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WO1990000399A1 - Peptides de liaison de protease retrovirale - Google Patents

Peptides de liaison de protease retrovirale Download PDF

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Publication number
WO1990000399A1
WO1990000399A1 PCT/US1989/002972 US8902972W WO9000399A1 WO 1990000399 A1 WO1990000399 A1 WO 1990000399A1 US 8902972 W US8902972 W US 8902972W WO 9000399 A1 WO9000399 A1 WO 9000399A1
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WO
WIPO (PCT)
Prior art keywords
amino
hydroxy
alk
oxo
methyl ester
Prior art date
Application number
PCT/US1989/002972
Other languages
English (en)
Inventor
Geoffrey Bainbridge Dreyer
William Francis Huffman
Thomas Downing Meek
Brian Walter Metcalf
Michael Lee Moore
Original Assignee
Smithkline Beckman Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Smithkline Beckman Corporation filed Critical Smithkline Beckman Corporation
Priority to FI910084A priority Critical patent/FI910084A0/fi
Publication of WO1990000399A1 publication Critical patent/WO1990000399A1/fr
Priority to NO91910053A priority patent/NO910053L/no
Priority to DK002691A priority patent/DK2691A/da
Priority to NO920319A priority patent/NO920319D0/no
Priority to NO920318A priority patent/NO920318D0/no

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K5/00Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0207Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)4-C(=0), e.g. 'isosters', replacing two amino acids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
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    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/30Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and unsaturated
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    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00Carboxylic acid amides
    • C07C233/01Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/02Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals
    • C07C233/09Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals with carbon atoms of carboxamide groups bound to carbon atoms of an acyclic unsaturated carbon skeleton
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    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/22Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
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    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06Esters of carbamic acids
    • C07C271/08Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/18Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by doubly-bound oxygen atoms
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    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06Esters of carbamic acids
    • C07C271/08Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/22Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
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    • C07DHETEROCYCLIC COMPOUNDS
    • C07D207/00Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/16Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
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    • C07FACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00Compounds containing elements of Groups 4 or 14 of the Periodic Table
    • C07F7/02Silicon compounds
    • C07F7/08Compounds having one or more C—Si linkages
    • C07F7/18Compounds having one or more C—Si linkages as well as one or more C—O—Si linkages
    • C07F7/1804Compounds having Si-O-C linkages
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    • C07FACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02Phosphorus compounds
    • C07F9/28Phosphorus compounds with one or more P—C bonds
    • C07F9/30Phosphinic acids [R2P(=O)(OH)]; Thiophosphinic acids ; [R2P(=X1)(X2H) (X1, X2 are each independently O, S or Se)]
    • C07F9/32Esters thereof
    • C07F9/3205Esters thereof the acid moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/3223Esters of cycloaliphatic acids
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K5/00Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/021Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)n-C(=0)-, n being 5 or 6; for n > 6, classification in C07K5/06 - C07K5/10, according to the moiety having normal peptide bonds
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    • C07KPEPTIDES
    • C07K5/00Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06Dipeptides
    • C07K5/06008Dipeptides with the first amino acid being neutral
    • C07K5/06017Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06052Val-amino acid
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K5/00Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08Tripeptides
    • C07K5/0802Tripeptides with the first amino acid being neutral
    • C07K5/0804Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K5/00Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08Tripeptides
    • C07K5/0802Tripeptides with the first amino acid being neutral
    • C07K5/0804Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/081Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K5/00Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10Tetrapeptides
    • C07K5/1002Tetrapeptides with the first amino acid being neutral
    • C07K5/1016Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K7/00Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02Linear peptides containing at least one abnormal peptide link
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K7/00Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04Linear peptides containing only normal peptide links
    • C07K7/06Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/10Dispersions; Emulsions
    • A61K9/127Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
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    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00Systems containing only non-condensed rings
    • C07C2601/06Systems containing only non-condensed rings with a five-membered ring
    • C07C2601/08Systems containing only non-condensed rings with a five-membered ring the ring being saturated

Definitions

  • Retroviruses that is, viruses within the family of Retroviridae, are a class of viruses which transport their genetic material as ribonucleic acid rather than
  • RNA-tumor viruses also known as RNA-tumor viruses, their presence has been associated with a wide range of diseases in humans and animals. They are beIleved to be the causative agents in pathological states associated with infection by Rous sarcoma virus (RSV), murine leukemia virus (MLV), mouse mammary tumor virus (MMTV), feline leukemia virus (FeLV), bovine leukemia virus (BLV), Mason-Pfizer monkey virus (MPMV), simian sarcoma virus (SSV), simian acquired immunodeficiency syndrome (SAIDS), human T-lymphotropic virus (HTLV-I, -II) and human immunodeficiency virus (HIV-1, HIV-2), which is the etiologic agent of AIDS (acquired immunodeficiency syndrome) and AIDS related complexes, and many others.
  • RSV Rous sarcoma virus
  • MMV murine leukemia virus
  • MMTV mouse mammary tumor virus
  • the pathogens have, in many of these cases, been isolated, no effective method for treating this type of infection has been developed.
  • the HTLV and HIV have been especially well characterized.
  • all retroviruses are rather similar in overall structure.
  • the extracellular virus particle is composed of an outer membrane studded with viral glycoproteins, a core of structural proteins, and a genome of single stranded ribonucleic acid.
  • the retroviral genome has a distinctive regional organization, referred to as the 5'-gag-pol-env-3' structure, wherein the gag region encodes the core structural proteins, the pol region encodes certain critical viral enzymes such as reverse transcriptase,
  • Viral replication occurs only within host cells and is dependent upon host cellular functions. Critical to this replication is the production of functional viral proteins. Protein synthesis is accomplished by
  • Retroviral proteases have not been well characterized. Both the proteases and their polyprotein substrates are recovered from virion particles in very low yield. To assess their activity, it is necessary to provide a substrate to express the proteolytic activity. Use of the
  • Proteases are enzymes which cleave peptide bonds in proteins and polypeptides. They are present in most
  • regulatory function is their ability to act selectively upon polypeptides. This selectivity is comprised of two factors, 1.) their ability to act upon specific substrates and 2.) their ability to hydrolyze only specific peptide bonds. On a structural level this substrate specificity is the result of the primary amino acid sequence, the resultant local
  • conformation of a large polypeptide may be significantly different than that of a small peptide, it is difficult to predict which amino acid sequence will bind optimally in a small peptide.
  • retroviruses and suggest a sequence preference for cleavage at X-Pro for the retroviral proteases, wherein X is usually either an aromatic (Phe, Tyr) or a large and hydrophobic (Met, Leu) amino acid. It is further suggested that this sequence is usually flanked on either side by a small and hydrophobic amino acid.
  • retroviral proteases may be acid proteases. This is based upon the observation that pepstatin, a known acid protease inhibitor, has been shown to be a weak inhibitor of the retroviral proteases associated with bovine leukemia virus, Moloney murine leukemia virus and human T-cell leukemia virus.
  • Kettner, et al. U.S. Patent No. 4,636,492 and Kettner, et al., U.S. Patent No. 4,644,055. Accordingly, tri- and tetra-peptides which substantially correspond to the amino acid sequence of the cleavage site of the substrate of the protease are modified to possess a C-terminal halomethyl ketone moiety. The halomethyl ketone, being a reactive moiety, is presumed to react with the protease once binding of the peptide has been effected.
  • Pepstatin a pentapeptide inhibitor isolated from a Streptomyces culture, has been shown to inhibit a wide range of acid proteases, and has attracted attention due to its ability to inhibit renin and pepsin. (See Umezawa, H. et al., J. Antibiot., Tokyo, 23, 259-263(1970).) Pepstatin contains the unusual amino acid, (3S, 4S)-4-amino-3-hydroxy-6-methylheptanoic acid (AHMHA), called statine, in two
  • This unusual amino acid is not an ⁇ -amino acid, but is beIleved to function as a
  • statine to probe the relationship between structure and inhibitory activity.
  • (3S, 4S)-4-amino-3- hydroxy-5-phenylpentanoic acid (AHPPA) showed activity similar to statine when incorporated into peptides in place of statine.
  • This invention comprises peptides, hereinafter
  • proteases are useful as substrates for assaying protease activity. In another respect, these peptides are inhibitors of viral protease and are useful for treating disease related to infection by these agents.
  • This invention is also a pharmaceutical composition, which comprises a compound of formula (I) and a
  • This invention further constitutes a method for treating viral disease, which comprises administering to a mammal in need thereof an effective amount of a compound of formula
  • This invention also provides a method for assaying viral protease activity.
  • this invention is a compound, as shown hereinafter as formula (lIb), which can be used as an intermediate in the preparation of the peptides of formula (I), which renders the peptide resistant to degradation by a viral protease.
  • A is BocNH, CbzNH, H, R' R"N, R"CONR' or DnsNH, or if a,b and c are 0 and Y is a covalent bond, then A is H, Boc, Cbz, R" or R"CO;
  • B is one or more D or L amino acids, ⁇ -Ala or is a covalent bond
  • C and D are the same or different and are Glx, Asx, Ala, ⁇ -Ala, Arg, Gly, Ile, Leu, Lys, Ser, Thr, Val, Met or His;
  • Q is a D or L amino acid and is Ser, Thr, Asp, His, Cys, Arg or Ala;
  • W is Pro or ⁇ 3-dehydro-Pro ;
  • X is Ala, Gly, Ile, Leu, Val, Met, Lys, Glx or Asx; Y is one or more D or L amino acids, or is a covalent bond;
  • Z is CO 2 R"", CONR' R"", COR', CH 2 OH, CH 2 NR'R”" or H, or if d and e are 0 and Y is a covalent bond, Z is OR"" or NR'R""; a, b, c, d and e are each independently 0 or 1, provided that c and e are not simultaneouly 0;
  • M is Cha, Phe(4'R a ) or -NHCHR 1 R 2 -; wherein:
  • R 1 is independently C 1-5 Alk, (CH 2 ) n SC 1-5 Alk,
  • R a is halogen, OR 1 , NO 2 , NH 2 or H;
  • R 2 is (CH 2 ) n -, CHR 3 (CH 2 ) m CO-, CO (CH 2 ) m CO-,
  • R 3 and R3' are each independently OH, H or NH 2 ;
  • m is independently 0, 1, 2 or 3;
  • n is independently 1 or 2;
  • p is 0, 1 or 2;
  • R' is H or C 1-5 Alk
  • R" is H or C 1-18 Alk
  • R" is H, C 1-5 Alk, C 3-6 Cycloalkyl, (CH 2 ) n C 6 H 5,
  • Prodrugs are considered to be any covalently bonded carriers which release the active parent drug according to formula (I) in vivo.
  • A comprises the terminal amino group of the peptide
  • Z comprises the terminal carboxyl group of the peptide.
  • the terminal residues of the peptide are "des-amino" and “descarboxy” amino acids respectively.
  • A comprises the terminal amino group of the residue corresponding to B; or, when B is a covalent bond, to Q; or, when a is 0 and B is a covalent bond, to C; or when B is a covalent bond and a and b are 0, to D.
  • B is a covalent bond and a, b, and c are 0, the amino group of M is substituted by an acyl or alkyl group, as specially provided by A in formula (I).
  • Z comprises the terminal carboxyl group of the amino acid residue corresponding to Y; or when Y is a covalent bond, to X; or When Y is a covalent bond and d and e are 0, the terminal carboxyl group of M is substituted by Z as specially provided in formula (I).
  • ⁇ -Ala refers to 3-amino propanoic acid.
  • Cha refers to cyclohexylalanine.
  • Boc refers to the t- butyloxycarbonyl radical
  • Dns refers to the dansyl radical, which is 1-dimethylamino napthylene-5-sulfonyl
  • Cbz refers to the carbobenzyloxy radical
  • BrZ refers to the o-bromobenzyloxycarbonyl radical
  • Clz is the p-chlorocarbobenzyloxy radical
  • CI 2 Z refers to the 2,4-dichlorocarbobenzyloxy radical
  • Bzl refers to the benyzl radical
  • MeBzl refers to the 4-methyl benzyl radical
  • Ac refers to acetyl
  • Alk or C 1-5 Alk refers to C 1-5 alkyl
  • Ph refers to phenyl
  • DCC refers to the residue constitutes ty
  • DMSO dimethylsulfoxide
  • DMF dimethyl formamide
  • THF tetrahydrofuran
  • HF hydrofluoric acid
  • TFA trifluoroacetic acid
  • C 1-5 alkyl as appIled herein is meant to include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tertiary butyl, pentyl and isopentyl.
  • Asp and Glu which have
  • carboxylic acid side chains encompass free carboxylic acids, C 1-5 alkyl and benzyl ester side chains.
  • C 1-18 Alk is intended to include any straight or branched chain alkyl group of 1 to 18 carbons.
  • the peptides of this invention bind to viral proteases in a manner which mimics the binding of the natural
  • the peptides are generally dodecapeptides or smaller. However, longer peptides which encompass the residues defined herein as -Q-C-D-M-W-X-, as given in formula (I), are also beIleved to be active and are considered within the scope of this invention.
  • X is preferably a neutral or acidic amino acid.
  • X is Ala, Gly, Ile, Leu, Val, Met, Lys, Glx or Asx. Valine is especially suitable.
  • D is preferably a neutral and hydrophobic amino acid, although certain hydrophilic residues such as Asp and Ser are
  • the residue corresponding to C may be a neutral, acidic or basic amino acid. Glu, Gln, Arg, Lys, Ser, Ala, ⁇ -Ala, Asn and Gly are suitable. Gln, Asn and Ala are especially preferred for C.
  • the residue Q is preferably a hydrophilic residue, such as D- or L-, Ser, Thr, Asp or His. Especially preferred are Thr and Ser.
  • B corresponds to one or more amino acids, which may be hydrophilic or hydrophobic, or it may be a covalent bond in the shorter peptides.
  • the identity of B is not critical and a residue may be chosen for the favorable physico-chemical and biochemical properties it confers on the overall peptide, such as water solubility and resistance to exopeptidases.
  • the choice of a D-amino acid often confers resistance to exopeptidases when the D-amino acid is at the terminus of the peptide.
  • Q may be advantageous for Q to be a D-amino acid.
  • B is preferably one or two D- or L-amino acids chosen from Ala, ⁇ -Ala, Gly, Ile, Val, Leu, Met, His, Lys, Arg, Glx, Asx, Cys,
  • B is preferably a covalent bond.
  • Y may correspond to one or more amino acids, or a covalent bond. If it is one or more amino acids, they may be hydrophilic or hydrophobic. One to three residues are preferred, but a longer chain is acceptable. In much the same manner as B, the residues of Y may be used to confer favorable biochemical or physio-chemical properties to the peptide. Thereby, the use of hydrophilic residues may be used to confer desirable solubility properties or D-amino acids at the carboxy terminus may be used to confer
  • Y is one to three amino acids chosen from Ala, Gly, Ile, Leu, Met, Val, Arg, Lys, Thr, Ser, Cys, Glx or Asx.
  • Y may be a covalent bond but a single amino acid is especially suitable.
  • Ala, Gly, Ile, Met, Arg, Asx and Val are preferred.
  • Valine is especially preferred.
  • Peptides wherein M is Cha or Phe(4'Ra) and d is 1 may act as substrates, and are hydrolyzed to smaller peptides.
  • the substrates may generally be of any length, 6-9 residues is preferred. These are conveniently used to assay for protease activity.
  • they may compete with the natural viral substrates and thereby serve to inhibit viral replication and, hence, disease progression in vivo; although, due to their metabolic instability, their duration of action may be short.
  • these peptides may be used in an assay for protease activity by subjecting the peptide to the protease in a suitably buffered medium.
  • Analysis of the activity is carried out in such a manner as to detect the hydrolytic cleavage of the peptide.
  • One such method constitutes the separation of at least one of the product peptides, or a derivative thereof, and its
  • chromatographic means can be used to effect the separation of the peptide products using conventional solid supports such as silica gel, octadecyl silane, Sephadex®, ion exchange resins or adsorption resins. Detection/quantitation of the products is effected by ultraviolet absorption or other spectroscopic analysis.
  • incorporation or substitution of a radioactive isotope in the peptide, such as tritium or 13 C label in one or more of the amino acids, provides for
  • incorporation of a fluorescent moiety, such as a dansyl group on the amino terminus of the peptide provides for fluorescent detection/quantitation.
  • Fluorogenic methods which would incorporate a quenching agent into the peptide, such as a 3-(4-N-methyl-pyridyl)-propyl oxy ester of the carboxy terminus, in addition to the fluorescent marker, would also be useful to assay protease activity. Such methods are disclosed, for example, by Dunn, et al., Anal. Biochem.. 129, 502(1983), and provide for continuous
  • protease binding activity of the peptides of this invention is demonstrated by their ability to act as substrates.
  • the following table illustrates the substrate kinetics of these peptides.
  • proteases and thereby inhibit the proteolytic activity.
  • Modifications of such peptides which bind to viral proteases so as to retard the hydrolysis of the peptide provide a means for effecting longer lived inhibition of protease activity.
  • another subgeneric group of compounds of this invention comprises the peptides wherein the residue M is -NHCHR 1 R 2 -.
  • These peptides contain an unnatural amino acid, which is not an ⁇ -amino acid.
  • These peptides do not possess a peptide bond in the same manner as a substrate and therefore, although they bind to the protease in a manner which mimics the binding of the natural substrate, are resistant to hydrolysis.
  • Peptides of 2-9 residues are suitable.
  • Peptides of 3-6 residues are preferred.
  • R 1 is CH 2 C 6 H 4 -R a , CH 2 C 6 H 11 or C 1-5 Alk.
  • R 2 is preferably CHR 3 CHR 3' (CH 2 ) m CO-, CHR 3 CHR 3' CHR 1 CO-,
  • R', R 3 , R 3' , m and n have the meanings defined in formula (I).
  • R 1 is CH 2 C 6 H 4 -Ra and R 2 is CH(OH) CH 2 CH 2 CO-.
  • the peptides of formula (I) have the partial structure -Ala-NHCH (CH 2 Ph) CH (OH) CH 2 CH 2 CO-Val-Val-.
  • retroviruses an inhibitor or substrate is likely to be broadly active over the range of retroviruses.
  • DNA viruses which are dependant upon virally encoded proteases, such as the hepatitis virus, may also be susceptible to such
  • the peptides of the invention are prepared by coupling the appropriate amino acid residues, optionally removing any protective groups and optionally modifying the amino or carboxy terminus of the peptide. They are prepared
  • amino acid or peptide is suitably protected as known in the peptide art.
  • carbobenzyloxy-group is preferred for protection of the amino group, especially at the ⁇ position.
  • a benzyl group or suitably substituted benzyl group is used to protect the mercapto group of cysteine, or other thiol containing amino acids; or the hydroxyl of serine or threonine.
  • the tosyl or nitro group may be used for protection of the guanidine of Arg or the imidazole of His, and a suitably substituted carbobenzyloxy group or benzyl group may be used for the hydroxyl group of Tyr, Ser or Thr, or the ⁇ -amino group of lysine.
  • Suitable substitution of the carbobenzyloxy or benzyl protecting groups is ortho and/or para substitution with chloro, bromo, nitro or methyl, and is used to modify the reactivity of the protective group.
  • Cysteine and other sulfur-containing amino acids may also be protected by formation of a disulfide with a thioalkyl or thioaryl group.
  • the protective groups are, most conveniently, those which are not removed by mild acid treatment. These protective groups are removed by such methods as catalytic hydrogenation, sodium in liquid ammonia or HF treatment as known in the art.
  • the peptide is built up sequentially starting from the carboxy terminus and working toward the amino terminus of the peptide.
  • Solid phase synthesis is begun by covalently attaching the C terminus of a protected amino acid to a suitable resin, such as a
  • MBHA chloromethyl resin
  • CMR chloromethyl resin
  • a BHA or MBHA support resin is used if the carboxy terminus of the product peptide is to be a carboxamide.
  • a CMR support is generally used if the carboxy terminus of the product peptide is to be a carboxyl group, although this may also be used to produce a
  • the amino group is hydrolyzed by mild acid treatment, and the free carboxyl of the second protected AA is coupled to this amino group. This process is carried out sequentially, without isolation of the
  • the completed peptide may then be deblocked and/or split from the carrying resin in any order.
  • HBr/acetic acid splits the peptide from the resin and produces the carboxy terminal amino acid as a carboxylic acid.
  • Treatment of a CMR supported peptide with ammonia or alkyl amines in an alcoholic solvent provides a carboxamide or alkyl carboxamide at the carboxy terminus.
  • the CMR resin may be treated with an appropriate alcohol, such as methyl, ethyl, propyl, butyl or benzyl alcohol, in the presence of triethylamine to cleave the peptide from the resin and produce the ester directly.
  • an appropriate alcohol such as methyl, ethyl, propyl, butyl or benzyl alcohol
  • Esters of the peptides of this invention may also be prepared by conventional methods from the carboxylic acid precursor.
  • the carboxylic acid is treated with an alcohol in the presence of an acid catalyst.
  • the carboxylic acid may be converted to an activated acyl intermediate, such as an acid halide or activated anhydride or ester, and treated with an alcohol, preferably in the presence of a base.
  • the preferred method for cleaving a peptide from the support resin is to treat the resin supported peptide with anhydrous HF in the presence of a suitable cation scavenger, such as anisole or dimethoxy benzene.
  • a suitable cation scavenger such as anisole or dimethoxy benzene.
  • Peptides hydrolyzed in this way from the CMR are carboxylic acids, those split from the BHA resin are obtained as carboxamides.
  • Modification of the terminal amino group of the peptide is accomplished by alkylation or acetylation as is generally known in the art. These modifications may be carried out upon the amino acid prior to incorporation into the peptide, or upon the peptide after it has been synthesized and the terminal amino group liberated, but before the protecting groups have been removed.
  • acetylation is carried out upon the free amino group using the acyl halide, anhydride or activated ester, of the corresponding alkyl acid, in the presence of a tertiary amine.
  • Mono-alkylation is carried out most
  • Dialkylation may be carried by treating the amino group with an excess of an alkyl halide in the presence of a base.
  • the longer chain alkyl and acyl groups of this invention are beIleved to have advantageous properties due to their affinity for lipid membranes.
  • Myristylation or stearylation is useful.
  • Solution synthesis of peptides is accomplished using conventional methods used to form amide bonds.
  • a protected Boc-amino acid which has a free carboxyl group is coupled to a protected amino acid which has a free amino group using a suitable carbodiimide coupling agent, such as N, N' dicyclohexyl carbodiimide (DCC), optionally in the presence of catalysts such as 1-hydroxybenzotriazole (HOBT) and dimethylamino pyridine (DMAP).
  • a suitable carbodiimide coupling agent such as N, N' dicyclohexyl carbodiimide (DCC)
  • catalysts such as 1-hydroxybenzotriazole (HOBT) and dimethylamino pyridine (DMAP).
  • HOBT 1-hydroxybenzotriazole
  • DMAP dimethylamino pyridine
  • a protected Boc-amino acid or peptide is treated in an anhydrous solvent, such as methylene chloride or tetrahydrofuran (THF), in the presence of a base, such as N-methyl morpholine, DMAP or a trialkyl amine, with isobutyl chloroformate to form the "activated anhydride", which is subsequently reacted with the free amine of a second protected amino acid or peptide.
  • anhydrous solvent such as methylene chloride or tetrahydrofuran (THF)
  • a base such as N-methyl morpholine, DMAP or a trialkyl amine
  • the protecting groups may be removed as hereinbefore described, such as by
  • hydrofluoric acid or alkali hydrofluoric acid or alkali.
  • Esters are often used to protect the terminal carboxyl group of peptides in solution synthesis. They may be converted to carboxylic acids by treatment with an alkali metal hydroxide or carbonate, such as potassium hydroxide or sodium carbonate, in an aqueous alcoholic solution. The acids may be converted to other esters via an activated acyl intermediate as previously described.
  • the amides and substituted amides of this invention are prepared from carboxylic acids of the peptides in much the same manner.
  • ammonia or a substituted amine may be reacted with an activated acyl intermediate to produce the amide.
  • Use of coupling reagents, such as DCC, is convenient for forming substituted amides from the carboxylic acid itself and a suitable amine.
  • methyl esters of this invention may be converted to the amides, or substituted-amides, directly by treatment with ammonia, or a substituted amine, in methanol solution.
  • a methanol solution of the methyl ester of the peptide is saturated with ammonia and stirred in a
  • Carboxamides are preferred embodiments of this invention due their enhanced stability relative to esters.
  • the amino acid which constitutes the M residue of the peptide comprises either phenylalanine, a derivative thereof, or an unnatural amino acid denoted as -NHCHR 1 R 2 -.
  • the derivatives of phenylalanine include tyrosine; (O-C 1- 5 alkyl)tyrosine, 4'-halophenylalanine, 4'-nitrophenylalanine and 4'-aminophenylalanine as are generally well known in the peptide art.
  • R 1 is independently C 1-5 Alk, (CH 2 ) n SC 1-5 Alk,
  • R a is halogen, OR', NO 2 , NH 2 or H;
  • R 2 is (CH 2 ) n -, CHR 3 (CH 2 ) m CO-, CO(CH 2 ) m CO-, CHR 3 CHR 3' (CH 2 ) m CO-, CHR 3 CHR 3' CHR 1 (CH 2 ) m CO-,
  • R 3 and R3' are each independently OH, H or NH 2 ;
  • m is independently 0, 1, 2 or 3;
  • n is independently 1 or 2;
  • p is 0, 1 or 2;
  • R 5 is H
  • R 6 is CH 3 CO, C 1-5 Alk, Dns, Cbz or Boc, or taken together R 5 and R 6 are phthalimido;
  • X is H, halogen, OR", OC 1-5 Alk, NHR'R" or OCOC 1-5 Alk; R' H or C 1-5 Alk; and
  • R" is H or C 1-18 Alk.
  • peptide substrate which binds to a protease may be converted to an inhibitor by replacement of one or both of the amino acids at the scissile site by a suitable compound of formula (Ila).
  • the unnatural amino acids are generally not ⁇ -amino acids, but are derived from ⁇ -amino acids.
  • identity of R 1 is established by the choice of an amino acid of formula (III), wherein R 1 is chosen as previously set forth.
  • aldehyde of formula (V) thereby provides a versatile
  • the N-methoxy-methyl amides of these ⁇ -amino acids are also useful intermediates for this use.
  • the ⁇ -amino acids may be obtained from natural sources, commercial sources or may be synthesized by a number of methods well known in the peptide art.
  • Alcohols of formula (IV) may be obtained from the diborane reduction of the corresponding protected amino acid.
  • Aldehydes of formula (V) may be obtained from the reduction of the methyl ester of the corresponding amino acid, for example with diisobutyl aluminum hydride (DIBAL).
  • DIBAL diisobutyl aluminum hydride
  • the alcohol of formula (IV) may be oxidized to the aldehyde using a mild oxidizing agent, such as the Dess Martin periodinane or using the Swern method (DMSO, oxalyl chloride, triethylamine).
  • a mild oxidizing agent such as the Dess Martin periodinane or using the Swern method (DMSO, oxalyl chloride, triethylamine).
  • amino acids alanine, valine, leucine and isoleucine are especially useful for producing chiral aldehydes.
  • ⁇ -Amino butyric acid, ⁇ -amino isobutyric acid and ⁇ -amino pentanoic acid are especially useful for producing chiral aldehydes.
  • any alkyl- ⁇ -amino acid can be synthesized by alkylation of a nitro-acetic acid ester with an appropriate alkyl halide in the presence of a base, such as triethylamine, DBU or other tertiary amine.
  • a base such as triethylamine, DBU or other tertiary amine.
  • Alkoxides such as sodium methoxide or ethoxide are also suitable bases.
  • Subsequent reduction of the ⁇ -nitro alkyl acid or ester with hydrogen and a palladium or platinum catalyst yields the amino acid.
  • it is common to produce ⁇ -amino acids by alkylation of an N-benzylidine glycine ester, such as a methyl or ethyl ester.
  • R 1 is aryl or aralkyl
  • amino acids in which R 1 is aryl or aralkyl are available commercially, or are well known in the art such as phenylalanine, 4'-halo and 4'-nitrophenylalanine, tyrosine, (O-alkyl) tyrosine, phenylglycine and (4'-hydroxy)phenylglycine.
  • Homologs of these compounds may be prepared by conventional techniques of amino acid synthesis. Thus, alkylation of a nitro acetic acid ester or a N-benzylidene glycine with 1-phenyl-2-bromoethane or 1-phenyl- 3-bromopropane, as described above, produces the homologous amino acids.
  • ⁇ -amino acids in which R 1 contains an oxygen or sulfur are obtained from serine, homo-serine, threonine, cysteine or methionine or alkylation or these amino acids, excepting methionine.
  • Alkylation is carried out by treating the corresponding hydroxyl or thiol compound with a base, such as triethylamine, DBU or sodium hydride, and an
  • alkyl halide such as methyl, ethyl, isopropyl or isobutylbromide.
  • di-homoanalogues are prepared by conventional techniques for producing amino acids as
  • an ester of nitro acetic acid is treated with a base, as previously described and acrolein in a Michael reaction.
  • the aldehyde produced is subsequently reduced by a mild reducing agent, such as sodium borohydride, to yield the desired hydroxy group.
  • a mild reducing agent such as sodium borohydride
  • Subsequent catalytic reduction of the nitro group yields the ⁇ -amino 5-hydroxy pentanoic acid.
  • the corresponding mercapto analogue may be synthesized from the hydroxy intermediate.
  • the hydroxyl group is converted to a halide, such as by treatment with phosphorous tribromide or oxalyl bromide or chloride in the presence of triethylamine or another tertiary amine, or by treatment with triphenyl phosphine and carbon tetrabromide.
  • a halide such as by treatment with phosphorous tribromide or oxalyl bromide or chloride in the presence of triethylamine or another tertiary amine, or by treatment with triphenyl phosphine and carbon tetrabromide.
  • Treatment of the halide with sodium sulfide or potassium thioacetate followed by saponification with sodium hydroxide yields the 5-mercapto-2-amino pentanoic acid.
  • This intermediate may be further alkylated in the same manner as cysteine or serine described herein above.
  • a hydrocarbon substituent at R 2 is suitably accomplished by reaction of the aldehyde of formula (V) with an organometallic hydrocarbon, such as a Grignard reagent or organo-lithium species.
  • organometallic hydrocarbon such as a Grignard reagent or organo-lithium species.
  • the incipient carboxylic acid functionality of R 2 is in protected form as in an ortho ester or disguised, as in an oxidizable olefin.
  • the organometallic species is a magnesium or lithium alkylide generated from lithium or magnesium and an alkylene halide, such as allyl bromide, 1-bromo-3-butene, 1-bromo-4-pentene, or 1-bromo-5-hexene, or an alkyl substituted derivative thereof .
  • Oxidation of the hydroxy- or keto-alkane is accomplished by conventional methods, such as with potassium permanganate or by ozonolysis followed by standard dimethyl sulfide workup and subsequent oxidation with potassium permanganate or Jones reagent. As would be obvious to one skilled in the art, this method is inapplicable when R 1 contains a sulfur substituent.
  • the hydroxyl may be further converted to chloride or bromide using a halogenating agent, such as phosphorous trichloride or phosphorous tribromide, and subsequently reduced to a methylene group with hydrogen and Raney® nickel.
  • a halogenating agent such as phosphorous trichloride or phosphorous tribromide
  • the keto group may be reductively aminated with sodium cyanoborohydride and an ammonium halide, such as ammonium chloride or bromide.
  • the carboxylic acid may be converted to an ester, amide, aldehyde, anhydride or acyl halide by common methods well known in the chemical art.
  • the introduction of unsaturation at R 2 may be derived from the amino acid products corresponding to formulas (VII). Reduction of the carbonyl group to a hydroxyl group, as previously indicated, and subsequent elimination of the hydroxyl group, provides the unsaturated compounds of this invention.
  • the hydroxyl group may be eliminated by
  • R1, R5, R6, X and n are as definded in formula (Ila), are prepared by a process which comprises reacting a compound of formula (V), under reducing conditions, with an ester or acid of proline, ⁇ 3-dehydro-proline or 2-carboxypiperidine, and thereafter, in any order, converting the ester to a carboxylic acid, ester, aldehyde or amide; or converting the acid to an ester, amide, anhydride or acyl halide.
  • a hydroxy-amino acid of formula (IV) is converted to the corresponding halide using a conventional reagent, such as phosphorous tribromide, phosphorous oxychloride or thionyl chloride in the presence of a base, such as triethylamine or pyridine, and treated with the thioalkanoic acid or ester again in the presence of a base.
  • a conventional reagent such as phosphorous tribromide, phosphorous oxychloride or thionyl chloride in the presence of a base, such as triethylamine or pyridine, and treated with the thioalkanoic acid or ester again in the presence of a base.
  • the sulfide produced in this way may be further oxidized to a sulfoxide by treatment with a mild oxidizing agent, such as sodium metaperiodate, or a sulfone, by treatment with potassium permanganate.
  • dimethyl oxaloacetate is treated with diethylamino sulfur trifluoride (DAST) to produce ⁇ -difluoro dimethyl succinate.
  • DAST diethylamino sulfur trifluoride
  • oxidized to the corresponding aldehyde using a mild oxidizing method such as Collins reagent, the Dess-Martin periodinane or the Swern method (DMSO, oxalyl chloride, triethylamine).
  • Collins reagent the Dess-Martin periodinane or the Swern method (DMSO, oxalyl chloride, triethylamine).
  • derivatives of these homologues may be prepared by alkylation of the initial ⁇ -keto diester with a base and alkyl halide prior to conducting the sequence.
  • alkylation of dimethyl oxalacetate with 2 equivalents of methyl iodide in the presence of DBU yields 2-keto-3, 3-dimethyl succinnate.
  • Further conversion by the above sequence to 2,2 dimethyl-3,3-difluoro-4-oxo-methyl butanoate, condensation with 2-phenyl nitroethane and reduction with hydrogen and 5% Pd/C yields 2,2 dimethyl-3,3-difluoro-4-hydroxy-5-amino-6-phenyl methyl hexanoate.
  • pyridine/chromium trioxide are suitable oxidants.
  • phosphinyl substituent at R is carried out by a condensation of benzyl carbamate with triphenyl phosphite and an aldehyde of the general formula R 1 CHO, such as phenylacetaldehyde, isobutyraldehyde or 2-methyl butyraldehyde, in acetic acid to yield a phosphonate of formula (IX).
  • organo metallic reagent such as the lithium or Grignard reagent derived from alkyl bromide, bromo-3-butene, 2-(2-methylpropenyl) chlorocyclopentane or 2- (2-methyl propenyl)-cyclohexane, yields the alkenyl methyl phosphinates of this invention as given by formula (Xa) and (Xb):
  • R 1 , R 5 , R 6 , R', R", m and n are as defined for formula (Ila) and R" ' is C 1-5 alkyl.
  • Subsequent oxidation, such as with potassium permanganate or by ozonolysis as herein described affords the corresponding alkyl phosphinate carboxylic acids of this invention. Accordingly, the compounds of formulas (XI):
  • R 1 , R 5 , R 6 , R', X and n are as defined as in formula (Ila), may be prepared by oxidizing a compound of formula
  • phosphinate to the phosphinic acid may be accomplished by treatment with a strong acid, such as hydrogen bromide.
  • a strong acid such as hydrogen bromide.
  • R 1 contains a sulfur substituent.
  • a further aspect of this invention relates to the preparation of completely novel amino acids which, when properly incorporated into peptides, provide a nonhydrolyzable imitation of a peptide bond.
  • These compounds are prepared as hereinbefore described and are further illustrated in the Examples which follow. They are
  • R 1 is independently C 1-5 Alk, -(CH 2 ) n SC 1-5 Alk,
  • R a is halogen, NO 2 , OH, OC 1-5 Alk, NH 2 , or H;
  • R 2 is (CH 2 ) n -X, (CH 2 ) n CO-X, CHR 3 CHR 1 CHR' (CH 2 ) m CO-, COCR 1 CHR' (CH 2 ) m CO-X, CHR 3 CF 2 (CH 2 ) m CO-X, COCF 2 CR'R" (CH 2 ) m CO-X, CHR 3 (CH 2 ) m CO-X, CO (CH 2 ) m CO-X,
  • R 3 is H, NH 2 or OH
  • R 5 is H
  • R 6 is CH 3 CO, C 1-5 Alk, Dns, Cbz or Boc, or taken together R 5 and R 6 are phthalimido;
  • X is H, halogen, OH, OC 1-5 Alk, NHR'R" or OCOC 1-5 Alk; R' and R" are H or C 1-5 Alk; m is independently 0, 1, 2 or 3; and n is independently 1 or 2; provided that R 2 is not CHR 3 CH 2 CO-X, COCH 2 CO-X, COCF 2 CO-X or CHR 3 CF 2 CO-X when R 1 is phenylmethylene, isobutyl or cyclohexylmethylene, and R 2 is not CH (OH) CH 2 CH 2 CO-X or
  • diastereomeric These diasteromers are usually separable by chromatography over silica gel or an octadecyl silane chromatographic support with a suitable mobile phase. If silica gel is used, a mixture of ethyl acetate and hexane or methanol and chloroform or methylene chloride are used to elute the diasteromers. If an octadecyl silane support is used, a mixture of water and methanol or acetonitrile may be used to purify the diastereomers.
  • the mobile phase may be buffered by the addition of .02 to .5 % of acetic acid, trifluoracetic acid or phosphate buffer. If the residue, designated as M, possesses only a single chiral center, or the diasteromers are difficult to separate, it may be preferable to
  • the unnatural amino acids of the M residue are N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • an acid addition salt may be
  • Acid addition salts of the peptides are prepared in a standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic, succinic or methanesulfonic.
  • an acid such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic, succinic or methanesulfonic.
  • the acetate salt form is especially useful. If the final peptide contains an acidic group, cationic salts may be prepared.
  • the parent compound such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic, succinic or methanesulfonic.
  • an alkaline reagent such as a hydroxide, carbonate or alkoxide, containing the appropriate cation.
  • Cations such as Na + , K + , Ca ++ and NH 4 + are examples of cations present in pharmaceutically
  • Certain of the compounds form inner salts or zwitterions which may also be acceptable.
  • the compounds of formula (I), wherein M is -HNCHR 1 R 2 -, are used to induce anti-viral activity in patients which are infected with susceptible viruses and require such treatment.
  • the method of treatment comprises the administration orally, parenterally, buccally, trans-dermally, rectally or by insufflation, of an effective quantity of the chosen
  • Dosage units of the active ingredient are selected from the range of .05 to 15 mg/kg. These dosage units may be
  • protease inhibiting properties of the peptides of this invention are demonstrated by their ability to inhibit the hydrolysis of a peptide
  • rHIV protease in the range of about 10 nM to about 250 ⁇ M.
  • the following table is representative of the inhibition constants of these peptides.
  • compositions of the peptides of this invention, or derivatives thereof, may be formulated as solutions or lyophilized powders for parenteral
  • Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use.
  • the liquid formulation is generally a buffered, isotonic, aqueous solution.
  • suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution.
  • Such formulation is especially suitable for parenteral administration, but may also be used for oral administration or contained in a metered dose inhaler or nebulizer for insufflation. It may be desirable to add excipients such as polyvmylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.
  • a preferred composition for parenteral administration may additionally be comprised of a quantity of the compound encapsulated in a liposomal carrier.
  • the liposome may be formed by dispersion of the peptides in an aqueous phase with phospholipids, with or without cholesterol, using a variety of techniques, including conventional handshaking, high pressure extrusion, reverse phase evaporation and
  • compositions is more fully disclosed in copending Application Serial No. 06/763,484 and is incorporated herein by
  • Such a carrier may be optionally directed toward its site of action by an immunoglobulin or protein reactive with the viral particle or infected cells.
  • the choice of such proteins would" of course be dependent upon the antigenic determinants of the infecting virus.
  • An example of such a protein is the CD-4 T-cell glycoprotein, or a derivative thereof, such as sCD-4 (soluble CD-4), which is reactive with the glycoprotein coat of the human immunodeficiency virus (HIV).
  • sCD-4 soluble CD-4
  • these peptides may be encapsulated
  • compositions may be added to enhance or stabilize the
  • Liquid carriers include syrup, peanut oil, olive oil,
  • Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin.
  • the carrier may also include a sustained release material such as glyceryl monostearate or glyceryl
  • the amount of solid carrier varies but, preferably, will be between about 20 mg to about 1 g per dosage unit.
  • the pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms.
  • a liquid carrier When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.
  • a pulverized powder of the peptides of this invention may be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository.
  • excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols
  • the pulverized powders may also be compounded with an oily preparation, gel, cream or emulsion, buffered or unbuffered, and administered through a transdermal patch.
  • Beneficial effects may be realized by co-administering, individually or in combination, other anti-viral agents with the protease inhibiting compounds of this invention.
  • anti-viral agents include nucleoside analogues,
  • Nucleoside analogues which include 2',3'-dideoxycytidine(ddC), 2',3'-dideoxyadenine (ddA) and
  • compositions comprise an anti-viral agent, a protease inhibiting peptide of this invention and a pharmaceutically acceptable carrier.
  • the enzyme used to assay the peptide of this invention was produced in this manner and purified from the cell pellet as follows.
  • the E. coli cell pellet was resuspended in a buffer consisting of 50 mM TrisHC1, pH 7.5; 1.0 mM each DTT, EDTA and PMSF
  • the protease was N85-95% pure.
  • immunoblot analysis >90% of the immunoreactive material was precipitated at the ammonium sulfate step.
  • activity assay the highest peak of activity was found in the fractions collected at 45 and 46 minutes.
  • the activity itself cannot be used to obtain reliable recovery data as it is influenced by high salt, i.e., with increasing salt, increasing levels of activity were obtained. Thus, with each step in the purification, more total activity was
  • reaction rates were linear over the time course of the
  • MENDT buffer 50 mM Mes (pH 6.0; 2-(N-morpholino) ethanesulfonic acid), 1
  • reaction mixtures 37°C were quenched after 10-20 minutes with an equal volume of cold 0.6 N trichloroacetic acid, and, following centrifugation to remove precipitated material, peptidolysis products were analyzed by reverse phase HPLC (Beckman
  • Peptide amides are synthesized by solid phase peptide synthesis using benzhydrylamine resin as the support.
  • Protected amino acids are added sequentially starting from the carboxyl terminus until the desired sequence has been obtained.
  • the t-butyloxycarbonyl (Boc) group is used for protection of the alpha-amino group.
  • Side chain functional groups are protected as follows: arginine and histidine, tosyl (Tos); cysteine, p-methylbenzyl (MeBzl); serine and threonine, benzyl ether (Bzl); lysine, p-chlorocarbobenzoxy (Clz); glutamic acid and aspartic acid, benzyl ester (OBzl); tyrosine,
  • p-bromocarbobenzoxy (BrZ). Removal of the Boc group is accomplished by treatment with 50% trifluoroacetic acid (TFA) in methylene chloride. Neutralization of the amine-TFA salt is accomplished by treatment with 7% diisopropylethylamine (DIEA) in methylene chloride. Amino acids are coupled to the growing peptide using 3 equiv. Boc-amino acid and 3 equiv. 1-hydroxybenzotriazole (HOBt) in DMF and 3 equiv. of
  • DCC dicyclohexylcarbodiimide
  • Boc-Arg (Tos) -Ala-Ser (Bzl)-Gln-Asn-Tyr (BrZ)-Pro-Val-Val-BHA was prepared in the usual way. After removal of the N-terminal Boc group with 50% TFA in
  • the peptide was cleaved from the resin with removal of the side chain protecting groups by treatment with anhydrous liquid HF (10 ml) in the presence of anisole (1 ml) at 0°C for 50 minutes. After removal of the HF under vacuum, the resin was washed with ethyl ether and air-dried. The resin was then extracted with 2 x 30 ml 1% HOAC/H 2 O followed by 2 x 30 ml 10% HOAC/H 2 O. The combined extracts were lyophilized to yield 476 mg crude peptide.
  • Boc-Phe-OMe (12.9 g, 46.4 mmol), prepared as above, was dissolved in dry toluene (50 mL) and cooled to -78°C.
  • Diisobutylaluminum hydride (25% in toluene, 77.3 mL, 116 mmol, 150% excess) was added over 2 min.
  • methanol (15 mL) was added slowly to control effervescence.
  • the mixture was poured into Rochelle salt (94 g potassium sodium tartrate in 1 L water, 150 mL) and shaken with ether (100 mL) until extractable.
  • the aqueous phase was Washed with ether (3 X 100 mL).
  • the organic phases were combined, dried (MgSO 4 ), and evaporated.
  • Example 3(f) By the same procedure used to prepare the compound of Example 3(f), except substituting isomer A (186 mg, 0.50 mmol) from Example 3 (e), the titled compound was obtained (135 mg, 0.37 mmol) as a white solid in 75% yield.
  • the 2-hydroxy configuration of isomer B was determined in the same manner as isomer A.
  • N-Boc-L-serine-O-benzyl ester (5.90 g, 20.0 mmol) and N-methylmorpholine (2.74 ml, 25.0 mmol) in THF (100 ml) at 40°C under argon. After 45 min. N-methylmorpholine (2.75 ml, 25.0 mmol) and solid L-alanine methyl ester hydrochloride (3.22 g, 23.0 mmoir were added. The mixture was allowed to stir with gradual warming to 20°C. After 18 hr. the mixture was diluted with ethyl acetate and washed successively with 5% HCl, 5% NaHCO 3 and brine. Filtration and removal of solvent under vacuum provided the titled compound (7.54 g, 19.8 mmol) as an oil in 99% yield.
  • Example 2(d) The compound of Example 2(d) (0.35 g, 1.0 mmol) was coupled to Val-Val-BHA (1.0 mmol) using DCC (0.21 g, 1.0 mmol) and HOBT (0.15 g, 1 mmol) in 50% CH 2 Cl 2 /DMF overnight.
  • Quantitative ninhydrin test indicated 83% coupling.
  • the peptide resin was acetylated with Ac 2 O (1 mL in 30 mL CH 2 C1 2 ) until the ninhydrin test was negative. The peptide was then completed and acetylated, according to the procedure of Example 1, using half of the resin.
  • TLC R f 0.34 (B:A:W 4 : 1 :1), 0.68 (B:E:A:W 1:1:1:1).
  • HPLC (Hamilton PRP-1 250 X 4.1 mm analytical HPLC column, water-acetonitrile- 0.1% TFA, 95:5 to 60:40 over 15 min.) k' 2.73.
  • Amino acid analysis Ser 0.57, Gln 1.00, Asn 1.00, Val 2.00.
  • Example 2(d) The compound of Example 2(d) (0.87 g, 2.5 mmol) was coupled to Val-Val-BHA (1.2 mmol) with DCC (0.52 g, 2.5 mmol) and HOBt (0.38 g, 2.5 mmol) in 30 mL of 50% CH 2 Cl 2 /DMF overnight. Ninhydrin test showed complete coupling.
  • the peptide was completed using 0.6 g (0.4 mmol) of the resin.
  • the peptide was cleaved from the resin using 15 mL HF with 1.5 mL anisole at 0°C for 1 h.
  • the peptide resin mixture was washed with ether (3X) followed by HOAc (4X). Lyophilization of the HOAc gave 192.5 mg crude peptide (62%).
  • the resin supported intermediate Ac-Ser (Bzl) -Gln-Asn- [3(RS)-AHPPA]-Pro-Val-Val-BHA was prepared on a 1 mm scale in the usual manner according to Example 1.
  • Boc-AHPPA used was racemic at the 3 position.
  • the peptide was cleaved from the resin and the benzyl group was removed from the serine hydroxyl by treatment at 0° with 10 ml of anhydrous HF and 1 ml of anisole for 60 min.
  • the HF was removed in vacuo at 0°.
  • the residue was triturated with diethyl ether, the peptide was extracted with acetic acid (3 x 20 ml) and lyophilized to yield 302 mg.
  • the resin supported intermediate Ac-Ser (Bz)-Gln-Asn- [3(RS)-AHPPA]-Val-Val-NH 2 was prepared in the usual manner on a .5 mm scale.
  • the Boc-AHPPA used was racemic in the 3 position.
  • the peptide was cleaved from the resin with removal of the benzyl protecting group by treatment with anhydrous HF (10 ml) in the presence of anisole (1 ml) at 0° for 60 min. After removal of the HF at 0° under vacuum, the resin was triturated with diethyl ether and air-dried. The peptide was extracted from the resin with acetic acid (4 x 20 ml) and lyophilized to yield 110 mg. The peptide was purified using counter current distribution (B:A:W, 4:1:5; 200 transfers). The major fractions were pooled,
  • the resin supported intermediate Boc-Ser (Bzl)-Gln-Asn- [3(R)-AHPPA]-Val-Val-BHA was prepared in the usual manner on a 1 mm scale. Omitting the acetylation step, the peptide was cleaved from the resin with removal of the benzyl protecting group by treatment with HF (10 ml) and anisole (1 ml) at 0° for 60 min. The HF was removed under vacuum at 0°. The residue was triturated with diethyl ether and the peptide was extracted from the residue with acetic acid (4 x 10 ml). The acetic acid extract was lyophilized to yield 85 mg. 40 mg of the crude peptide was purified by preparative HPLC
  • the resin supported peptide intermediate Boc-Ser (Bz)-Gln-Asn-[3(S)-AHPPA]-Val-Val-NH 2 was prepared in the usual manner on a 1 mm scale.
  • the peptide was cleaved from the resin, deblocked and purified in the same manner as in
  • Example 21(d) The compound of Example 21(d) (5 mg, 6.2 mmol) was dissolved in trifluoroacetic acid (.25 ml). After 90 min., the solution was concentrated under vacuum, the residue was dissolved in methanol (.5 ml) and concentrated HCl (about .025 ml) was added. The solution was concentrated, the resulting gum was triturated with ether and dried under vacuum to afford the titled compound (4.5 mg.) as a white solid.
  • Example 23(f) The product of Example 23(f) (1.3 mg, 1.6 mmol), was dissolved in trifluoroacetic acid. After 90 min. the solution was concentrated to dryness under vacuum. The residue was triturated with ether and the ether was removed under vacuum to yield the titled hexapeptide as a white solid.
  • Example 23(f) Using the procedure of Example 23(f), the hexapeptide of Example 23(e) (23 mg, 26 mmol) was debenzylated to provide the titled compound as a white solid (18.9 mg, 23.5 mmol; 90% yield).
  • Example 27 The compound of Example 25 (1.2 mg, 1.5 mmol) was dissolved in trifluoroacetic acid. After 90 min. the solution was concentrated to dryness under vacuum. The residue was triturated with ether to yield a white solid.
  • Example 27
  • Example 27(d) The product of Example 27(d) (12 mg, 15 mmol) was dissolved in trifluoroacetic acid (0.5 ml). After 90 min the solution was concentrated under vacuum; the residue was dissolved in methanol (1 ml) and cone. HCl (ca .05 ml) was added. The solution was concentrated, the resulting gum was triturated with ether and dried under vacuum to afford the titled compound (11 mg) as a white powder.
  • Example 27 Using the procedure of Example 27 (d), the above compound of Example 32(c) (23 mg, 24 mmol) was hydrogenolyzed to provide the titled compound (19.1 mg, 22
  • Trimethylsilyl bromide (4.0 ml, 30 mmol) was added to a solution of the compound of Example 32(d) (9.6 mg, 11.3 mmol) in dry CH 2 Cl 2 (1 ml). After 4.5 hr 1:1 water:acetic acid
  • N-methyl morpholine (0.27 ml, 2.5 mmol)
  • isobutyl chloroformate 0.246 ml, 1.9 mmol
  • Example 35(b) The tripeptide of Example 35(b) (115 mg, .179 mmol) is treated with trifluoroacetic acid and HCl according to the procedure of Example 34 (c).
  • the resulting hydrochloride salt was coupled to Boc-Ser (Bzl)-Ala-Ala (83 mg, .19 mmol) using N-methyl morpholine (50 mg, .51 mmol), and
  • hexapeptide was obtained as white crystals (40 mg; 25% yield) .
  • Example 35(c) 39 mg, 40.6 mmol was deprotected by the hydrogenolysis procedure of Example 34 (d).
  • the titled compound was obtained as white crystals (2.3 mg;

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Abstract

Des mimes peptidiques du substrat de polyprotéine de protéase de rétrovirus se lient à des protéases virales et sont utiles pour déterminer l'activité de la protéase ou pour inhiber son activité et dans le traitement de maladies virales.
PCT/US1989/002972 1988-07-08 1989-07-07 Peptides de liaison de protease retrovirale WO1990000399A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
FI910084A FI910084A0 (fi) 1988-07-08 1989-07-07 Retrovirala proteaser bindande peptider.
NO91910053A NO910053L (no) 1988-07-08 1991-01-07 Retrovirusprotease-bindende peptider.
DK002691A DK2691A (da) 1988-07-08 1991-01-07 Peptider, der binder retroviral protease
NO920319A NO920319D0 (no) 1988-07-08 1992-01-23 Retrovirusprotease-bindende peptider
NO920318A NO920318D0 (no) 1988-07-08 1992-01-23 Retrovirusprotease-bindende peptider

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US21617888A 1988-07-08 1988-07-08
US216,178 1988-07-08
US32193789A 1989-03-10 1989-03-10
US321,937 1989-03-10
US37432689A 1989-06-29 1989-06-29
US374,326 1989-06-29

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AU (1) AU3964489A (fr)
DK (1) DK2691A (fr)
FI (1) FI910084A0 (fr)
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WO (1) WO1990000399A1 (fr)

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5142056A (en) * 1989-05-23 1992-08-25 Abbott Laboratories Retroviral protease inhibiting compounds
EP0610744A1 (fr) * 1993-02-09 1994-08-17 Bayer Corporation Dérivés sulfonamidométhyléniques comme agent immunosuppressif
EP0610745A3 (en) * 1993-02-09 1994-09-28 Miles Inc Novel aminomethylene derivatives as immunosuppressants.
WO1995001958A1 (fr) * 1993-07-08 1995-01-19 Merrell Pharmaceuticals Inc. Analogues de la difluorostatone
FR2714621A1 (fr) * 1994-01-06 1995-07-07 Centre Nat Rech Scient Procédé de préparation de liposomes sans utilisation de solvant organique.
US5516786A (en) * 1989-05-13 1996-05-14 Bayer Aktiengesellschaft Fungicidal substituted amino acid amides
US5554728A (en) * 1991-07-23 1996-09-10 Nexstar Pharmaceuticals, Inc. Lipid conjugates of therapeutic peptides and protease inhibitors
US5559256A (en) * 1992-07-20 1996-09-24 E. R. Squibb & Sons, Inc. Aminediol protease inhibitors
US5580984A (en) * 1989-05-23 1996-12-03 Abbott Laboratories Retroviral protease inhibiting compounds
US5691368A (en) * 1995-01-11 1997-11-25 Hoechst Marion Roussel, Inc. Substituted oxazolidine calpain and/or cathepsin B inhibitors
US5716973A (en) * 1991-01-02 1998-02-10 Merrell Pharmaceuticals Inc. Anti-viral compounds
US5831094A (en) * 1993-09-09 1998-11-03 Merrell Pharamceuticals Inc. Difluoro statone antiviral analogs
US5969132A (en) * 1994-02-04 1999-10-19 Merrell Pharmaceuticals Inc. Macrocyclic difluorostatone derivatives useful as antiviral agents
US6114380A (en) * 1995-12-18 2000-09-05 Merrell Pharmaceuticals Inc. Difluoro statone analogs
EP1138673A3 (fr) * 1994-03-25 2001-10-17 Vertex Pharmaceuticals Incorporated Nouveaux carbamates et urées utilisés pour modifier la résistance multi-medicamenteuse
US20110039786A1 (en) * 2008-04-30 2011-02-17 Kyoto University Metastin derivative and use thereof
CN105294535A (zh) * 2015-11-13 2016-02-03 弓保成 一种治疗盆腔炎的药物组合物
JP2016523238A (ja) * 2013-06-12 2016-08-08 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツングMerck Patent Gesellschaft mit beschraenkter Haftung 関節症の処置のためのヒドロキシエチレン誘導体
WO2025089973A1 (fr) * 2023-10-24 2025-05-01 Universidad Peruana De Ciencias Aplicadas S.A.C. Protéine artificielle de détection d'activité et d'inhibition de protéases

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3833764A1 (de) * 1988-10-05 1990-04-12 Merck Patent Gmbh Arzneimittel zur behandlung von durch retroviren verursachte erkrankungen
CA2036413C (fr) * 1990-02-23 2000-12-12 Paul Cates Anderson Inhibiteurs de la protease du vih
CA2036398C (fr) * 1990-02-23 2000-06-13 Boehringer Ingelheim (Canada) Ltd./ Boehringer Ingelheim (Canada) Ltee Agents d'inhibition de la protease du vih
CA2036397C (fr) * 1990-02-23 2000-12-12 Paul Cates Anderson Inhibiteurs de la protease du vih, renfermant des unites d'amino-acides

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US4016148A (en) * 1975-01-27 1977-04-05 Hoffmann-La Roche Inc. Peptide derivatives of phosphonic and phosphinic acids and intermediates therefor
US4128542A (en) * 1976-07-13 1978-12-05 Hoffmann-La Roche Inc. Peptide derivatives
US4143134A (en) * 1976-07-21 1979-03-06 Hoffmann-La Roche Inc. Halo-phosphonopeptides
US4250085A (en) * 1977-12-23 1981-02-10 Hoffmann-La Roche Inc. Acyl derivatives
US4629783A (en) * 1985-04-29 1986-12-16 Genetic Systems Corporation Synthetic antigen for the detection of AIDS-related disease
US4798787A (en) * 1984-09-19 1989-01-17 Cetus Corporation Peptide antibodies and their use in detecting oncogene products
US4816561A (en) * 1983-05-09 1989-03-28 Todaro George J Biologically active polypeptides
US4816441A (en) * 1985-11-15 1989-03-28 Novo Industri A/S Peptides and compositions
US4818748A (en) * 1986-03-12 1989-04-04 Bayer Aktiengesellschaft Renin inhibitors and aminoacid and aminoaldehyde derivatives

Patent Citations (9)

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Publication number Priority date Publication date Assignee Title
US4016148A (en) * 1975-01-27 1977-04-05 Hoffmann-La Roche Inc. Peptide derivatives of phosphonic and phosphinic acids and intermediates therefor
US4128542A (en) * 1976-07-13 1978-12-05 Hoffmann-La Roche Inc. Peptide derivatives
US4143134A (en) * 1976-07-21 1979-03-06 Hoffmann-La Roche Inc. Halo-phosphonopeptides
US4250085A (en) * 1977-12-23 1981-02-10 Hoffmann-La Roche Inc. Acyl derivatives
US4816561A (en) * 1983-05-09 1989-03-28 Todaro George J Biologically active polypeptides
US4798787A (en) * 1984-09-19 1989-01-17 Cetus Corporation Peptide antibodies and their use in detecting oncogene products
US4629783A (en) * 1985-04-29 1986-12-16 Genetic Systems Corporation Synthetic antigen for the detection of AIDS-related disease
US4816441A (en) * 1985-11-15 1989-03-28 Novo Industri A/S Peptides and compositions
US4818748A (en) * 1986-03-12 1989-04-04 Bayer Aktiengesellschaft Renin inhibitors and aminoacid and aminoaldehyde derivatives

Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5516786A (en) * 1989-05-13 1996-05-14 Bayer Aktiengesellschaft Fungicidal substituted amino acid amides
US5670675A (en) * 1989-05-23 1997-09-23 Abbott Laboratories Retroviral protease inhibiting compounds
US5142056A (en) * 1989-05-23 1992-08-25 Abbott Laboratories Retroviral protease inhibiting compounds
US5580984A (en) * 1989-05-23 1996-12-03 Abbott Laboratories Retroviral protease inhibiting compounds
US5545750A (en) * 1989-05-23 1996-08-13 Abbott Laboratories Retroviral protease inhibiting compounds
US5716973A (en) * 1991-01-02 1998-02-10 Merrell Pharmaceuticals Inc. Anti-viral compounds
US5804552A (en) * 1991-07-23 1998-09-08 Nexstar Pharmaceuticals, Inc. Lipid conjugates of therapeutic peptides and protease inhibitors
US5554728A (en) * 1991-07-23 1996-09-10 Nexstar Pharmaceuticals, Inc. Lipid conjugates of therapeutic peptides and protease inhibitors
US5760036A (en) * 1992-07-20 1998-06-02 E. R. Squibb & Sons, Inc. Aminediol protease inhibitors
US5559256A (en) * 1992-07-20 1996-09-24 E. R. Squibb & Sons, Inc. Aminediol protease inhibitors
US5776933A (en) * 1992-07-20 1998-07-07 E. R. Squibb & Sons, Inc. Method of inhibiting protease
US5633277A (en) * 1993-02-09 1997-05-27 Miles Inc. Sulfonamide aminomethylene derivatives as immunosuppressants
EP0610745A3 (en) * 1993-02-09 1994-09-28 Miles Inc Novel aminomethylene derivatives as immunosuppressants.
EP0610744A1 (fr) * 1993-02-09 1994-08-17 Bayer Corporation Dérivés sulfonamidométhyléniques comme agent immunosuppressif
US5717093A (en) * 1993-07-08 1998-02-10 Merrell Pharmaceuticals Inc. Difluoro statone analogs
WO1995001958A1 (fr) * 1993-07-08 1995-01-19 Merrell Pharmaceuticals Inc. Analogues de la difluorostatone
US5831094A (en) * 1993-09-09 1998-11-03 Merrell Pharamceuticals Inc. Difluoro statone antiviral analogs
US5948778A (en) * 1993-09-09 1999-09-07 Merrel Pharmaceuticals Inc. Difluoro statone antiviral analogs
US6103259A (en) * 1994-01-06 2000-08-15 Capsulis Process for the preparation of liposomes without the use of an organic solvent
WO1995018601A1 (fr) * 1994-01-06 1995-07-13 Centre National De La Recherche Scientifique (C.N.R.S.) Procede de preparation de liposomes sans utilisation de solvant organique
FR2714621A1 (fr) * 1994-01-06 1995-07-07 Centre Nat Rech Scient Procédé de préparation de liposomes sans utilisation de solvant organique.
US5969132A (en) * 1994-02-04 1999-10-19 Merrell Pharmaceuticals Inc. Macrocyclic difluorostatone derivatives useful as antiviral agents
EP1138673A3 (fr) * 1994-03-25 2001-10-17 Vertex Pharmaceuticals Incorporated Nouveaux carbamates et urées utilisés pour modifier la résistance multi-medicamenteuse
US5691368A (en) * 1995-01-11 1997-11-25 Hoechst Marion Roussel, Inc. Substituted oxazolidine calpain and/or cathepsin B inhibitors
US6114380A (en) * 1995-12-18 2000-09-05 Merrell Pharmaceuticals Inc. Difluoro statone analogs
US20110039786A1 (en) * 2008-04-30 2011-02-17 Kyoto University Metastin derivative and use thereof
US8592379B2 (en) * 2008-04-30 2013-11-26 Kyoto University Metastin derivative and use thereof
JP2016523238A (ja) * 2013-06-12 2016-08-08 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツングMerck Patent Gesellschaft mit beschraenkter Haftung 関節症の処置のためのヒドロキシエチレン誘導体
CN105294535A (zh) * 2015-11-13 2016-02-03 弓保成 一种治疗盆腔炎的药物组合物
WO2025089973A1 (fr) * 2023-10-24 2025-05-01 Universidad Peruana De Ciencias Aplicadas S.A.C. Protéine artificielle de détection d'activité et d'inhibition de protéases

Also Published As

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JPH03505875A (ja) 1991-12-19
FI910084A7 (fi) 1991-01-07
HU894124D0 (en) 1991-09-30
DK2691D0 (da) 1991-01-07
AU3964489A (en) 1990-02-05
FI910084A0 (fi) 1991-01-07
HUT58764A (en) 1992-03-30
DK2691A (da) 1991-03-06

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