WO1993012076A1 - Reactifs pour la synthese automatisee d'analogues peptidiques - Google Patents

Reactifs pour la synthese automatisee d'analogues peptidiques Download PDF

Info

Publication number
WO1993012076A1
WO1993012076A1 PCT/US1991/009388 US9109388W WO9312076A1 WO 1993012076 A1 WO1993012076 A1 WO 1993012076A1 US 9109388 W US9109388 W US 9109388W WO 9312076 A1 WO9312076 A1 WO 9312076A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound according
carbon atoms
peptide
optionally substituted
cyclohexylene
Prior art date
Application number
PCT/US1991/009388
Other languages
English (en)
Inventor
Thomas Roy Webb
Original Assignee
Corvas International Inc.
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 Corvas International Inc. filed Critical Corvas International Inc.
Priority to PCT/US1991/009388 priority Critical patent/WO1993012076A1/fr
Priority to AU13390/92A priority patent/AU1339092A/en
Publication of WO1993012076A1 publication Critical patent/WO1993012076A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/003—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by transforming the C-terminal amino acid to amides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C281/00—Derivatives of carbonic acid containing functional groups covered by groups C07C269/00 - C07C279/00 in which at least one nitrogen atom of these functional groups is further bound to another nitrogen atom not being part of a nitro or nitroso group
    • C07C281/06—Compounds containing any of the groups, e.g. semicarbazides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C281/00—Derivatives of carbonic acid containing functional groups covered by groups C07C269/00 - C07C279/00 in which at least one nitrogen atom of these functional groups is further bound to another nitrogen atom not being part of a nitro or nitroso group
    • C07C281/06—Compounds containing any of the groups, e.g. semicarbazides
    • C07C281/08—Compounds containing any of the groups, e.g. semicarbazides the other nitrogen atom being further doubly-bound to a carbon atom, e.g. semicarbazones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C281/00—Derivatives of carbonic acid containing functional groups covered by groups C07C269/00 - C07C279/00 in which at least one nitrogen atom of these functional groups is further bound to another nitrogen atom not being part of a nitro or nitroso group
    • C07C281/16—Compounds containing any of the groups, e.g. aminoguanidine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers

Definitions

  • This invention relates to methods for the automated solid-phase synthesis of peptide analogs and to novel reagents useful therein.
  • the present invention is directed to a process which uses novel reagents which comprise novel heterobifunctional semi- carbazide (I) , or semicarbazone (II) or (III) linker moieties which may be attached to insoluble resins (or supports) , via a pendant carboxylic acid group to give a support reagent suitable for automated solid phase synthesis of peptide analogs.
  • the resulting support reagent is suitable for use in a conventional automated or semi-automated peptide synthesizer using protected a ino acids or amino acid analogs, to give a protected peptide (or peptide analog) aldehyde, attached to the support reagent.
  • the product peptide aldehyde or peptide analog is cleaved from the support and deprotected to give the desired peptide analog in good yield.
  • peptide aldehydes and analogs can be rapidly and efficiently produced. These peptide analogs are useful as enzyme inhibitors and have potential as pharmaceutical agents.
  • Analogs which utilize the catalytic mechanism of an enzyme have been suggested as enzyme inhibitors, however it has only been recently that this idea has been explored.
  • a major problem has been the difficulty in synthesizing the target peptide analog molecules.
  • One candidate group are analogs having the aldehyde group. These peptide analogs are particularly attractive in that they can be prepared from naturally occurring amino acids. Highly specific and potent peptide transition-state analog enzyme inhibitors would be of interest as therapeutic agents.
  • Solution methods for the synthesis of peptide aldehydes have been developed, however, their preparation remains a tedious, labor intensive, and time consuming process.
  • the serine proteinases may be suitable targets for inhibition by peptide transition-state analogs.
  • the trypsin sub-family is composed of serine proteinases which hydrolyse peptide bonds that follow an arginine or lysine residue. Trypsin-like enzymes play a physiological role in digestion, coagulation, fibrinolysis, blood pressure regulation, fertility, and inflammation (see: "Design of Enzyme Inhibitors as Drugs” Eds. Sandier, M. , Smith, H.J. , Oxford Science Publications, 1989) . Selective inhibitors of members of this family of enzymes may therefore be useful in the intervention of many disease states.
  • the catalytic mechanism of serine proteinases involves the attack of the active-site serine on the carbonyl bearing the sissue amide bond of the substrate, to give a tetra- hedral intermediate. It has been reported that peptide analogs which are stable mimics of this tetrahedral intermediate (i.e., transition-state analogs) can be selective enzyme inhibitors (see Delbaere, L.T.J., Brayer, G.D., J. Mol. Biol. 183:89-103, 1985 and Aoyagi, T., ⁇ ezawa, H. , Eds., Proteases and Biological Control, Cold Spring Harbor Laboratory Press, 429-454, 1975).
  • Peptide aldehydes were initially discovered as natural products produced by a number of actinomycete strains. Some of these derivatives have been reported to be selective inhibitors of various types of serine and cysteine proteinases (see Aoyagi, T. et al.. cited above) .
  • the peptide alaninal elastatinal is a potent elastase inhibitor, while not inhibiting trypsin or trypsin-like enzymes (see Hassall, CH. et al.. FEBS Lett., 183:201-5, 1985).
  • the peptide arginal leupeptin has been reported to be a selective inhibitor of trypsin-like enzymes (see Aoyagi, T. , Umezawa, H. , Eds., "Structures and activities of protease inhibitors of microbial origin", Proteases and Biological Control, Cold Spring Harbor Laboratory Press, 429-454, 1975).
  • Leupeptin, along with naturally occurring variants and synthetic analogs, has been reported to be potent inhibitors of several trypsin like enzymes in the coagulation cascade. Synthetic peptide analogs have been prepared which are reported to show a marked selectivity for particular coagulation factors.
  • the present invention relates to methods for the automated synthesis of peptide analogs and for reagents useful for such methods. Such syntheses can be performed on conventional automated peptide synthesizers, using the novel solid insoluble support reagents (hereinafter referred to as "semicarbazone (or semicarbazide) amino acid aldehyde supports” or "SAAA supports”) of the present invention.
  • SAAA supports novel solid insoluble support reagents
  • the present invention also relates to novel linker moieties used to prepare the SAAA supports. These linker moieties have the general structure: (IV)
  • A is a divalent spacer group which comprises a non-reactive divalent hydrocarbyl group having from 2 to about 15 carbon atoms;
  • Pr is a protecting group removable under non- adverse conditions
  • R is hydrogen, or alkyl of 1 to 12 carbon atoms, cycloalkyl of 5 to 8 carbon atoms, aryl or aralkyl of about 7 to about 15 carbon atoms, all optionally substituted with 1 to 3 groups independently selected from hydroxy, sulfhydryl, alkylthio, carboxyl, amide, amine, alkylamine, idolyl, 3-N-formylindolyl, benzylozy, halobenzyloxy, guanido, nitroguanido or imidazolyl optionally substituted with alkoxyalkyl; alk is an alkylene group of about 3 to about 12 carbon atoms optionally substituted with 1 to 3 substituents indepen ⁇ dently selected from hydroxy, alkyl, aryl or guanido and provided that any functional groups of R, or alk which are reactive under conditions of peptide synthesis are option ⁇ ally protected
  • Suitable protecting groups, Pr include t- butoxycarbonyl (BOC) , 9-fluorenylmethyloxycarbonyl (FMOC) and other suitable protecting groups.
  • Suitable non-reactive hydrocarbyl groups, A are those which are substantially inert (and may have suitably protected functional groups) under conditions for auto ⁇ mated and semi-automated peptide synthesis.
  • A has from about 5 to about 10 carbon atoms.
  • A is prefer ⁇ ably a divalent C 5 -C 8 cycloalkylene group optionally substituted with 1 to about 5 alkyl groups such as a 1,4- cyclohexylene group, or a divalent C 5 -C 8 arylene or aralkylene group optionally substituted with 1 to about 5 alkyl groups such as a 1,3- or 1,4-phenylene radical, an alkyl-1,3- or 1,4-phenylene radical or the like.
  • R 1 is hydrogen, or a protected or unprotected amino acid side chain.
  • Particularly suitable amino acid side chains include side chains selected from those of the following amino acids: glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, histidine, phenylalanine, tyrosine, and tryptophan.
  • Preferred alk groups include propylene to give the side chain of the amino acid pro- line.
  • amino acid side chain contains functional groups which are reactive under conditions for peptide synthesis
  • those groups are preferably protected by suitable protecting groups which are removable under non- adverse conditions.
  • suitable protecting groups include the side chains of the following amino acids: hydroxyproline, norleucine, 3-phosphoserine, homoserine, O-phosphohomoserine, dihydroxyphenylalanine, 5-hydro- xytryptophan, 1-methylhistidine, 3-methylhistidine, and ⁇ - aspartyl phosphate.
  • R. groups include ⁇ -amino-adipic acid, cysteine sulfonic acid, cysteic acid and ornithine.
  • preferred alk groups include propylene, optionally substituted with hydroxy to give a proline or hydroxyproline analog.
  • the carbon of the C-terminal carboxyl group is attached to a solid support or resin or such that the hydroxy group of the C-terminal carboxyl group is replaced by -X wherein -X is independently selected from -NH-Sp, -O-Sp, and -CH 2 -Sp, wherein Sp denotes an insoluble support, preferably a suitably functionalized 1% cross- linked polystyrene.
  • the support reagents of the present invention are conveniently prepared using protecting groups suitable for chemically extending the peptide chain by conventional automated solid phase techniques.
  • protecting groups include, but are not limited to t-butoxycarbonyl (BOC) and 9-fluorenylmethyloxy-carbonyl (FMOC) , allyloxycarbonyl and the like. See for example. Green, T. ; “Protecting Groups in Organic Synthesis,” (John Wiley and Sons, 1981).
  • For a general description of peptide syntheses see Greenstein, J.P., Winitz, M. "Chemistry of the Amino Acids," pages 763-1268. (John Wiley and Sons: New York, 1986) .
  • the analog can be cleaved from the support by mild acid/formaldehyde treatment.
  • the pro ⁇ tecting groups on the N-terminus and side-chains of the amino acid components of the analog are selected so that they are not affected by cleavage of the peptide analog from the support. If desired, acid or base sensitive protecting groups can be removed before cleavage.
  • a deprotection step involving catalytic hydrogenation after cleavage of the peptide analog from the support may be used, since many protecting groups (e.g., benzyl, benzyloxycarbonyl, benzyloxymethyl, halobenzyloxycarbonyl, and nitor) are readily removed by mild hydrogenolysis without affecting functionality of the desired final product (see, e.g.. Example 8) .
  • protecting groups e.g., benzyl, benzyloxycarbonyl, benzyloxymethyl, halobenzyloxycarbonyl, and nitor
  • the present invention is directed to peptide analogs of the formula:
  • R t is hydrogen; or alkyl, cycloalkyl, aryl or aralkyl, optionally substituted with 1 to 3 substituents independently selected from hydroxy, alkoxy, sulfhydryl, alkythio, carboxyl, amide, amino, alkyla ino, indolyl, 3- N-forylindolyl, benzyloxy, halobenzyloxy, guanido, nitro- guanido- or optionally substituted imidazolyl substituted with alkoxy-alkyl; and alk is an alkylene group of about 3 to about 12 carbon atoms optionally substituted with 1 to 3 substituents independently selected from hydroxy, alkyl, aryl or guanido; A is a non-reactive hydrocarbyl group of 2 to about 15 carbon atoms; and X is indepen ⁇ dently -NH-Sp, O-Sp, or -CH 2 -Sp, where Sp is an
  • a groups are those having 5 to 10 carbon atoms. More preferably, A is a divalent C 5 -C 8 cycloalkylene group, a C 5 -C 8 divalent arylene group or a C 5 - C 8 divalent aralkylene group, all optionally substituted with 1 to 5 alkyl groups. Especially preferred A groups include 1,3-cyclohexylene, 1,4-cyclohexylene, 1,3- phenylene, 1,4-phenylene, 1,3-xylylene, 1,4-xylylene and the like.
  • R, and alk groups are those which correspond to the side chains of the amino acids typically found in proteins, i.e., glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, glutamic acid, aspartic acid, glutamate, aspartate, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan and proline. Also preferred are amino acid residues, Res, having R. or alk groups which comprise those amino acid side chains.
  • n is less than 10 (to give a decamer after cleavage) or more preferably less than 5 (to give a pentamer) .
  • An additional aspect of the present invention is directed to methods of preparing the above peptide analog (V) using the linker moieties and support moieties of the present invention. After cleavage and deprotection, a peptide aldehyde of the formula
  • amino acid residue refers to radicals having the structure (i) -C(0)RNH- wherein R typically is -CH(R 1 )- and R. is H or a carbon containing substituent
  • alk is an alkylene group.
  • the amino acids used in the application of this invention are those naturally occurring amino acids found in proteins, or the naturally occurring anabolic or catabolic products of such amino acids which contain amino and carboxyl groups. Also included are the D and L stereoisomers of such amino acids when the structure of the amino acid admits of stereoisomeric forms.
  • a named amino acid shall be construed to include both the D or L stereoisomers, preferably the L stereoisomer.
  • hydrocarbyl denotes an organic radical composed of carbon and hydrogen which may be aliphatic (including alkyl, alkenyl, and alkynyl groups and groups which have a mixture of saturated and unsaturated bonds) , alicyclic (carbocyclic) , aryl (aromatic) or combinations thereof; and may refer to straight-chained, branched- chain, or cyclic structures or to radicals having a combination thereof, as well as to radicals substituted with halogen atom(s) or heteroatoms, such as nitrogen, oxygen and sulfur and their functional groups (such as amino, alkoxy, aryloxy, carboxyl, ester, amide, carbamate or lactone groups, and the like) , which are commonly found in organic compounds and radicals.
  • hydrocarbylcarbonyl refers to the group R'C- wherein R' is a hydrocarbyl group.
  • alkyl refers to saturated aliphatic groups, including straight, branched and carbocyclic groups.
  • aryl refers to aromatic hydrocarbyl and heteoaromatic groups which have at least one aromatic ring.
  • alkyl refers to an alkyl group which has been substituted with an aromatic (or aryl) group, and includes, for example, groups such as benzyl.
  • alkylene refers to straight and branched- chain alkylene groups which are biradicals, and includes, for example, groups such as ethylene, propylene, 2- methylpropylene (e.g.
  • 3-methylpentylene (e.g., -CH 2 CH 2 CHCH 2 CH 2 -) and the like.
  • arylene refers to aromatic groups which are biradicals.
  • aralkylene refers to aralykyl groups which are biradicals.
  • esters refers to a group having a -C II-O- linkage, and includes both acyl ester groups and carbonate ester groups.
  • halo or halogen refers to fluorine, chlorine, bromine and iodine.
  • non-adverse conditions describes condi- tions of reaction or synthesis which do not substantially adversely affect the skeleton of the peptide analog and/or its amino acid (and/or amino acid analog) components.
  • One skilled in the art can readily identify functionalities, coupling procedures, deprotection procedures and cleavage conditions which meet these criteria.
  • support refers to a solid particulate, insoluble material to which a linker moiety of the present invention is linked and from which a peptide analog may be synthesized.
  • Supports used in synthesizing peptide analogs are typically substantially inert and nonreactive with the reagents used in the synthesis of peptide analogs and include resins such as that included in the SAAA support reagents of the present invention.
  • peptide analog refers oligomers of amino acids (or amino acid residues) which are linked by peptide linkages wherein either the C-terminal carboxyl or the N- terminal amino has been chemically modified to another functional group or replaced with a different functional group.
  • the C-terminal carboxyl group may be replaced with an aldehyde group.
  • automated synthesis or “automated peptide synthesis” refers to the synthesis of peptides or peptide analogs using an instrument which carries out the indivi ⁇ dual steps of each addition cycle to add a amino acid or amino acid analog to the growing peptide chain occurs without manual manipulation.
  • semi-automated synthesis refers to the synthesis of peptides or peptide analogs where in each addition cycle to add an amino acid or amino acid analog to a growing peptide chain, the coupling step is done manually and other individual steps occur without manual manipulation.
  • semi-automated synthesis differs from automated synthesis in that some of the coupling steps are carried out manually.
  • FIG. 1 depicts general reaction schemes for the preparation of certain some reagents according to the present invention.
  • i represents benzyl 12/1 alcohol/p-toluenesulfonic acid, reflux in toluene;
  • ii represents carbonyl diimidazole/DMF.
  • Figure lb depicts Procedure A and iii represents compound (2.) /triethylamine; iv represents H 2 /Pd; v represents trifluoroacetic acid/ 0°C; vi represents MBHA resin/benzotriazol-1-yloxy-tris- (dimethylamino) -phosphoniu hexafluorophosphate; and vii represents Boc-N-nitro-L-arginal (l.)/NaOAc, reflux.
  • Figure IC depicts Procedure B and v represents trifluoroacetic acid /0°C; vi represents MBHA resin/benzotriazol-1-yloxy-tris- (dimethylamino) - phosphonium hexafluorophosphate; and vii represents Boc-N 8 - nitro-arginal (i) / NaOAc, reflux.
  • FIG. 2A and 2B depict reaction schemes for the preparation of support reagents of the present invention.
  • FIG. 3 depicts a reaction scheme for the preparation of ⁇ -protected amino acid analogs which may be used in the preparation of the support reagents of the present inven ⁇ tion.
  • aspects of the present invention are directed to processes for the synthesis of support reagents which comprise novel linker moieties and the pep ⁇ tide analogs derived therefrom, as well as processes whereby transition-state peptide analogs are prepared using said support reagents.
  • the peptide chain of the peptide analog to be synthesized may be extended using solid-phase synthesis methods, such as that generally described by Merrifield, J. Am. Chem. Soc. .85:2149 (1963); however other chemical syntheses protocols known in the art may be used.
  • Solid- base synthesis is initiated from the C-terminus of the peptide aldehyde or analog to be synthesized by coupling a protected ⁇ -amino acid to a suitable SAAA support.
  • supports or resins which are suitable for the preparation of SAAA supports of the present invention include MBHA, BHA, aminomethyl phenyl resins and the like.
  • the preparation of the hydroxymethyl resin is described by Bodansky et al.. Chem. Ind. 3_8:1597-1598 (London) 1966. Chioromethylated resins are commercially available from BioRad Laboratories, Richmond, CA and from Lab. Systems, Inc. The preparation of resins is described by Stewart et al.. "Solid Phase Peptide Synthesis," Chapter 1, pages 1-6 (Freeman & Co., San Francisco 1969).
  • BHA and MBHA resin supports are commercially available, but have conven ⁇ tionally been used only when the desired polypeptide being synthesized has an unsubstituted amide at the C-terminus.
  • the peptide chain is extended by coupling additional amino acids to the chain using known techniques for the formation of peptide bonds.
  • One suitable method comprises converting the ⁇ -amino protected amino acid to be added to the peptide chain to an "activated" derivative wherein the carboxyl group is rendered more susceptible to reaction with the free N-terminal ⁇ -amino group of the peptide fragment.
  • the amino acid can be converted to a mixed anhydride by reaction of a protected amino acid with ethyl choloroformate, pivaloyl chloride or like acid chlorides.
  • the amino acid can be converted to an active ester such as a 2,4,5-trichloropheyl ester, a pentachlorophenol ester, a pentafluorophenyl ester, a p- nitrophenyl ester, a N-hydroxysuccinimide ester, or an ester formed from 1-hydroxybenzotriazole.
  • Another coupling method involves use of a suitable coupling agent such as N,N'-dicyclohexylcarbodiimide or N,N'diisopropyl-carbodiimide.
  • a suitable coupling agent such as N,N'-dicyclohexylcarbodiimide or N,N'diisopropyl-carbodiimide.
  • Other appropriate coupling agents are disclosed in E. Gross & J. Gonhofer, The Peptides: Analysis. Structure. Biology. Vol. I: Major Methods of Peptide Bond Formation (Academic Press, New York, 1979) .
  • ⁇ -amino groups of amino acids (monomers) employed in the peptide synthesis are protected during the coupling 15 reaction to prevent side reactions involving the reactive, if unprotected, ⁇ -amino function.
  • certain amino acids contain reactive side-chain functional groups (e.g., sulfhydryl, amino, carboxyl, and hydroxyl) which must also be protected with suitable protecting groups to prevent chemical reaction of those groups from occurring during both the initial and subsequent coupling steps.
  • suitable protecting groups known in the art, are described in E. Gross & J. Meienhofer, The Peptides: Analysis, Structure, Biology. Vol. 3: Protection of Func ⁇ tional Groups in Peptide Synthesis (Academic Press, NEw York, 1981) .
  • An ⁇ -amino protecting group (a) should render the ⁇ -amino function inert under the conditions employed in the coupling reaction, (b) should be readily removable after the coupl ⁇ ing reaction under conditions that will not remove side- chain protecting groups and will not alter the structure of the peptide fragment, and (c) should eliminate the possibility of racemization upon activation immediately prior to coupling.
  • An amino acid side-chain protecting group (a) should render the side chain functional group inert under the conditions employed in the coupling reac ⁇ tion, (b) should be stable under the conditions employed in removing the ⁇ -amino protecting group, and (c) should be readily removable upon completion of the desired amino acid peptide aldehyde under reaction conditions that will not alter the structure of the peptide analog chain.
  • protecting groups known to be useful for peptide synthesis will vary in reactivity with the agents employed for their removal.
  • certain protecting groups such as triphenylmethyl and 2-(p-biphenylyl) isopropyl- oxycarbonyl are very labile and can be cleaved under mild acid conditions.
  • protecting groups such as t- 16 butyloxycarbonyl (BOC) , t-amyloxycarbonyl, adamantyl- oxycarbonyl, and p-methoxybenxyloxycarbonyl are less labile and require moderately strong acids, such as trifluoroacetic, hydrochloric, or boron trifluoride in acetic acid, for their removal.
  • BOC butyloxycarbonyl
  • t-amyloxycarbonyl t-amyloxycarbonyl
  • adamantyl- oxycarbonyl p-methoxybenxyloxycarbonyl
  • p-methoxybenxyloxycarbonyl are less labile and require moderately strong acids, such as trifluoroacetic, hydrochloric, or boron trifluoride in acetic acid, for their removal.
  • Still other protecting groups such as benxyloxy-carbonyl (CBZ) , halobenxyloxycarbonyl, p-nitrobenzyloxycarbonyl cycloalkyloxycarbonyl, and isopropyloxycarbonyl, are even less labile and require stronger acids, such as hydrogen fluoride, hydrogen bromide, or boron trifluoroacetate in trifluoroacetic acid, for their removal.
  • CBZ benxyloxy-carbonyl
  • halobenxyloxycarbonyl p-nitrobenzyloxycarbonyl cycloalkyloxycarbonyl
  • isopropyloxycarbonyl are even less labile and require stronger acids, such as hydrogen fluoride, hydrogen bromide, or boron trifluoroacetate in trifluoroacetic acid, for their removal.
  • Example of amino acid protecting groups include: (1) for an ⁇ -amino group, (a) aromatic urethane-type protecting groups , such as fluorenylmethyloxycarbonyl (FMOC) ; (b) aliphatic urethane-type protecting groups, such as BOC, t- amyloxycarbonyl, isopropyloxycarbonyl, 2-(p- biphenylyl) -isopropyloxycarbonyl , allyloxycarbonyl and the like; (c) cycloalkyl urethane-type protecting groups, such as cyclo- pentyloxycarbonyl, adamantyloxycarbonyl, and cyc1ohexy1oxy-carbony1 ; and (d) allyloxycarbonyl.
  • the preferred ⁇ -amino protecting groups are BOC or FMOC (2) for the side chain amino group present in Lys, protecting groups - include any of the groups mentioned above in (1) such as BOC, p- chloro
  • protecting groups preferably include nitro, or 2,2,5,7,8- pentamethylchroman-6-sulfonyl or 2,3,6- triroethyl-4-methoxyphenylsulfonyl.
  • protecting include, for example, t-butyl; benzyl (BZL) ; substituted BZL groups, such as p- methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl, o- chlorobenzyl, and 2,6-dichlorobenzyl. 17
  • protecting groups include, for example, by esterification using groups such as t-butyl, or preferably benzyl.
  • suitable protecting groups include the benzyloxymethyl group
  • protecting groups such as tetrahydropyranyl, tert-butyl, trityl, benzyl, chlorobenzyl, 4- bromobenzyl, and 2,6-dichlorobenzly are suitably employed.
  • the preferred protecting group is bromo-benzyloxycarbonyl.
  • trityl is preferably employed as a protecting group.
  • the ⁇ -amino protecting group is removed, such as by using trifluoroacetic acid (TFA) in methylene chloride or TFA alone.
  • TFA trifluoroacetic acid
  • the deprotection is carried out at a temperature of from about O'C to about ambient temperature.
  • suitable cleaving reagents such as HC1 in dioxane, and conditions for removal of specific ⁇ -amino protecting groups are described in Shroder & Lubke, supra, Chapter I, pages 72-75.
  • the remaining ⁇ -amino and side-chain protected amino acids are coupled stepwise in the desired order.
  • some may be coupled to one another prior to their addition to the solid-base synthesizer so as to give a dipeptide or tripeptide analog.
  • the selection of an appropriate coupling reagent is within the skill of the art. Particularly suitable as a coupling reagent is N,N'- dicyclohexyl carbodiimide or diispropylcarbodiimide.
  • Each protected amino acid or amino acid sequence to be coupled to the growing peptide sequence or chain is 18 introduced into the solid-phase reactor in excess, and the coupling is suitably carried out in a medium of suitable solvent such as dimethylformamide (DMF) or CH 2 C1 2 or mix ⁇ tures thereof. If incomplete coupling occurs, the coupl- ing procedure is repeated before removal of the ⁇ -amino protecting group of the product peptide sequence prior to the coupling of the next amino acid.
  • the success of the coupling reaction at each stage of the synthesis may be monitored.
  • a preferred method of monitoring the synthesis uses by the ninhydrin reaction, as described by Kaiser et al.. Anal. Biochem 34: 595 (1970) .
  • the coupling reactions can be performed automatically using well known methods and instruments, for example, a Biosearch 9550 Peptide Synthesizer or Applied Biosystems Model 430A peptide synthesizer.
  • the protected peptide aldehyde is cleaved from the resin support, and protecting groups are removed.
  • the cleavage reaction and removal of the protecting groups may be suit- ably accomplished simultaneously or stepwise.
  • the protecting groups present on the N-terminal ⁇ -amino groups may be removed preferen ⁇ tially either before or after the protected peptide is cleaved from the support.
  • Purification of the polypeptide analogs prepared using the reagents of the present invention is typically achieved using conventional procedures such as preparative HPLC (including reversed phase HPLC) or other known chromatography, affinity chromatography (including mono- clonal antibody columns) or countercurrent distribution.
  • FIG. 2 depicts alternative generalized reaction schemes for the preparation of support reagents of the present invention.
  • Blocking group, Bl denotes a protecting group which is removable under non-adverse conditions. 19
  • the protecting group, Pr, of protected semicarbazide moiety (2L) is removed under non ⁇ adverse conditions.
  • Deprotected semicarbazide moiety (27) is reacted with ⁇ -amino protected amino acid aldehyde (25) or (25a) to give semicarbazone intermediate (2J3) or (28a) , as appropriate.
  • the resulting intermediate is reacted with support (22.) to give the appropriate support regent (26) or (26a) in a reaction which maintains the stereo ⁇ chemistry of the chiral center of amino acid aldehyde (25) .
  • the stereochemistry of the chiral center will be conserved.
  • Figure 3 depicts one preferred reaction scheme for preparing aldehydes of formula (25.) and 25a) .
  • blocking group, Bl denotes a protecting group which is removable under non-adverse conditions.
  • Figure 1 depicts a general reaction scheme for the synthesis of certain preferred semicarbazide and semicar ⁇ chili-one linker moieties and the support reagents incorpo ⁇ rating those moieties.
  • the reaction of t-butylcarbazate with carbonyldiimidazole gives an intermediate (having presumed structure 3 ) which is then allowed to react directly with epsilon amino ester (2.) .
  • Epsilon amino ester (2.) may be conveniently prepared from the commer ⁇ cially available trans-4-aminomethylcyclohexane carboxylic acid.
  • the resulting protected semicarbazide (4.) can be isolated by chromatography or alternatively may be con- verted directly to crystalline carboxylic acid (5_) , without further isolation in an approximately 62% overall yield.
  • the free semicarbazide salt is prepared by allowing trifluoroacetic acid (TFA) to react with (5_) .
  • carboxylic acid (5_) is allowed to react with an insoluble amine resin such as methylbenzhydrylamine (MBHA) .
  • MBHA methylbenzhydrylamine
  • the reaction product is treated successively with TFA and then with the protected amino acid aldehyde ⁇ -t-butoxycarbonyl-N 9 -nitroarginal (3L) to give support (! .
  • other suitably pro- tected amino acid aldehydes may be substituted for (1) .
  • Procedure B depicts an alternative protocol for the preparation of support regents of the present invention.
  • preformed semicarbazone (2) is allowed to react directly with the resin (for example an amine resin such as MBHA) .
  • the resin for example an amine resin such as MBHA
  • Procedure B may be preferred 21 since support reagents having high substitution of semi ⁇ carbazone linking moiety are readily obtained.
  • polymeric resins having one or more of the following characteristics are particularly suitable:
  • Resins which are insoluble in polar aprotic solvents such as dimethyl formamide (DMF) , N-methylpyrrolidone, tetrahydrofuran (THF) and other solvents conventionally used in solid-phase peptide synthesis (such as dichloromethane or methanol) .
  • polar aprotic solvents such as dimethyl formamide (DMF) , N-methylpyrrolidone, tetrahydrofuran (THF) and other solvents conventionally used in solid-phase peptide synthesis (such as dichloromethane or methanol) .
  • Resins which are stable in the presence of reagents such as TFA, diisopropylethyl amine, dicyclohexylcarbodiimide (DCC) and other reagents conventionally used in solid phase peptide synthesis.
  • Suitable resins having the above properties include commercially available resins which include P-methyl- (benzhydrylamine) (MBHA) or aminomethylated 1% divinylbenzene crosslinked polystyrene resins.
  • MBHA P-methyl- (benzhydrylamine)
  • Other suitable functionalized supports include pellicular and macroporous insoluble supports known to those skilled in the art (See, e.g.. G. Barany and R.B. Merrifield, "Solid- Phase Peptide Synthesis," in The Peptides. Volume 2, pages (1-2984 (Academic Press, New York 1980)). 22
  • SAAA resin (!) is added to the reaction vessel of an automated or semi-automated synthesizer.
  • the t-butoxycarbonyl (BOC) protecting group on an ⁇ -amino group is removed with an acid such as TFA to give the resulting free amine.
  • the amino group is coupled to a suitably activated carboxyl group of a blocked amino acid, using a suitable reagent such as DCC.
  • the resulting resin undergoes a series of washing steps between each reaction. The addition cycle can then be repeated using the next ⁇ - blocked amino acid until the desired peptide sequence is complete.
  • the peptide aldehyde or analog may be released from the support (resin) by cleavage methods such as by treatment with aqueous formaldehyde and dilute acid to give the protected peptide aldehyde (or analog) . If the product peptide analog has side chain or N-terminal protecting or blocking groups, these groups may be removed by conventional procedures such as by treatment with hydrogen and palladium catalysts (See Example 8) . The peptide aldehydes may be further purified using procedures such as HPLC (See Examples 8 et sec.) .
  • BOC-N 9 -nitroarginine was obtained from Calbiochem.
  • N- methylpiperidine, N,O-dimethylhydroxylamine hydrochloride and isobutylchloroformate, and lithium aluminum hydride were obtained from Aldrich Chemical Company, Inc.
  • Dichloromethane, ethyl acetate, methanol and tetrahydrofuran were obtained from Fisher Scientific Company.
  • a flask was placed under a nitrogen atmosphere and cooled to -50°C, then charged with 70 mL (70 mmole) of 1 N lithium hydride (in tetrahydrofuran) and 500 mL of dry tetrahydrofuran. 50 mL of a solution containing 66 mmole ofBOC-N 9 -nitroarginineN-methyl-O-methylcarboxamide indry tetrahydrofuran was slowly added while the temperature of the reaction mixture was maintained at -50'C.
  • DMF dimethylformamide
  • the above-identified compound was prepared using 500 mg SAAA support !, ⁇ -BOC amino acids and standard solid phase peptide synthesis as described above.
  • the BOC pro ⁇ tecting groups were removed using a solution of 50% TFA/DCM.
  • the resin (support) was neutralized with 10% diisopropylethylamme in DCM.
  • Coupling of amino acids to support reagent (and growing amino-acid-support chain) was performed in DMF with DCC and 1-hydroxybenztriazole.
  • To 470 mg of the resulting peptide analog resin 5 mL tetra ⁇ hydrofuran (THF) , 1 mL acetic acid, 1 mL formaldehyde and 100 ⁇ l IN HCl are combined and stirred for about one hour.
  • THF tetra ⁇ hydrofuran
  • the solution is filtered and washed with 10 L THF.
  • the filtrate is diluted with 100 mL water and extracted with ethyl acetate.
  • the ethyl acetate phase is washed with brine, dried (magnesium sulfate) , and concentrated.
  • the nitro protecting group and other hydrogen-removable pro- tecting groups are removed by hydrogenation in 10 mL 10% water/methanol with 300 ⁇ l IN HCl and 200 mg palladium on carbon at 5 psi for 45 minutes.
  • the mixture is filtered through a fine fritted filter with Celite, washed with methanol/water and concentrated to give the crude peptide aldehyde.
  • the resulting peptide aldehyde can then be further isolated by C-18 reverse phase HPLC, using an aqueous/ acetonitrile (0.01% TFA) system to give the corresponding TFA salts.
  • the product is then purified using reverse 34 phase high performance chromatography on a 10 micron 300 angstrom pore size C-18 packing. The column was eluted with an aqueous gradient of 0.1% trifluoroacetic and acetonitrile, gradient going from 5% to 40% acetonitrile containing 0.01% trifluoroacetic acid. Lyophilization of the appropriate fractions gave the above-identified pep ⁇ tide aldehyde as the trifluoroacetate salt.
  • the above-identified compound was prepared using SAAA support 8, using ⁇ -BOC amino acids, O-benzyl protection for serine, and standard solid phase peptide synthesis as described above.
  • the resin was cleaved, and deprotected, as described below.
  • the nitro group and other hydrogen removable protecting groups are removed, when it is desirable, by hydrogenation in 10 mL 10% H 2 0/MeOH with 300 ⁇ l 1 N HCl and 200 mg activated palladium on carbon at 5 psi for 45 minutes.
  • the mixture is filtered through a fine fritted filter with Celite, washed with MeOH/water and concentrated to give the crude peptide.
  • the resulting peptide aldehyde can then be purified by C-18 reverse phase HPLC, using an aqueous/acetonitrile (0.01% TFA) system to give the corresponding TFA salts.
  • a 1-L, three-necked, round-bottom flask is equipped with a mechanical stirrer, an electronic digital thermometer, and a graduated additional funnel.
  • the flask is charged with 39.1 g (0.4 moles) of N,0-dimethylhydroxy- lamine hydrochloride (Aldrich Chemical Co.) and 236 L methylene chloride.
  • the suspension is stirred and cooled to 2'C with ah ice-water bath.
  • N-methyl-piperidine (Aldrich Chemical Co.), 48.8 L (0.41 moles)
  • a clear, colorless solution results which is kept cold and is used in the following reaction.
  • a 5-L, three-necked, round-bottomed flask is equipped with a mechanical stirrer, thermometer, and an addition funnel with drying tube, the flask is charged with 100 g (0.4 moles) of ⁇ -t-butoxylcarbonyl-L-leucine hydrate (Bachem, Inc.), 458 mL tetrahydrofuran (Fisher Scientific Co.) and 1.8 L methylene chloride.
  • a clear solution results on stirring, which is cooled to -20*C ( ⁇ 2") by immersing the flask in a dry ice-2-propanol bath.
  • N- methyl-piperidine 48.8 mL (0.41 moles) is placed in the addition funnel and is added rapidly to the mixture, while the temperature is allowed to rise to about -12"C ( ⁇ 2°).
  • Methyl chloroformate Aldrich Chemical Co.
  • 31 mL 0.4 moles
  • the cooling bath is removed and the clear solution is allowed to warm to room tempera ⁇ ture over about 4 hours (and may be stirred overnight for convenience).
  • the solution is then cooled to about 5°C and extracted with two 500 mL portions of aqueous 0.2 N hydrochloric acid and two 500 mL portions of aqueous 0.5 N sodium hydroxide, while maintaining the organic phase at 40 about 5°C to about 15°C during the extractions.
  • the solu ⁇ tion is washed with 500 mL of saturated aqueous sodium chloride solution, dried over magnesium sulfate and con ⁇ centrated on a rotary evaporator at a bath temperature of about 30-35°C
  • the residue is further evacuated on a Varian 5500 instrument (using a 250 mm x 4.6 mm I.D. Alltech C-18 column with 60:40 methanol:0.5 M NH 4 H 2 P0 4 as the mobile phase, UV detection at 210 nm) .
  • the flask is immersed in a dry ice-2-propanol bath and the suspension is cooled to about -45'C
  • a solution of the ⁇ -t-butoxycarbonyl-L-leucine N- methyl-O-methylcarboxamide (prepared according to para ⁇ graph A above) , in 300 mL anhydrous ethyl ether is placed in the addition funnel and added to the lithium aluminum hydride suspension (which is cooled to about -45"C before the addition) in a steady stream while maintaining the reaction temperature at about -35'C ( ⁇ 3°).
  • the cooling bath is removed and the mixture is stirred and is allowed to warm to about ⁇ 5°.
  • the mixture is once again cooled to about -35'C and a solution of 96.4 g (0.171 moles) of sodium bisulfate (Matheson, Coleman and Bell, a saturated aqueous solution is obtained after stirring overnight) in 265 mL deionized water is placed in the addition funnel.
  • the sodium bisul- 41 fate solution is added cautiously at first and then rapidly, while the temperature is allowed to rise to about -2'C ( ⁇ 3").
  • the cooling bath is removed and the mixture is stirred for about one hour.
  • the reaction mixture is filtered through a 2 inch pad of celite. The filter cake is washed with two 500 mL portions of ethyl ether.
  • the combined ether layers are washed in sequence with three 350 mL portions of cold (about 5'C) IN hydrochloric acid, two 350 mL portions of saturated aqueous sodium bicarbon ⁇ ate solution, and 350 L saturated sodium chloride solu ⁇ tion.
  • the organic solution is dried over magnesium sulfate and evaporated on a rotary evaporator (bath at 30*C), to give a residual, slightly cloudy syrup.
  • the product is stored in a freezer (about -17'C) prior to use, since it may racemize if stored at room temperature.
  • Example 7B The procedure of Example 7B is followed except that an equamolar amount of ⁇ -N-(t-butoxycarbonyl)-leucinal- semicarbazonyl-trans-4-methyl-cyclohexane-carboxylic acid (the product of Example 19) is used in place of the product of Example 5. This procedure gives a solid support suitable for the synthesis of peptide C-terminal leucinals. 43
  • Example 7B In preparing the above-identified product, the proce ⁇ dure described in Example 7B is followed except that an equimolar amount of the product of Example 21 is used in place of compound 7 (the product of Example 6) .
  • This procedure gives a solid support product suitable for the synthesis of peptide C-terminal alaninals, in 98-99.5% coupling yield (by ninhydrin) .
  • Example 7B In preparing the above-identified product, the procedure described in Example 7B is followed except that an equimolar amount of the product of Example 23 is used in place of compound 7 (the product of Example 6) . This procedure gives a solid support product suitable for the synthesis of peptide C-terminal valinals, in 98-99.5% coupling yield (by ninhydrin) . 46
  • Example 7B In preparing the above-identified product, the procedure described in Example 7B is followed except that an equimolar amount of the product of Example 25 is used in place of compound 7 (the product of Example 6) .
  • This procedure gives a solid support product suitable for the synthesis of peptide C-terminal phenylalaninals in 98- 99.5% coupling yield (by ninhydrin) .
  • the title compound was prepared using 500 mg valinal SAAA support (product of Example 24) , ⁇ -BOC-L-proline, ⁇ -
  • TFA/DCM was used to remove the BOC groups.
  • the resin was 48 neutralized with 10% diisopropylethylamme in DCM. Coupling was performed in DMF with DCC and 1-hydroxy- benztriazole.
  • To 470 mg of resulting peptide aldehyde resin (from above) 5 L THF, 1 L acetic acid, 1 mL formaldehyde, and 100 ⁇ l IN HCl are combined and stirred for 1 hour. The solution is filtered and washed with 10 mL THF and the filtrate is diluted with 100 mL of water and extracted with ethyl acetate. The ethyl acetate phase is washed with brine, dried (MgSO and concentrated.
  • the resulting peptide aldehyde can then be purified by C-18 reverse phase HPLC, using an aqueous/acetonitrile (0.1% TFA) system to give the corresponding TFA salts.
  • the crude product was purified using reverse phase high performance chromatography on a 10 micron 300 angstrom pore size C-18 packing. The column was eluted with a aqueous gradient of 0.01% trifluoroacetic acid gradient going from 5% to 60% acetonitrile containing 0.01% trifluoroacetic acid. Lyophilization of the appropriate fractions gave the title compound as its trifluoroacetate salt.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

On décrit des réactifs destinés à être utilisés dans la synthèse d'analogues peptidiques faisant appel à la technique de synthèse peptidique automatisée. L'invention concerne également des procédés de synthèse d'analogues peptidiques.
PCT/US1991/009388 1991-12-13 1991-12-13 Reactifs pour la synthese automatisee d'analogues peptidiques WO1993012076A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/US1991/009388 WO1993012076A1 (fr) 1991-12-13 1991-12-13 Reactifs pour la synthese automatisee d'analogues peptidiques
AU13390/92A AU1339092A (en) 1991-12-13 1991-12-13 Reagents for automated synthesis of peptide analogs

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1991/009388 WO1993012076A1 (fr) 1991-12-13 1991-12-13 Reactifs pour la synthese automatisee d'analogues peptidiques

Publications (1)

Publication Number Publication Date
WO1993012076A1 true WO1993012076A1 (fr) 1993-06-24

Family

ID=22226047

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/009388 WO1993012076A1 (fr) 1991-12-13 1991-12-13 Reactifs pour la synthese automatisee d'analogues peptidiques

Country Status (2)

Country Link
AU (1) AU1339092A (fr)
WO (1) WO1993012076A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0675899A1 (fr) * 1992-12-15 1995-10-11 Corvas International, Inc. NOUVEAUX INHIBITEURS DU FACTEUR Xa
WO2000005243A2 (fr) * 1998-07-24 2000-02-03 Corvas International Inc. Procede de derivatisation d'une resine et utilisations du produit obtenu
FR2825095A1 (fr) * 2001-05-28 2002-11-29 Dev Des Antigenes Combinatoire Dispositif de presentation de polypeptides, utilisable comme "puce" pour la detection miniaturisee de molecules
US6872827B2 (en) 2002-04-26 2005-03-29 Chembridge Research Laboratories, Inc. Somatostatin analogue compounds
US7358273B2 (en) 1998-03-19 2008-04-15 Vertex Pharmaceuticals Incorporated Inhibitors of caspases
EP2270005A1 (fr) 2000-05-19 2011-01-05 Vertex Pharmceuticals Incorporated Promédicament inhibiteur d'ECI
US9116157B2 (en) 2010-11-05 2015-08-25 Brandeis University Ice-cleaved alpha-synuclein as a biomarker
US11021514B2 (en) 2016-06-01 2021-06-01 Athira Pharma, Inc. Compounds

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4307094A (en) * 1979-07-23 1981-12-22 Hoffmann-La Roche Inc. Triazolopyridazine derivatives

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4307094A (en) * 1979-07-23 1981-12-22 Hoffmann-La Roche Inc. Triazolopyridazine derivatives

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0675899A1 (fr) * 1992-12-15 1995-10-11 Corvas International, Inc. NOUVEAUX INHIBITEURS DU FACTEUR Xa
EP0675899A4 (fr) * 1992-12-15 1996-01-24 Corvas Int Inc NOUVEAUX INHIBITEURS DU FACTEUR Xa.
US5883077A (en) * 1992-12-15 1999-03-16 Corvas International, Inc. Inhibitors of factor Xa
EP2261235A2 (fr) 1998-03-19 2010-12-15 Vertex Pharmaceuticals Incorporated Inhibiteurs de caspase
EP2261234A2 (fr) 1998-03-19 2010-12-15 Vertex Pharmaceuticals Incorporated Inhibiteurs de caspase
US8691848B2 (en) 1998-03-19 2014-04-08 Vertex Pharmaceuticals Incorporated Inhibitors of caspases
EP2261233A2 (fr) 1998-03-19 2010-12-15 Vertex Pharmaceuticals Incorporated Inhibiteurs de caspase
EP2261232A2 (fr) 1998-03-19 2010-12-15 Vertex Pharmaceuticals Incorporated Inhibiteurs de caspase
US7358273B2 (en) 1998-03-19 2008-04-15 Vertex Pharmaceuticals Incorporated Inhibitors of caspases
EP2011800A2 (fr) 1998-03-19 2009-01-07 Vertex Pharmaceuticals Incorporated Inhibiteurs de caspase
WO2000005243A2 (fr) * 1998-07-24 2000-02-03 Corvas International Inc. Procede de derivatisation d'une resine et utilisations du produit obtenu
WO2000005243A3 (fr) * 1998-07-24 2000-04-20 Corvas Int Inc Procede de derivatisation d'une resine et utilisations du produit obtenu
EP2270005A1 (fr) 2000-05-19 2011-01-05 Vertex Pharmceuticals Incorporated Promédicament inhibiteur d'ECI
US9994613B2 (en) 2000-05-19 2018-06-12 Vertex Pharmaceuticals Incorporated Prodrug of an ICE inhibitor
WO2002097442A3 (fr) * 2001-05-28 2003-11-06 Dev Des Antigenes Soc D Et Dispositif de presentation de polypeptides, utilisable comme 'puce' pour la detection miniaturisee de molecules.
FR2825095A1 (fr) * 2001-05-28 2002-11-29 Dev Des Antigenes Combinatoire Dispositif de presentation de polypeptides, utilisable comme "puce" pour la detection miniaturisee de molecules
US6872827B2 (en) 2002-04-26 2005-03-29 Chembridge Research Laboratories, Inc. Somatostatin analogue compounds
US9116157B2 (en) 2010-11-05 2015-08-25 Brandeis University Ice-cleaved alpha-synuclein as a biomarker
US11021514B2 (en) 2016-06-01 2021-06-01 Athira Pharma, Inc. Compounds

Also Published As

Publication number Publication date
AU1339092A (en) 1993-07-19

Similar Documents

Publication Publication Date Title
JP2875834B2 (ja) ウレタン保護アミノ酸‐ｎ‐カルボキシ無水物
CA2155931C (fr) Inhibiteurs de la thrombose
US5602231A (en) Process for making peptides
IE60128B1 (en) Hydroxylamine derivatives,their preparation and use as medicaments
JPH10510261A (ja) タンパク質：ファルネシルトランスフェラーゼの置換テトラーおよびペンタペプチド阻害剤
CA2128711C (fr) Inhibiteurs de la trypsine
US5032675A (en) Process for the production of glutamine derivatives
US6235876B1 (en) Liquid phase process for the preparation of GNRH peptides
US5283293A (en) Reagents for automated synthesis of peptide analogs
CA2036997C (fr) Peptides fixateurs de leu3a
JPH0354957B2 (fr)
WO1993012076A1 (fr) Reactifs pour la synthese automatisee d'analogues peptidiques
CA1104156A (fr) Analogue de la bestatine
US5367072A (en) Reagents for automated synthesis of peptide analogs
HU185229B (en) Process for preparing pharmaceutically active peptides and acetates thereof
FR2557114A1 (fr) Nouveaux derives de la gonadoliberine, procede pour leur preparation et compositions pharmaceutiques les contenant
JPH10505575A (ja) ペプチジルアルギニナールの合成方法
JPH06510281A (ja) ペプチドアミドの製造
JPH0689024B2 (ja) ポリペプチド類の製造方法
JPH05508859A (ja) ペプチド合成方法
Pajpanova et al. Canavanine derivatives useful in peptide synthesis
JPH0533718B2 (fr)
JPS5828867B2 (ja) 新テトラペプチド誘導体類
JPS61155396A (ja) ニユーロテンシン様ペプチド及びその製法
EP0896001A1 (fr) Procédé pour la préparation de dérivés d'antagonistes de l'oxytocin, produits intérmédiares pour la préparation de dérivés d'antagonistes de l'oxytocin et procédé pour la préparation de produits intermédiares

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA FI JP KR NO

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU MC NL SE

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA