throbber
WO 94/22494
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`PCTIUSS4/03256
`
`mixture, and formulation into an efficacious therapeutic
`
`'
`agent,_
`The term "substituted", as used herein, means that
`
`an one or more hydrogen on the designated atom is
`
`replaced with a selection from the indicated group,
`
`provided that the designated atom's normal valency
`
`not exceeded, and that the substitution results in
`
`stable compound. When a substitent is keto (i.e.,
`
`then 2 hydrogens on the atom are replaced.
`
`As used herein, "alkyl" is intended to include both
`
`branched and straight—chain saturated aliphatic
`
`hydrocarbon groups having the specified number of carbon
`
`atoms;
`
`"haloalkyl" is intended to include both branched
`
`and straight-chain saturated aliphatic hydrocarbon
`
`groups having the specified number of carbon atoms,
`substituted with 1 or more halogen (for example -CVFH
`
`where v = 1 to 3 and w
`
`l to (2v+1)); "alkoxy"
`
`represents an alkyl group of indicated number of carbon
`
`atoms attached through an oxygen bridge; "cycloalkyl" is
`
`intended to include saturated ring groups,
`
`including
`
`mono—,bi- or poly-cyclic ring systems, such as
`cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
`cycloheptyl, cyclooctyl and adamantyl; and "biycloalkyl"
`
`is intended to include saturated bicyclic ring groups
`
`such as [3.3.0]bicyclooctane,
`
`[4.3.0]bicyclononane,
`
`[4.4.0]bicyclodecane (decalin),
`
`[2.2.2]bicyclooctane,
`
`"Alkenyl" is intended to include
`and so forth.
`hydrocarbon chains_of either a straight or branched
`configuration and one or more unsaturated carbon—carbon
`bonds which may occur in any stable point along the
`
`chain, such as ethenyl, propenyl and the like; and
`
`"alkynyl" is intended to include hydrocarbon chains of
`
`either a straight or branched configuration and one or
`
`LUPIN EX. 1023
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`
`more triple carbon—carbon bonds which may occur in any
`
`stable point along the chain, such as ethynyl, propynyl
`and the like.
`
`The phrase "boronic acid" as used herein means a
`group of the formula —B(R34)(R35), wherein R34 and R35
`are independently selected from: —OH; -F;
`-NR13R14; or
`C1-C5-alkoxy; or R34 and R35 can alternatively be taken
`
`together to form: a cyclic boron ester where said chain
`
`or ring contains from 2 to 20 carbon atoms and,
`optionally, 1-4 heteroatoms independently selected from
`
`N, S, or O; a divalent cyclic boron amide where said
`
`chain or ring contains from 2 to 20 carbon atoms and,
`
`optionally, 1-4 heteroatoms independently selected from
`
`N, S, or O; a cyclic boron amide-ester where said chain
`
`or ring contains from 2 to 20 carbon atoms and,
`
`optionally, 1-4 heteroatoms independently selected from
`
`N, S, or 0.
`
`Such cyclic boron esters, boron amides, or
`
`boron amide-esters may also be optionally substituted
`
`with 1-5 groups independently selected from R11.
`Boron esters include boronic acid protecting
`
`groups,
`
`including moieties derived from diols,
`
`for
`
`example pinanediol and pinacol to form pinanediol
`boronic acid ester and the pinacol boronic acid,
`
`respectively. Other illustrations of diols useful for
`
`deriving boronic acid esters are perfluoropinacol,
`ethylene glycol, diethylene glycol, 1,2-ethanediol,
`l,3—propanedio1,.l,2¥propanediol,
`l,2—butanediol,
`l,4—butanediol,,2,3-butanediol, 2,3—hexanediol,
`
`1,2—hexanediol, catechol, 1,2-diisopropylethanediol,
`
`5,6—decanediol, 1,2—dicyclohexylethanediol.
`
`"Halo" or "halogen" as used herein refers to
`
`fluoro, chloro, bromo and iodo; and "counterion" is used
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`
`to represent a small, negatively charged species such as
`
`chloride, bromide, hydroxide, acetate, sulfate and the
`like.
`~
`
`As used herein, "aryl" or "aromatic residue" is
`
`intended to mean phenyl or naphthyl.
`
`As used herein,
`
`"carbocycle" or "carbocyclic residue" is intended to
`
`mean any stable 3- to 7- membered monocyclic or bicyclic
`
`or 7- to l4—membered bicyclic or tricyclic or an up to
`
`26-membered polycyclic carbon ring, any of which may be
`
`saturated, partially unsaturated, or aromatic. Examples
`
`of such carbocyles include, but are not limited to,
`
`cyclopropyl, cyclopentyl, cyclohexyl, phenyl, biphenyl,
`
`indanyl, adamantyl, or tetrahydronaphthyl
`naphthyl,
`(tetralin).
`
`As used herein,
`
`the term "heterocycle" or
`
`"heterocyclic ring system" is intended to mean a stable
`
`5- to 7- membered monocyclic or bicyclic or 7- to 10—
`
`membered bicyclic heterocyclic ring which may be
`
`saturated, partially unsaturated, or aromatic, and which
`consists of carbon atoms and from 1 to 4 heteroatoms
`
`selected independently from the group consisting of N, O
`
`and S and wherein the nitrogen and sulfur heteroatoms
`
`may optionally be oxidized, and the nitrogen may
`
`optionally be quaternized, and including any bicyclic
`
`group in which any of the above—defined heterocyclic
`
`rings is fused to a benzene ring.
`
`The heterocyclic ring
`
`may be attached to its pendant group at any heteroatom
`or carbon atom which results in a stable structure.
`The
`
`heterocyclic rings described herein may be substituted
`
`on carbon or on a nitrogen atom if the resulting
`
`compound is stable. Examples of such heterocycles
`
`include, but are not limited to, benzopyranyl,
`
`thiadiazine, tetrazolyl, benzofuranyl, benzothiophenyl,
`
`indolene, quinoline,
`
`isoquinolinyl or benzimidazolyl,
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`piperidinyl, 4—piperidone, 2—pyrrolidone,
`
`!
`tetrahydroquinoline,
`tetrahydrofuran,
`tetrahydroisoquinoline, decahydroquinoline,
`
`octahydroisoquinoline, azocine,
`
`triazine‘1including
`
`1,2,3—,
`
`l,2,4—, and 1,3,5-triazine), 6H-1,2,5-
`
`thiadiazine, 2H,6H-1,5,2-dithiazine,
`
`thiophene,
`
`tetrahydrothiophene,
`
`thianthrene, furan, pyran,
`
`isobenzofuran, chromene, xanthene, phenoxathiin,
`
`2H—pyrrole, pyrrole,
`
`imidazole, pyrazole,
`
`thiazole,
`
`isothiazole, oxazole (including 1,2,4- and 1,3,4-
`
`oxazole),
`
`isoxazole, triazole, pyridine, pyrazine,
`
`pyrimidine, pyridazine,
`
`indolizine,
`
`isoindole, 3H-
`
`indole,
`
`indole,
`
`lH—indazole, purine, 4H-quinolizine,
`
`isoquinoline, quinoline, phthalazine, naphthyridine,
`
`quinoxaline, quinazoline, cinnoline, pteridine,
`
`4aH-carbazole, carbazole, B-carboline, phenanthridine,
`
`acridine, perimidine, phenanthroline, phenazine,
`
`phenarsazine, phenothiazine,
`
`furazan, phenoxazine,
`
`isochroman, chroman, pyrrolidine, pyrroline,
`
`imidazolidine,
`
`imidazoline, pyrazolidine, pyrazoline,
`
`piperazine,
`
`indoline,
`
`isoindoline, quinuclidine, or
`
`morpholine. Also included are fused ring and spiro
`compounds containing, for example,
`the above
`
`heterocycles.
`
`As used herein,
`
`the term "any group that, when
`
`administered toga mammalian subject, cleaves to form a
`
`free hydroxyl, amino or sulfhydryl" means any group
`bonded to an O, N, or S atom, respectively, which is
`cleaved from the O, N, or S atom when the compound is
`administered to a mammalian subject to provide a
`
`compound having a remaining free hydroxyl, amino, or
`
`sulfhydryl group, respectively. Examples of groups
`
`that, when administered to a mammalian subject, are
`
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`cleaved to form a free hydroxyl, amino or sulfhydryl,
`
`include but are not limited to, C1-C5 alkyl substituted
`
`with 0-3 R11, C3-C5 alkoxyalkyl substituted with 0-3
`
`R11, C1-C5 alkylcarbonyl substituted with 0-3 R11, C1-C5
`
`alkoxycarbonyl substituted with 0-3 R11, C1-C5
`
`alkylaminocarbonyl substituted with 0-3 R11, benzoyl
`
`substituted with 0-3 R12, phenoxycarbonyl substituted
`
`with 0-3 R12, phenylaminocarbonyl substituted with 0-3
`
`R12. Examples of groups that, when administered to a
`
`mammalian subject, are cleaved to form a free hydroxyl,
`
`amino or sulfhydryl,
`
`include hydroxy, amine or
`
`sulfhydryl protecting groups, respectively.
`
`As used herein,
`
`the term “amine protecting group"
`
`means any group known in the art of organic synthesis
`
`for the protection of amine groups.
`
`Such amine
`
`protecting groups include those listed in Greene,
`
`"Protective Groups in Organic Synthesis" John Wiley &
`
`Sons, New York (1981) and "The Peptides: Analysis,
`
`Sythesis, Biology, Vol. 3, Academic Press, New York
`
`the disclosure of which is hereby incorporated
`(1981),
`by reference. Any amine protecting group known in the
`art can be used. Examples of amine protecting groups
`include, but are not limited to,
`the following: 1) acyl
`
`types such as formyl, trifluoroacetyl, phthalyl, and
`
`p-toluenesulfonyl; 2) aromatic carbamate types such as
`
`benzyloxycarbonyl
`
`(Cbz or Z) and substituted
`
`benzyloxycarbonyls, 1-(p-biphenyl)-l-
`
`methylethoxycarbonyl, and 9-fluorenylmethyloxycarbonyl
`
`(Fmoc); 3) aliphatic carbamate types such as tert-
`
`butyloxycarbonyl
`
`(Boc), ethoxycarbonyl,
`
`diisopropylmethoxycarbonyl, and allyloxycarbonyl; 4)
`
`cyclic alkyl carbamate types such as
`
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`
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`
`cyclopentyloxycarbonyl and adamantyloxycarbonyl;
`
`5)
`
`alkyl types such as triphenylmethyl and benzyl; 6)
`
`trialkylsilane such as trimethylsilane; and 7)
`
`thiol
`
`containing types such as phenylthiocarbonyl and
`
`dithiasuccinoyl. Also included in the term "amine
`
`protecting group" are acyl groups such as azidobenzoyl,
`
`p—benzoylbenzoyl, o-benzylbenzoyl, p-acetylbenzoyl,
`
`dansyl, glycyl-p-benzoylbenzoyl, phenylbenzoyl,
`
`m-benzoylbenzoyl, benzoylbenzoyl.
`
`As used herein, "pharmaceutically acceptable salts"
`
`refer to derivatives of the disclosed compounds wherein
`
`the parent compound of formula (I)
`
`is modified by making
`
`acid or base salts of the compound of formula (1).
`
`Examples of pharmaceutically acceptable salts include,
`
`but are not
`
`limited to, mineral or organic acid salts of
`
`basic residues such as amines; alkali or organic salts
`
`of acidic residues such as carboxylic acids; and the
`like.
`
`Pharmaceutically acceptable salts of the compounds
`
`of the invention can be prepared by reacting the free
`
`acid or base forms of these compounds with a
`
`stoichiometric amount of the appropriate base or acid in
`
`water or in an organic solvent, or in a mixture of the
`
`two; generally, nonaqueous media like ether, ethyl
`
`acetate, ethanol,
`
`isopropanol, or acetonitrile are
`
`preferred. Lists of suitable salts are found in
` . 17th ed-. Mack
`
`Publishing Company, Easton, PA, 1985, p. 1418,
`
`the
`
`disclosure of which is hereby incorporated by reference.
`The term "amino acid" as used herein means an
`
`organic compound containing both a basic amino group and
`
`an acidic carboxyl group.
`
`Included within this term are
`
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`
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`
`modified and unusual amino acids, such as those
`
`disclosed in, for example, Roberts and Vellaccio (1983)
` , 5: 342-429,
`the teaching of"which is
`hereby incorporated by reference. Modified or unusual
`
`amino acids which can be used to practice the invention
`
`include, but are not limited to, D—amino acids,
`
`hydroxylysine, 4—hydroxyproline, ornithine,
`
`2,4-diaminobutyric acid, homoarginine, norleucine,
`
`N—methy1aminobutyric acid, naphthylalanine,
`
`phenylglycine, B-phenylproline,
`
`tert—leucine,
`
`4—aminocyc1ohexy1alanine, N—methyl-norleucine,
`
`3,4-dehydroproline, 4-aminopiperidine-4-carboxylic acid,
`
`6-aminocaproic acid,
`
`trans-4-(aminomethyl)—
`
`cyclohexanecarboxylic acid, 2-, 3-, and 4-(aminomethyl)—
`
`benzoic acid, 1-aminocyclopentanecarboxylic acid,
`
`1-aminocyclopropanecarboxylic acid, and 2-benzyl-5-
`
`aminopentanoic acid.
`The term "amino acid residue" as used herein means
`
`that portion of an amino acid (as defined herein) that
`
`is present in a peptide.
`
`The term "peptide".as used herein means a linear
`
`compound that consists of two or more amino acids (as
`defined herein) that are linked by means of a peptide
`
`bond.
`
`The term "peptide" also includes compounds
`
`containing both peptide and non-peptide components, such
`
`as pseudopeptide or peptide mimetic residues or other
`
`non—amino acid components.
`
`Such a compound containing
`
`both peptide and non-peptide components may also be
`referred to as a "peptide analog".
`
`A "pseudopeptide" or "peptide mimetic" is a
`
`compound which mimics the structure of an amino acid
`
`residue or a peptide, for example, by using linking
`
`groups other than amide linkages between the peptide
`mimetic and an amino acid residue (pseudopeptide bonds)
`
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`
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`
`PCTlUS94l032S6
`
`and/or by using non-amino acid substituents and/or a
`U:
`
`modified amino acid residue.
`
`A "pseudopeptide residue" means that portion of an
`
`pseudopeptide or peptide mimetic (as defined herein)
`
`that is present in a peptide.
`
`The term "peptide bond" means a covalent amide
`
`linkage formed by loss of a molecule of water between
`
`the carboxyl group of one amino acid and the amino group
`of a second amino acid.
`
`The term "pseudopeptide bonds" includes peptide
`
`bond isosteres which may be used in place of or as
`
`substitutes for the normal amide linkage. These
`
`substitute or amide "equivalent" linkages are formed
`
`from combinations of atoms not normally found in
`
`peptides or proteins which mimic the spatial
`
`requirements of the amide bond and which should
`
`stabilize the molecule to enzymatic degradation.
`
`The terms "Ln", "linking group" and "linker", used
`
`interchangeably throughout, designate the group of atoms
`separating Q from the metal chelator, Ch.
`
`The terms "activated Ln group", "activated Ln",
`
`"activated linking group" and "activated linker", used
`
`interchangeably throughout, refer to a linking group
`
`that bears one or more reactive group capable of
`
`reacting with, and forming a bond with, a chelator or a
`Q.
`
`The terms "Ch", "metal chelator", and "chelator"
`
`are used interchangeably throughout to designate a
`
`chemical moiety capable of binding to or complexing with
`a metal nuclide.
`
`The term "cyclizing moiety" means the intermediate
`
`compound that serves as the precursor to the R31 group
`of Q.
`
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`
`The term "ring substituted cyclizing moiety" is a
`
`cyclizing moiety bearin a substituent group one or more
`
`of its carbocyclic or heterocyclic rings.
`
`The term "linker modified cyclizing moiety" refers
`
`to a cyclizing moiety that bears an activated Ln group.
`
`The term "cyclic compound intermediate" means the
`
`intermediate compound that serves as the precursor to
`
`the Q group in the claimed compounds.
`
`The term "linker modified cyclic compound
`
`intermediate" means a cyclic compound intermediate that
`
`bears an activated Ln group.
`
`The compounds of the present invention can be
`
`prepared in a number of ways well known to one skilled
`in the art of organic synthesis. Preferred methods
`include but are not limited to those methods described
`below.
`.
`
`The following abbreviations are used herein:
`
`Acm
`
`D—Abu
`
`5—Aca
`
`acetamidomethyl
`
`D—2—aminobutyric acid
`
`5-aminocaproamide(5-aminohexanamide)
`
`b—Ala, b-Ala or
`
`bAla
`
`Boc
`
`3—aminopropionic acid
`
`t-butyloxycarbonyl
`
`Boc-iodo-Mamb .
`
`t-butyloxycarbonyl-3—aminomethy1—4-iodo—
`benzoic acid
`
`Boc—Mamb
`acid
`
`Boc-ON
`
`'
`
`tFbutyloxycarbonyl-3—aminomethylbenzoic
`
`[2-(tert-butyloxycarbonyloxylimino)-2-
`
`phenylacetonitrile
`
`Cl2Bzl
`
`dichlorobenzyl
`
`CBZ, Cbz or Z Carbobenzyloxy
`
`DCC
`
`dicyclohexylcarbodiimide
`
`9 of 532
`
`

`
`“K)%MZM94
`
`PCT/US94/03256
`
`DIEA
`
`di—NMeOrn
`DMAP
`.
`HBTU
`
`NMeArg
`
`MeArg
`NMeAmf
`
`NMeAsp
`
`NMeGly
`
`MeGly
`NMe-Mamb
`
`NMM
`
`OcHex
`
`OBzl
`
`oSu
`
`pNP
`TBTU
`
`diisopropylethylamine
`
`N—aMe-N—gMe-ornithine
`
`4—dimethylaminopyridine
`
`2-(1H—Benzotriazol-1-yl)-1,1,3,3-
`
`tetramethyluronium hexafluorophosphate
`
`a—N—methy1 arginine
`
`N—Methylaminomethylphenylalanine
`
`a-N-methyl aspartic acid
`
`N—methy1 glycine
`
`N—methyl-3—aminomethylbenzoic acid
`
`N—methy1morpho1ine
`
`O—cyclohexyl
`
`O—benzyl
`
`O-succinimidyl
`
`.p—nitrophenyl
`
`2-(1H—Benzotriazol—1—yl)-1,1,3,3-
`
`tetramethyluronium
`tetrafluoroborate
`
`2-(Trimethylsilyl)ethyloxycarbonyl
`
`tosyl
`
`trityl
`
`The following conventional three-letter amino acid
`abbreviations are used herein;
`the conventional one-
`
`letter amino acid abbreviations are ngt used herein:
`
`Ala
`
`Arg
`
`Asp
`
`Cys
`
`alanine
`
`arginine
`asparagine
`
`aspartic acid
`
`cysteine
`
`glutamine
`
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`
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`
`glutamic acid
`
`glycine
`histidine
`
`isoleucine
`
`leucine
`
`lysine
`methionine
`
`norleucine
`
`phenylalanine
`
`phenylglycine
`
`proline
`serine
`
`threonine
`
`tryptophan
`
`tyrosine
`valine
`
`The compounds of the present
`
`invention can be
`
`synthesized using standard synthetic methods known to
`those skilled in the art. Preferred methods include but
`
`are not limited to those methods described below.
`
`Generally, peptides are elongated by deprotecting
`the a-amine of the C-terminal residue and coupling the
`
`next suitably protected amino acid through a peptide
`
`linkage using the methods described. This deprotection
`
`and coupling procedure is repeated until the desired
`
`sequence is obtained. This coupling can be performed
`with the constituent amino acids in a stepwise fashion,
`
`or condensation of fragments (two to several amino
`
`acids), or combination of both processes, or by solid
`
`phase peptide synthesis according to the method
`originally described by Merrifield, J. Am. Chem. Soc.,
`
`11 of 532
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`
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`
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`
`85, 2149-2154 (1963),
`
`the disclosure of which is hereby
`
`incorporated by reference.
`
`_
`
`The compounds of the invention may also be
`
`synthesized using automated peptide synthesizing
`
`equipment.
`
`In addition to the foregoing, procedures for
`
`peptide synthesis are described in Stewart and Young,
`"Solid Phase Peptide Synthesis", 2nd ed, Pierce Chemical
`Co., Rockford,
`IL (1984); Gross, Meienhofer, Udenfriend,
`
`Eds.,
`
`"The Peptides: Analysis, Synthesis, Biology, Vol.
`
`1, 2, 3, 5, and 9, Academic Press, New York,
`
`(1980-
`
`1987); Bodanszky, "Peptide Chemistry: A Practical
`
`Textbook", Springer-Verlag, New York (1988);
`
`and
`
`Bodanszky et al.
`
`"The Practice of Peptide Sythesis"
`
`Springer—Ver1ag, New York (1984),
`
`the disclosures of
`
`which are hereby incorporated by reference.
`
`The coupling between two amino acid derivatives. an
`
`amino acid and a peptide,
`
`two peptide fragments, or the
`
`cyclization of a peptide can be carried out using
`
`standard coupling procedures such as the azide method,
`
`mixed carbonic acid anhydride (isobutyl chloroformate)
`method, carbodiimide (dicyclohexylcarbodiimide,
`
`diisopropylcarbodiimide, or water-soluble carbodiimides)
`
`method, active ester (p—nitropheny1 ester, N-
`
`hydroxysuccinic imido ester) method, Woodward reagent K
`method, carbonyldiimidazole method, phosphorus reagents
`
`such as BOP-Cl, or oxidation—reduction method.
`
`some of
`
`these methods (especially the carbodiimide) can be
`
`enhanced by the addition of 1-hydroxybenzotriazole.
`
`These coupling reactions may be performed in either
`
`solution (liquid phase) or solid phase.
`
`The functional groups of the constituent amino
`
`acids must be protected during the coupling reactions to
`
`avoid undesired bonds being formed.
`
`The protecting
`
`groups that can be used are listed in Greene,
`
`12 of 532
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`
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`
`PCT/US94/03256
`
`"Protective Groups in Organic Synthesis" John Wiley &
`
`Sons, New York (1981) and "The Peptides: analysis,
`
`Sythesis, Biology, Vol. 3, Academic Press, New York
`
`(1981),
`
`the disclosure of which is hereby incorporated
`
`by reference .
`
`'
`
`The a-carboxyl group of the C-terminal residue is
`
`usually protected by an ester that can be cleaved to
`
`give the carboxylic acid. These protecting groups
`
`include: 1) alkyl esters such as methyl and t-butyl, 2)
`
`aryl esters such as benzyl and substituted benzyl, or 3)
`esters which can be cleaved by mild base treatment or
`
`mild reductive means such as trichloroethyl and phenacyl
`
`the C-terminal amino
`In the solid phase case,
`esters.
`acid is attached to an insoluble carrier (usually
`
`polystyrene). These insoluble carriers contain a group
`which will react with the carboxyl group to form a bond
`
`which is stable to the elongation conditions but readily
`
`cleaved later. Examples of which are: oxime resin
`
`(DeGrado and Kaiser
`
`(1980) J. Org. Chem.
`
`45, 1295-1300)
`
`chloro or bromomethyl resin, hydroxymethyl resin,
`
`and
`
`'
`
`aminomethyl resin. Many of these resins are
`
`commercially available with the desired C-terminal amino
`acid already incorporated.
`7
`The a—amino group of each amino acid must be
`
`protected. Any protecting group known in the art can be
`
`used. Examples of these are: 1) acyl types such as
`
`formyl, trifluoroacetyl, phthalyl, and p-
`
`toluenesulfonyl; 2) aromatic carbamate types such as
`
`benzyloxycarbonyl
`
`(Cbz) and substituted
`
`benzyloxycarbonyls,
`
`l-(p-biphenyl)-1-
`
`methylethoxycarbonyl, and 9-fluorenylmethyloxycarbonyl
`
`(Fmoc); 3) aliphatic carbamate types such as tert-
`
`butyloxycarbonyl
`
`(Boc), ethoxycarbonyl,
`
`diisopropylmethoxycarbonyl, and allyloxycarbonyl; 4)
`
`13 of 532
`
`

`
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`
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`
`cyclic alkyl carbamate types such as
`
`cyclopentyloxycarbonyl and adamantyloxycarbonyl;
`
`5)
`
`alkyl types such as triphenylmethyl and benzyl; 6)
`
`trialkylsilane such as trimethylsilane; and 7)
`
`thiol
`
`containing types such as phenylthiocarbonyl and
`
`dithiasuccinoyl.
`
`The preferred a-amino protecting group
`
`is either Boc or Fmoc. Many amino acid derivatives
`
`suitably protected for peptide synthesis are
`
`commercially available.
`
`The a-amino protecting group is cleaved prior to
`
`the coupling of the next amino acid. When the Boc group
`
`is used,
`
`the methods of choice are trifluoroacetic acid,
`
`neat or in dichloromethane, or HCl in dioxane.
`
`The
`
`resulting ammonium salt is then neutralized either prior
`
`to the coupling or in situ with basic solutions such as
`
`aqueous buffers, or tertiary amines in dichloromethane
`
`or dimethylformamide. When the Fmoc group is used,
`
`the
`
`reagents of choice are piperidine or substituted
`
`piperidines in dimethylformamide, but any secondary
`
`amine or aqueous basic solutions can be used.
`
`The
`
`deprotection is carried out at a temperature between 0
`
`°C and room temperature.
`
`Any of the amino acids bearing side chain
`
`functionalities must be protected during the preparation
`
`of the peptide using any of the above—identified groups.
`
`Those skilled in the art will appreciate that the
`
`selection and use of appropriate protecting groups for
`
`these side chain functionalities will depend upon the
`
`amino acid and presence of other protecting groups in
`
`the peptide.
`
`The selection of such a protecting group
`
`is important
`
`in that it must not be removed during the
`
`deprotection and coupling of the a-amino group.
`
`For example, when Boc is chosen for the a-amine
`
`protection the following protecting groups are
`
`14 of 532
`
`

`
`WO 94122494
`
`PCTlUS94I03256
`
`acceptable: p-toluenesulfonyl
`
`(tosyl) moieties and nitro
`
`for arginine; benzylcxycarbcnyl, substituted
`
`benzyloxycarbonyls,
`
`tosyl or trifluoroacetyl for lysine;
`
`benzyl or alkyl esters such as cyclopentyl for glutamic
`
`and aspartic acids; benzyl ethers for serine and
`
`threonine; benzyl ethers, substituted benzyl ethers or
`
`2—bromobenzyloxycarbcny1 for tyrosine; p-methylbenzyl,
`
`p-methoxybenzyl, acetamidomethyl, benzyl, or t-
`
`butylsulfonyl for cysteine; and the indole of tryptophan
`
`can either be left unprotected or protected with a
`
`formyl group.
`
`when Fmoc is chosen for the a-amine protection
`
`usually tert—butyl based protecting groups are
`
`acceptable.
`
`For instance, Boc can be used for lysine,
`
`tert—butyl ether for serine,
`
`threonine and tyrosine, and
`
`tert-butyl ester for glutamic and aspartic acids.
`
`Once the elongation and cyclization of the peptide
`
`is completed all of the protecting groups are removed.
`
`For the liquid phase synthesis the protecting groups are
`removed in whatever manner as dictated by the choice of
`
`protecting groups. These procedures are well known to
`those skilled in the art.
`
`when a solid phase synthesis is used,
`
`the peptide
`
`should be removed from the resin without simultaneously
`
`removing protecting groups from functional groups that
`
`might interfere with the cyclization process. Thus, if
`
`the cleavage
`the peptide is to be cyclized in solution,
`conditions need to be chosen such that a free a-
`
`carbcxylate and a free a—amino group are generated
`
`without simultaneously removing other protecting groups.
`
`Alternatively,
`
`the peptide may be removed from the resin
`
`by hydrazinolysis, and then coupled by the azide method.
`
`Another very convenient method involves the synthesis of
`
`peptides on an oxime resin,
`
`followed by intramolecular
`
`15 of 532
`
`

`
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`
`PCT/US94/03256
`
`nucleophilic displacement from the resin, which
`
`generates a cyclic peptide (Osapay, Profit, and Taylor
`
`(1990) Tetrahedron Letters 43, 6121-6124). When the
`
`oxime resin is employed,
`
`the Boo protection scheme is
`
`generally chosen.
`
`Then,
`
`the preferred method for
`
`removing side chain protecting groups generally involves
`
`treatment with anhydrous HP containing additives such as
`
`dimethyl sulfide, anisole,
`
`thioanisole, or p—cresol at 0
`
`°C.
`
`The cleavage of the peptide can also be
`
`accomplished by other acid reagents such as
`
`trifluoromethanesulfonic acid/trifluoroacetic acid
`
`mixtures.
`
`Unusual amino acids used in this invention can be
`
`synthesized by standard methods familiar to those
`skilled in the art
`("The Peptides: Analysis, Sythesis,
`
`Biology, Vol. 5, pp. 342-449, Academic Press, New York
`
`(1981)). N—Alkyl amino acids can be prepared using
`
`procedures described in previously (Cheung et al.,
`(1977) Can. J. Chem.
`55, 906; Freidinger et al.,
`
`(1982)
`
`J. Org. Chem.
`
`48, 77 (1982)), which are incorporated
`
`here by reference.
`
`The compounds of the present
`
`invention may be
`
`prepared using the procedures further detailed below.
`
`Representative materials and methods that may be
`
`used in preparing the compounds of the invention are
`
`described further below.
`
`Manual solid phase peptide synthesis was performed
`
`in 25 mL polypropylene filtration tubes purchased from
`
`BioRad Inc., or in 60 mL hour—glass reaction vessels
`
`Oxime resin
`purchased from Peptides International.
`(substitution level = 0.96 mmol/9) was prepared
`
`according to published procedures (DeGrado and Kaiser
`
`(19801 J. Org. Chem. 45, 1295), or was purchased from
`
`16 of 532
`
`

`
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`
`PCTIUS94/03256
`
`Novabiochem (substitution level = 0.62 mmol/g). All
`
`chemicals and solvents (reagent grade) were used as
`
`supplied from the vendors cited without further
`
`purification.
`
`t-Butyloxycarbonyl
`
`(Boc) amino acids and
`
`other starting amino acids may be obtained commercially
`
`from Bachem Inc., Bachem Biosciences Inc.
`
`(Philadelphia,
`
`PA), Advanced ChemTech (Louisville, KY), Peninsula
`
`Laboratories (Belmont, CA), or Sigma (St. Louis, MO).
`
`2-(1H—Benzotriazol—1-yl)-1,1,3,3—tetramethy1uronium
`
`hexafluorophosphate (HBTU) and TBTU were purchased from
`
`Advanced ChemTech. N-methylmorpholine (NM), m-cresol,
`
`D—2—aminobutyric acid (Abu),
`
`trimethylacetylchloride,
`
`diisopropylethylamine (DIEA), 3-cyanobenzoic acid and
`
`[2-(tert—butyloxycarbonyloxylimino)-phenylacetonitrile]
`
`(Boc—ON) were purchased from Aldrich Chemical Company.
`
`Dimethylformamide (DME), ethyl acetate, chloroform
`
`(CHCl3), methanol
`
`(MeOH), pyridine and hydrochloric acid
`
`(HCl) were obtained from Baker. Acetonitrile,
`
`dichloromethane (DCM), acetic acid (HOAC),
`
`trifluoroacetic acid (TFA), ethyl ether,
`
`triethylamine,
`
`acetone, and magnesium sulfate were purchased from EM
`Science. Palladium on carbon catalyst
`(10% Pd) was
`
`purchased from Fluka Chemical Company. Absolute ethanol
`was obtained from Quantum Chemical Corporation.
`Thin
`
`layer chromatography (TLC) was performed on Silica Gel
`
`60 F254 TLC plates (layer thickness 0.2 mm) which were
`
`purchased from EM Separations.
`
`TLC visualization was
`
`accomplished using UV light,
`
`iodine,
`
`ninhydrin spray
`
`and/or Sakaguchi spray. Melting points were determined
`
`using a Thomas Hoover or Electrothermal 9200 melting
`
`point apparatus and are uncorrected.
`
`HPLC analyses were
`
`performed on either a Hewlett Packard 1090, Waters Delta
`Prep 3000, Rainin, or DuPont 8800 system.
`NMR spectra
`were recorded on a 300 MHz General Electric QB-300,
`
`17 of 532
`
`

`
`WO 94/22494
`
`PCT/US94/03256
`
`Varian 300, or varian 400 spectrometer.
`
`Fast atom
`
`bombardment mass spectrometry (FAB-MS) was performed on
`
`a VG Zab—E double-focusing mass spectrometer using a
`
`Xenon FAB gun as the ion source or a Finnigan MAT 8230.
`
`Boc-D—2—aminobutyric acid (Boc-D-Abu) was prepared
`
`by a modification of procedures previously reported in
`the literature (Itoh, Hagiwara, and Kamiya (1975) Iett.
`
`Lett., 4393), as shown in the scheme below.
`
`NH;
`
`OH
`
`O
`
`aoc-on
`pH = 9, Et3N
`
`D-2-amlnobutyrlc acid
`
`0
`/lk
`
`NH
`
`+ 0
`
`O
`
`D—2-aminobutyric acid (1.0 g, 9.70 mmol) was
`
`dissolved in 20 ml H20 and a solution of Boc-ON (2.62 g,
`
`10.6 mmol)
`
`in 20 ml acetone was added.
`
`A white
`
`precipitate formed which dissolved upon addition of
`
`to give a pale yellow
`triethylamine (3.37 ml, 24.2 mmol)
`The solution was
`solution (pH =‘9, wet pH paper).
`stirred at room temperature overnight at which time the
`
`acetone was removed under reduced pressure.
`
`The
`
`remaining aqueous layer was extracted with ether three
`
`times, acidified to pH 2 with concentrated Hcl, and then
`
`extracted with ethyl acetate three times.
`
`The combined
`
`organic layers were dried over anhydrous magnesium
`
`sulfate and evaporated under reduced pressure to give t-
`butyloxycarbonyl—D—2—aminobutyric acid as an oil
`(2.05
`
`g,greater than quantitative yield, contains solvent),
`which was used without further purification.
`1H NMR
`
`(CDCl3) 0.98 (t, 3H), 1.45 (s, 9H), 1.73 (m, 1H), 1.90
`
`(m, 1H), 4.29 (m, 1H), 5.05 (m, 1H).
`
`18 of 532
`
`

`
`W0 94/22494
`
`I
`
`PCT/US94/03256
`
`S
`
`. E 1.
`
`u
`
`u
`
`a
`
`This section teaches the synthesis of certain
`
`cyclizing moieties that serve as intermediates to the
`
`5
`
`R31 groups in Q. Later sections teach the synthesis of
`
`other cyclizing moieties.
`
`Boo-aminomethylbenzoic acid derivatives useful as
`
`cyclizing moieties in the synthesis of the,compounds of
`
`the invention are prepared using standard procedures,
`
`for example, as described in Tett. Lett., 4393 (1975);
`
`Modern Synthetic Reactions, H.0. House (1972); or
`
`Harting et al. J. Am. Chem. Soc., 50: 3370 (1928), and
`as shown schematically below.
`
`0
`
`OH
`
`°
`
`°”
`
`i
`
`0
`
`on
`
`//N WP‘
`I
`‘éEfiMdT"
`\
`
`/Z\""2—"'°'
`\\ I
`
`soc-on
`1;;j;:;—*
`" '
`
`//NH—BOC
`|
`\
`
`3—Cyanobenzoic acid (10.0 g, 68 mmol) was dissolved in
`200 ml ethanol by heating in a 35-50°C water bath.
`Concentrated Hcl
`(6.12 ml, 73 mmol) was added and the
`
`solution was transferred to a 500 ml nitrogen-flushed
`
`round bottom flask containing palladium on carbon
`
`catalyst
`
`(1.05 g,
`
`10% Pd/C).
`
`The suspension was stirred
`
`under an atmosphere of hydrogen for 38 hours, filtered
`
`19 of 532
`
`

`
`WO 94122494
`
`PCT/US94/03256
`
`through a scintered glass funnel, and washed thoroughly
`with H30.
`The ethanol was removed under reduced
`‘
`w
`
`pressure and the remaining aqueous layer, which
`
`contained a white solid, was diluted to 250 ml with
`
`additional H20. Ethyl ether (250 ml) was added and the
`
`suspension was transferred to a separatory funnel. Upon
`
`vigorous shaking, all solids dissolved and the aqueous
`
`layer was then washed two times with ether, evaporated
`
`under reduced pressure to a volume of 150 ml, and
`
`lyophilized to give the title compound (3-
`
`aminomethylbenzoic acid-HCl)
`
`(8.10 g, 64%) as a beige
`
`solid.
`
`1}; NMR (D20)
`
`4.27 (s, 2H), 7.60 (t, 1H), 7.72
`
`(d,1H), 8.06 (d, 2H).
`
`The title compound was prepared according to a
`
`modification of standard procedures previously reported
`
`in the literature (Itoh, Hagiwara, and Kamiya (1975)
`
`Tett. Lett., 4393).
`
`3—Aminomethylbenzoic acid
`
`(hydrochloride salt)
`
`(3.0 g, 16.0 mmol) was dissolved in
`
`60 ml H20.
`
`To this was added a solution of Boc—ON (4.33
`
`g, 17.6 mmol)
`
`in
`
`60 ml acetone followed by
`
`triethylamine (5.56 ml, 39.9 mmol).
`
`The solution
`
`turned yellow and the pH was adjusted to 9
`
`(wet pH
`
`paper) by adding an additional 1.0 ml
`
`(7.2 mmol)
`
`triethylamine.
`
`The solution was stirred overnight at
`
`room temperature at which time the acetone was removed
`
`under reduced pressure and the remaining aqueous layer
`
`was washed three times with ether.
`
`The aqueous layer
`
`was then acidified to pH 2 with 2N HCl and then
`
`extracted three times with ethyl acetate.
`
`The combined
`
`organic layers were washed three times with H20, dried
`
`over anhydrous magnesium sulfate, and evaporated to
`
`20 of 532
`
`

`
`WO 94/22494
`
`PCTfUS94I03256
`
`dryness under reduced pressure.
`
`The material was
`
`recrystallized from ethyl acetate/ hexane,to give two
`
`crops of the title compound (2.58 g,
`
`64%) as an off-
`
`white solid.
`
`mp 123-125°C ,-11-1 NMR (CDCl3)
`
`1.47 (s,
`
`9
`
`H), 4.38 (br s, 2 H), 4.95 (br S, 1H), 7.45 (t, 1H),
`
`7.55 (d;
`
`IH): 8.02 (d, 2H).
`
`t-Butyloxycarbonyl-3—aminopheny1acetic acids useful
`
`as intermediates in the synthesis of the compounds of
`
`the invention are prepared using standard procedures,
`
`for example, as described in Collman and Groh (1982) J.
`
`Am. Chem. Soc., 104:
`below.
`
`l39l, and as shown schematically
`
`co,H
`NI-I40!-I
`-—-——-———}
`FO5O4'7H2O
`
`co,H
`I-BuO;COCOgt-Bu
`--:—e-é—-}
`DIEA
`
`N02
`
`Ha Nsfl
`
`co,H
`
`N
`
`H’
`
`‘co,t-au
`
`A solution of 3—aminophenylacetic acid (Aldrich, 10
`
`g, 66 mmol), di-tert-butyl dicarbonate (15.8 g, 72
`
`mmol), and DIEA (8.6 g, 6

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