EP1100782A1 - Support solide d'amino-aldehyde, sa sequence de liaison et ses methodes de preparation et d'utilisation - Google Patents

Support solide d'amino-aldehyde, sa sequence de liaison et ses methodes de preparation et d'utilisation

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Publication number
EP1100782A1
EP1100782A1 EP99938805A EP99938805A EP1100782A1 EP 1100782 A1 EP1100782 A1 EP 1100782A1 EP 99938805 A EP99938805 A EP 99938805A EP 99938805 A EP99938805 A EP 99938805A EP 1100782 A1 EP1100782 A1 EP 1100782A1
Authority
EP
European Patent Office
Prior art keywords
peptide
solid support
aldehyde
resin
carbon atoms
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP99938805A
Other languages
German (de)
English (en)
Inventor
Daniel V. Siev
J. Edward Semple
Christopher A. Roberts
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dendreon Pharmaceuticals LLC
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
Publication of EP1100782A1 publication Critical patent/EP1100782A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings 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
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings 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
    • C07D211/56—Nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • 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
    • C07K1/045—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers using devices to improve synthesis, e.g. reactors, special vessels
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures

Definitions

  • This invention provides a novel method for solid-phase peptide synthesis wherein a cyclic or linear amino aldehyde is derivatized through linkage to a solid-support, and employed in the production of biologically significant peptides, peptidyl aldehydes and derivatives and analogs thereof.
  • a linker comprising ethyl glycolate was employed for immobilization of an argininal cyclol to a solid support.
  • a linker comprising a methylene (-C11 2 -) was disclosed.
  • the present invention provides an improved linker and method oi ' synthesis thereof wherein the linker comprises between about one to fifteen, and preferably about five methylene units, and variants thereof.
  • the present invention provides improved methodology for achieving and cleavage of this linkage.
  • the present disclosure applies this technology to the linkage to solid supports of linear and cyclic aldehydes for synthesis of peptides, peptide analogs and peptidomimetic compounds.
  • This invention provides a novel method for solid-phase peptide synthesis wherein a cyclic or linear amino aldehyde is derivatized through linkage to a solid-support, and employed in the production of biologically significant peptides, peptidyl aldehydes and derivatives and analogs thereof.
  • the invention is a method (Method I) for making a solid support having the formula (I):
  • RI is derived from RI ', a moiety having a cyclic amino aldehyde or derivative thereof at its carboxy-terminus, wherein the moiety I optionally comprises a protecting group at one or more positions thereof;
  • n is an integer from 1 to about 15, provided that n is at least two if both X and Y are bonds, and n is preferably about 5;
  • X and Y are independently a bond or -[Z] p -, wherein: p is an integer between 1 and 5, provided that the combination of X, Y and -(CH 2 ) n - represents a chain equivalent in length to a linear chain of about two to fifteen, and preferably about five carbon atoms; Z is -CH2CII2O-, or -C(A)(B)-, wherein A and B may vary in each occurrence of Z, and are independently selected from the group consisting of hydrogen, alkyl of one to about six carbon atoms, alkenyl of about three to about ten carbon atoms, aryl of about six to about ten carbons, and wherein in combination, A and B may form a five to seven membered ring; said method comprising the steps of
  • the invention is a method (Method II) for making a solid support having a formula (II): R3[-O-X-(CH 2 )m-Y-CO-NH-SS]2 (II); wherein:
  • R3 is a moiety derived from R3' having a linear chain amino aldehyde, or a derivative thereof, at its carboxy-terminus, wherein the moiety R3' optionally comprises a protecting group at one or more positions;
  • m is an integer of between about 1 and 15, provided that m is at least two if both X and Y are bonds, and m is preferably about 5;
  • X and Y are independently a bond or -[Z] -, wherein p is an integer between 1 and 5, provided that the combination of X, Y and -(CII 2 ) n ⁇ - represents a chain equivalent in length to a linear chain of one to fifteen, and preferably about five carbon atoms;
  • Z is -CH 2 CH 2 O-, or -C(A)(B)-, wherein A and B may vary in each occurrence of Z, and are independently selected from the group consisting of hydrogen, alkyl of one to about six carbon atoms, alkenyl of about three to about ten carbon atoms, aryl of about six to about ten carbons, and wherein in combination, A and B may form a five to seven membered ring; said method comprising the steps of:
  • a further object of this invention is to provide a method for derivatization of a cyclic amino aldehyde.
  • a further object of this invention is to provide novel resins for solid-phase peptide synthesis.
  • Figure 1 is a How chart representing the linkage of a cyclic aldehyde to a solid support following the procedure of Examples 2 through 7, and synthesis of a growing peptide chain thereon, following the procedures of Examples 8 through 12.
  • Figure 2 is a flow chart representing the linkage of a cyclic aldehyde to a solid support following the procedure of Examples 13 through 19, and synthesis of a growing peptide chain thereon, following the procedures of Examples 8 through 12.
  • Figure 3 is a flow chart representing the linkage of a cyclic aldehyde to a solid support which has already been reacted with a linker according to the method of this invention.
  • Alloc is allyloxycarbonyl
  • Alloc-Cl is allyl chloroformate A.M. resin is amino-methylated polystyrene resin
  • Boc is tert-butoxycarbonyl
  • Bom is benzyloxymethyl
  • BOP is benzotriazole-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate
  • CbZ is benzyloxycarbonyl or carbobenzyloxy 2-C1Z is 2-chlorobenzyloxycarbonyl
  • DIEA is N, N-diisopropylethylamine
  • DMSO dimethylsulfoxide
  • EDC l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt
  • Fmoc is 9-f uorenylmethyloxycarbonyl
  • I IF is hydrogen fluoride
  • FIOBT or HOBt is 1 -hydroxybenzotriazole onohydrate
  • HPLC high pressure liquid chromatography
  • LAH lithium aluminum hydride
  • MS is mass spectrometry
  • Mts is mesitylene-2-sulphonyl
  • NMM is N-methylmorpholine(also refe ⁇ ed to as 4-methylmorpholine)
  • NMR nuclear magnetic resonance spectroscopy
  • PG protecting group
  • PMC 2,2,5,7,8-pentamethylchroman-6-sulfonyl
  • PyBOP is Benzotriazole-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate
  • TeOC is Me 3 Si(CH 2 )OCO
  • TFA is trifluoroacetic acid
  • THF is tetrahydrofuran
  • TLC or tic is thin layer chromatography
  • TFMSA is trifluoromethylsulfonic acid, also commonly referred to as "triflic acid"
  • TMSOTf is trimethylsilyltrifluoro methane sulfonate
  • Tos is p-toluenesulfonly also referred to as Tozyl or Ts
  • Troc is trichloroethoxycarbonyl (an amine protecting group removable with zinc)
  • SS is a polymeric solid support that is stable in the presence of acids, bases and/or other reagents; a “solid support” is any form of bead or resin typically used in the art of peptide synthesis to provide a “handle” whereby a growing synthetic peptide chain may be made available for synthetic manipulation without the risk of loss in peptide yield typically experienced when such syntheses are conducted in solution; the terms “solid support” and “resin” are used interchangeably.
  • solid support solid support
  • SS or “support” refer to a solid particulate, insoluble material lo which a linker moiety of the present invention is linked and from which a peptide or peptide analog may be synthesized.
  • Supports used in synthesizing peptides and peptide analogs are typically substantially inert and nonreactive with the reagents used in the synthesis of peptides and peptide analogs, particularly once an initial linkage between an aldehyde has been established (i.e., a PI position has been established), according to the method of this invention.
  • a solid support is a cross-linked resin, such as polystyrene.
  • NH 2 -SS is a SS comprising at least one functional amino group available for formation of an amide (peptide) bond.
  • a cyclic aldehyde is a cyclic ⁇ -amino aldehyde, such as a hemiaminal (for arginine), hemiacetal (for Asp/homoSer) and thiohemiacetal (for homoCys); a molecule having a cyclic configuration either bearing an aldehyde moiety or bearing a nascent aldehyde moiety; a prototypical example of this type of molecule is an arginine aldehyde or a peptidyl arginine aldehyde, wherein it has been recognized that arginine aldehyde exists in solution in one of four principal equilibrium states: as the open arginine aldehyde, as the arginine aldehyde hydrate, and as two amino cycl ⁇ l forms (i.e., cyclic aldehyde derivatives; see U.S. Patent No. 5,703,208, column 9, herein incorporated by reference; see also
  • a cyclic aldehyde derivative is any derivative of a cyclic aldehyde; preferably, for purposes of this invention, a cyclic aldehyde derivatized with a blocking group.
  • a linear aldehyde is any molecule wherein the aldehyde moiety, -CHO, is not cyclized.
  • amino acid refers to natural amino acids or unnatural amino acids, and amino acid analogs in their D and L stereoisomers if their structure allows such stereoisomeric forms.
  • Natural amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (Flis), isoleucine (He), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val).
  • Unnatural amino acids include, but are not limited to azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2- aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminoisobutyric acid, des osine, 2,2'-diaminopimelic acid, 2,3- diaminopropionic acid, n-ethylglycine, N-ethylasparagine, hydroxylysine, all- hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N- methylglycine, N-methylisoleucine, N-methylvaline, norvaline, norleucine, orni
  • Amino acid analogs include, but are not limited to, the natural and unnatural amino acids which are chemically blocked, reversibly or irreversibly, or modified on their N-terminal amino group or their side-chain groups, as for example, methionine sulfoxide, ethionine sulfone, S-(carboxymethyl)-cysteine, S- (carboxymethyl)-cysteine sulfoxide and S-(carboxymethyl)-cysteine sulfone, aspartic acid-(beta methyl ester), N-ethylglycine, alanine carboxamide.
  • the natural and unnatural amino acids which are chemically blocked, reversibly or irreversibly, or modified on their N-terminal amino group or their side-chain groups, as for example, methionine sulfoxide, ethionine sulfone, S-(carboxymethyl)-cysteine, S- (carboxymethyl)-cystein
  • amino acid residue refers to radicals having the structure (1) NI I-R-C(O), wherein R typically is -CI I(R*)-, wherein R* is I I or a carbon containing substituent; or
  • p is 1 , 2, or 3, representing the azetidinecarboxylic acid, proline or pipecolic acid residues, respectively.
  • a peptide or peptide analog is a molecule comprising at least two amino acids or amino acid analogs linked through peptide (amide) linkages.
  • a peptidomimetic compound is any compound which structurally resembles or mimics a natural peptidyl array, and compounds comprising such residues; compounds which, although not a natural peptide, in the sense that it either contains no amino acids or contains amino acid analogs, exhibits a biological activity of a known peptidyl compound.
  • good leaving group or "leaving group” are used herein lo define a molecular substituent which, when used in conducting chemical syntheses, exhibits the desirable properties of being labile under defined synthetic conditions, and of being easily separated from synthetic products under defined conditions.
  • leaving groups include, but are not limited to, hydrogen, hydroxyl radicals, halogen atoms, p- nitrophenoxide, water, methyl groups, and the like.
  • protecting group is used herein to refer to well known moieties which have the desirable property of preventing specific chemical reactions at a site on a molecule undergoing chemical modification intended to be left unaffected by the particular chemical modification, while at the same time being easily removed from the molecule under conditions that do not adversely affect other sites in the modified molecule.
  • protecting groups include, but are not limited to, Cbz, Boc, Alloc, Fmoc, Troc, Teoc (Me Si(CH 2 ) 2 OCO), PMC, and the like, and others disclosed herein.
  • alkyl refers to saturated aliphatic groups including straight-chain, branched- chain and cyclic groups.
  • alkenyl refers lo unsalurated aliphatic groups having at leasl one double bond.
  • aryl refers to aromatic groups which have at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may optionally be substituted with a substituent selected from, but not limited to, lower alkyl of one to ten carbon atoms; alkenyl; nitro; cyano; halo; -S(O) M -, wherein, for purposes of this definition, q is 0, 1 , or 2; carboxylic acid or carboxylic acid derivatives, esters amides, and ihe like.
  • aralkyl refers to an alkyl group substituted with an aryl group.
  • exemplary aralkyl groups include, but are not limited to, benzyl, picolyl, and the like, which may optionally be substituted wilh a substiluenl selected from, but not limited to, lower alkyl of one to ten carbon atoms; alkenyl; nitro; cyano; halo; -S(O) t
  • cycloalkyl refers to an alkyl in which at least a portion of the molecule is in a closed ring configuration.
  • exemplary cycloalkyl groups include, but are not limited to, cyclohexyl, cyclopropyl, cyclopentyl and cycloheptyl.
  • heterocycle refers to any compound having a closed cyclic structure in which at least one atom thereof is other than a carbon atom.
  • cyclic alkyl, cyclic aryl, cyclic aralkyl compounds containing a nitrogen, oxygen or sulfur atom in the cyclic structure are heterocycles.
  • halo or halogen refers to fluorine, chlorine, bromine and iodine.
  • non-adverse conditions describes conditions 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.
  • a spacer in figure 1 , a pentane spacer is shown
  • a spacer comprising about a five carbon atom linear chain, or a variant thereof having substantially the same spatial effect, substituted or not, along with other enhancements described herein, affords enhanced efficiency in the overall chemical process described herein.
  • the term "about a five carbon atom linear chain” includes linkers wherein two, three, four, five, six or seven carbon atoms, or variants thereof, are linked in a linear chain. Linkers shorter than two or longer than about seven have been found to be less efficient and result in reduced yield of the synthetic process.
  • FIG 1 there is depicted a reaction sequence according to one aspect of this invention, with reference being made to specific exemplary support (see the various example numbers, shown as “Ex.” and the relevant example number, at various stages throughout the reaction scheme) provided herein below.
  • a cyclic aldehyde 100 (exemplified in the reaction scheme by N- -t-butoxycarbonyl-N s nitro-argininal; those skilled in the art will recognize that solvent, protecting group or reaction condition variations may be made without departing from the procedure defined herein) exhibiting a Boc protected ⁇ -amino group is contacted with a preferred linker of this invention (represented by 6-ethylhydroxyhexanoate) in the presence of an acidic catalyst.
  • a preferred linker of this invention represented by 6-ethylhydroxyhexanoate
  • excess linker is capped by reaction with acetic anhydride and pyridine, or the like, to facilitate purification of the desired product.
  • an intermediate 110 is provided, with the cyclic aldehyde bonded to the linker.
  • Reduction e.g., via palladium catalyzed hydrogenation
  • the intermediate 120 pennits N-omega Alloc protection of the cyclic aldehyde, and hydrolysis of the alkyl group (e.g. ethyl in Figure 1 ) in subsequent steps, to produce the free-carboxylic acid containing intermediate 130.
  • Contacting 130 with a resin bearing at least one reactive amino group results in formation of an amide bond between the linker and the resin to form compound 140.
  • Acid- catalyzed Boc deprotection of the amino group permits standard, solid-phase peptide chemistry to be conducted to produce a peptide, peptide derivative or analog 150.
  • Deprotection and final cleavage from the resin permits release of the peptide, peptide derivative or analog thereof 160 having a free carboxy-terminal aldehyde functionality.
  • the thus-synthesized compound 160 may then be used directly.
  • the aldehyde functionality may be converted through standard chemical means to a carboxylic acid or other desirable functionality.
  • an arginine aldehyde is obtained from the parent carboxylic acid 200 by appropriate omega- group protection (e.g., PMC).
  • omega- group protection e.g., PMC
  • Alloc or like protection of the ⁇ -amino group and conversion to ihe N-methoxy-N'-methylamide (i.e., Weinreb amide formation from the carboxylic acid moiety) by treatment with O,N-diemthylhdoxylamine or like reagent results in production of compound 210.
  • a reaction scheme wherein a starting material 300 representing a trimethylsilyl (TMS) or like protected linker is directly linked with an amino resin and deprotected to form the derivatized resin 310 exhibiting a free, reactive hydroxyl group.
  • nascent cyclic aldehyde 320 prepared, for example, by treatment of compound 210 from figure 2 with LAH/THF, THF -78°C, results in production of the immobilized cyclic aldehyde 330.
  • Deprotection of the aldehyde produces the reactive species 340, with which solid-phase peptide chemistry may be conducted, including but not limited to production of peptide, peptidomimetic protease inhibitors, and preparation of a library of compounds through combinatorial chemical variations in a plurality of different reaction vessels using different monomeric, oligo eric or polymeric condensations with the product 340.
  • the invention is a method (Method I) for making a solid support having the formula (I): (I) R] -O-X-(CH 2 ) n -Y-CO-Nl I-SS wherein:
  • R I is derived from RI ', a moiety having a cyclic amino aldehyde or derivative thereof at its carboxy-terminus, wherein the moiety RI optionally comprises a protecting group at one or more positions thereof;
  • n is an integer from about 1 lo about 15, provided that n is at least two if both X and Y are bonds, and n is preferably about 5;
  • X and Y are independently a bond or -
  • the solid support produced according to the method of this invention may be used to advantage in the synthesis of known and novel inhibitors of proteases, including but not limited to serine proteases, aspartyl proteases, and cysteine proteases.
  • Peptides, peptide analogs and peptidomimetic compounds significant to inhibition of enzymes relevant to the blood coagulation pathway may be prepared to advantage according to the methods disclosed herein.
  • novel and known serine protease inhibitors, aspartic acid protease inhibitors, and cysteine protease inhibitors may be prepared using the solid support produced according to the method of this invention. Accordingly, inhibitors of a wide variety of biologically and medically relevant proteolytic enzymes are produced according to the method of this invention.
  • protease inhibitors are selected from, but are not limited to: thrombin inhibitors, urokinase inhibitors, inhibitors of factor Xa, elastase inhibitors, inhibitors of Hepatitis C enzymes, chymase inhibitors, trypsin inhibitors, and tryptase inhibitors.
  • RI is a moiety having a cyclic amino aldehyde, or derivative thereof, at its carboxy-terminus.
  • Suitable RI moieties include single cyclic amino aldehydes and derivatives thereof, as well as peptide or peptidomimetic side-chains of two or more atoms thai have a cyclic amino aldehyde, or derivative thereof, at the carboxy-terminus of the chain.
  • RI is an arginine aldehyde, an aspartic acid aldehyde, a homoserine aldehyde or a homocysteine aldehyde, and derivatives thereof.
  • Common derivatives of such amino aldehydes are those having the ⁇ -side chain protected with a protecting group (PG) such as Boc, Alloc, PMC, Fmoc, or Teoc.
  • PG protecting group
  • Other protecting groups disclosed herein or known in ihe art may be used when appropriate.
  • the alpha and omega heteroatoms of R I are blocked with PGl and PG2, respectively, prior to step (i) (see Example 1 through 16 and Figure 2).
  • the alpha nitrogen of RI is blocked with PG l prior to step (i) and the omega nitrogen of RI is blocked with PG2 after step (ii) (see Example 1 through 5 and Figure 1).
  • PGl and PG2 are protecting groups selected to be orthogonal. That is, the selection of PGl and PG2 is made after considering the intended use of the solid support, such that PG l or PG2 can be selectively removed. Removal of one PG group avails that deprotected heteroatom for subsequent reaction, while the protected nitrogen remains unavailable for undesired reactions. Examples 8 and 19 describe the removal of the PG from the alpha nitrogen, while the PG on the omega nitrogen is retained to protect that nitrogen during subsequent reactions (see Examples 10 and 20).
  • Preferred combinations of PGl and PG2 include the following PGs, which can be either PGl or PG2: Boc in combination with Fmoc, Alloc, or Teoc; Fmoc in combination with Alloc, Teoc, or PMC; Alloc in combination with Teoc or PMC.
  • a preferred NH 2 -SS is a resin having a functional amine group, such as amino methylated polystyrene resin, benzhydrylamine resin, and 4-methylbenzhydrylamine resin.
  • a functional amine group such as amino methylated polystyrene resin, benzhydrylamine resin, and 4-methylbenzhydrylamine resin.
  • Other resins known in the art having at least one functional amine group may be used according to the methods disclosed herein.
  • step (i) set forth above proceeds under acid-catalyzed conditions, such as those disclosed in figures 1 and 2, Examples 3 through 5, Example 16, and elsewhere throughout this disclosure and according to methods known in the art.
  • Step (ii) comprises hydrolysis, which is conducted under conditions such as those reflected in Example 6, Example 17, and according to methods known in the art for this type of chemical transformation.
  • Step (iii) comprises dehydration conditions, such as those disclosed in Example 7, Example 1 , and conditions known in the art for this type of chemical transformation.
  • R2 groups include but are not limited to lower (about one to ten carbon atoms) alkyl, alkenyl, and aralkyl.
  • An especially preferred R2 group is ethyl.
  • An especially preferred linker is OIl-(CH 2 ) -C0 -ethyl.
  • X and Y are not bonds
  • either or both moieties may be alkyl or substituted alkyl groups, provided that if substituted, reactive moieties likely to interfere in standard peptide synthetic regimens are not preferred.
  • X, Y and the repeating methylene unit combine to form a spacer that is inert and which provides a length similar to that which an alkyl spacer of about five carbon atoms would provide.
  • Method II for making a solid support having a formula (II): R3[-O-X-(CI I 2 ), press-Y-CO-NI I-SSJ 2 (II); wherein:
  • R3 is a moiety derived from R3' having a linear chain amino aldehyde, or a derivative thereof, at its carboxy-terminus, wherein the moiety R3' optionally comprises a protecting group at one or more positions;
  • m is an integer of between about 1 and 15, provided that m is at least two if both X and Y are bonds;
  • X and Y are independently a bond or -
  • Z is -CH 2 CH 2 0-, or -C(A)(B)-, wherein A and B may vary in each occurrence of Z, and are independently selected from the group consisting of hydrogen, alkyl of one to about six carbon atoms, alkenyl of about three to about ten carbon atoms, aryl of about six to about ten carbons, and wherein in combination, A and B may form a five to seven membered ring; said method comprising the steps of:
  • R2 is selected from the group consisting o -NH-SS, if the linker has previously been reacted with a solid support, or -H, alkyl of one to about six carbon atoms, alkenyl of about three to about ten carbon atoms, aryl of about six to about ten carbon atoms, and aralkyl of about six to about fifteen carbon atoms, under acid catalyzed conditions permitting reaction of the carbonyl of R3' with the hydroxyl of the linker, to form: R3f-0-X-(CH 2 ) m -Y-CO 2 -R2] 2 ,
  • Preferred R2 groups include but are not limited to lower (about one to ten carbon atoms) alkyl, alkenyl, and aralkyl.
  • An especially preferred R2 group is ethyl.
  • An especially preferred linker is OI I-(C1 I 2 )s-CO -ethyl.
  • X and Y are not bonds, either or both moieties may be alkyl or substituted alkyl groups, provided that if substituted, reactive moieties likely to interfere in standard peptide synthetic regimens are not preferred.
  • X, Y and the repeating methylene unit combine to form a spacer that is inert and which provides a length similar to that which an alkyl spacer of about five carbon atoms would provide.
  • the solid support prepared according to the method of this invention may be used to advantage in the synthesis of known and novel inhibitors of proteases, including, but not limited to, proteases significant to the blood coagulation pathway. Accordingly, novel and known thrombin inhibitors, inhibitors of factor Xa, urokinase inhibitors, Hepatitis C enzymes, chymase, prostate specific antigen (PSA), Factor Vila, elastase, trypsin, and the like may be prepared using the solid support produced according to the method of this invention.
  • argininal moiety wherein the side chain has a guanidino group
  • single or double protection of the side chain may be desirable or essential in order to achieve a desired reaction at another side, e.g., at the amino-terminal nitrogen, without production of a plurality of unwanted side-chain reactions.
  • appropriate side-chain protection may be employed to keep the side chain in a linear form.
  • ⁇ -amino protecting group In practicing the methods of the present invention, the following considerations apply to the selection of ⁇ -amino protecting groups, omega side chain protecting groups, and carboxy protecting groups.
  • ⁇ -amino protecting group PG l
  • ⁇ -amino protecting group should:
  • a suitable ⁇ -amino protecting group may be selected from the group consisting of acid labile ⁇ -amino protecting groups (cleavage conditions are noted, as well, in brackets or text):
  • Triphenylmethyl (trityl) group is cleaved under very mild acid conditions [1% TFA].
  • Benzyloxycarbonyl (CBz), 2-chlorobenzyloxycarbonyl (2-C1Z), cycloalkyloxycarbonyl, and isopropyloxycarbonyl require stronger acids, such as hydrogen lluoride, hydrogen bromide or boron Irilluoroacelate in trifiuoro acetic acid for their removal.
  • the CBz and the 2-C1Z groups may most conveniently be cleaved 99
  • a suitable ⁇ -amino protecting group also may be selected from the group consisting of base labile ⁇ - amino protecting groups.
  • Fluorenylmethyloxycarbonyl Fmoc
  • Allyloxycarbonyl Alloc
  • Pd (0) catalyst transfer of the ally] group to an acceptor nucleophile such as morpholine, dimedone, tributyl tin hydride and N- methyl aniline.
  • Preferred ⁇ -amino protecting groups (PG) include Boc, Fmoc, Alloc, and Cbz.
  • amino acid side-chain protecting group should:
  • a suitable amino acid side chain protecting group may be selected from the group consisting of (methods for cleavage of these protecting groups are shown in brackets []):
  • the preferred protecting groups include nitro [H2/Pd/C, HF], benzyloxycarbonyl (CBz) [HF, TFMSA, TMSOTf, H2/Pd/C], tert-butyloxycarbonyl (Boc) [TFA], 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) [TFA], 2,3,6-trimethyl-4-methoxyphenylsulfonyl (Mtr) [TFA], p-toluenesulfonyl (Tos) [HF, TFMSA], mesitylene-2-sulphonyl (Mts) [HF, TFMSA], allyloxycarbonyl
  • protecting groups include trilyl 1 1 % TT ⁇
  • protecting groups such as tert-butyl [TFA], trityl [1% TFA], benzyl, 2-bromobenzyl and 2,6-dichlorobenzyl, all cleaved by the same reagents [HF, TFMSA, H2/Pd/C], are suitably employed.
  • protecting groups include methyl [OIL, II+], ethyl [Oil-, H+], t-butyl [TFA], ally. [Pd(0), morpholine], cyclohexyl [HF, TMSOTf], or benzyl groups [HF, TFMSA, TMSOTf, H2/Pd/C].
  • protecting groups include trityl [TFA] and xanthyl [TFA].
  • suitable protecting groups include 2,4- dinitrophenyl (Dnp) [thiophenol], trityl [TFA], benzyloxymethyl (Bom) [HF, TFMSA, TMSOTf, H2/Pd/C], p-toluene sulfonyl (Tos) [HF, TFMSA], and benzyloxycarbonyl (Cbz) [HF, II2/Pd/C].
  • suitable protecting groups include trityl [TFA], 4-methylbenzyl (pMeBzl) [I IF, TFMSAJ, 4-methoxybenzyl (pMeOBzl) [HF,
  • TFMSA acetamidomethyl
  • tBu tert-Butyl
  • suitable protecting groups include formyl [10% piperidine in DMF, followed by HF] and tert-butyloxycarbonyl (Boc) [TFA].
  • a carboxy terminus protecting group should:
  • suitable protecting groups include the esters methyl [OI L, I I+], ethyl [OI L, I I+], tert-butyl [TFA], benzyl [OI L, I I2/Pd/C] and allyl [Pd(O), morpholine
  • Solid-phase synthesis is useful for the production of relatively small amounts of certain compounds. Solid-phase synthesis also is useful for the synthesis of a library of compounds, each differing from the other in one or more variables.
  • reactors for the solid-phase synthesis of peptidyl aldehydes, including peptide argininals are comprised of a reactor vessel with at least one surface permeable to solvent and dissolved reagents, but not permeable to synthesis resin of the selected mesh size.
  • Such reactors include glass solid phase reaction vessels with a sintered glass frit, poylpropylene tubes or columns with frits, or like reaction vessels known in the art or commercially available. The type of reactor chosen depends on volume of solid-phase resin needed, and different reactor types might be used at di fferent stages of a synthesis.
  • the linker and solid supports prepared according to Methods I and II may be employed in the solid phase synthesis of peptide or peptidyl aldehyde compositions.
  • Preferred uses include use for the synthesis of peptide or peptidyl aldehyde inhibitors of one or more serine proteases.
  • An especially preferred use of the invented solid supports is use for the synthesis of thrombin inhibitors, Factor Xa inhibitors, and urokinase inhibitors (see Example 12 for preparation of a thrombin inhibitor, Example 21 for preparation of a factor Xa inhibitor, and Example 9 for preparation of a urokinase inhibitor using the solid support of this invention and the methods disclosed herein for preparation thereof).
  • ihe linker of formula (1) or (II) comprises an ether linkage
  • a further aspect of this invention is the preparation of a derivatized resin by linking to the group RI or R3 one or more amino acids, amino acid analogs, peptides or peptide analogs to form a peptide or peptidomimetic chain of amino acids or amino acid derivatives comprising the cyclic aldehyde or nascent aldehyde RI or R3 at the carboxy terminus thereof.
  • N- ⁇ -t-butoxycarbonyl-N-omega-allyloxycarbonyl-argininal (6-hexanoyl- aminomethylated polystyrene resin) cyclol; N h -Alloc-Arginylcyclo-O-6-hydroxylhexanoyl-amino methylated polystyrene;
  • N-omega-allyloxycarbonyl-argininal (6-hexanoyl-aminomethylated polystyrene resin) cyclol;
  • N- ⁇ -Fmoc-alanyl-N-omega-allyloxycarbonyl-argininal (6-hexanoyl-aminomethylated polystyrene resin) cyclol
  • alanyl-N-omega-allyloxycarbonyl-argininal (6-hexanoyl-aminomethylated polystyrene resin) cyclol
  • D-seryl(O-t-butyl)-alanyl-N-omega-allyloxycarbonyl-argininal (6-hexanoyl- aminomethylated polystyrene resin) cyclol; carbamate analogs of D-seryl(O-t-butyl)-alanyl-N-omega-allyloxycarbonyl-argininal (6- hexanoyl-aminomethylated polystyrene resin) cyclol; isobutyloxycarbonyl-D-seryl(O-t-butyl)-alanyl-N-d-allyloxycarbonyl-argininal (6- hexanoyl-aminomelhylated polystyrene resin) cyclol; carbamate analogs of D-seryI(O-t-bulyl)-alanyl- argininal (6-hexanoyl-aminomethylated polystyrene resin)
  • N- ⁇ -Alloc-N°-PMC- Arginylcyclo-O-6-hydroxylhexanoyl-amino methylated polystyrene resin Nd-PMC-Arginylcyclo-O-6-hydroxyhexanoyl-amino methylated polystyrene resin
  • Benzylsulfonamido-dArg(N ⁇ 2)-Sarcosine-Gly- N°-PMC-Arginylcyclo-0-6- hydroxyhexanoyl amino methylated polystyrene resin N- ⁇ -Alloc-N°-PMC- Arginylcyclo-O-6-hydroxylhexanoyl-amino methylated polystyrene resin.
  • the invented solid supports and linker also are useful in the field of combinatorial chemistry and development of chemical libraries.
  • preferred methods within this aspect of the invention result in the development of combinatorial libraries having an aldehyde in the PI position, especially argininals:
  • peptides or peptide analogs of any desired length may be prepared according to this method by repeating the coupling steps as many times as necessary.
  • a library is designed wherein the P
  • multiple reactions are carried out in parallel.
  • site is first incorporated onto the resin bearing the linker as disclosed herein, followed by the variations in the P 4 , P 3 , and P residues, thereby forming a library of peptide or peptidomimetic compounds available for structure-activity analyses in any of a number of in vitro or in vivo assay systems, including protease inhibition assays.
  • this process of library formation and parallel synthesis may be carried out in a number of known formats.
  • the synthesis is conducted in Whatman mini columns, or ihe like, wherein standard peptide synthetic methods known in the art are used to extend the peptide chain, with each subsequent coupling being achieved at the carboxy terminus of each added residue.
  • the multiple peptide variants are cleaved from the resin, isolated, and tested for biological activity.
  • automated synthesis of a library of peptides or peptide analogs may be conducted in any commercially available peptide synthesizer.
  • this aspect of the invention represents a method for making a library of peptides or peptide analogs comprising the steps of: (a) in each of a series of separate containers or reaction vessels, contacting an aldehyde with the free hydroxyl of the linker as disclosed herein under acid conditions;
  • step (b) hydrolyzing the product of step (a) to produce a free carboxylic acid moiety at the terminus of the linker;
  • step (c) contacting the product of step (b) with a resin comprising at least one functional amino group to form an amide bond, thereby immobilizing said aldehyde as a PI residue linked to said resin through said linker;
  • step (e) repeating step (d) as many times as required to generate a peptide or peptide analog of the desired number of residues, with appropriate intermediate steps of protection and deprotection of reactive groups present on the growing peptide or peptide analog chain.
  • Standard methods for cleavage of peptide aldehyde or peptide aldehyde analogs prepared according to the method of this invention are not preferred.
  • Standard cleavage conditions including for example, treatment with HF; TFA; TFA:I I 2 O (e.g. 9: 1); TFA:DCM:H 2 0 (e.g. 5:4: 1 ), while utilizable, result in lower than optimal yields due to dehydration of the aldehyde moiety, such as conversion of a cyclic argininal tautomer to a covalently cyclized dehydration adduct.
  • Serine residue dehydration may also occur under standard cleavage conditions, and this, too, is undesirable.
  • the title compound can be made following the procedure set forth in Example 2 of U.S. 5,731 ,413.
  • acetic anhydride (55.1 g, 539.6 mmol, 5 equiv.) and pyridine (42.7 g, 539.6 mmol, 5 equiv.) were added into the reaction to cap the excess hydroxyesler-linker. The reaction was allowed to continue overnight. The residue was evaporated. The residue was taken up in ethyl acetate and washed with IN HCl (1 X), water (1 X), saturated sodium bicarbonate (1 X), water (1 X) and dried (magnesium sulfate), filtered, and concentrated.
  • This Example describes a general procedure for solid-phase syntheses of an arginine aldehyde having urokinase inhibitory activity, using the resin of Example 7. The procedures of this Example were also followed lo synlhesize a library of compounds, wherein eacli member comprised an arginine aldehyde but differed from the oilier members of the library in one or more other substituents.
  • Step 1 Preparation of N-omega-allyloxycarbonyl-argininal (6-hexanoyl- aminomethylated polystyrene resin) cyclol
  • Example 7 To a 60mL solid phase reaction vessel was added 2.0 g of the compound of Example 7 (0.6-0.7 meq/g substitution), and a mixture of dichloromethane (12mL), trifiuoroacetic acid (6mL), and thioanisole (2mL). Nitrogen gas was bubbled for 15 min. The reactants were drained from the resin and the resin was washed successively with dichloromethane(2X20mL), diisopropylethylamine (20mL), dichloromethane (2X20mL), diisopropylethylamine (20mL), dichloromethane (2X20mL), and diethyl ether (2X20mL). The title compound was stored under vacuum. A ninhydrin test of the resin showed a dark blue color characteristic of the free amine produced by the removal of the t-butoxycarbonyl group.
  • Step 2 Preparation of N- ⁇ -Fmoc-alanyl-N-ome ⁇ a-allyloxycarbonyl-areininal (6- hexanoyl-aminomethylated polystyrene resin) cyclol
  • Step 1 The compound of Step 1 (2.1 g) was placed in a solid phase reaction vessel to which were added Fmoc-alanine (lg, 3.2 mmol), 1 -hydroxybenzotriazole (0.5g, 3.2 mmol), TBTU ( 1.024g, 3.2 mmol) and diisopropylethylamine (600 ⁇ L, 3.4 mmol) in dimethylfbrmamide ( 15-20mL). Nitrogen gas was bubbled through the reactor at room temperature for 2 h.
  • Fmoc-alanine lg, 3.2 mmol
  • 1 -hydroxybenzotriazole 0.5g, 3.2 mmol
  • TBTU 1.024g, 3.2 mmol
  • diisopropylethylamine 600 ⁇ L, 3.4 mmol
  • Step 4 N- ⁇ -Fmoc-D-seryl(O-t-butyl)-alanyl-N-omeaa-allyloxycarbonyl-argininal (6- hexanoyl-aminomethylated polystyrene resin) cyclol
  • the compound of Step 3 (100 mg) was placed in a reaction vessel, such as a Kan 1 M reaction vessel (1RORI, San Diego, CA).
  • the reaction vessel was placed in a 20mL vial containing dimethylformamide (4mL), N- ⁇ -Fmoc-D-serine(O-t-butyl) (184mg, 0.48mmol), 1 -hydroxybenzotriazole (73mg, 0.48mmol), TBTU ( 154mg, 0.48mmol), and diisopropylethylamine (84 ⁇ l, 0.48mmol).
  • the reaction vessel was agitated for 3 h on a shaker table.
  • the reaction vessel was drained, washed successively with dimethylformamide (2X3mL), dichloromethane(2X3mL), dimethylformamide(2X3mL), dichloromethane(2X3mL), isopropanol(2X3mL), dichloromethane(2X3mL), isopropanol(2X3mL), and diethyl ether(2X3mL).
  • the resin was dried under vacuum to give the title compound.
  • Step 5 D-seryl(Q-t-butyl)-alanyl-N-omega-allyloxycarbonyl-argininal (6-hexanoyl- aminomethylated polystyrene resin) cyclol
  • Step 4 The reaction vessel containing the compound of Step 4 was treated with 50% piperidine in dimethylformamide (5mL) in a 20mL vial for 45 min al room temperature while agitated on a shaker table. The resin was washed as above and vacuum dried lo give the compound of Step 5. A ninhydrin assay on a small ali uot gave a ai k blue resin and solution indicating a high yield for the deprotection.
  • Step 6 carbamate analogs of D-servKO-t-bulvD-alanyl-N-omega-allyloxycarbonyl- argininal (6-hexanoyl-aminomethylated polystyrene resin) cyclol
  • the reaction vessel containing the compounds of Step 5 was placed in a 20mL vial with 0.12M isobutylchloroformate, in dimethylformamide (5-10mL). Diisopropylethylamine (105-21 O ⁇ l, 0.6-1.2 mmol) was added, and the vial was shaken for 2.5 h.
  • the reaction vessel was drained and washed successively with dimethylformamide (2X3mL), dichloromethane(2X3mL), dimethylformamide(2X3mL), dichloromethane(2X3mL), isopropanol(2X3mL), dichloromethane(2X3mL), isopropanol(2X3mL), and diethyl ether(2X3mL).
  • the reaction vessel containing isobutyloxycarbonyl-D-seryl(O-t-butyl)- alanyl-N-d-allyloxycarbonyl-argininal (6-hexanoyl-aminomethylated polystyrene resin) cyclol was vacuum dried.
  • reaction vessels containing related resin-bound analogs were combined in the 20 ml vial in Step 6, with various individual chloroformates added, and treated in parallel for the remainder of Steps 7 and 8.
  • Step 7 carbamate analogs of l ) -seryl( () -l-bulyl)-alanyl- argininal (6-hexanoyl- aminomethylated polystyrene resin) cyclol
  • Removal of the allyloxy protecting group from the product of Step 5 was accomplished by placing a collection of 38 reaction vessels, including the reaction of interest, in a 250mL polypropylene bottle and adding a mixture of methylsulfoxide (l OmL), tetrahydrofuran (l OmL), I N HCl (2.5mL), and morpholine (25mL). Tetrakis triphenylphosphine palladium (0.87g) was then added, and ihe bottle was shaken for 4 h at room temperature.
  • the reaction vessel including the one containing isobutyloxycarbonyl-D-scryl(()-t-bulyl)-alanyl-argininal (6-hexanoyl-aminomclhylated polystyrene resin) cyclol, were drained, washed successively with dimethylformamide (2X3mL), dichloromethane(2X3mL), dimethylformamide(2X3mL), dichloromethane(2X3mL), isopropanol(2X3mL), dichloromethane(2X3mL), isopropanol(2X3mL), and diethyl ether(2X3rnL), and vacuum dried to give the title compound.
  • Step 8 isobutyloxycarbonyl-D-seryl-alanyl-argininal
  • reaction vessel containing isobutyloxycarbonyl-D-seryl(0-t-butyl)-alanyl-argininal (6-hexanoyl-aminomethylated polystyrene resin) cyclol was emptied into a Whatman polypropylene mini-column containing trifiuoroacetic acid/dichloromethane/water

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Abstract

La présente invention concerne une nouvelle méthode de synthèse de peptides en phase solide, un amino-aldéhyde cyclique ou linéaire étant dérivatisé par liaison à un support solide et utilisé dans la production de peptides, dérivés de peptides, composés peptidomimétiques et leurs analogues biologiquement importants. L'invention concerne le procédé de liaison et de synthèse des peptides, y compris en chimie combinatoire.
EP99938805A 1998-07-24 1999-07-23 Support solide d'amino-aldehyde, sa sequence de liaison et ses methodes de preparation et d'utilisation Withdrawn EP1100782A1 (fr)

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AU2001247697B2 (en) 2000-03-22 2006-06-22 Solulink, Incorporated Hydrazine-based and carbonyl-based bifunctional crosslinking reagents
US6511973B2 (en) 2000-08-02 2003-01-28 Bristol-Myers Squibb Co. Lactam inhibitors of FXa and method

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