyl groups are esters such as tert-butyl ester (tBu), allyl ester (All), triphenylmethyl ester (Trt ester), cyclohexyl ester (cHx), benzyl.
  • esters such as tert-butyl ester (tBu), allyl ester (All), triphenylmethyl ester (Trt ester), cyclohexyl ester (cHx), benzyl.
  • Base (Bzl), o-nitrobenzyl ester, p-nitrobenzyl ester, p-methoxybenzyl ester, trimethylsilylethyl ester, 2-phenylisopropyl ester, Mercaptomethyl ester (Fm), 4-(N-[1-(4,4-dimethyl-2,6-dioxocyclohexylene)-3-methylbutyl]amino)benzyl ester (Dmab), in particular; preferred protective groups of the invention are All, tBu, cHx, Bzl and Trt esters.
  • the side chain of the trifunctional amino acid can be protected by a temporary or permanent protective group orthogonal to the N-terminal and C-terminal protective groups during the course of the synthesis.
  • the hydroxyl group of the tyrosine side chain can be protected with a 2-bromobenzyloxycarbonyl group (2-BrZ), tBu, All, Bzl or 2,6-dichlorobenzyl (2,6-diClZ).
  • the histidine side chain can be protected with a protective group selected from the group consisting of Tos, Dnp, methyl (Me), Boc, benzyloxymethyl (Bom), Bzl, Fmoc, Mts, Trt and Mtt.
  • the amide groups of the glutamine and asparagine side chains can be protected by the Trt group or the xanthene group (Xan) or can be used without protection.
  • esters such as tBu ester, All ester, triphenylmethyl ester (Trt ester), cHx ester, Bzl ester, o-nitrobenzyl ester can be used.
  • Trt ester triphenylmethyl ester
  • cHx ester Bzl ester
  • o-nitrobenzyl ester o-nitrobenzyl ester
  • p-nitrobenzyl ester p-methoxybenzyl ester, trimethylsilylethyl ester, 2-phenylisopropyl ester, Fm ester or Dmab ester, and the like.
  • the arginine side chain can be protected by a protective group selected from the group consisting of: Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), Alloc, nitro, 2, 2, 4 6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) and 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc).
  • the oxime group of the tryptophan side chain can be protected by a formyl group (For), Boc, Mts or can be used without protection.
  • An amide can be used to protect the amino group of the lysine side chain, such as acetic acid amide, benzoic acid amide, pivalic acid amide; carbamates such as Cbz or Z, ClZ, pNZ, Boc, Troc, Teoc, Fmoc or Alloc , Trt, Mtt, Dnp, Dde, ivDde, Adpoc, etc.
  • the methionine side chain can be used in the form of a sulfoxide, in the form of a sulfone, or unprotected. The methionyl (sulfoxide) and methionyl (sulfone) side chains are unprotected.
  • the protective group strategy used is a strategy in which the amino group is protected by Boc, the carboxyl group is protected by Bzl, cHx or All, and the tyrosine side chain is protected by 2-BrZ or Bzl,
  • the side chain of the acid is protected by a Tos or Bom group
  • the aspartic acid and glutamic acid side chains are protected by Bzl, cHx or All
  • glutamine and asparagine are not protected by a side chain.
  • the methionine is not used.
  • side chain protection the arginine side chain is protected by Tos, the tryptophan side chain is protected by For or Mts and the lysine side chain is protected by ClZ, Fmoc or Alloc.
  • the protective group strategy employed is a strategy wherein the amino group is protected by Fmoc, the carboxyl group is protected by tBu, All or Trt ester, and the tyrosine side chain is protected by tBu, group
  • the amino acid side chain is protected by a Trt or Mtt group
  • the aspartic acid and glutamic acid side chains are protected by tBu or All
  • glutamine and asparagine are protected by a side chain Trt group
  • methionine is not added side
  • the arginine side chain is protected by Pmc or Pbf
  • the tryptophan side chain is protected by Boc or unprotected
  • the lysine side chain is protected by Boc, Trt or Alloc.
  • protective group also includes polymeric carriers in solid phase synthesis.
  • the possible solid support for use in the process of the invention involves a polystyrene support, a polyethylene glycol grafted to polystyrene, etc., such as, but not limited to, a-- Methyl benzhydrylamine resin (MBHA) [Matsueda GRet al., "A p-methylbenzhydrylamine resin for improved solid-phase synthesis of peptide amides", (1981), Peptides, 2, 4550], 2-chlorotriphenyl Base resin [Barlos K.
  • MBHA Methyl benzhydrylamine resin
  • MERS Middle East respiratory syndrome
  • polypeptide As defined herein, the terms “polypeptide”, “protein”, “peptide” and “amino acid sequence” are used interchangeably herein to refer to a polymer of amino acid residues of any length.
  • the polymer may be linear or branched, it may contain modified amino acids or amino acid analogs, and may be interrupted by chemical moieties other than amino acids.
  • the term also encompasses amino acid polymerizations that have been modified naturally or artificially (eg, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a label or biologically active component). Things.
  • the term “peptide” includes two or more naturally occurring or synthetic amino acids joined by a covalent bond, such as an amide bond.
  • amino acid is defined as having at least one primary, secondary, tertiary or quaternary amino group and at least one acid group, wherein the acid group can be a carboxylic acid, a sulfonic acid or a phosphoric acid or a mixture thereof.
  • the amino group may be " ⁇ ", “ ⁇ ", “ ⁇ ” to " ⁇ ” with respect to the acid group.
  • Suitable amino acids include, but are not limited to, the 20 common naturally occurring amino acids found in peptides (eg, A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P) , D, and L-isomers of S, T, W, Y, V (as known from single-letter or three-letter abbreviations) and naturally occurring and non-naturally produced by organic synthesis or other metabolic pathways Amino acids present.
  • peptides eg, A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P
  • D and L-isomers of S, T, W, Y, V (as known from single-letter or three-letter abbreviations) and naturally occurring and non-naturally produced by organic synthesis or other metabolic pathways Amino acids present.
  • the backbone of the "amino acid” may be substituted with one or more groups selected from the group consisting of halogen, hydroxy, thiol, and heterocyclic groups.
  • amino acid also includes within its scope glycine, alanine, valine, leucine, isoleucine, norleucine, methionine, valine, phenylalanine, color Amino acid, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamine, lysine, arginine, histidine, taurine , betaine, N-methyl alanine and the like. Amino acids in the (L) and (D) forms are included.
  • amino acid side chain refers to a moiety attached to the a-carbon of an amino acid.
  • amino acid side chain of alanine is a methyl group
  • amino acid side chain of phenylalanine is a phenylmethyl group
  • amino acid side chain of cysteine is a thiomethyl group
  • amino acid side chain of aspartic acid is Carboxymethyl
  • amino acid side chain of tyrosine is 4-hydroxybenzyl, and the like.
  • other non-naturally occurring amino acid side chains such as those naturally occurring (e.g., amino acid metabolites) or those prepared synthetically (e.g., alpha-disubstituted amino acids).
  • olefinic and its synonymous variant (eg, olefin or alkenyl), as used herein, denotes a monovalent group derived from a straight or branched hydrocarbon moiety having at least one carbon-carbon double bond by removal of a single hydrogen atom. .
  • the alkenyl moiety contains a specified number of carbon atoms.
  • C 2 -C 10 means that the group may have 2 to 10 carbon atoms. This means that it has C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 or C 10 alkenyl groups.
  • lower alkenyl refers to a C 2 -C 8 alkenyl chain.
  • alkenyl is a chain (straight or branched) having from 2 to 20 carbon atoms.
  • the olefinic group includes, for example, a vinyl group, a propenyl group, a butenyl group, a 1-methyl-2-buten-1-yl group and the like which may have one or more substituents.
  • the olefinic group substituents include, but are not limited to, any of the substituents described herein that result in the formation of a stable moiety.
  • substituents include, but are not limited to, the following groups: aliphatic group, alkyl group, alkenyl group, alkynyl group, heteroaliphatic group, heterocyclic group, aryl group, heteroaryl group, acyl group, oxo group, imino group, sulfur Oxyl, cyano, isocyano, amino, azido, nitro, alkylamino, heteroalkylamino, alkylaryl, arylalkyl, aliphatic oxy, heteroaliphatic oxy, Alkoxy, heteroalkoxy, aryloxy, heteroaryloxy, aliphatic thiooxy, heteroaliphatic thiooxy, alkylthiooxy, heteroalkylthiooxy, arylthiooxy, Heteroarylthiooxy groups, each of which may or may not be further substituted.
  • acyclic aliphatic group encompasses linear or branched alkyl, alkenyl and alkynyl groups.
  • alkyl refers to a linear or branched saturated group having from 1 to 24, preferably from 1 to 16, more preferably from 1 to 14, even more preferably from 1 to 12, still more preferably 1, 2, 3, 4 , 5 or 6 carbon atoms and bonded to the remainder of the molecule by simple bonds, including, for example and without limitation, methyl, ethyl, isopropyl, isobutyl, tert-butyl, heptyl, octyl, decyl, Dodecyl, lauryl, hexadecyl, octadecyl, pentyl, 2-ethylhexyl, 2-methylbutyl, 5-methylhexyl, and the like.
  • alkenyl group refers to a linear or branched group having from 2 to 24, preferably from 2 to 16, more preferably from 2 to 14, even more preferably from 2 to 12, still more preferably 2, 3, 4, 5 or 6 carbon atoms having one or more carbon-carbon double bonds, preferably having 1, 2 or 3 carbon-carbon double bonds, the bonds being conjugated or unconjugated, which are bonded to the rest of the molecule by simple bonds
  • alkynyl group refers to a linear or branched group having from 2 to 24, preferably from 2 to 16, more preferably from 2 to 14, even more preferably from 2 to 12, still more preferably 2, 3, 4, 5 or 6 carbon atoms having one or more carbon-carbon triple bonds, preferably 1, 2 or 3 carbon-carbon triple bonds, the bonds being conjugated or unconjugated, which are bonded to the rest of the molecule by simple bonds And includes, for example and without limitation, an ethynyl group, 1-propynyl, 2-propynyl, 1-butyl, 2-butyl, 3-butyl, pentyl, such as 1-pentyl, and the like.
  • the alkynyl group can also contain one or more carbon-carbon double bonds including, for example and without limitation, the group but-1-en-3-ynyl, pent-4-en-1-ynyl and the like.
  • alicyclic group is used in the present invention to encompass, for example and without limitation, a cycloalkyl or cycloalkenyl or cycloalkynyl group.
  • cycloalkyl refers to a saturated monocyclic or polycyclic aliphatic group having from 3 to 24, preferably from 3 to 16, more preferably from 3 to 14, even more preferably from 3 to 12, still more preferably 3,4. , 5 or 6 carbon atoms and bonded to the rest of the molecule by simple bonds, including, for example and without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclohexyl, dimethyl Cyclohexyl, octahydroquinone, decahydronaphthalene, dodecahydrophenene, and the like.
  • cycloalkenyl refers to a non-aromatic monocyclic or polycyclic aliphatic group having from 5 to 24, preferably from 5 to 16, more preferably from 5 to 14, even more preferably from 5 to 12, still more preferably 5 or 6 carbon atoms having one or more carbon-carbon double bonds, preferably 1, 2 or 3 carbon-carbon double bonds, the bonds being conjugated or unconjugated, which are bonded to the rest of the molecule by simple bonds, including For example, it is not limited to a cyclopent-1-en-1-yl group or the like.
  • cycloalkynyl refers to a non-aromatic monocyclic or polycyclic aliphatic group having from 8 to 24, preferably from 8 to 16, more preferably from 8 to 14, even more preferably from 8 to 12, still more preferably 8 or 9 carbon atoms having one or more carbon-carbon triple bonds, preferably 1, 2 or 3 carbon-carbon triple bonds, the bonds being conjugated or unconjugated, which are bonded to the rest of the molecule by simple bonds, including For example, it is not limited to a cyclooct-2-yn-1-yl group or the like.
  • the cycloalkynyl group can also contain one or more carbon-carbon double bonds including, for example and without limitation, cyclooct-4-en-2-ynyl groups and the like.
  • aryl group refers to an aromatic group having from 6 to 30, preferably from 6 to 18, more preferably from 6 to 10, still more preferably 6 or 10 carbon atoms, including 1, 2, 3 or 4
  • the aromatic ring is bonded or fused by a carbon-carbon bond, and includes, for example and not limited to, a phenyl group, a naphthyl group, a diphenyl group, a decyl group, a phenanthryl group or a fluorenyl group; or an aralkyl group.
  • aralkyl refers to an alkyl group substituted with an aromatic group, having from 7 to 24 carbon atoms and including, for example and without limitation, -(CH 2 ) 1-6 -phenyl, -(CH 2 ) 1- 6- (1-Naphthyl), -(CH 2 ) 1-6 -(2-naphthyl), -(CH 2 ) 1-6 -CH(phenyl) 2 and the like.
  • heterocyclyl group refers to a 3-10 membered hydrocarbonated ring wherein one or more of the atoms in the ring, preferably 1, 2 or 3 of the atoms in the ring are elements other than carbon such as nitrogen, Oxygen or sulfur, and may be saturated or unsaturated.
  • a heterocyclic ring can be a monocyclic, bicyclic or tricyclic ring system which can include a fused ring system; and a nitrogen, carbon or sulfur atom in the residue heterocyclic ring can be optionally oxidized; the nitrogen atom can be Optionally quaternized; and the residue heterocyclic group may be partially or fully saturated or aromatic.
  • heterocyclic group most preferably refers to a 5 or 6 membered ring.
  • saturated heterocyclic groups are dioxane, piperidine, piperazine, pyrrolidine, morpholine and thiomorpholine.
  • aromatic heterocyclic groups also referred to as heteroaromatic groups, are pyridine, pyrrole, furan, thiophene, benzofuran, imidazoline, hydroquinone, quinoline, pyridazine and naphthyridine.
  • heteroarylalkyl group refers to an alkyl group substituted with a substituted or unsubstituted aromatic heterocyclyl group having from 1 to 6 carbon atoms and an aromatic heterocyclic group having 2 to 24 carbon atoms and 1 to 3 non-carbon atoms, and include, for example and without limitation, -(CH 2 ) 1-6 -imidazolyl, -(CH 2 ) 1-6 -triazolyl, -(CH 2 ) 1-6 -thienyl, -(CH 2 ) 1-6 -furanyl, -(CH 2 ) 1-6 -pyrrolidinyl and the like.
  • halogen or variant, such as “halide” or “halo”, as used herein, refers to fluoro, chloro, bromo and iodo.
  • heteroatom or variant such as “hetero-” as used herein refers to O, N, NH and S.
  • alkoxy refers to a straight or branched alkoxy group. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy and the like.
  • amino refers to a group in the form -NR a R b , wherein R a and R b are independently selected from, but not limited to, hydrogen, optionally substituted alkyl, optionally substituted alkenyl, A group of optionally substituted alkynyl groups and optionally substituted aryl groups.
  • the compounds of the invention as described herein may be substituted with any number of substituents or functional moieties.
  • substituted (whether or not it is after the term “optionally) and the substituents contained in the formula of the invention all refer to a given structure with a group of a specified substituent.
  • the substituents may be the same or different at each position.
  • substituted as used herein is intended to include substitutions with all of the permissible substituents of the organic compound, any of the substituents described herein.
  • substituents include, but are not limited to, the following groups which result in the formation of a stable moiety: an aliphatic group, an alkyl group, an alkenyl group, an alkynyl group, a heteroaliphatic group, a heterocyclic group, an aryl group, a heteroaryl group, an acyl group, an oxo group.
  • a hetero atom such as nitrogen may have a hydrogen substituent and/or any suitable substituent as described herein that satisfies the valence of the hetero atom and results in the formation of a stable moiety.
  • the compound may contain one or more asymmetric centers and thus exist as a racemate and a racemic mixture, a single enantiomer, an individual diastereomer and a mixture of diastereomers. All such isomeric forms of these compounds are expressly included herein.
  • the compounds may also be represented in a variety of tautomeric forms, in which case all tautomeric forms of the compounds described herein are expressly included herein (eg, alkylation of the ring system may result in multiple sites) Alkylation, all of which are explicitly included herein). All such isomeric forms of such compounds are expressly included herein. All crystal forms of the compounds described herein are expressly included herein.
  • the term "functionally equivalent variant”, as used in the present invention, refers to all those compounds derived from the sequences of the compounds of the invention which are modified by substitution of one or two amino acids, provided that the compounds mentioned are maintained on a corresponding basis. At least 20%, at least 50%, at least 80% of the function of the modified compound. In a particular embodiment, the function of the compounds of the invention and their functionally equivalent variants are determined by quantifying their ability to inhibit neuronal exocytosis, particularly by quantifying the inhibition of SNARE complex formation.
  • the primary function of the SNARE fusion complex is to allow the neurotransmitter-filled vesicles to approach and contact the presynaptic cell membrane and enable calcium-mediated fusion of those vesicles, which results in neurotransmitter release.
  • concentration of the released neurotransmitter is reduced relative to cells that have not been treated with an inhibitory compound or a functionally equivalent variant.
  • modifications of the compounds of the invention for the production of functionally equivalent variants are carried out by replacing one or two amino acids of the peptide with one of its corresponding characteristically equivalent amino acids.
  • a compound or functionally equivalent variant of the invention is considered to inhibit the formation of a SNARE complex by reducing the formation of the SNARE complex by at least 15%, at least 20%, at least 30%, relative to a cell control to which any inhibitory compound has been added. At least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%.
  • the compounds of the present invention can exist as stereoisomers or as mixtures of stereoisomers; for example, the amino acids constituting them can have a configuration L-, D- or racemic independently of each other.
  • isomeric mixtures as well as racemic mixtures or mixtures of diastereomers, or pure diastereomers or enantiomers, depending on the number of asymmetric carbons and isomers or isomeric mixtures Asymmetric carbon present.
  • Preferred structurally pure isomers of the compounds of the invention i.e. enantiomers or diastereomers.
  • AA 1 can be described as -Lys-, it is understood that AA 1 is selected from -L-Lys-, -D-Lys- or a mixture of the two, either racemic or non-racemic.
  • the preparation procedures described in this document allow one skilled in the art to obtain the respective stereoisomers of the compounds of the invention by selecting the amino acids of the correct configuration.
  • compositions of the peptides of the invention are also within the scope of the invention.
  • pharmaceutically acceptable salt means a salt whose use in animals and more particularly in humans is recognized, and includes salts for the formation of base addition salts, whether they are inorganic or organic salts, Inorganic salts such as, but not limited to, lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum, or organic salts such as, but not limited to, ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, refined ammonia Acid, lysine, histidine or piperazine, etc.; or acid addition salts, whether they are organic or inorganic salts, organic salts such as, but not limited to, acetate, citrate, lactate, malonic acid Salt, maleate, tartrate, fumarate, benzoic acid, aspartate, glutamate, succinate, oleate, trifluoroacetate, oxalate,
  • the nature of the salt is not critical provided that it is cosmetically or pharmaceutically acceptable.
  • the pharmaceutically acceptable salts of the peptides of the invention can be obtained by conventional methods well known in the art (Berge SM et al, "Pharmaceutical Salts", (1977), J. Pharm. Sci., 66, 119, which is incorporated by reference in its entirety. Into this article).
  • the solid phase synthesis carrier Rink amide resin used in the examples is Tianjin Nankai Synthetic Co., Ltd.; HBTU, HOBt, DIEA and Fmoc protected natural amino acids or D-type unnatural amino acids are Shanghai Jill Biochemical Co., Ltd. and Beijing Oukanas Technology Co., Ltd. .
  • N-methylpyrrolidone (NMP) is a product of ACROS; trifluoroacetic acid (TFA) is a product of Beijing Bomaijie Technology Co., Ltd.; DMF and DCM are products of South Korea's Samsung; chromatographically pure acetonitrile is a product of Fisher. Other reagents are domestically produced pure products if they are not described.
  • 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 lysate was trifluoroacetic acid (TFA), and the crude peptide was dissolved in water and stored by lyophilization. Separated and purified by medium pressure liquid chromatography or high pressure liquid chromatography (HPLC), the pure peptide content is greater than 90%.
  • Matrix-assisted laser desorption time-of-flight mass spectrometry MALDI-TOF-MS was used to determine the molecular weight of the peptide sequence.
  • Peptide sequences were synthesized using a CEM microwave polypeptide synthesizer.
  • Blocking reagent 20% v/v acetic anhydride in DMF solution.
  • Rink Amide resin 0.23g (0.1mmol) of Rink Amide resin was weighed into the CEM microwave peptide synthesizer reactor, and then the amino acid, activator, activated base, deprotecting reagent, and blocking reagent were arranged at the above concentrations, and then fully automated with CEM microwave.
  • the peptide synthesizer performs the 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 1.25 g of a peptide resin.
  • peptide resin Pyrolysis of peptide resin: 1.25 g of a peptide resin synthesized by a microwave synthesizer was weighed, placed in a 250 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, reacted in ice bath for 30 minutes, and then removed. The reaction was completed by stirring the ice bath for 90 min. The reaction was completed.
  • the crude peptide was purified by medium pressure or high pressure chromatography.
  • the column is a C18 column, the eluent is acetonitrile, water and a small amount.
  • the column was previously equilibrated with 200 ml of 15% acetonitrile/water/0.1% glacial acetic acid solution.
  • 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, most of the solvent was removed by rotary evaporation, and the pure polypeptide was lyophilized.
  • the content of HPLC was more than 90%, and the molecular weight was confirmed by MALDI-TOF.
  • the polypeptide synthesis method is the same as in Example 1, except that after the completion of the linear polypeptide, the solid phase olefin metathesis reaction is carried out, and the side chain olefin group is covalently linked to synthesize the binding peptide.
  • the specific steps are as follows: the polypeptide resin is transferred to an eggplant-shaped bottle protected from light, protected by nitrogen for 5 min, and 25 g of a Grubbs catalyst is weighed, dissolved in 5 ml of dichloroethane, added to the reaction system, and stirred under nitrogen for 6 h. Repeat once after the reaction is completed. After completion of the reaction, the mixture was washed with DMF for 3 times, then with anhydrous methanol, and dried at room temperature under vacuum to obtain 1.02 g of a peptide resin.
  • the purification method was the same as in Example 1.
  • the method for synthesizing the polypeptide is the same as that in the first embodiment.
  • the dosage of the catalyst needs to be increased.
  • the specific steps are as follows: the polypeptide resin is transferred to an eggplant-shaped bottle protected from light, and protected by nitrogen for 5 min, and weighed.
  • Example 70 compounds inhibit MERS-CoV S2 subunit-mediated cell fusion activity evaluation (IC 50)
  • the cells can express strong fluorescence, and the subsequent fusion inhibition test can be performed.
  • Huh-7 cells were digested, and a cell suspension was prepared, and 4 to 5 ⁇ 10 4 cells were added to each well. Incubate at 37 ° C.
  • Example 71 Determination of the conformation of a compound by circular dichroism
  • X a of the formula (I) can be any hydrophobic amino acid without affecting the inhibitory activity of the polypeptide compound.
  • Compound 86 shown in Table 6 below was prepared by the same method as described in the above Examples 1-69, and the inhibition of the MERS-CoV S2 subunit-mediated cell fusion activity of the compound was tested according to the method described in Example 70. (IC 50 ), the results are shown in Table 6.
  • B is ⁇ -aminoisobutyric acid
  • the present inventors also performed a pseudovirus activity test on the polypeptide compound of the present invention to demonstrate that the short peptide was able to inhibit MERS-CoV infection from the virus level, and the effect was comparable to that of the positive control HR2P-M2 (i.e., Compound 70) (Table 8).
  • Huh-7 cells were digested 12 h earlier, and a single cell suspension was prepared using DMEM and the cell concentration was adjusted.
  • the target cell Huh-7 was digested 16 h before the experiment to prepare a cell suspension, and 10 4 cells were added to each well after adjusting the cell concentration.
  • the peptide drug polypeptide was diluted 2 times in DMEM medium containing 10% FBS, 60 ⁇ L per well; be careful to add the polypeptide slowly, and stir while adding to prevent the polypeptide from being precipitated due to the addition of a large amount of polypeptide in a short time.

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Abstract

提供了与通式(I)具有至少80％同一性的多肽化合物： R 1-U-Leu-Asp-Leu-Thr-Tyr-Glu-X a-Leu-X 1-Leu-Gln-X 2-Val-Val-Lys-X 3-Leu-Asn-Glu-X 4-Tyr-W-R 2 (I)，所述化合物可抗MERS-CoV感染。还提供了所述化合物的立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体，含有上述多肽化合物、其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体的药物组合物，以及上述多肽化合物、其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体的药物组合物在治疗或预防MERS-CoV感染相关疾病，尤其是中东呼吸综合征中的用途。

Description

抑制MERS-CoV感染的多肽 技术领域
本发明属于生物医药领域，涉及抗MERS-CoV感染的多肽化合物，具体地，涉及与通式(I)具有至少80％同一性的多肽化合物：
R 1-U-Leu-Asp-Leu-Thr-Tyr-Glu-X a-Leu-X 1-Leu-Gln-X 2-Val-Val-Lys-X 3-Leu-Asn-Glu-X 4-Tyr-W-R 2(I)，
其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体。本发明还涉及含有这样的多肽化合物、其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体的药物组合物，以及这样的多肽化合物、其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体的药物组合物治疗或预防MERS-CoV感染相关疾病尤其是中东呼吸综合征的用途。
背景技术
2012年，一种被称为中东呼吸综合征冠状病毒(MERS-CoV)的新型冠状病毒在中东地区流行。该病毒可引起SARS样的疾病，导致多器官功能的衰竭，在人群中造成约40％的病死率。由于MERS-CoV具有人传人的能力，因而存在在人群中大规模流行的可能性，因此，急需尽快对MERS-CoV进行深入研究，以寻找其药物设计靶点并开发相应的抗MERS-CoV药物，用于患者的临床治疗及预防MERS-CoV潜在的大流行可能。
MERS-CoV融合蛋白主要由S1亚基和S2亚基构成。其中，S1亚基负责与宿主细胞表面的CD26受体特异性识别结合；S2亚基负责介导病毒与靶细胞膜之间的融合。S2亚基又可进一步分为融合肽(fusion peptide，FP)、N末端七联重复序列(N-terminal heptad repeat，NHR，也称为HR1)和C末端七联重复序列(C-terminal heptad repeat，CHR，也称为HR2)功能区。S1亚基与CD26受体结合后，S2亚基发生构象改变，将其FP插入到宿主细胞膜中，同时诱导HR1和HR2区域相互作用形成六股α螺旋束(6HB)结构，从而介导病毒与宿主细胞膜的融合。晶体结构研究表明，MERS-CoV HR2功能区中L1262到Y1280为α螺旋区。该区域对6HB的形成至关重要。
在过去的几十年中，化学家和生物学家采用了各种巧妙的方法来增强α-螺旋结 构，包括基于非共价键和共价键的策略，其中一种是多肽装订技术。
多肽装订技术是“固定螺旋”(helix stabilization)行之有效的策略之一。共价桥连的多肽装订技术可以更有效地“锁定”多肽在溶液中的α螺旋构象。利用该技术构建的装订肽(stapled peptide)可以用于选择性地调控蛋白间相互作用。多肽装订技术的总体思路是将α螺旋中不与靶蛋白相互作用一侧多肽序列的i→i+3或i→i+4或i→i+7位的氨基酸残基侧链通过正交反应进行共价桥连，从而锁定多肽的α螺旋构象。
发明内容
本发明人基于MERS-CoV HR2 α螺旋区多肽序列，构建了高活性、代谢稳定的MERS-CoV融合抑制剂，由此提供了治疗及预防MERS-CoV的解决方案。
因此，在一个方面，本发明涉及与通式(I)具有至少80％同一性的多肽化合物：
R 1-U-Leu-Asp-Leu-Thr-Tyr-Glu-X a-Leu-X 1-Leu-Gln-X 2-Val-Val-Lys-X 3-Leu-Asn-Glu-X 4-Tyr-W-R 2(I)，
其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体；
其中X a为能够与MERS-COV HR2发生疏水相互作用的氨基酸残基，其选自以下D型或L型疏水性氨基酸：丙氨酸(Ala)、缬氨酸(Val)、亮氨酸(Leu)、异亮氨酸(Ile)、脯氨酸(Pro)、苯丙氨酸(Phe)、色氨酸(Trp)、正亮氨酸(n-Leu)和蛋氨酸(Met)；
X 1、X 2、X 3和X 4各自是独立选择的D型或L型氨基酸；
U和W不存在或各自是独立选择的一个或多个附加的D型或L型氨基酸；
R 1选自：H，衍生自聚乙二醇的聚合物，非环状取代的或未经取代的脂族基团，取代的或未经取代的脂环基，取代的或未经取代的杂环基，取代的或未经取代的杂芳基烷基，取代的或未经取代的芳基，取代的或未经取代的芳烷基和R 5-CO-，其中R 5选自：H，非环状取代的或未经取代的脂族基团，取代的或未经取代的脂环基，取代的或未经取代的芳基，取代的或未经取代的芳烷基，取代的或未经取代的杂环基和取代的或未经取代的杂芳基烷基；
R 2选自：-NR 3R 4，-OR 3和-SR 3，其中R 3和R 4独立地选自：H，衍生自聚乙二醇的聚合物，非环状取代的或未经取代的脂族基团，取代的或未经取代的脂环基，取代的或未经取代的杂环基，取代的或未经取代的杂芳基烷基，取代的或未经取代的芳基，和取代的或未经取代的芳烷基；和
R 1和R 2不是D型或L型氨基酸。
在优选的实施方案中，本发明涉及与通式(I)具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的多肽化合物或其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体。
在优选的实施方案中，X a选自如下D型或L型的氨基酸：甲硫氨酸，亮氨酸，异亮氨酸，缬氨酸和正亮氨酸。
在优选的实施方案中，R 1选自：H，乙酰基，月桂酰基，肉豆蔻酰基和棕榈酰基。在优选的实施方案中，R 1选自：H或R 5-CO-；其中R 5选自：H或者取代的或未经取代的C 1-C 24烷基。
在优选的实施方案中，R 2选自：-NR 3R 4和-OR 3，其中R 3和R 4独立地选自H，甲基，乙基，己基，十二烷基和十六烷基。在优选的实施方案中，R 2选自：-NR 3R 4或-OR 3，其中R 3和R 4独立地选自：H或者取代的或未经取代的C 1-C 24烷基。
不受理论约束，据信在本发明的多肽化合物处于α螺旋构象时，通式(I)中的X 1、X 2、X 3和X 4位于不与靶蛋白相互作用的一侧，而是暴露在与溶剂接触的一面。进而，本发明人令人惊讶地发现，在X 1、X 2、X 3和X 4中引入一个或多个α螺旋稳定氨基酸可以帮助本发明的多肽化合物稳定在α螺旋构象，从而能够有效地抑制MERS-CoV融合，并适合用于治疗及预防MERS-CoV。
因此，在一些实施方案中，X 1、X 2、X 3和X 4中的一个或多个独立地是α螺旋稳定氨基酸。在优选的实施方案中，X 1、X 2、X 3和X 4中的两个或更多个独立地是α螺旋稳定氨基酸。在优选的实施方案中，X 1与X 2、X 1与X 3、X 2与X 3和/或X 3与X 4是α螺旋稳定氨基酸。在优选的实施方案中，X 1、X 2与X 3是α螺旋稳定氨基酸。在优选的实施方案中，X 1、X 3与X 4是α螺旋稳定氨基酸。在优选的实施方案中，X 2、X 3与X 4是α螺旋稳定氨基酸。在优选的实施方案中，X 1、X 2、X 3与X 4是α螺旋稳定氨基酸。
在这样的实施方案中，在X 1、X 2、X 3和X 4中不是α螺旋稳定氨基酸的氨基酸残基可以各自独立地选自任何氨基酸，优选地，选自以下D型或L型氨基酸：谷氨酰胺、丙氨酸和丝氨酸。
如本文所用的“α螺旋稳定氨基酸”意指能为MERS-CoV融合抑制剂肽的α螺旋构象增加稳定性的氨基酸。α螺旋稳定氨基酸是本领域中所公知。适合本文目的的合适的α螺旋稳定氨基酸包括，但不限于：丙氨酸、正缬氨酸、正亮氨酸、α-氨基丁酸、 α-氨基异丁酸(AIB)、亮氨酸、异亮氨酸、缬氨酸等。在一些实施方案中，α螺旋稳定氨基酸为天然存在的蛋白的α螺旋的一部分的任何氨基酸，例如Leu、Phe、Ala、Met、Gly、Ile、Ser、Asn、Glu、Asp、Lys、Arg。
在优选的实施方案中，α螺旋稳定氨基酸是α,α-双取代的氨基酸。在具体的实施方案中，α,α-双取代氨基酸包含第一取代基和第二取代基，第一取代基和第二取代基各自键合至α碳，其中第一取代基和第二取代基各自独立地选自任选地用羟基、酰胺、巯基、卤基所取代的C1-C4烷基，或第一取代基和第二取代基与它们所连接的α碳形成环(例如C3-C8环)。在示例性实施方案中，第一取代基和第二取代基各自独立地选自：甲基、乙基、丙基以及正丁基，或第一取代基和第二取代基形成环辛烷或环庚烷(例如1-氨基环辛烷-1-羧酸)。在示例性实施方案中，第一取代基与第二取代基相同。在示例性实施方案中，第一取代基与第二取代基不同。在示例性实施方案中，第一取代基和第二取代基各自为C1-C4烷基。在一些实施方案中，第一取代基和第二取代基各自为C1或C2烷基。在示例性实施方案中，第一取代基和第二取代基各自为甲基，使得所述α,α-双取代氨基酸为α-氨基异丁酸(AIB)。在其他示例性实施方案中，α,α-双取代氨基酸为1-氨基环丙烷-1-羧酸(ACPC)。在其他示例性实施方案中，α,α-双取代氨基酸还可以是具有烯属侧链的α,α-双取代的氨基酸。具有烯属侧链的α,α-双取代的氨基酸的非限制性实例可以包括例如(R)-2-(4’-戊烯基)丙氨酸(R 5)、(S)-2-(4’-戊烯基)丙氨酸(S 5)、(S)-2-(4’-辛烯基)丙氨酸(S8)、(R)-2-(4’-辛烯基)丙氨酸(R8)和2-氨基-2-(4-戊烯基)-6-庚烯酸(B 5)。
在优选的实施方案中，本发明涉及与SEQ ID NO.1-37中的任一个具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的多肽化合物或其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体。
在另一方面，本发明人还利用多肽装订技术将本发明的多肽化合物中的两个或更多个氨基酸残基交联起来，以形成所谓的装订肽。
如本文所用，术语“装订肽”是指通过分子内交联体而被稳定在所需构象的肽。在优选的实施方案中，装订肽是通过分子内交联体而被稳定在α螺旋构象的肽。关于装订肽的描述，可参见例如Verdine GL,Hilinski GJ(2012),"Stapled peptides for intracellular drug targets",Methods in Enzymology.503:3–33；Walensky LD,Bird GH(2014),"Hydrocarbon-stapled peptides:principles,practice,and progress", Journal of Medicinal Chemistry,57(15):6275–88；Blackwell Helen E.,Grubbs Robert H,(1998),"Highly Efficient Synthesis of Covalently Cross-Linked Peptide Helices by Ring-Closing Metathesis",Angewandte Chemie International Edition,37(23):3281–3284，其通过引用整体并入本文。
本发明人令人惊讶地发现，基于MERS-CoV HR2 α螺旋区多肽序列构建的在其中特定氨基酸位点上包含分子内交联体的装订肽是具有非常高活性的MERS-CoV融合抑制剂，其能够有效地治疗和预防MERS-CoV感染以及与MERS-CoV感染相关的疾病。
具体地，本发明人发现在通式(I)的多肽序列中，在X 1、X 2、X 3和X 4中的两个或更多个之间形成交联体可以产生具有非常高活性的MERS-CoV融合抑制剂，其能够有效地治疗和预防MERS-CoV感染以及与MERS-CoV感染相关的疾病。
因此，在一个方面，本发明涉及与通式(I)具有至少80％同一性的多肽化合物：
R 1-U-Leu-Asp-Leu-Thr-Tyr-Glu-X a-Leu-X 1-Leu-Gln-X 2-Val-Val-Lys-X 3-Leu-Asn-Glu-X 4-Tyr-W-R 2(I)，
其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体；
其中X a为能够与MERS-COV HR2发生疏水相互作用的氨基酸残基，其选自以下D型或L型疏水性氨基酸：丙氨酸(Ala)、缬氨酸(Val)、亮氨酸(Leu)、异亮氨酸(Ile)、脯氨酸(Pro)、苯丙氨酸(Phe)、色氨酸(Trp)、正亮氨酸(n-Leu)和蛋氨酸(Met)；
X 1、X 2、X 3和X 4各自是独立选择的D型或L型氨基酸，并且在X 1、X 2、X 3和X 4中的两个或更多个之间形成交联体；
U和W不存在或各自是独立选择的一个或多个附加的D型或L型氨基酸；
R 1选自：H，衍生自聚乙二醇的聚合物，非环状取代的或未经取代的脂族基团，取代的或未经取代的脂环基，取代的或未经取代的杂环基，取代的或未经取代的杂芳基烷基，取代的或未经取代的芳基，取代的或未经取代的芳烷基和R 5-CO-，其中R 5选自：H，非环状取代的或未经取代的脂族基团，取代的或未经取代的脂环基，取代的或未经取代的芳基，取代的或未经取代的芳烷基，取代的或未经取代的杂环基和取代的或未经取代的杂芳基烷基；
R 2选自：-NR 3R 4，-OR 3和-SR 3，其中R 3和R 4独立地选自：H，衍生自聚乙二醇的聚合物，非环状取代的或未经取代的脂族基团，取代的或未经取代的脂环基，取代 的或未经取代的杂环基，取代的或未经取代的杂芳基烷基，取代的或未经取代的芳基，和取代的或未经取代的芳烷基；和
R 1和R 2不是D型或L型氨基酸。
在优选的实施方案中，本发明涉及与通式(I)具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的多肽化合物或其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体。
在优选的实施方案中，X a选自如下D型或L型的氨基酸：甲硫氨酸，亮氨酸，异亮氨酸，缬氨酸和正亮氨酸。
在优选的实施方案中，R 1选自：H，乙酰基，月桂酰基，肉豆蔻酰基和棕榈酰基。在优选的实施方案中，R 1选自：H或R 5-CO-；其中R 5选自：H或者取代的或未经取代的C 1-C 24烷基。
在优选的实施方案中，R 2选自：-NR 3R 4和-OR 3，其中R 3和R 4独立地选自H，甲基，乙基，己基，十二烷基和十六烷基。在优选的实施方案中，R 2选自：-NR 3R 4或-OR 3，其中R 3和R 4独立地选自：H或者取代的或未经取代的C 1-C 24烷基。
在优选的实施方案中，在X 1与X 2、X 1与X 3、X 2与X 3和/或X 3与X 4之间形成交联体。在优选的实施方案中，在X 1、X 2与X 3之间形成交联体。在优选的实施方案中，在X 1、X 3与X 4之间形成交联体。在优选的实施方案中，在X 2、X 3与X 4之间形成交联体。在优选的实施方案中，在X 1、X 2、X 3与X 4之间形成交联体。
在优选的实施方案中，本发明的多肽化合物包含一个或多个在X 1与X 2、X 1与X 3、X 2与X 3和X 3与X 4之间形成的交联体。
不受理论约束，据信在本发明的多肽化合物处于α螺旋构象时，通式(I)中的X 1、X 2、X 3和X 4位于不与靶蛋白相互作用的一侧，而是暴露在与溶剂接触的一面，并且通过将X 1、X 2、X 3和X 4中的两个或更多个交联起来，可以帮助本发明的多肽化合物稳定在α螺旋构象。因此，在本发明的一些实施方案中，X 1、X 2、X 3和X 4可以选自任何氨基酸，只要其能够通过如本文所述的交联体彼此连接。根据所需的具体交联形式选择合适的天然或非天然氨基酸在本领域技术人员的能力范围之内。在这样的实施方案中，在X 1、X 2、X 3和X 4中未形成交联的氨基酸残基可以各自独立地选自任何氨基酸，优选地，选自以下D型或L型氨基酸：谷氨酰胺、丙氨酸和丝氨酸。
如本文所用，术语“交联体”或其语法变体是指两个肽结构域(即，例如螺旋肽的两个环)的分子内连接。当肽具有螺旋二级结构时，交联体可以是大环，其是外部(非 螺旋肽结构的一部分)的核心或固有(非交联)的螺旋肽结构。大环可以包括全碳氢连接环，并且包含与肽的至少两个氨基酸的α-碳连接的侧链。交联的肽具有至少一个交联体。在各种实例中，交联的肽具有1、2或3个或至少1、2或3个交联体。在一个实例中，本文公开的肽仅包含一个交联体。
可以使用本领域已知的任何交联体，只要其能够将通式(I)的X 1、X 2、X 3和X 4位置的两个或更多个氨基酸连接起来。
在一个实施方案中，交联体可以是非共价或共价交联体。
可以使用本领域已知的任何非共价交联体，只要其能够将通式(I)的X 1、X 2、X 3和X 4位置的两个或更多个氨基酸连接起来。在优选的实施方案中，非共价交联体可以选自但不限于包含氢键、静电相互作用或金属螯合的交联体(参见例如，V.Azzarito,K.Long,N.S.Murphy and A.J.Wilson,Nat.Chem.,2013,5,161–173；V.Haridas,Eur.J.Org.Chem.,2009,5112–5128.；其均通过引用整体并入本文)。
还可以使用本领域已知的任何共价交联体，只要其能够将通式(I)的X 1、X 2、X 3和X 4位置的两个或更多个氨基酸连接起来。在优选的实施方案中，共价交联体可以选自但不限于包含烃键联、分子内酰胺键、二硫键、硫醚键或Cu(I)-催化的叠氮-炔烃环加成(CuAAC)反应的交联(参见例如，YH Lau等人，“Peptide stapling techniques based on different macrocyclisation chemistries”，Cheminform,2015,46(15)；White CJ,Yudin AK,“Contemporary strategies for peptide macrocyclization”,Nature chemistry,2011,3(7):509；其均通过引用整体并入本文)。
在一个实施方案中，还可以使用接头来将通式(I)的X 1、X 2、X 3和X 4位置的两个或更多个氨基酸间接连接起来，从而形成包含接头的交联体。适合于本文目的的这样的接头是本领域公知的，并且可以是任何合适的接头。这样的接头的实例可以包括例如α,ω-二氨基烷烃接头、1,4-二氨基丙烷接头或1,5-二氨基戊烷接头可光切换的接头、官能化的双点击接头(基于CuAAC反应)等((参见例如，YH Lau等人(同上)；White CJ等人(同上))。
在优选的实施方案中，所述包含烃键联的交联体是全碳氢交联体。在优选的实施方案中，在X 1、X 2、X 3和X 4中形成所述全碳氢交联的氨基酸残基是具有烯属侧链的α,α-双取代的氨基酸。应理解，在本发明的多肽化合物中，X 1、X 2、X 3和X 4可以是任何具有烯属侧链的α,α-双取代的氨基酸，只要其彼此之间可以通过烯属侧链形成全碳氢交联体。这样的具有烯属侧链的α,α-双取代的氨基酸的非限制性实例可以包括例 如(R)-2-(4’-戊烯基)丙氨酸(R 5)、(S)-2-(4’-戊烯基)丙氨酸(S 5)、(S)-2-(4’-辛烯基)丙氨酸(S8)、(R)-2-(4’-辛烯基)丙氨酸(R8)和2-氨基-2-(4-戊烯基)-6-庚烯酸(B 5)。关于全碳氢交联体的具体描述，可参见例如Loren D.Walensky and Gregory H.Bird,“Hydrocarbon-Stapled Peptides:Principles,Practice,and Progress”,J Med Chem.2014Aug 14；57(15):6275–6288，其通过引用整体并入本文。
在优选的实施方案中，本发明涉及与SEQ ID NO.38-69中的任一个具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的多肽化合物或其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体。
本发明人已发现，本发明的多肽化合物能够有效地抑制MERS-CoV与细胞的融合。
因此，在优选的实施方案中，本发明的多肽化合物可用于抑制MERS-CoV融合。在优选的实施方案中，本发明的多肽化合物可用于治疗或预防MERS-CoV感染相关疾病。在优选的实施方案中，所述MERS-CoV感染相关疾病为中东呼吸综合征。
因此，在一个方面，本发明还涉及药物组合物，其包含至少一种本发明的多肽化合物或其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体，以及至少一种药学上可接受的载体、稀释剂、赋形剂或辅助剂。所述肽本身可以众多种形式中的任何一种存在于所述组合物中。例如，两种或更多种肽可仅仅混合在一起，或可通过复合、结晶或离子键合或共价键合更紧密地缔合。
在一个方面，本发明涉及MERS-CoV融合抑制剂，其包含至少一种本发明的多肽化合物或其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体。
如本文所述的肽或其药物组合物或其MERS-CoV融合抑制剂可以许多方式施用，这取决于期望局部施用还是全身性施用并且取决于待治疗的区域。在一些实施方案中，可经以下方式向患者施用所述肽或其药物组合物或其MERS-CoV融合抑制剂：口腔或直肠、或经粘膜、或肠内、或肌内、或皮下、或髓内、或鞘内、或直接心室内、或静脉内、或玻璃体内、或腹膜内、或鼻内、或眼内。
合适的药学上可接受的载体的例子包括但不限于无菌水、盐水、缓冲液、磷酸缓冲盐水、缓冲氯化钠、植物油、最小必需培养基(MEM)、具有HEPES的MEM，等等。
在一个方面，本发明涉及本发明的多肽化合物或其立体异构体、其混合物、其药 学上可接受的盐和/或其功能等价的变体用于抑制MERS-CoV融合的用途。
在一个方面，本发明涉及本发明的多肽化合物或其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体用于治疗或预防MERS-CoV感染相关疾病的用途。
在优选的实施方案中，所述MERS-CoV感染相关疾病为中东呼吸综合征。
在一个方面，本发明涉及本发明的多肽化合物或其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体在制备MERS-CoV融合抑制剂中的用途。
在一个方面，本发明涉及本发明的多肽化合物或其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体在制备用于治疗或预防MERS-CoV感染相关疾病的药物中的用途。
在优选的实施方案中，所述MERS-CoV感染相关疾病为中东呼吸综合征。
在一个方面，本发明涉及制备本发明的多肽化合物的方法，其包括：a.合成与通式(I)具有至少80％同一性的多肽，和b.将X 1、X 2、X 3和X 4中的两个或更多个通过交联连接起来。在优选的实施方案中，通过交联连接X 1与X 2、X 1与X 3、X 2与X 3和/或X 3与X 4。在优选的实施方案中，通过交联连接X 1、X 2与X 3。在优选的实施方案中，通过交联连接X 1、X 3与X 4。在优选的实施方案中，通过交联连接X 2、X 3与X 4。在优选的实施方案中，通过交联连接X 1、X 2、X 3与X 4。
在本发明的实施方案中，如本文所述的多肽化合物、它们的立体异构体、其混合物、它们的药学上可接受的盐和/或其功能等价的变体的合成能够根据现有技术已知的任何常规方法进行，比如用固相肽合成方法[Stewart J.M.y Young J.D.,"Solid Phase Peptide Synthesis,2nd edition",(1984),Pierce Chemical Company,Rockford,Illinois；Bodanzsky M.y Bodanzsky A.,"The practice of Peptide Synthesis",(1994),Springer Verlag,Berlin；LloydWilliams P.et al.,"Chemical Approaches to the Synthesis of Peptides and Proteins",(1997),CRC,Boca Raton,FL,USA]，溶液中的合成、酶促合成[Kullmann W."Proteases as catalysts for enzymic syntheses of opioid peptides",(1980),J.Biol.Chem.,255(17),82348238]或其任意组合。化合物还能够通过经过目的在于产生所希望序列的基因工程修饰的或未修饰的细菌菌株的发酵来获得，或者通过动物、真菌或优选植物来源的蛋白的游离含有至少所希望序列的肽段的受控水解来获得。例如，可以使用编码如本文所述的多肽氨基酸序列的核酸序列以及任选地进行适当的氨基酸修饰来产生本发明的化合物。
仅作为示例，获得本发明的多肽化合物、它们的立体异构体和其混合物的方法可以包括下述阶段：
-将与N-末端保护的和C-末端游离的氨基酸与N-末端游离的和C-末端保护或结合至固体载体的氨基酸偶联；
-消除保护N-末端的基团；
-重复偶联程序并且消除保护N-末端的基团直至获得所希望的肽序列；
-消除保护C-末端的基团或裂解固体载体；
-任选地，将肽序列中的两个或更多个氨基酸通过适当的交联体交联。
优选，C-末端结合至固体载体并且该过程在固相中进行，因此包括将N-末端保护的和C-末端游离的氨基酸与N-末端游离的和C-末端结合至聚合物载体的氨基酸偶联；消除保护N-末端的基团；和按必要次数重复该程序以获得所希望长度的化合物，最终随后从最初的聚合物载体的裂解合成的化合物。
在整个合成当中，氨基酸侧链的官能团保持用暂时或永久保护性基团方便地保护，并且能够与从聚合物载体裂解肽的过程同时地或正交地脱保护。
另选地，固相合成能够用会聚策略进行：将肽与聚合物载体偶联或者与预先结合至聚合物载体的肽或氨基酸偶联。会聚合成策略是本领域技术人员广泛已知的并且描述于Lloyd-Williams P.et al.,"Convergent Solid-Phase Peptide Synthesis",(1993),Tetrahedron,49(48),11065-11133。
在采用现有技术已知的标准程序和条件的情况下，本发明过程能够以无差别顺序包括额外的N-末端和C-末端脱保护和/或从聚合物载体裂解肽的阶段；在此之后这些末段官能团能够加以修饰。在式(I)肽固定至聚合物载体时或一旦肽已从聚合物载体分开，则能够进行N-末端和C-末端的任选修饰。
任选地，R 1能够这样引入：在适当碱和溶剂存在下，通过亲核取代反应，将本发明化合物N-末端与R 1-X化合物反应，其中R 1具有前述含义和X是离去基团比如且不限于甲苯磺酰基基团、甲磺酰基基团和卤素基团等；其中具有并不牵涉于N-C键形成中的官能团的片段用暂时或永久保护性基团加以适宜保护。
任选地和/或额外地，R 2残基能够这样引入：在适当溶剂和碱比如N,N-二异丙基乙胺(DIEA)或三乙胺或添加剂比如1-羟基苯并三唑(HOBt)或1-羟基氮杂苯并三唑(HOAt)和脱水剂比如碳二亚胺、脲鎓盐、鏻盐或脒鎓盐等存在下，将化合物HR 2，其中R 2是-OR 3、-NR 3R 4或-SR 3，与互补片段反应相应于式(I)化合物，其中R 2是-OH， 或者通过与例如亚硫酰氯预先形成酰卤，和由此获得通式(I)的根据本发明的肽，其中片段具有并不牵涉于N-C键形成中官能团用暂时或永久保护性基团加以适宜保护；或者另选地其它R 2残基可以通过同时掺入肽从聚合物载体裂解的过程来引入。
本领域技术人员会容易地理解C-末端和N-末端的脱保护/裂解步骤和它们随后的衍生化能够根据现有技术已知的过程以不同顺序进行。
术语"保护性基团"涉及阻塞有机官能团的和能够在受控条件下除去的基团。保护性基团、它们的相对反应性和它们保持惰性的条件是本领域技术人员已知的。
氨基基团的代表性保护性基团的实例尤其是酰胺比如酰胺乙酸酯，酰胺苯甲酸，酰胺特戊酸酯；氨基甲酸酯类比如苄氧基羰基(Cbz或Z)，2-氯苄基(CIZ)，对-硝基苄氧基羰基(pNZ)，叔丁氧基羰基(Boc)，2,2,2-三氯乙氧羰基(Troc)，2-(三甲基甲硅烷基)乙基氧基羰基(Teoc)，9-芴基甲基氧基羰基(Fmoc)或烯丙基氧基羰基(Alloc)，三苯甲基(Trt)，甲氧基三苯甲基(Mtt)，2,4-二硝基苯基(Dnp)，N-1-(4,4-二甲基-2,6-二氧代环己-1-亚基)乙基(Dde)，1-(4,4-二甲基-2,6-二氧代-亚环己基)-3-甲基丁基(ivDde)，1-(1-金刚烷基)-1-甲基乙氧基羰基(Adpoc)，优选Boc或Fmoc。
羧基基团代表性保护性基团的实例是酯，比如叔丁基酯(tBu)，烯丙基酯(All)，三苯基甲基酯(Trt酯)，环己基酯(cHx)，苄基酯(Bzl)，邻-硝基苄基酯，对-硝基苄基酯，对-甲氧基苄基酯，三甲基甲硅烷基乙基酯，2-苯基异丙基酯，芴基甲基酯(Fm)，4-(N-[1-(4,4-二甲基-2,6-二氧代亚环己基)-3-甲基丁基]氨基)苄基酯(Dmab)，尤其；本发明的优选保护性基团是All，tBu，cHx，Bzl和Trt酯。
三官能氨基酸的侧链能够在合成过程期间用与N-末端和C-末端保护性基团正交的暂时或永久保护性基团加以保护。
酪氨酸侧链的羟基基团能够用2-溴苄氧基羰基基团(2-BrZ)，tBu，All，Bzl或2,6-二氯苄基(2,6-diClZ)等来保护。组氨酸侧链能够用选自的保护性基团来保护：Tos，Dnp，甲基(Me)，Boc，苄氧基甲基(Bom)，Bzl，Fmoc，Mts，Trt和Mtt。谷氨酰胺和天冬酰胺侧链的酰胺基团能够通过Trt基团或呫吨基基团(Xan)保护或能够不加未保护地使用。为了保护天冬氨酸和谷氨酸侧链的羧基基团能够使用酯比如tBu酯，All酯，三苯基甲基酯(Trt酯)，cHx酯，Bzl酯，邻-硝基苄基酯，对-硝基苄基酯，对-甲氧基苄基酯，三甲基甲硅烷基乙基酯，2-苯基异丙基酯，Fm酯或Dmab酯等。精氨酸侧链可以通过选自的保护性基团保护：Tos，4-甲氧基-2,3,6-三甲基苯磺酰基(Mtr)，Alloc，硝基，2,2,4,6,7-五甲基二氢苯并呋喃-5-磺酰基(Pbf)和2,2,5,7,8-五甲基色满-6- 磺酰基(Pmc)。色氨酸侧链的吲哚基团能够通过甲酰基基团(For)，Boc，Mts保护或者能够不加保护地使用。为了保护赖氨酸侧链的氨基基团能够使用酰胺，比如乙酸酰胺，苯甲酸酰胺，特戊酸酰胺；氨基甲酸酯类比如Cbz或Z，ClZ，pNZ，Boc，Troc，Teoc，Fmoc或Alloc，Trt，Mtt，Dnp，Dde，ivDde，Adpoc等。甲硫氨酸侧链能够以亚砜形式、以砜形式保护或不加保护地使用。甲硫氨酰基(亚砜)和甲硫氨酰基(砜)侧链是未加保护的。
在优选实施方式中，所用的保护性基团策略是如下的策略：氨基基团通过Boc保护，羧基基团通过Bzl、cHx或All保护，酪氨酸侧链通过2-BrZ或Bzl保护，组氨酸侧链通过Tos或Bom基团保护，天冬氨酸和谷氨酸侧链通过Bzl、cHx或All保护，谷氨酰胺和天冬酰胺未加侧链保护地使用，甲硫氨酸未加侧链保护地使用，精氨酸侧链通过Tos保护，色氨酸侧链通过For或Mts保护并且赖氨酸侧链通过ClZ、Fmoc或Alloc保护。
在又一优选的实施方式中，所用的保护性基团策略是如下的策略：氨基基团通过Fmoc保护，羧基基团通过tBu、All或Trt酯保护，酪氨酸侧链通过tBu保护，组氨酸侧链通过Trt或Mtt基团保护，天冬氨酸和谷氨酸侧链通过tBu或All保护，谷氨酰胺和天冬酰胺通过侧链Trt基团保护，甲硫氨酸未加侧链保护地使用，精氨酸侧链通过Pmc或Pbf保护，色氨酸侧链通过Boc保护或未加保护地使用，并且赖氨酸侧链通过Boc、Trt或Alloc保护。
这些和其它保护性基团的实例，它们的引入和除去，能够参考文献[Atherton B.and Sheppard R.C.,"Solid Phase Peptide Synthesis:A practical approach",(1989),IRL Oxford University Press]。术语"保护性基团"也包括固相合成中的聚合物载体。
在合成完全或部分发生在固相中的情况下，用于本发明过程中的可能固体载体牵涉聚苯乙烯载体，接枝至聚苯乙烯的聚乙二醇等，比如且不限于对-甲基二苯甲基胺树脂(MBHA)[Matsueda G.R.et al.,"A p-methylbenzhydrylamine resin for improved solid-phase synthesis of peptide amides",(1981),Peptides,2,4550]，2-氯三苯甲基树脂[Barlos K.et al.,"Darstellung geschützter PeptidFragmente unter Einsatz substituierter TriphenylmethylHarze",(1989),Tetrahedron Lett.,30,39433946；Barlos K.et al.,"Veresterung von partiell geschützten PeptidFragmenten mit Harzen。Einsatz von 2-Chlorotritylchlorid zur Synthese von Leu1Gastrin I"，(1989)，Tetrahedron Lett.，30，39473951]，Tenta
Figure PCTCN2018079474-appb-000001
树脂(Rapp Polymere GmbH)， Chem
Figure PCTCN2018079474-appb-000002
树脂(Matrix Innovation，Inc)等，其可以或可以不包括不稳定连接体比如5-(4-氨甲基-3,5-二甲氧基苯氧基)戊酸(PAL)[Albericio F.et al.,"Preparation and application of the 5-(4-(9-fluorenylmethyloxycarbonyl)aminomethyl-3,5-dimethoxy-phenoxy)valeric acid(PAL)handle for the solid-phase synthesis of C-terminal peptide amides under mild conditions",(1990),J.Org.Chem.,55,37303743]，2-[4-氨甲基-(2,4-二甲氧基苯基)]苯氧基乙酸(AM)[Rink H.,"Solid-phase synthesis of protected peptide fragments using a trialkoxy-diphenyl-methylester resin",(1987),Tetrahedron Lett.,28,3787-3790],Wang [Wang S.S.,"p-Alkoxybenzyl Alcohol Resin and p-Alkoxybenzyloxycarbonylhydrazide Resin for Solid Phase Synthesis of Protected Peptide Fragments",(1973),J.Am.Chem.Soc.,95,1328-1333]等，其使得可以同时脱保护和从聚合物载体裂解肽。
定义
在本发明中使用的缩写具有下面的含义：
Ala(Alanine,A)                                     丙氨酸
Asn(Asparagine,N)                                 天冬酰胺
Asp(Aspartic acid,D)                            天冬氨酸
B 5(2-amino-2-(4-pentenyl)-6-heptenoic acid)2-氨基-2-(4-戊烯基)-6-庚烯酸
DCM(Dichloromethane)                            二氯乙烷
DMF(N,N-Dimethyl malonate)                    二甲基甲酰胺
Fmoc(Fluorenylmethoxycarbonyl)             芴甲氧羰基
Gln(Glutamine,Q)                                  谷氨酰胺
Glu(Glutamic acid,E)                            谷氨酸
Gly(Glycine,G)                                     甘氨酸
HBTU            2-(1H-1-羟基苯并三唑)-1,1,3,3-四甲基脲六氟磷酸
His(Histidine,H)                                   组氨酸
HoBt(1-Hydroxylbenzotriazole anhydrous)       1-羟基苯并三氮唑
HR1(N-terminal heptad repeat,NHR)              N端重复序列
HR2(C-terminal heptad repeat,CHR)          C端重复序列
HPLC(High performance liquid chromatography)  高效液相色谱
Ile(Isoleucine,I)                                 异亮氨酸
Leu(Leucine,L)                                    亮氨酸
Lys(Lysine,K)                                     赖氨酸
MERS(Middle East respiratory syndrome)    中东呼吸综合征
MERS-CoV                             中东呼吸综合征冠状病毒
Met(Methionine,M)                               甲硫氨酸
nIle(Norleucine,Z)                                正亮氨酸
R 5((R)-2-(4’-pentenyl)alanine)                (R)-2-(4’-戊烯基)丙氨酸
S 5((S)-2-(4’-pentenyl)alanine)               (S)-2-(4’-戊烯基)丙氨酸
S 8((S)-2-(4’-octyl)alanine)                     (S)-2-(4’-辛烯基)丙氨酸
Ser(Serine,S)                                   丝氨酸
TFA(trifluoroacetic acid)                       三氟乙酸
Thr(Threonine,T)                                苏氨酸
Tyr(Tyrosine,Y)                                  酪氨酸
Val(Valine,V)                                    缬氨酸
如本文所定义的，术语“多肽”，“蛋白质”，“肽”和“氨基酸序列”在本文中可互换使用，是指任何长度的氨基酸残基的聚合物。该聚合物可以是直链或支链的，它可以包含修饰的氨基酸或氨基酸类似物，并且可以被非氨基酸的化学部分打断。该术语还包括已被天然或人工修饰(例如二硫键形成，糖基化，脂化，乙酰化，磷酸化或任何其他操作或修饰，例如与标记或生物活性组分缀合)的氨基酸聚合物。术语“肽”包括通过共价键(例如酰胺键)连接的两个或更多个天然存在的或合成的氨基酸。
在本公开内容的上下文中，术语“氨基酸”被定义为具有至少一个伯、仲、叔或季氨基和至少一个酸基，其中酸基可以是羧酸、磺酸或磷酸或其混合物。氨基相对于酸基可以是“α”、“β”、“γ”至“ω”。合适的氨基酸包括但不限于在肽中发现的20种常见天然存在的氨基酸(例如A，R，N，C，D，Q，E，G，H，I，L，K，M，F，P，S，T，W，Y，V(如由单字母或三字母缩写所已知的))的D-和L-异构体以及通过有机合成或其他代谢途径制备的天然存在的和非天然存在的氨基酸。
“氨基酸”的主链可以被选自卤素、羟基、胍基、杂环基团的一个或多个基团取代。因此术语“氨基酸”在其范围内还包括甘氨酸，丙氨酸，缬氨酸，亮氨酸，异亮氨酸，正 亮氨酸，甲硫氨酸，脯氨酸，苯丙氨酸，色氨酸，丝氨酸，苏氨酸，半胱氨酸，酪氨酸，天冬酰胺，谷氨酰胺，天冬氨酸，谷氨酰胺，赖氨酸，精氨酸，组氨酸，牛磺酸，甜菜碱，N-甲基丙氨酸等。(L)和(D)形式的氨基酸被包括在内。
术语“氨基酸侧链”是指连接至氨基酸的α-碳的部分。例如，丙氨酸的氨基酸侧链是甲基，苯丙氨酸的氨基酸侧链是苯基甲基，半胱氨酸的氨基酸侧链是硫代甲基，天冬氨酸的氨基酸侧链是羧甲基，酪氨酸的氨基酸侧链是4-羟基苯甲基，等等。还包括其他非天然存在的氨基酸侧链，例如天然存在的那些(例如氨基酸代谢物)或合成制备的那些(例如α-双取代的氨基酸)。
如本文所用，术语“烯属”及其同义变体(例如烯烃或烯基)表示通过除去单个氢原子从具有至少一个碳-碳双键的直链或支链烃部分衍生的单价基团。烯基部分含有指定数量的碳原子。例如，C 2-C 10表示该基团可能具有2至10个碳原子。这意味着它具有C 2，C 3，C 4，C 5，C 6，C 7，C 8，C 9或C 10烯基。术语“低级烯基”是指C 2-C 8烯基链。这意味着它具有C 2，C 3，C 4，C 5，C 6，C 7或C 8低级烯基。在没有任何数字名称的情况下，“烯基”是其中具有2至20个碳原子的链(直链或支链)。
烯属基团包括例如可带有一个或多个取代基的乙烯基，丙烯基，丁烯基，1-甲基-2-丁烯-1-基等。烯属基团取代基包括但不限于本文所述的导致形成稳定部分的任何取代基。取代基的实例包括但不限于以下基团：脂族基，烷基，烯基，炔基，杂脂族基，杂环基，芳基，杂芳基，酰基，氧代，亚氨基，硫代氧基，氰基，异氰基，氨基，叠氮基，硝基，烷基氨基，杂烷基氨基，烷基芳基，芳基烷基，脂族氧基，杂脂肪族氧基，烷氧基，杂烷氧基，芳氧基，杂芳氧基，脂族硫氧基，杂脂肪族硫氧基，烷硫基氧基，杂烷硫基氧基，芳硫基氧基，杂芳基硫氧基，其中每一个都可以或可以不被进一步取代。
如本文所用，术语"非环状脂族基团"涵盖线性或支化的烷基，烯基和炔基基团。
术语"烷基"是指线性或支化的饱和基团，其具有1至24，优选1至16，更优选1至14，甚至更优选1至12，还更优选1、2、3、4、5或6个碳原子和通过简单键结合至分子其余部分，包括例如且不限于，甲基，乙基，异丙基，异丁基，叔丁基，庚基，辛基，癸基，十二烷基，月桂基，十六烷基，十八烷基，戊基，2-乙基己基，2-甲基丁基，5-甲基己基等。
术语"烯基基团"是指线性或支化的基团，其具有2至24，优选2至16，更优选2至14，甚至更优选2至12，还更优选2、3、4、5或6个碳原子，具有一个或多个碳-碳双键，优选具有1，2或3个碳-碳双键，键是共轭或不共轭的，其通过简单键结合至分 子其余部分，包括例如且不限于乙烯基(-CH 2＝CH 2)，烯丙基(-CH 2-CH＝CH 2)，油烯基，亚油烯基等基团。
术语"炔基基团"是指线性或支化的基团，其具有2至24，优选2至16，更优选2至14，甚至更优选2至12，还更优选2、3、4、5或6个碳原子，具有一个或多个碳-碳三键，优选1，2或3个碳-碳三键，键是共轭或不共轭的，其通过简单键结合至分子其余部分，包括例如且不限于乙炔基基团，1-丙炔基，2-丙炔基，1-丁基，2-丁基，3-丁基，戊基，比如1-戊基，等。炔基基团还能够含有一个或多个碳-碳双键，包括例如且不限于基团丁-1-烯-3-炔基，戊-4-烯-1-炔基等。
术语"脂环基基团"在本发明中用于涵盖例如且不限于环烷基或环烯基或环炔基基团。
术语"环烷基"是指饱和的单环或多环脂族基团，其具有3至24，优选3至16，更优选3至14，甚至更优选3至12，还更优选3，4，5或6个碳原子并且通过简单键结合至分子其余部分，包括例如且不限于，环丙基，环丁基，环戊基，环己基，环庚基，甲基环己基，二甲基环己基，八氢茚，十氢萘，十二氢非那烯等。
术语"环烯基"是指非芳族单环或多环脂族基团，其具有5至24，优选5至16，更优选5至14，甚至更优选5至12，还更优选5或6个碳原子，具有一个或多个碳-碳双键，优选1，2或3个碳-碳双键，键是共轭或不共轭的，其通过简单键结合至分子其余部分，包括例如且不限于环戊-1-烯-1-基基团等。
术语"环炔基"是指非芳族单环或多环脂族基团，其具有8至24，优选8至16，更优选8至14，甚至更优选8至12，还更优选8或9个碳原子，具有一个或多个碳-碳三键，优选1、2或3个碳-碳三键，键是共轭或不共轭的，其通过简单键结合至分子其余部分，包括例如且不限于环辛-2-炔-1-基基团等。环炔基基团还能够含有一个或多个碳-碳双键，包括例如且不限于环辛-4-烯-2-炔基基团等。
术语"芳基基团"是指芳族基团，其具有6至30，优选6至18，更优选6至10，还更优选6或10个碳原子，其包含1、2、3或4个芳族环，通过碳-碳键结合或稠合，包括例如且不限于苯基，萘基，二苯基，茚基，菲基或蒽基等；或者芳烷基基团。
术语"芳烷基"是指被芳族基团取代的烷基，具有7至24个碳原子和包括例如且不限于-(CH 2) 1-6-苯基，-(CH 2) 1-6-(1-萘基)，-(CH 2) 1-6-(2-萘基)，-(CH 2) 1-6-CH(苯基) 2以及类似物。
术语"杂环基基团"是指3-10元的烃化的环，其中环中原子的一个或多个，优选环中原子的1、2或3个是不同于碳的元素比如氮、氧或硫，并且可以是饱和或不饱和的。出于本发明意图，杂环能够是单环、双环或三环系统，其可以包括稠环系统；和残基杂环中的氮、碳或硫原子可以被任选地氧化；氮原子可以被任选地季铵化；和残基杂环基可以是部分或完全饱和的或是芳族的。术语杂环基最优选是指5或6元环。饱和的杂环基基团的实例是二噁烷，哌啶，哌嗪，吡咯烷，吗啉和硫吗啉。芳族杂环基基团，也称为杂芳族基团的实例是吡啶，吡咯，呋喃，噻吩，苯并呋喃，咪唑啉，对苯二酚，喹啉，哒嗪和萘啶。
术语"杂芳基烷基基团"是指被取代的或未经取代的芳族杂环基基团取代的烷基，烷基具有1至6个碳原子和芳族杂环基基团具有2至24个碳原子和1至3个非碳的原子，并且包括例如且不限于-(CH 2) 1-6-咪唑基，-(CH 2) 1-6-三唑基，-(CH 2) 1-6-噻吩基，-(CH 2) 1-6-呋喃基，-(CH 2) 1-6-吡咯烷基等。
本文使用的术语“卤素”或变体如“卤化物”或“卤代”是指氟，氯，溴和碘。
如本文所用的术语“杂原子”或变体如“杂-”是指O、N、NH和S。
如本文所用的术语“烷氧基”是指直链或支链烷氧基。实例包括甲氧基，乙氧基，正丙氧基，异丙氧基，叔丁氧基等。
如本文所用的术语“氨基”是指-NR aR b形式的基团，其中R a和R b独立地选自包括但不限于氢、任选取代的烷基、任选取代的烯基、任选取代的炔基和任选取代的芳基的组。
应理解，如本文所述的本发明的化合物均可被任何数目的取代基或官能部分取代。通常，术语"(经)取代(的)"(无论其是否在术语"任选地"之后)和含于本发明的式中的取代基都是指用指定取代基的基团对给定结构中的氢基的替代。当任何给定结构中的超过一个位置可被超过一个选自指定基团的取代基取代时，所述取代基在每个位置可相同或不同。如本文所用的术语"经取代的"预期包括用有机化合物的所有可允许取代基、本文所述的任何取代基进行的取代。
例如，取代基包括但不限于导致形成稳定部分的以下基团：脂肪族基、烷基、烯基、炔基、杂脂肪族基、杂环基、芳基、杂芳基、酰基、氧代、亚氨基、硫羰基、氰基、异氰基、氨基、叠氮基、硝基、羟基、硫醇基和卤代以及它们的任何组合，包括但不限于以下基团：脂肪族氨基、杂脂肪族氨基、烷基氨基、杂烷基氨基、芳基氨基、杂芳基氨基、烷基芳基、芳基烷基、脂肪族氧基、杂脂肪族氧基、烷氧基、杂烷氧基、芳氧基、杂芳氧基、脂肪族硫基、杂脂肪族硫基、烷硫基、杂烷硫基、芳硫基、杂芳硫基、酰氧 基等。本发明涵盖任何和所有的此类组合以得到稳定的取代基/部分。出于本发明的目的，例如氮的杂原子可具有氢取代基和/或满足杂原子的化合价并且导致形成稳定部分的如本文所述的任何合适的取代基。
化合物可含有一个或多个不对称中心，因此以外消旋体和外消旋混合物、单一对映异构体、个体非对映异构体和非对映异构体混合物存在。本文明确包括这些化合物的所有这些异构形式。化合物也可以以多种互变异构形式表示，在这种情况下，本文明确地包括本文所述的化合物的所有互变异构形式(例如，环体系的烷基化可导致多个位点的烷基化，本文明确地包括所有这样的反应产物)。本文明确地包括了这种化合物的所有这些异构形式。本文明确包括在此描述的化合物的所有晶体形式。
术语"功能等价的变体"，如本发明中所用，是指通过替换一个或两个氨基酸修饰的衍生自本发明化合物序列的全部那些化合物，条件是所提及的化合物维持基于相应的非修饰化合物的功能的至少20％，至少50％，至少80％的功能。在特别的实施方式中，本发明化合物的功能和其功能等价的变体通过定量其抑制神经元胞吐的能力来确定，特别是通过定量SNARE复合物形成的抑制来确定。SNARE融合复合物的主要功能是使得充满神经递质的囊泡接近并接触突触前细胞膜并且使得可以进行那些囊泡的钙介导的融合，这导致神经递质释放。然而，在加入抑制SNARE复合物形成的化合物或功能等价的变体的情况下，释放的神经递质的浓度相对未用抑制性化合物或功能等价的变体处理的细胞有所下降。在本发明的上下文中，为产生功能等价的变体的本发明化合物的修饰如下进行：将肽的一个或两个氨基酸用其相应特性等价的氨基酸之一替换。下述氨基酸视为特性等价：谷氨酸(E)和天冬氨酸(D)；苏氨酸(T)和丝氨酸(S)；缬氨酸(V)，亮氨酸(L)，异亮氨酸(I)和正亮氨酸(n-L)；天冬酰胺(N)和谷氨酰胺(Q)；赖氨酸(K)，精氨酸(R)和组氨酸(H)和最后的芳族氨基酸苯丙氨酸(F)，色氨酸(W)和酪氨酸(Y)。
本发明化合物或功能等价的变体视为抑制SNARE复合物的形成的条件是：相对已加入任何抑制性化合物的细胞对照，SNARE复合物的形成减少至少15％，至少20％，至少30％，至少40％，至少50％，至少60％，至少70％，至少80％，至少90％，或100％。
本发明化合物能够作为立体异构体或立体异构体的混合物存在；例如，构成它们的氨基酸能够相互独立地具有构型L-、D-或外消旋。因此，可能的是，获得异构混合物以及外消旋混合物或非对映体的混合物，或纯的非对映体或对映体，取决于不对称 碳的数目和异构体或异构混合物所存在的不对称碳。本发明化合物的优选结构纯异构体，也即对映体或非对映体。
例如，在描述AA 1能够是-Lys-的情况下，应理解AA 1选自-L-Lys-，-D-Lys-或两者的混合物，是外消旋或非外消旋的。描述于该文献的制备程序使得本领域技术人员可以通过选择正确构型的氨基酸获得本发明化合物各自的立体异构体。
本发明肽的药学上可接受的盐也属于本发明的领域之内。术语"药学上可接受的盐"意指其在动物中和更特别地在人类中的用途得到承认的盐，并且包括用以形成碱加成盐的盐，无论它们是无机盐还是有机盐，无机盐比如且不限于锂、钠、钾、钙、镁、锰、铜、锌或铝等，或有机盐比如且不限于乙胺、二乙胺、乙二胺、乙醇胺、二乙醇胺、精氨酸、赖氨酸、组氨酸或哌嗪等；或者酸加成盐，无论它们是有机盐还是无机盐，有机盐比如且不限于乙酸盐，柠檬酸盐，乳酸盐，丙二酸盐，马来酸盐，酒石酸盐，富马酸盐，苯甲酸，天冬氨酸盐，谷氨酸盐，琥珀酸盐，油酸酯，三氟乙酸盐，草酸盐，双羟萘酸盐或葡糖酸盐等，或无机盐比如且不限于氯化物，硫酸盐，硼酸盐或碳酸盐等。盐的性质不是关键，条件是它是化妆上或药学上可接受的。本发明肽的药学上可接受的盐能够通过现有技术中熟知的常规方法获得(Berge S.M.等人,"Pharmaceutical Salts",(1977),J.Pharm.Sci.,66,119，其通过引用整体并入本文)。
实施例
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例所用固相合成载体Rink酰胺树脂为天津南开合成责任有限公司；HBTU、HOBt、DIEA以及Fmoc保护的天然氨基酸或D型的非天然氨基酸为上海吉尔生化公司以及北京欧凯纳斯科技有限公司。N-甲基吡咯烷酮(NMP)为ACROS公司产品；三氟乙酸(TFA)为北京博迈杰科技有限公司产品；DMF、DCM为韩国三星公司产品；色谱纯乙腈为Fisher公司产品。其他试剂如无说明均为国产分析纯产品。
实施例1-37：化合物1-37的制备
多肽合成采用标准的Fmoc固相方法。选用Rink Amide树脂，肽链由C端向N端延长。缩合剂为HBTU/HOBt/DIEA。脱保护剂为哌啶/DMF溶液。裂解液为三氟乙 酸(TFA)，粗肽水溶解后冻干保存。用中压液相色谱法或高压液相色谱法(HPLC)分离纯化，纯肽含量大于90％。基质辅助激光解析飞行时间质谱(MALDI-TOF-MS)确定肽序列分子量。
利用CEM微波多肽合成仪合成肽序列。
合成条件如下：
保护氨基酸：0.2M的DMF溶液，
活化剂0.45M HBTU/HOBt的DMF溶液，
活化碱：2M DIEA的NMP溶液，
脱保护剂：20％v/v哌啶的DMF溶液，
封闭试剂：20％v/v乙酸酐的DMF溶液。
称取Rink Amide树脂0.23g(0.1mmol)置于CEM微波多肽合成仪反应器中，然后将氨基酸，活化剂，活化碱，脱保护试剂，封闭试剂按上述浓度配置好后，用CEM微波全自动多肽合成仪进行合成。完成后肽树脂用DMF洗涤3遍后用无水甲醇收缩，室温真空干燥，得肽树脂1.25g。
裂解液(体积百分比)：三氟乙酸：间甲酚：苯甲硫醚：水＝85:5:5:5。
肽树脂的裂解：称取微波合成仪合成好的肽树脂1.25g，放入250ml茄形瓶中，冰浴，电磁搅拌。按1克肽树脂加入10ml的量配制裂解液。TFA需预先冰浴降温30min或者预先存放于冰箱中使用；将配好的裂解液加入到冰浴条件下的肽树脂中，电磁搅拌，树脂变橙红色，冰浴条件下反应30min，然后撤掉冰浴室温下继续搅拌反应90min，反应完成，剧烈搅拌下加入冷乙醚200ml，析出白色沉淀，继续搅拌30min；用G4砂芯漏斗滤出析出物，用冷乙醚反复洗涤3遍，晾干。加入双蒸水50ml，乙腈5ml使固体充分溶解，抽滤，滤液冻干得粗肽1.04g。
粗肽的纯化：粗肽用中压或高压色谱进行纯化。色谱柱为C18柱，洗脱剂为乙腈，水及少量一算。具体操作步骤：称取粗肽1.00g，加水20ml，乙腈5ml使固体溶解，离心10min(3000转/分钟)，取上清液上样。色谱柱预先用15％乙腈/水/0.1％冰乙酸溶液200ml平衡。上样后继续用15％乙腈/水/0.1％冰乙酸溶液200ml冲洗，高效液相检测洗脱液成分。根据液相检测结果逐渐升高乙腈含量，直至所纯化的多肽主峰被洗脱出来。合并洗脱液，旋转蒸发取出大部分溶剂，冻干纯多肽，HPLC检测含量大于90％，MALDI-TOF确证分子量。
实施例38-57：化合物38-57的制备
Figure PCTCN2018079474-appb-000003
多肽合成方法同实施例1，只是在线性多肽完成之后，进行固相烯烃复分解反应，将侧链烯烃基团进行共价连接合成装订肽。具体步骤如下：将多肽树脂转移至避光保护的茄形瓶中，通氮气保护5min，称取一代Grubbs催化剂25mg，溶于5ml二氯乙烷中，加入反应体系，在氮气保护下搅拌6h，反应完毕后重复一次。反应完成后用DMF洗涤3遍后用无水甲醇收缩，室温真空干燥，得肽树脂1.02g，裂解纯化方法同实施例1。
实施例58-69：化合物58-69的制备
Figure PCTCN2018079474-appb-000004
多肽合成方法同实施例1，之后的固相烯烃复分解反应中，需要加大催化剂的投料量，具体步骤如下：将多肽树脂转移至避光保护的茄形瓶中，通氮气保护5min，称取一代Grubbs催化剂38mg，溶于8ml二氯乙烷中，加入反应体系，在氮气保护下搅拌6h，反应完毕后重复一次。反应完成后用DMF洗涤3遍后用无水甲醇收缩，室温真空干燥，得肽树脂1.01g，裂解纯化方法同实施1。
实施例70：化合物抑制MERS-CoV S2亚基介导的细胞融合活性评价(IC 50)
1)第一天：消化293T并制备细胞悬液，6孔板中每孔中加入2～4×10 5个细胞。
2)第二天：培养12～24h后，待密度在80％左右进行转染，其中一孔转染pAAV-IRES-EGFP-MERS-S质粒(293T/MERS/EGFP)用于融合实验；一孔转染pAAV-IRES-EGFP(293T/EGFP)用作阴性对照。
3)转染8～12h后，更换新鲜DMEM细胞培养液。
4)第四天：转染36～48h后细胞能够表达较强的荧光，可进行后续融合抑制试验。
5)融合前6～12h，消化Huh-7细胞，制备细胞悬液，每孔中加入4～5×10 4个细胞。37℃培养备用。
6)配制梯度稀释的多肽，每孔55μL反应体系。
7)将293T/MERS/EGFP细胞，以及293T/EGFP细胞用EDTA进行消化。制备单个细胞悬液。
8)药物板中每孔加入10 4个293T/MERS/EGFP细胞。同时设置无药物的培养孔 做阳性对照(293T/MERS/EGFP+Huh-7)，293T/EGFP细胞孔做阴性对照(293T/EGFP+Huh-7)。终体积控制在110μL。
9)室温孵育30min，使药物与细胞充分作用。
10)抽弃Huh-7细胞的培养基，取100μL药物与细胞的混合液加入到Huh-7细胞之上。
11)24h观察融合情况，待阳性对照组(293T/MERS/EGFP+Huh-7)细胞有明显的融合，加入等体积的4％多聚甲酸固定，终止融合。
12)荧光显微镜或者高内涵筛选系统进行拍照分析。计算多肽对细胞融合的抑制率，并使用CalcuSyn软件计算多肽的IC 50值。
按照上述方法，活性测试结果见下面的表1(其中，序号38-69的序列中斜体表示的残基代表交联后的非天然氨基酸残基)。
表1：对MERS-CoV S2蛋白介导的细胞融合的抑制活性(IC 50)
Figure PCTCN2018079474-appb-000005
Figure PCTCN2018079474-appb-000006
Figure PCTCN2018079474-appb-000007
Figure PCTCN2018079474-appb-000008
Figure PCTCN2018079474-appb-000009
由表1活性结果可知，所有肽化合物显示了抑制MERS-CoV细胞融合活性，其中化合物36-38，46-48，67抑制MERS-CoV融合活性达到低μM水平，与阳性对照HR2P-M2(化合物70)相当。
实施例71：圆二色谱测定化合物的构象
取少量多肽溶解于50mM PBS溶液中(pH＝7.2)，充分溶解后取上清液，标定多肽母液浓度，稀释至终浓度50μM，用圆二色谱仪测定椭圆率，以222nm处的摩尔椭圆率峰值等于33000作为100％的α-螺旋来计算样品螺旋含量百分比。结果如下表2所示。
表2：
化合物 序列 α-螺旋度(％)
1 LDLTYEM LSLQQVV KALNESY 无规卷曲
2 LDLTYEI LSLQQVV KALNESY 21.3
3 LDLTYEZ LSLQQVV KALNESY 15.6
4 LDLTYEL LSLQQVV KALNESY 28.3
5 LDLTYEV LSLQQVV KALNESY 55.6
6 LDLTYEM LSLQS 5VV KS 5LNESY 37.4
7 LDLTYEI LSLQS 5VV KS 5LNESY 56.8
8 LDLTYEZ LSLQS 5VV KS 5LNESY 45.8
9 LDLTYEL LSLQS 5VV KS 5LNESY 22.5
10 LDLTYEV LSLQS 5VV KS 5LNESY 44.4
11 LDLTYEM LSLQR 5VV KR 5LNESY 25.4
12 LDLTYEI LSLQR 5VV KR 5LNESY 21.7
13 LDLTYEZ LSLQR 5VV KR 5LNESY 34.5
14 LDLTYEL LSLQR 5VV KR 5LNESY 42.2
15 LDLTYEV LSLQR 5VV KR 5LNESY 11.2
16 LDLTYEM LSLQQVV KS 5LNES 5Y 35.6
17 LDLTYEI LSLQQVV KS 5LNES 5Y 13.2
18 LDLTYEZ LSLQQVV KS 5LNES 5Y 45.2
19 LDLTYEL LSLQQVV KS 5LNES 5Y 55.2
20 LDLTYEV LSLQQVV KS 5LNES 5Y 44.8
21 LDLTYEM LSLQQVV KR 5LNER 5Y 55.5
22 LDLTYEI LSLQQVV KR 5LNER 5Y 28.6
23 LDLTYEZ LSLQQVV KR 5LNER 5Y 36.8
24 LDLTYEL LSLQQVV KR 5LNER 5Y 41.2
25 LDLTYEV LSLQQVV KR 5LNER 5Y 21.3
26 LDLTYEM LSLQR 5VV KB 5LNES 5Y 56.3
27 LDLTYEI LSLQR 5VV KB 5LNES 5Y 55.6
28 LDLTYEZ LSLQR 5VV KB 5LNES 5Y 54.6
29 LDLTYEL LSLQR 5VV KB 5LNES 5Y 45.6
30 LDLTYEV LSLQR 5VV KB 5LNES 5Y 58.9
31 LDLTYEM LSLQS 5VV KB 5LNER 5Y 45.2
32 LDLTYEI LSLQS 5VV KB 5LNER 5Y 23.3
33 LDLTYEZ LSLQS 5VV KB 5LNER 5Y 21.5
34 LDLTYEL LSLQS 5VV KB 5LNER 5Y 56.6
35 LDLTYEV LSLQS 5VV KB 5LNER 5Y 61.4
36 LDLTYEM LS 8LQQVV KB 5LNES 5Y 65.6
37 LDLTYEMLR 8LQQVV KB 5LNER 5Y 54.5
38 LDLTYEM LSLQS 5VV KS 5LNESY 53.7
39 LDLTYEI LSLQS 5VV KS 5LNESY 42.6
40 LDLTYEZ LSLQS 5VV KS 5LNESY 32.3
41 LDLTYEL LSLQS 5VV KS 5LNESY 23.6
42 LDLTYEV LSLQS 5VV KS 5LNESY 42.1
43 LDLTYEM LSLQR 5VV KR 5LNESY 16.5
44 LDLTYEI LSLQR 5VV KR 5LNESY 18.6
45 LDLTYEZ LSLQR 5VV KR 5LNESY 25.2
46 LDLTYEL LSLQR 5VV KR 5LNESY 54.2
47 LDLTYEV LSLQR 5VV KR 5LNESY 26.6
48 LDLTYEM LSLQQVV KS 5LNES 5Y 46.5
49 LDLTYEI LSLQQVV KS 5LNES 5Y 58.9
50 LDLTYEZ LSLQQVV KS 5LNES 5Y 56.5
51 LDLTYEL LSLQQVV KS 5LNES 5Y 54.4
52 LDLTYEV LSLQQVV KS 5LNES 5Y 54.1
53 LDLTYEM LSLQQVV KR 5LNER 5Y 12.8
54 LDLTYEI LSLQQVV KR 5LNER 5Y 47.7
55 LDLTYEZ LSLQQVV KR 5LNER 5Y 48.2
56 LDLTYEL LSLQQVV KR 5LNER 5Y 54.0
57 LDLTYEV LSLQQVV KR 5LNER 5Y 12.3
58 LDLTYEM LSLQR 5VV KB 5LNES 5Y 54.6
59 LDLTYEI LSLQR 5VV KB 5LNES 5Y 33.3
60 LDLTYEZ LSLQR 5VV KB 5LNES 5Y 42.2
61 LDLTYEL LSLQR 5VV KB 5LNES 5Y 21.1
62 LDLTYEV LSLQR 5VV KB 5LNES 5Y 20.8
63 LDLTYEM LSLQS 5VV KB 5LNER 5Y 63.3
64 LDLTYEI LSLQS 5VV KB 5LNER 5Y 61.8
65 LDLTYEZ LSLQS 5VV KB 5LNER 5Y 23.3
66 LDLTYEL LSLQS 5VV KB 5LNER 5Y 25.6
67 LDLTYEV LSLQS 5VV KB 5LNER 5Y 14.2
68 LDLTYEM LS 8LQQVV KB 5LNES 5Y 28.9
69 LDLTYEMLR 8LQQVV KB 5LNER 5Y 48.7
从表2的结果可知，本发明的肽化合物均展现出一定程度的α螺旋度。
实施例72：化合物71-78的制备以及活性检测
采用上述实施例1-69中所述相同的方法制备下表3所示的化合物71-78，并根据实施例70所述的方法测试这些化合物对MERS-CoV S2亚基介导的细胞融合活性的抑制(IC 50)，结果如表3所示。
表3：对MERS-CoV S2蛋白介导的细胞融合的抑制活性(IC 50)
化合物 序列 IC 50(μM)
71 L-LDLTYEMLSLQS 5VVKS 5LNESY 3.75±0.75
72 L-LDLTYEMLSLQQVVKS 5LNES 5Y 0.63±0.12
73 TL-LDLTYEM LSLQS 5VVKS 5LNESY 4.56±1.03
74 TL-LDLTYEM LSLQQVVKS 5LNES 5Y 2.68±0.92
75 LDLTYEMLSLQS 5VVKS 5LNESY-I 5.24±1.32
76 LDLTYEMLSLQQVVKS 5LNES 5Y-I 2.65±0.51
77 LDLTYEMLSLQS 5VVKS 5LNESY-ID 0.87±0.14
78 LDLTYEMLSLQQVVKS 5LNES 5Y-ID 1.02±0.33
从表3的结果可知，在实施例1-69的21肽化合物的N端或C端添加额外的氨基酸不会影响其抑制活性。
实施例73：化合物79-81的制备以及活性检测
采用上述实施例1-69中所述相同的方法制备下表3所示的化合物79-81，并根据实施例70所述的方法测试这些化合物对MERS-CoV S2亚基介导的细胞融合活性的抑制(IC 50)，结果如表4所示。
表4：对MERS-CoV S2蛋白介导的细胞融合的抑制活性(IC 50)
化合物 序列 IC 50(μM)
79 LDLTYEM LSLES 5VV KS 5LNESY 1.75±0.26
80 LDLTYEM LSKQQVV KS 5LNES 5Y 2.85±0.33
81 LDLEYEM KSLES 5VV KS 5LEESY 7.02±1.98
从表4的结果可知，在实施例1-69的21肽化合物中非对应于式(I)中的X位置的氨基酸进行氨基酸置换不会影响其抑制活性。
实施例74：化合物82-85的制备以及活性检测
采用上述实施例1-69中所述相同的方法制备下表5所示的化合物82-85，并根据实施例70所述的方法测试这些化合物对MERS-CoV S2亚基介导的细胞融合活性的抑制(IC 50)，结果如表5所示。
表5：对MERS-CoV S2蛋白介导的细胞融合的抑制活性(IC 50)
化合物 序列 IC 50(μM)
82 LDLTYEZ LSLQS 5VV KS 5LNESY 0.641±0.005
83 LDLTYEI LSLQS 5VV KS 5LNESY 0.248±0.009
84 LDLTYEL LSLQS 5VV KS 5LNESY 2.233±0.004
85 LDLTYEV LSLQS 5VV KS 5LNESY 3.219±0.017
根据表5以及结合表1的结果可知，式(I)的X a可以为任何疏水性氨基酸而不影响多肽化合物的抑制活性。
实施例75：化合物86的制备以及活性检测
采用上述实施例1-69中所述相同的方法制备下表6所示的化合物86，并根据实施例70所述的方法测试该化合物对MERS-CoV S2亚基介导的细胞融合活性的抑制(IC 50)，结果如表6所示。
表6：对MERS-CoV S2蛋白介导的细胞融合的抑制活性(IC 50)
化合物 序列 IC 50(μM)
86 LDLTYEE LSLQS 5VV KS 5LNESY >50
根据表6的结果可知，当式(I)的X a为非疏水性氨基酸时，多肽化合物的抑制活性丢失。这样的结果证明，本发明的式(I)的X a选自疏水性氨基酸而不能选自非疏水性氨基酸。
实施例76：化合物87-89的制备以及活性检测
采用上述实施例1-69中所述相同的方法制备下表6所示的化合物87-89，并根据实施例70所述的方法测试这些化合物对MERS-CoV S2亚基介导的细胞融合活性的抑 制(IC 50)，结果如表7所示。
表7：对MERS-CoV S2蛋白介导的细胞融合的抑制活性(IC 50)
化合物 序列 IC 50(μM)
87 LDLTYEM LSLQBVV KBLNESY 2.54±0.38
88 LDLTYEM LBLQBVV KALNESY 6.66±2.15
89 LDLTYEM LSLQQVV KBLNEBY 3.04±1.41
其中：B为α-氨基异丁酸
实施例76：假病毒活性测试
本发明人还对本发明的多肽化合物进行了假病毒活性测试，以从病毒水平证明短肽能够抑制MERS-CoV感染，其效果与阳性对照HR2P-M2(即化合物70)相当(表8)。
MERS-CoV假病毒的包装
1.转染前16h消化293T细胞，在10cm的组织培养皿中铺板(2×10 6/dish)。
2.转染前2h给细胞更换预温的新鲜DMEM培养基。
3.采用磷酸钙转染试剂共转染编码MERS-CoV S蛋白真核表达质粒pcDNA3.1-MERS-S，以及编码缺失Env、表达luciferase的HIV-1基因组质粒(pNL4-3.luc.RE)，每种质粒转染20μg。
4.转染8～10h后，更换10mL含10％FBS和1％青霉素链霉素的新鲜DMEM培养液。
5. 72h后收集含有MERS-CoV假病毒的转染上清液。
6. 4000rpm离心4min去除细胞碎片，0.45μm无菌过滤器过滤，分装并储存在-80℃备用。
MERS-CoV假病毒滴度检测
1.提前12h消化Huh-7细胞，使用DMEM制备单个细胞悬液并调整细胞浓度。
2. 96孔板中每孔中加入104个细胞。37℃5％CO 2培养12h备用。
3.使用DMEM培养基在96孔板中2倍梯度稀释假病毒。
4.去除96孔板中Huh-7的细胞培养上清，加入100μL稀释后的病毒稀释液。
5. 37℃5％CO 2培养12h后，去除病毒液，更换200μL新鲜DMEM培养基。
6.加入病毒72h后抽弃上清，测定萤光值。
7.选择萤光值大于空白对照1000倍的病毒稀释度进行后续假病毒中和实验。
萤火虫酶(luciferase)检测
1.使用ddH2O稀释5×细胞裂解液至1×工作浓度。平衡至室温。
2.小心吸弃96孔板中的待测细胞的培养基，使用PBS洗细胞一次，动作轻柔以防止细胞脱落，并尽可能将PBS去除。
3.每孔中加入1×细胞裂解液20μL，之后置涡旋混匀仪上震荡混匀30～60min直至细胞完全裂解。
4.抽取20μL裂解液至96孔不透明白色酶标板中。
5.每孔中加入100μL萤火虫酶检测试剂。
6.多功能酶标仪中检测萤光
多肽活性检测
1.使用DMSO溶解多肽并测定多肽浓度。
2.实验前16h消化靶细胞Huh-7，制备细胞悬液，调整细胞浓度后每孔中加入10 4个细胞。
3.96孔板中使用含10％FBS的DMEM培养液2倍梯度稀释多肽药物多肽，每孔60μL；注意要缓慢加入多肽，边加入边搅拌，防止因短时间内加入大量多肽导致多肽析出。
4.根据之前测定的MERS-CoV假病毒滴度进行稀释，在药物稀释板中每孔加入60μL稀释后的假病毒。
5.室温作用30min，使药物与病毒充分作用。
6.之后抽弃细胞的培养上清，每孔中加入药物与病毒混合液100μL。
7. 37℃培养12h后更换含10％FBS的新鲜DMEM培养基。
8.继续培养，72h后测定萤火虫酶。
9.根据萤光值与药物浓度的对应关系，计算制作抑制率曲线，并计算药物的半数有效量IC 50。
按照上述方法，活性检测的结果见下面的表8。
表8：对MERS-CoV假病毒感染的抑制活性
化合物 序列 IC 50(μM)
38 LDLTYEM LSLQS 5VV KS 5LNESY 3.03±0.29
48 LDLTYEM LSLQQVV KS 5LNES 5Y 0.97±0.08
70 SLTQINTTLLDLEYEMKKLEEVVKKLEESYIDLKEL 1.07±0.21
尽管本发明的具体实施方式已经得到了详细的阐述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围有所附权利要求及其任何等同物给出。

Claims (16)

  1. 与通式(I)具有至少80％同一性的多肽化合物：
    R 1-U-Leu-Asp-Leu-Thr-Tyr-Glu-X a-Leu-X 1-Leu-Gln-X 2-Val-Val-Lys-X 3-Leu-Asn-Glu-X 4-Tyr-W-R 2(I)，
    其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体；
    其中X a为能够与MERS-COV HR2发生疏水相互作用的氨基酸残基，其选自以下D型或L型疏水性氨基酸：丙氨酸(Ala)、缬氨酸(Val)、亮氨酸(Leu)、异亮氨酸(Ile)、脯氨酸(Pro)、苯丙氨酸(Phe)、色氨酸(Trp)、正亮氨酸(n-Leu)和蛋氨酸(Met)；
    X 1、X 2、X 3和X 4各自是独立选择的D型或L型氨基酸；
    U和W不存在或各自是独立选择的一个或多个附加的D型或L型氨基酸；
    R 1选自：H，衍生自聚乙二醇的聚合物，非环状取代的或未经取代的脂族基团，取代的或未经取代的脂环基，取代的或未经取代的杂环基，取代的或未经取代的杂芳基烷基，取代的或未经取代的芳基，取代的或未经取代的芳烷基和R 5-CO-，其中R 5选自：H，非环状取代的或未经取代的脂族基团，取代的或未经取代的脂环基，取代的或未经取代的芳基，取代的或未经取代的芳烷基，取代的或未经取代的杂环基和取代的或未经取代的杂芳基烷基；
    优选地，R 1选自：H，乙酰基，月桂酰基，肉豆蔻酰基和棕榈酰基；
    R 2选自：-NR 3R 4，-OR 3和-SR 3，其中R 3和R 4独立地选自：H，衍生自聚乙二醇的聚合物，非环状取代的或未经取代的脂族基团，取代的或未经取代的脂环基，取代的或未经取代的杂环基，取代的或未经取代的杂芳基烷基，取代的或未经取代的芳基，和取代的或未经取代的芳烷基；
    优选地，R 2选自：-NR 3R 4和-OR 3，其中R 3和R 4独立地选自H，甲基，乙基，己基，十二烷基和十六烷基；
    和
    R 1和R 2不是D型或L型氨基酸。
  2. 权利要求1的多肽化合物，其中X a选自如下D型或L型的氨基酸：甲硫氨酸，亮氨酸，异亮氨酸，缬氨酸和正亮氨酸。
  3. 权利要求1或2的多肽化合物，其中X 1、X 2、X 3和X 4中的一个或多个独立地是α螺旋稳定氨基酸。
    优选地，X 1、X 2、X 3和X 4中的一个或多个独立地是α,α-双取代的氨基酸，
    优选地，X 1、X 2、X 3和X 4中的一个或多个各自独立地选自以下D型或L型氨基酸：α-氨基异丁酸、1-氨基环丙烷-1-羧酸、(R)-2-(4’-戊烯基)丙氨酸(R 5)、(S)-2-(4’-戊烯基)丙氨酸(S 5)、(S)-2-(4’-辛烯基)丙氨酸(S8)、(R)-2-(4’-辛烯基)丙氨酸(R8)和2-氨基-2-(4-戊烯基)-6-庚烯酸(B 5)，
    优选地，在X 1、X 2、X 3和X 4中不是α螺旋稳定氨基酸的氨基酸残基各自独立地选自以下D型或L型氨基酸：谷氨酰胺、丙氨酸和丝氨酸。
  4. 权利要求1或2的多肽化合物，其还包含在X 1、X 2、X 3和X 4中的两个或更多个之间形成的交联体，
    优选地，在X 1与X 2、X 1与X 3、X 2与X 3和/或X 3与X 4之间形成交联，
    优选地，所述交联体是非共价或共价交联体，
    优选地，所述交联体是包含接头的交联体。
  5. 权利要求4的多肽化合物，
    优选地，所述非共价交联体选自包含氢键、静电相互作用或金属螯合的交联体，
    优选地，所述共价交联体选自包含烃键联、分子内酰胺键、二硫键、硫醚键或Cu(I)-催化的叠氮-炔烃环加成反应的交联体。
  6. 权利要求4或5的多肽化合物，所述包含烃键联的交联体是全碳氢交联体，并且在X 1、X 2、X 3和X 4中形成所述全碳氢交联体的氨基酸残基是具有烯属侧链的α,α-双取代的氨基酸，
    优选地，在X 1、X 2、X 3和X 4中形成所述全碳氢交联的氨基酸残基各自独立地选自(R)-2-(4’-戊烯基)丙氨酸(R 5)、(S)-2-(4’-戊烯基)丙氨酸(S 5)、(S)-2-(4’-辛烯基)丙氨酸(S8)、(R)-2-(4’-辛烯基)丙氨酸(R8)和2-氨基-2-(4-戊烯基)-6-庚烯酸(B 5)。
  7. 权利要求4-6中任一项的多肽化合物，其中在X 1、X 2、X 3和X 4中未形成交联体的氨基酸残基各自独立地选自以下D型或L型氨基酸：谷氨酰胺、丙氨酸和丝氨酸。
  8. 权利要求1-7中任一项的多肽化合物，其是与SEQ ID NO.1-69中的任一个具有至少80％同一性的多肽化合物或其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体。
  9. 权利要求1-8中任一项的多肽化合物，
    优选地，其用于抑制MERS-CoV融合，
    优选地，其用于治疗或预防MERS-CoV感染相关疾病，
    优选地，所述MERS-CoV感染相关疾病为中东呼吸综合征。
  10. 一种药物组合物，其包含至少一种根据权利要求1-9中任一项所述的多肽化合物或其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体，以及至少一种药学上可接受的载体、稀释剂、赋形剂或辅助剂。
  11. 一种MERS-CoV融合抑制剂，其包含至少一种根据权利要求1-9中任一项所述的多肽化合物或其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体。
  12. 权利要求1-9中任一项的多肽化合物或其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体用于抑制MERS-CoV融合的用途。
  13. 权利要求1-9中任一项的多肽化合物或其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体用于治疗或预防MERS-CoV感染相关疾病的用途，
    优选地，所述MERS-CoV感染相关疾病为中东呼吸综合征。
  14. 权利要求1-9中任一项的多肽化合物或其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体在制备MERS-CoV融合抑制剂中的用途。
  15. 权利要求1-9中任一项的多肽化合物或其立体异构体、其混合物、其药学上可接受的盐和/或其功能等价的变体在制备用于治疗或预防MERS-CoV感染相关疾病的药物中的用途，
    优选地，所述MERS-CoV感染相关疾病为中东呼吸综合征。
  16. 制备权利要求1-2和4-9中任一项的多肽化合物的方法，其包括：
    a.合成与通式(I)具有至少80％同一性的多肽，和
    b.将X 1、X 2、X 3和X 4中的两个或更多个通过交联体连接起来，
    优选地，通过交联体连接X 1与X 2、X 1与X 3、X 2与X 3和/或X 3与X 4。
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