NZ234373A - Acylated n-terminal tetrapeptides having an esterified or amidified aspartic acid residue and optionally substituted at the c-terminal by 5-1h-tetrazolyl; use in treatment of herpes virus infections - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £34373 23 4 3 7 3 Priority Datejs):....^^.* ...

Complete Specification Filed: Class: (8)CQl.^»|l.Ql.>a5;..Afe>.^/.®7.

Publication Dots: ..jr.,4.J.UiU99Z.

P.O. Journal No: NO DRAWINGS Patents Form No. 5 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION IHERPES TETRAPEPTIDE DERIVATIVES HAVING A SUBSTITUTED ASPARTIC ACID SIDE CHAIN WE, BIO-MEGA INC., a Canadian company of 2100 Rue Cunard, Laval, Quebec, CANADA H7S 2G5 hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statements (followed by page la) 234373 * 55 603.306 Antiherpes Tetrapeptide Derivatives Having a Substituted Aspartic Acid Side Chain This invention relates to peptide derivatives having antiviral 5 properties and to means for using the derivatives to treat viral infections. More specifically, the invention relates to peptide derivatives (hereinafter called "peptides") exhibiting activity against herpes viruses, to pharmaceutical compositions comprising the peptides, and to a method of using the peptides to treat herpes 10 infections.

The family of herpes viruses is responsible for a wide range of infections that afflict humans and many important domestic animals. The diseases caused by these viruses range from bothersome cold sores to highly destructive infections of the central nervous 15 system (encephalitis). The more common members of this family include herpes simplex virus (types 1 and 2) responsible for cold sores and genital lesions; varicella zoster virus which causes chicken pox and shingles; and Epstein-Barr virus which causes infectious mononucleosis. Although some significant advances have been 20 made in the last decade in antiviral therapy, the need for effective, safe therapeutic agents for treating herpes viral infections continues to exist. For a recent review of current therapeutic agents in this area, see M.C. Nahata, "Antiviral Drugs: Pharmacokinetics, Adverse Effects and Therapeutic Use", J. Pharm. Technol., 3, 100 (1987). (followed by page 2) 234 373 2 The present application discloses a group of peptide derivatives having activity against herpes viruses. The relatively selective action of these peptides against herpes viruses, combined with a wide margin of safety, renders the peptides as desirable 5 agents for combating herpes infections.

The association of peptides with anti-herpes activity is uncommon. Instances of reports of such an association include B.M. Dutia et al., Nature, 321. 439 (1986), E.A. Cohen et al., Nature, 321. 441 (1986), J.H. Subak-Sharpe et al., UK patent 10 application 2185024, published July 8, 1987, E.A. Cohen et al., European patent application 246630, published November 25,1987, R. Freidinger et al., European patent application 292255, published November 23,1988, and R. Freidinger et al., U.S. patent 4,814,432, issued March 21, 1989. The subject peptides of the previous 15 reports can be distinguished from the peptides of the present application by characteristic structural and biological differences.

The peptides of this invention are represented by formula 1 X-NH-CHR1-C(W1)-NR2-CH[CH2C(0)-Y]-C(W2)-NH-CH[CR3(RA)-COOH]-C(W3)-NH-CHR*-Z 1 # wherein X is (C1.10)alkanoyl; (C^Jalkanoyl monosubstituted with halo, hydroxy or lower alkoxy; (C,.10)alkoxycarbonyl; benzoyl; benzoyl monosubstituted or disubstituted with a substituent selected from halo, hydroxy, lower alkyl, lower alkoxy, phenyl, 2-carboxy-phenyl or benzyl; 2,2-diphenylacetyl; phenyKQ.^alkanoyl; phenyl(C2.10)alkanoyl monosubstituted or disubstituted on the aromatic portion thereof with a substituent selected from halo, hydroxy, lower alkyl, lower alkoxy or phenyl; phenyl(C.j.10) alkenoyl; (lower cycloalkyl)carbonyl; (lower cycloalkyl)carbonyl substituted with one to four substituents selected from halo or lower ;lkyl; cyclohexylcarbonyl substituted at position 2 with lower olkanoyl, phenyl(lower)alkanoylorphenyl(lower)alkoxycarbonyl;3,6-dimethyl-2-(phenylethoxycarbonyl)cyclohexylcarbonyl; or a straight or branched chain 1,4-dioxoalkyl containing from five to eleven carbon atoms; R1 is lower alkyl, hydroxy(lower)alkyl, mercapto(lower)alkyl, methoxy(lower)alkyl, methylthio(lower)alkyl, benzyloxy(lower)alkyl, benzylthio^!nwer)alkyl, carboxy(lower)alkyl, lower cycloalkyl, (lower cycloalkyl)methyl, phenyl, phenylmethyl, 2-thienyl or 2-thienylmethyl; R2 is hydrogen, lower alkyl or phenyl(lower)alkyl; R3 and R4 each independently is hydrogen or lower alkyl, or R3 and R4 together with the carbon atom to which they are attached form a lower cycloalkyl; R5 is lower alkyl, lower cycloalkyl, or (lower cycloalkyl)methyl; W1, W2, and W3 each independently is oxo or thioxo; Y is a. (C1.„)alkoxy, (3-14)alkenyloxy, CH3(0CH2CH2)n-0 wherein n is the integer 1, 2 or 3, lower cycloalkyloxy, lower alkoxy monosubstituted with a lower cycloalkyl, phenoxy, phenoxy monosubstituted with hydroxy, halo, lower alkyl or lower alkoxy, phenyl(lower)alkoxy or phenyl(lower)alkoxy in which the aromatic portion thereof is substituted with hydroxy, halo, lower alkyl or lower alkoxy, or b. NR"R7 wherein Re is lower alkyl and R7 is lower alkoxy, or c. NR6R7 wherein R8 is hydrogen or lower alkyl and R7 is (C^Jalkyl, lower cycloalkyl, lower alkyl monosubstituted with a lower cycloalkyl; phenyl, phenyl monosubstituted with halo, lower alkyl or lower alkoxy; phenyl(lower)alkyl, phenyl(lower)alkyl in which the aromatic portion thereof is substituted with halo, lower alkyl or lower alkoxy; or (Het)- lower alkyl wherein Het represents a five or six membered heterocyclic radical containing one or two heteroatoms selected from nitrogen, oxygen or sulfur, or d. NR6R7 wherein Re and R7 together with the nitrogen to which they are attached form a pyrrolidino, piperidino, morpholino, thiomorpholino, piperazino or 4-(lower alkyl)piperazino; and Z is hydrogen; COOH; CH2COOH; CH2CH2COOH; CH2OH; 5-lH-tetrazolyl; COOR8 wherein RB is lower alkyl; CONR8R10 wherein R9 and R10 each independently is hydrogen or lower alkyl; or CON(R11)OH wherein R11 is hydrogen or lower alkyl; with the provisos that (1) when X is a (Q.^Jalkanoyl containing one or two carbon atoms (i.e. formyl or acetyl) then R2 is lower alkyl or phenyl lower alkyl, and that (2) when Z is hydrogen then R3 is hydrogen or lower alkyl and R4 is lower alkyl or R3 or R4 together with the carbon atom to which they are attached form a lower cycloalkyl; or a therapeutically acceptable salt thereof.

A preferred group of the peptides of this invention is represented by formula 1 wherein X is (C1.10)alkanoyl; (C^Jalkanoyl monosubstituted with chloro, fluoro, hydroxy or methoxy; benzoyl monosubstituted with phenyl, 2-carboxyphenyl or benzyl; phenyl(C2.10)alkanoyl; phenyl(C2.10)alkanoyl monosubstituted on the aromatic portion thereof with a substitutent selected from halo, hydroxy, lower alkyl, lower alkoxy or phenyl; phenyl(C3.10)alkenoyl; (lower cycloalkyl)carbonyl; (lower cycloalkyl)carbonyl monosubstituted, disubstituted, trisubstituted or tetrasubstituted with methyl; cyclohexylcarbonyl substituted^ position 2 with a phenyl(lower)alkanoy); la,2a,3B,6B-3,6-dimej/iyl-2-(phenylethoxycarbonyl)cyclohexanecarbonyl or 6-meth^=2-(J.-methylethyl)-l,4-dioxoheptyl; R1 is as defined hereinabove; O hydrogen or lower alkyl; R3 and R4 each independently is hydrogen or lower alkyl or R3 and R4 together with the carbon atom to which they are joined form a lower cycloalkyl; R5 is 1-methylethyl, 1,1-dimethylethyl, 1-methylpropyl, 2-methylpropyl, 2,2-dimethylpropyl, 5 cyclopentyl, cyclopentylmethyl, cyclohexyl or cyclohexylmethyl; W1, W2 and W3 are as defined hereinabove; Y is (C1.14)alkoxy, (Cg. 14)alkenyloxy, CH3(0CH2CH2)3-0, lower cycloalkyloxy, lower cyclo-alkylmethoxy, phenyl(lower)alkoxy, NR6R7 wherein R6 is lower alkyl and R7 is lower alkoxy, or NR6R7 wherein R6 is hydrogen or lower 10 alkyl and R7 is (C1.14)alkyl, lower cycloalkyl, lower cycloalkylmethyl, phenyl, phenyl monosubstituted with halo, lower alkyl or lower alkoxy, phenyI(lower)alkyl, phenyl(lower)alkyl monosubstituted with halo, lower alkyl or lower alkoxy, (Het)-lower alkyl wherein Het is a heterocyclic radical selected from 2-pyrrolyl, 2-pyridinyl, 4-pyri-15 dinyl, 2-furyl, 2-isoxazolyl and 2-thiazolyl, or NR6R7 wherein Ra and R7 together with the nitrogen atom to which they are attached form a pyrrolidino, piperidino or morpholino; and Z is as defined hereinabove; with the provisos that (1) when X is a (C,.10)alkanoyl containing one. or two carbon atoms then R2 is methyl, and that (2) 20 when Z is hydrogen then R3 is hydrogen, methyl or ethyl and R4 is methyl or ethyl, or R3 and R4 together with the carbon atom to which they are attached form a lower cycloalkyl; or a therapeutically acceptable salt thereof.

A more preferred group of the peptides is represented by 25 formula 1 wherein X and R5 are as defined in the last instance; R1 is lower alkyl, hydroxy(lower)alkyl, methoxy(lower)alkyl, benzyloxy(iower)nlkyl, lower cycloalkyl, (lower cycloalkyl)methyl, phenyl, phenylmethyl or 2-thienyl; R2 is hydrogen or methyl R4 each independently is hydrogen or lower alkyl or R 30 together with the carbon atom to which they are attachadforn?&$0y lower cycloalkyl; W\ W2 and W3 are oxo, Y is (C^Jal (G,.14)alkenyloxy, CP^OCHaCHgVO, lower cycloalkyloxy, lower cycloalkylmethoxy, phenyl(lower)alkoxy, NR'R7 wherein R6 is lower alkyl and R7 is lower alkoxy, or NR8R7 wherein R6 is hydrogen or lower alkyl and R7 is (C^Jalkyl, lower cycloalkyl, lower cycloalkylmethyl, phenyl, phenyl(lower)alkyl or pyridinyl(lower alkyl), or NR8R7 wherein R8 and R7 together with the nitrogen to which they are attached form a pyrrolidine, piperidino or morpholino; and Z is hydrogen, COOH, CH2COOH, CH2OH, 5-lH-tetrazolyl, CONR'R10 wherein R9 and R10 each independently is hydrogen or lower alkyl, or CON(R11)OH wherein R11 is hydrogen or methyl; or a therapeutically acceptable salt thereof; with the provisos (1) and (2) noted in the preceding paragraph being applicable.

A most preferred group of the peptides is represented by formula 1 wherein X is 2-ethylbutanoyl, 3-methylbutanoyl, 4-methylpentanoyl, octanoyl, 2-hydroxy-3-methylbutanoyl, 2-biphenylylcarbonyl,phenylacetyl,phenylpropionyl,2-(l-methylethyl)-6-phenylhexanoyl, 2-(l-methylethyl)-6-phenyl-3-hexenoyl, cyclopropylcarbonyl, 2,2,3,3-tetramethylcyclopropylcarbonyl, cyclo-hexylcarbonyl, 2-methylcyclohexylcarbonyl, 2,6-dimethyl-cyclohexylcarbonyl,2-(3-phenyl-l-oxopropyl)cyclohexanecarbonylor lcr,2a,3B,6B-3,6-dimethyl-2-(phenylethoxycarbonyl)cyclo-hexylcarbonyl; R1 is lower alkyl, hydroxymethyl, 1-hydroxyethyl, 1-benzyloxyethyl, cyclopentyl, cyclohexyl, cyclohexylmethyl, phenyl phenylmethyl or 2-thienyl; R2 is hydrogen or methyl; R3 and R4 each independently is hydrogen or lower alkyl or R3 and R4 together with the carbon atom to which they are attached form a lower cycloalkyl; R5 is 1-methylpropyl, 2-methylpropyl, 2,2-dimethylpropyl or cyclohexylmethyl, W1, W2 and W3 are oxo, Y is hexyloxy, 1-methylheptyloxy, octyloxy, decyloxy, trans-3-heptenyloxy, cis-3-octenyloxy, CH3(0CH2CH2)3-0, cyclopentyloxy, cyclohexyloxy, 234 373 7 cyclohexylmethoxy, phenylpropoxy, N (Me)OMe, ethylamino, phenyl-amino, phenylethylamino, N-methyl-N-phenylethylamino, 2-pyridinyl-ethyl,N,N-dimethylamino,N,N-diethylamino,N,N-diisopropylamino, N-methyl-N-octylamino, pyrrolidino, piperidino or morpholino; and 5 Z is hydrogen, COOH, CH2COOH, 5-lH-tetrazolyl, CH2OH or CONR®R10 wherein RB and R10 each independently is hydrogen, methyl, ethyl or propyl; with the proviso that when Z is hydrogen then R3 is hydrogen, methyl or ethyl and R4 is methyl or ethyl, or R3 and R4 together with the carbon atom to which they are 10 attached form a lower cycloalkyl; or a therapeutically acceptable salt thereof.

Still another preferred group of the peptides is represented by formula 1 wherein X is 2-ethylbutanoyl, R1 is 1,1-dimethylethyl or l-methylpropyl, R2 is hydrogen, R3, R4, Rs, W1, "W2, W3 and Y 15 are as defined in the last instance, and Z is hydrogen, COOH or CHjjOH; with the proviso that when Z is hydrogen then R3 and R4 each is methyl or ethyl, or R3 and R4 together with the carbon atom to which they are attached form a lower cycloalkyl; or a therapeutically acceptable salt thereof.

According to a further aspect of the present invention there is provided a pharmaceutical or cosmetic composition comprising an anti-herpes virally effective amount of a peptide of formula 1 (as hereinbefore defined), or a therapeutically acceptable salt thereof, together with at least one pharmaceutical^ or veterinarily 25 acceptable carrier or excipient.

According to a further aspect of the present invention there is provided a pharmaceutical or cosmetic composition comprising a peptide of formula 1 (as hereinbefore defined), or a therapeu- 234 373 8 tieally acceptable salt thereof, together with at least one physiologically acceptable carrier suitable for topical application.

According to a further aspect of the present invention there is provided a method of treating, or prophylaxis of, a herpes viral 5 infection in a mammal by administering to the said mammal an anti-herpes virally effective amount of a compound of formula 1 (as hereinbefore defined), of a therapeutically acceptable salt thereof.

According to a further aspect of tne present invention there is provided a method of inhibiting the replication of herpes virus 10 comprising contacting the virus with a herpes viral ribonucleotide reductase inhibiting amount of a compound of formula 1 (as hereinbefore defined), or a therapeutically acceptable salt thereof.

According lc a further aspect of the present invention'there is provider.5 the use of a peptide of formula 1 (as hereinbefore 15 defined), or a therapeutically acceptable salt thereof, for the manufacture of a p<vrmaceutical or cosmetic composition for the treatment of herpes virus infections.

According to a further aspect of the present invention there is provided the use of a peptide of formula 1 (as hereinbefore 20 defined), or a therapeutically acceptable salt thereof, for the manufacture of a composition for inhibiting the replication of herpes virus.

Processes for preparing the peptides of formula 1 are described hereinafter, and comprise a further aspect of the present 25 invention. 234 373 Details of the Invention Alternatively, formula 1 can be illustrated as: .cov The term 'residue' with reference to an amino acid or amino acid derivative means a radical derived from the corresponding a-amino acid by eliminating the hydroxyl of the carboxy group and one hydrogen of the a-amino group.

In genera], the abbreviations used herein for designating the amino acids and the protective groups are based on recommendations of the IUPAC-IUB Commission of Biochemical Nomenclature, see European Journal of Biochemistry 138. 9 (1984). For instance, Gly, Val, Thr, Ala, He, Asp, Ser and Leu represent the residues of glycine, L-valine, L-threonine, L-alanine, L-isoleucine, L-aspartic acid, L-serine and L-leucine, respectively.

The asymmetric carbon atoms residing in the principal linear axis (i.e. the backbone) of the peptides of formula 1, exclusive of the terminal groups, have an § configuration. Asymmetric carbon atoms residing in the side chain of an amino acid or derived amino acid residue, including those in terminal groups, may also have the E configuration. Furthermore, with respect to disubstituted benzoyl, disubstituted phenyl(C1.10)alkanoyl, and disubstituted and trisubstituted cyclohexanecarbonyl, as defined for X of peptides of formula 1, the substituents are selected on the basis that they do not interfere with each others presence. 23 4 3 7 The symbol "Tbg" represents the amino acid residue of 2(S)-amino-3,3-dimethylbutanoic acid. The symbol "Cpg" represents the amino acid residue of (S)-a-aminocyclopentaneacetic acid. The symbol "Phg" represents the amine acid residue of (S)-a-aminophenylacetic acid. Thr(OBzl) for the residue of 03-benzyl-L-threonine.

Other symbols used herein are: Asp(cyBu) for the residue of (S)-a-amino-l-carboxycyclobutaneacetic acid; Asp(cyPn) for the residue of (S)-a-amino-l-carboxycyclopentaneacetic acid; Asp(pyrrolidino) for the residue of the amide 2(S)-amino-4-oxo-4-pyrrolidinobutanoic acid; and Asp(morpholino), Asp(NEt2) and Asp(N-Me-N-octyl) similarly represent the residues of the corresponding amides wherein the pyrrolidino is replaced with morpholino, diethylamino and N-methyl-N-octylamino, respectively. The symbols "Asp(diMe)" represents ihe residue of 2(S)-amino-3,3-dimethylbutanedioic acid, i.e. 3,3-dimethyl-L-aspartic acid. Similarly, Asp(diEt), Asp(Bu) and Asp(Me) represent the residues of 3,3-diethyl-L-aspartic acid, 3-butyl-L-aspartic acid and 3-methyl-L-aspartic acid, respectively.

The term 'halo' as used herein means a halo radical selected from bromo, chloro, fluoro or iodo.

The term "lower alkyl" as used herein, either alone or in combination with a radical, means straight chain alkyl radicals containing one to six carbon atoms and branched chain alkyl radicals containing three to six carbon atoms and includes methyl, ethyl, propyl, butyl, hexyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl and 1,1-dimethylethyl. 23 4 3 11 The term "lower alkenyl" as used herein means straight chain alkenyl radicals containing two to six carbon atoms and branched chain alkenyl radicals containing three to six carbon atoms and includes vinyl, 1-propenyl, 1-methylethenyl, 2-methyl-1-propenyl, 2-5 methyl-2-propenyl and 2-butenyl.

The term 'lower cycloalkyl" as used herein, either alone or in combination with a radical, means saturated cyclic hydrocarbon radicals containing from three to six carbon atoms and includes cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The term "lower alkoxy" as used herein means straight chain alkoxy radicals containing one to four carbon atoms and branched chain alkoxy radicals containing three to four carbon atoms and includes methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy and 1,1-dimethylethoxy. The latter radical is known commonly as tertiary-15 butyloxy.

The term "lower alkanoyl" as used herein means straight chain 1-oxoalkyl radicals containing from one to six carbon atoms and branched chain 1-oxoalkyl radicals containing four to six carbon atoms and includes acetyl, 1-oxopropyl, 2-methyl-l-oxopropyl, 1-20 oxohexyl and the like.

The term "(C^Jalkyl" as used herein means straight and branched chain alkyl radicals containing from one to fourteen carbon atoms, respectively. The terms "(C^Jalkoxy" and "(C1.i4)alkoxy" as used herein, either alone or in combination with 25 a radical, mean straight and branched chain alkoxy radicals con taining from one to ten carbon atoms and one to fourteen carbon atoms, respectively. The term "(C-^Jalkenyloxy" means straight and branched chain alkenyloxy radicals containing from three to 234373 12 fourteen carbon atoms, and in which the double bond may be cis or tmn- and is positioned more than one carbon atom away from the oxygen atom of the radical; for example, 2-propenyloxy, 3-h^ptenyloxy and 3-octenyloxy. The term "(C1.10)alkanoyl" as used S herein means straight or branched chain 1-oxoalkyl radicals containing from one to ten carbon atoms; for example, acetyl, 4-methyl-l-oxopentyl or (4-methylpentanoyl) or 1-oxooctyl (or octanoyl). The term "(C^^Jalkanoyl" as used herein means straight or branched chain 1-oxoalkyl radicals containing from three to ten 10 carbon atoms. The term "pheny^C^alkanoyl" as used herein means phenyl substituted 1-oxoalkyl radicals wherein the 1-oxoalkyl portion thereof is a straight or branched chain 1-oxoalkyl containing from two to ten carbon atoms; for example, l-oxo-3-phenylpropyl and l-oxo-5-methyl-6-phenylhexyl. The term"phenyl(C3.10)alkenoyl" 15 as used herein means phenyl substituted 1-oxoalkenyl radicals wherein the 1-oxoalkenyl portion thereof is a straight or branched chain 1-oxalkenyl containing from three to ten carbon atoms; for example, 2-methyl-l-oxo-3-phenyl-3-pentenyl.

The symbol "»[CSNH]" used between the three letter 20 representations of two amino acid residues means that the normal amide bond between those residues in the peptide, being represented, has been replaced with a thioamide bond.

The term "pharmaceutically acceptable carrier" or "veterinarily acceptable carrier" as use herein means a non-toxic, 25 generally inert vehicle for the active ingredient which does not adversely affect the ingredient.

The term "physiologically acceptable carrier" as used herein means an acceptable cosmetic vehicle of one or more non-toxic 13 excipients which do not react with or reduce the effectiveness of the active ingredient contained therein.

The term "veterinarily acceptable carrier" as used herein means a physiologically acceptable vehicle for administering drug substances to domestic animals comprising one or more non-toxic pnarmaceutically acceptable excipients which do not react with the drug substance or reduce its effectiveness.

The term "effective amount" means a predetermined antiviral amount of the antiviral agent, i.e. an amount of the agent sufficient to be effective against the viral organisms in vivo.

The term "coupling agent" as used herein means an agent capable of effecting the dehydrative coupling of an amino acid or peptide free carboxy group with a free amino group of another amino acid or peptide to form an amide bond between the reactants. Similarly, such agents can effect the coupling of an acid and an alcohol to form corresponding esters. The agents promote or facilitate the dehydrative coupling by activating the carboxy group. Descriptions of such coupling agents and activated groups are included in general text books of peptide chemistry; for instance, E. Schroder and K.L. Lubke, 'The Peptides", Vol. 1, Academic Press, New York, N.Y., 1965, pp 2-128, and K.D. Kopple, "Peptides and Amino acids", W.A. Benjamin, Inc., New York, N.Y., 1966, pp 33-51. Examples of coupling agents are thionyl chloride, diphenylphosphoryl azidr, l,l'-carbonyldiimidazole, dicyclohexylcarbodiimide, N-hydroxysuccinimide, or 1-hydroxy-benzotriazole in the presence of dicyclohexylcarbodiimide. A very practical and useful coupling agent is (benzotriazol-l-yloxy)tris-(dimethylamino)-phosphonium hexafluorophosphate, described by B. Castro et al., Tetrahedron Letters, 1219 (1975), see also D. 234373 14 Hudson, J. Org. Chem., 53, 617 (1988), either by itself or in the presence of 1-hydroxybenzotriazole.

The peptides of formula 1 can be prepared by processes which incorporate therein methods commonly used in peptide syn-5 thesis such as classical solution coupling of amino acid residues and/or peptide fragments, and if desired solid phase techniques. Such methods are described, for example, by E. Schroder and K. Lubke, cited above, in the textbook series, "The Peptides: Analysis, Synthesis, Biology", E. Gross et al., Eds., Academic Press, New 10 York, N.Y., 1979-1987, Volumes 1 to 8, and by J.M. Stewart and J.D. Young in "Solid Phase Peptide Synthesis", 2nd ed., Pierce Chem. Co., Rockford, IL, USA, 1984.

A common feature of the aforementioned processes for the peptides is the protection of the reactive side chain groups of the IS various amino acid residues or derived amino acid residues with suitable protective groups which will prevent a chemical reaction from occurring at that site until the protective group is ultimately removed. Usually also common is the protection of an a-amino group on an amino acid or a fragment while that entity reacts at the 20 carboxy group, followed by the selective removal of the a-amino protective group to allow subsequent reaction to take place at that location. Usually another common feature is the initial protection of the C-terminal carboxyl of the amino acid residue or peptide fragment, if present, which is to become the C-terminal function of 25 the peptide, with a suitable protective group which will prevent a chemical reaction from occurring at that site until the protective group is removed after the desired sequence of the peptide has been assembled. 234373 In genera], therefore, a peptide of formula 1 can be prepared by the stepwise coupling in the order of the sequence of the peptide of the amino acid or derived amino acid residues, or fragments of the peptide, which if required are suitably protected, and eliminating all protecting groups, if present, at the completion of the stepwise coupling to obtain the peptide of formula 1. More specific processes are illustrated in the examples hereinafter.

The peptide of formula 1 of this invention can be obtained in the form of a therapeutically acceptable salt.

In the instance where a particular peptide has a residue which functions as a base, examples of such salts are those with organic acids, e.g. acetic, lactic, succinic, benzoic, salicylic, methane-sulfonic or p-toluenesulfonic acid, as well as polymeric acids such as tannic acid or carboxymethyl cellulose, and also salts with inorganic 15 acids such as hydrohalic acids, e.g. hydrochloric acid, or sulfuric acid, or phorphoric acid. If desired, a particular acid addition salt is converted into another acid addition salt, such as a non-toxic, pharmaceutically acceptable salt, by treatment with the appropriate ion exchange resin in the manner described by R.A. Boissonnas et 20 al., Helv. Chim. Acta, 43, 1849 (1960).

In the instance where a particular peptide has one or more free carboxy groups, examples of such salts are those with the sodium, potassium or calcium cations, or with strong organic bases, for example, triethylamine or N-methylmorpholine.

The antiviral activity of the peptides of formula 1 can be demonstrated by biochemical, microbiological and biological procedures showing the inhibitory effect of the compounds on the 234 373 16 replication of herpes simplex viruses, types 1 and 2 (HSV-1 and HSV-2), and other herpes viruses, for example, varicella zoster virus (VZV), Epstein-Ban- virus (EBV), equine herpes virus (EH V) and cytomegalovirus.

Noteworthy is the fact that all of the aforementioned viruses are dependent on their own ribonucleotide reductase to synthesize deoxyribonucleotides for their replication. Although this fact may not be directly linked with the antiviral activity found for the present peptides, the latter compounds have been shown so far to 10 have antiviral properties against all viruses dependent on ribonucleotide reductase to synthesize DNA for their replication.

In the examples hereinafter, the inhibitory effect on herpes ribonucleotide reductase is noted for exemplary peptides of formula 1. Noteworthy, in the connection with this specific inhibition of 15 herpes ribonucleotide reductase, is the relatively minimal effect or absence of such an effect of the peptides on cellular ribonucleotide reductase activity required for normal cell replication.

A method for demonstrating the inhibitory effect of the peptides of formula 1 on viral replication is the cell culture 20 technique; see, for example, T. Spector et al., Proc. Natl. Acad. Sci.

USA, 82, 4254 (1985).

The therapeutic effect of the peptides can be demonstrated in laboratory animals, for example, by using an assay based on genital herpes infection in Swiss Webster mice, described by E.R. 25 Kern, et al., Antiviral Research, 2, 253 (1983).

When a peptide of this invention, or one of its therapeutically acceptable salts, is employed as an antiviral agent, it is administered topically or systemically to warm-blooded animals, 234 373 17 e.g. humans, pigs or horses, in a vehicle comprising one or more pharmaceutical acceptable carriers, the proportion of which is determined by the solubility and chemical nature of the peptide, chosen route of administration and standard biological practice.

For topical administration, the peptide can be formulated in pharmaceutically accepted vehicles containing 0.1 to 10 percent, preferably 0.5 to 5 percent, of the active agent. Such formulations can be in the form of a solution, cream or lotion.

For systemic administration, the peptide of formula 1 is 10 administered by either intravenous, subcutaneous or intramuscular injection, ir* compositions with pharmaceutically acceptable vehicles or carriers. For administration by injection, it is preferred to use the peptide in solution in a sterile aqueous vehicle which may also contain other solutes such as buffers or preservatives as well as IS sufficient quantities of pharmaceutically acceptable salts or of glucose to make the solution isotonic.

Suitable vehicles or carriers for the above noted formulations are described in standard pharmaceutical texts, e.g. in "Remington's Pharmaceutical Sciences", 16th ed, Mack Publishing Company, 20 Easton, Penn., 1980.

The dosage of the peptide will vary with the form of administration and the particular active agent chosen. Furthermore, it will vary with the particular host under treatment. Generally, treatment is initiated with small increments until the optimum effect 2S under the circumstances is reached. In general, the peptide is most desirably administered at a concentration level that will generally afford antivirally effective results without causing any harmful or deleterious side effects. / 23 4 3 7 3 18 With reference to topical application, the peptide is administered cutaneously in a suitable topical formulation to the infected area of the body e.g. the skin or part of the oral or genital cavity, in an amount sufficient to cover the infected area. The treatment should be repeated, for example, every four to six hours until lesions heal. Healing results usually within 3 to 4 days. No contraindications have been observed.

With reference to systemic administration, the peptide of formula 1 is administered at a dosage of 10 meg to 1000 meg per 10 kilogram of body weight per day, although the aforementioned variations will occur. However, a dosage level that is in the range of from about 50 meg to 500 meg per kilogram of body weight per day is most desirably employed in order to achieve effective results.

Another aspect of this invention comprises a therapeutic or 15 cosmetic composition comprising a herpes viral prophylactic amount of the peptide of formula 1, or a therapeutically acceptable salt thereof, together with a physiologically acceptable cosmetic carrier. Additional components, for example, skin softeners, may be included in the formulation. The cosmetic formulations of this 20 invention are used prophylactically to prevent the outbreak of herpetic lesions of the skin. The formulation can be applied nightly to susceptible areas of the skin. Generally, the cosmetic composition contains less of the peptide than corresponding pharmaceutical compositions for topical application. A preferred range 25 of the amount of the peptide in the cosmetic composition is 0.01 to 02 percent by weight.

Although the formulation disclosed hereinabove are effective and relatively safe medications for treating herpes viral infections, the possible concurrent administration of these formulations with 234 373 19 other antiviral medications or agents to obtain beneficial results is not excluded. Such other antiviral medications or agents include acyclovir and antiviral surface active agents or antiviral interferons such as those disclosed by S.S. Asculai and F. Rapp in U.S. patent 5 4,507,281, March 26, 1985.

The following non-limiting examples illustrate further this invention. Solution percentages or ratios express volume to volume relationship, unless stated otherwise. Abbreviations used in the examples include Ac: acetyl; Boc: t-butyloxycarbonyl; BOP: 10 (benzotriazol-l-yloxy)tris(dimethylamino)-phosphoniumhexafluoro- phosphate; Bzl: benzyl; CH2C12: methylenedichloride; DIPEA: diisopropylethylamine; DCC: N,N-dicyclohexylcarbodiimide; DMF: dimethyl formamide; E^O: diethyl ether; EtOH: ethanol; HOBt: 1-hydroxybenzotriazole; HPLC: high performance liquid chromato-15 graphy; MeOH: methanol; TFA: trifluoroacetic acid; THF: tetra- hydrofuran. Temperatures are given in degrees centrigrade.

Example 1 Preparation of the Intermediate Boc-Aspf 1 (SVmethvlheptvloxv]-OH A solution of Boc-Asp-OBzl (10.2g, 31.6 mmoi) in 20 acetonitrile was added at 0°C to a mixture of N,N'-carbonyIdiimi- dazole (5.6g, 34.7 mmol), DIPEA (8 ml, 46 mmol) and 2(S)-octanol (6 ml, 37.9 mmol) and 4-dimethylaminopyridine (200 mg). The mixture was stirred for 3 h and then concentrated to dryness. The residue was dissolved in EtOAc. The solution was washed with IN 25 aqueous HCI, IN aqueous NaHC03, dried (MgS04) and concentrated. The resultant oil was purified by chromatography (SiOg, eluent: hexane-EtOAc, 7:3) to give Boc-Asp[l(S)-methyl-heptyloxy]-OBzl (92% yield). Hydrogenation of the latter 234 373 compound in the presence of 20% Pd(OH)j/C in ethanol solution afforded a quantitative yield of the title compound as a solid. NMR(200 MHz, CDC13)S 0.9(m,3H), 1.25(m,10H), 1.45(s,9H), 2.8(dd,lH), 3.0(dd,lH), 4.6(m,lH), 4.95 (m,lH) and 5.55(d,lH).

Analogous esters of Boc-Asp-OH were prepared in the same manner.

Example 2 PrepTOipn Qf the Intermediate Boc-Asp(Nptg)-OH BOP (2.20g, 5.0 mmol) was added under N2 to a cooled 10 (0°C) solution of Boc-Asp-OBzl (1.90g, 4.6 mmol) in CH2C12 (50 ml). After 3 min NHEt^HCl (0.55g, 5.0 mmol) and DIPEA (2.4 ml, 1.38 mmol) were added. The resultant solution was stirred at 20-22°C for 18 h. The solution was washed with 10% aqueous citric acid (2 X), 10% aqueous NaHC03 (2 X) and brine (2 X). The 15 organic layer was dried (MgS04) and concentrated to give an oil.

After SiOa chromatography of the oil using hexane-EtOAc (7:3) as the eluent, Boc-Asp^Et^-OBzl (1.55g, 89%) was obtained as an oil. Under a N2 atmosphere, a solution of the latter compound (i.55g, 4.09 mmol) in MeOH (100 ml) was mixed with 5% Pd/C 20 (155 mg). The mixture was shaken on a Parr apparatus under H2 (50 psi) for 90 min. The mixture was filtered through a 45 jum membrane and the filtrate concentrated to give Boc-Asp(NEt2)-OH (1.15g, 98%) as an oil. The structure of the product was confirmed by NMR.

In the same manner, corresponding N-substituted asparagine analogs were obtained by replacing NHEt^HCl with the appropriate amine or amine salt (e.g. pyrrolidine or N,0-dimethylhydro-xylamine hydrochloride). 234 373 21 The intermediates of examples 1 and 2 or their analogs can be incorporated into corresponding peptides of formula 1 according to the procedure of example 6.

Example 3 Preparation of the Intermediate Boc-AspfOBzlU[CSNH]Leu-OBzl A stirred mixture of Boc-Asp(OBzl)Leu-OBzl (2.90g, 5.51 mmol) and Lawesson's reagent (1.12g, 2.7mmol), see "U. Pederson et al., Tetrahedron, 3267 (1982), in toluene (30ml) was heated at reflux for 2h. Column chromatography with Si02(3.5 X 30 cm) 10 and elution with CH2C12 gave the title compound (2.0g), MS: 543(M+H)+, as a yellow oil (major fraction).

Analogous thioamides were prepared in the same manner and incorporated into the appropriate peptides of formula ] according to conventional solution phase peptide synthesis.

Example 4 Preparation of d.l-cis-6-f 3-Phenvl-1 -oxopropvn-3-cvclohexene-1 -carboxvlic acid A Grignard reagent was prepared in THF (40ml) from phenylethyl bromide (2.75 ml, 20 mmol) and Mg turnings (0.51 g, 20 21 mmol) while the mixture was heated at reflux temperature for 1 h. A solution of 3a,4,7,7a-tetrahydro-l,3-isobenzofurandione (3.04g, 20 mmol) in E^O (100 ml) was cooled to -40°C. The solution of the Grignard reagent was added dropwise via a cannula over 15 min to the stirred cooled solution. The reaction mixture 25 was allowed to warm to room temperature over 15 min and then stirred at that temperature for 2 h. The reaction mixture was quenched at 0°C with IN aqueous HC1 and then extracted with 234373 22 EtOAc. The organic phase was extracted with IN aqueous NaOH. The latter aqueous phase was washed with EtOAc, rendered acidic with 6N aqueous HC1 and extracted with EtOAc. The latter extract was dried (MgS04) and concentrated to dryness. The crude 5 product was purified by chromatography (Si02; eluent: hexane- EtOAc, 1:1) to give the title compound (2.99 g, 58%). The NMR of the product was in agreement with the assigned structure.

The title compound was incorporated as a N-terminal residue into the pentultimate protected intermediate of the desired 10 peptide of formula 1 according to the procedure of example 6.

Subsequent hydrogenolysis simultaneously removed any benzyl protecting groups of the pentultimate intermediate and reduced the double bond in the terminal residue to afford the corresponding peptide of formula 1 wherein X is d,l-cis-2-(3-phenyl-l-oxopropyl)-15 cyclohexanecarbonyl. The diastereoisomers can be separated by HPLC.

Example 5 Preparationofla.2a.3B.6B-3.6-Dimethvlcvclohexane-1.2-dicarboxvlic Acid Monophenvlethvl Ester A solution of trans,trans-2,4-hexadiene (1.10 g, 13.4 mmol) and maleic anhydride (1.31g, 13.4 mmol) in benzene (15 ml) was heated at 85°C for 4 h. The mixture was cooled and diluted with EtOAc. The organic phase was washed with HaO, dried (MgS04) and concentrated to give 3aa,4B,7B,7aa-tetrahydro-4,7-dimethyl 1,3-25 isobenzofurandione (2.00 g, 83%) as a white solid. A solution of the latter compound (500 mg) in THF was added to an excess of magnesium phenylethoxide in THF at -20°C. The stirred reaction mixture was allowed to come to room temperature and then stirred 234 373 23 for 18 h at that temperature. Isolation of the product in the manner described in example 4 gave la,2a,3B,6B-3,6-dimethyl-4-cyclohexene-l,2-dicarboxylic acid monophenylethyl ester (310 mg). Subsequent hydrogenation [H2, 20% Pd(OH)2/C,EtOH,3h] yielded 5 the title compound in quantitative yield. The NMR spectrum was in agreement with the assigned structure for the product.

The title compound was incorporated as a N-terminal reside according to the procedure of example 6 to yield desired peptides of formula 1 as a mixture of diastereoisomers which can be 10 separated by HPLC.

Example 6 Preparation of 3-Alkvl- or 3.3-Dialkvl-L-aspartic Acid Intermediates and fSVa-Amino-1 -carboxvcvcloalkvlacetic Acid Intermediates These intermediates, which can be used to prepare peptides of formula 1 in which R3 and R4 are other than hydrogen, can be prepared according to the method of M. Bochenska and J.F. Biernat, Rocz. Chem., 50,1195 (1976); see Chem. Abstr., 86,43990r (1977).

More specifically exemplified, (± )-Boc-Asp(cyPn)(OBzl)-OH was prepared as follows: To a solution of 1-bromocyclopentane-carboxylic acid ethyl ester [17.1g, 77.3 mmol, described by D.N. Harpp et al., J. Org. Chem., 46, 3420 (1975)] and freshly distilled ethyl isocyanoacetate (12.7 g, 122 mmol) in a mixture of 25 dimethylsulfoxide and Et,0 (1:1,120 ml) was added sodium hydride (4.5 g, 60% dispersion in mineral oil, 122 mmol) in small portions over 5 h. The resulting red slurry was stirred at room temperature for 16 h after which time it was treated with a saturated aqueous 234 3 7. 24 solution of ammonium chloride (5 ml). The mixture was diluted with water (500 ml). The resulting mixture was extracted (2X) with ethyl acetate. The ethyl acetate layers were combined and washed with water (2X) and then with brine. Drying tMgS04), filtering and 5 concentration of the extract afforded a dark red oil. This material was flash chromatographed through a 5 x 25 cm column of silica gel [eluent: ethyl acetate-hexane (1:10)]. Concentration of the appropriate fractions provided a-cyano-1 -carboxycyclopentaneacetic acid diethyl ester as a clear colorless viscous liquid (13 g, 66 %).

The latter compound (13 g, 51 mmol) was mixed with 6 N aqueous HC1 (60 ml) at 0°C. After dissolution, the reaction mixture was heated in a oil bath at 120°C for 24h. After this time water was removed from the mixture using a dry ice rotory evaporator. The resulting white solid was dried under high vacuum for 18 h. 15 The dried material was dissolved in a mixture of dioxane (50 ml) and 3N aqueous NaOH (52 ml). A solution of di(tertiarybutyl) dicarbonate (14.6 g, 67 mmol) in dioxane (25 ml) was added to the solution. The mixture was stirred at room temperature for 16 h. Additional 3N aqueous NaOH was added at intervals insuring a pH 20 of about 10. The mixture was diluted with water (500 ml) ^nd extracted (2X) with E^O (200 ml). The aqueous phase was rendered acidic (pH = 3) with solid citric acid and extracted (2X) with ethyl acetate (300 ml). The combined ethyl acetate extracts were washed with water (3x) and brine. Drying, filtering and 25 concentration of the extract afforded Boc-Asp(cyPn)-OH as a white solid (14 g, 96%).

To a solution of the latter compound (7.2 g, 25 mmol) in dry DMF (50 ml) was added KjC03 (7.6 g, 55 mmol) and benzyl bromide (6.6 ml, 55 mmol). The reaction mixture was stirred at 30 room temperature for about 7 h. Thereafter, the reaction mixture 23 4 3 7 J was poured into a mixture of water (500 ml) and ethyl acetate (350 ml). The organic phase was washed with water (2X) and brine. Drying, filtering and concentration of the extract provided a pale yellow viscous liquid. This material was flash chromatographed 5 through a 5 x 20 cm column of silica gel, eluting with hexane-ethyl acetate (12:1). Concentration of the appropriate fractions provided the dibenzyl derivative of Boc-Asp-(cyPn)-OH as a low melting white solid (11 g, 94%). The dibenzyl product was dissolved in THF (100 ml) and an aqueous solution of LiOH (23.5 10 ml, IN) was added. After 4 h, the reaction mixture was poured into water and extracted (3X) with EtzO. The aqueous phase was rendered acidic with 10% aqueous citric acid and extracted (2X) with ethyl acetate. The ethyl acetate layers were combined, dried (MgS04), filtered and concentrated to provide Boc-15 Asp(cyPn)(OBzl)-OH as a clear color less gum (7.3 g, 82%).

Example 7 General Procedure for the Solid Phase Preparation of Peptides of Formula 1 A modified version of the solid phase method of R.B.

Merrifield, J. Am. Chem. Soc., 85. 2149 (1963) was used to prepare the peptides preferably using a BHA-photoresin such as [4-(2-chloropropionyl)phenoxy]acetamidomethyl-copoly(styrene-1% divinyl-benzene) resin, sec D. Bellof and M. Mutter, Chemia, 25 22, 317 (1985). Protection of free carboxy groups and hydroxy gioups was provided by the Bzl protective group. Typically, a Boc-amino acid, representing the C-terminal unit of the desired peptide, e.g. Boc-Leu-OH, was linked to the above noted BHA-photoresin by the potassium fluoride method of K. Horiki et al., Chem. Lett., 30 165 (1978), using 9 molar equivalents of KF and 3.6 molar equivalents of Boc-Leu-OH, for example in DMF at 70° C for 24 234 373 26 hours, to give {4-{2-{Boc-leucine}propionyl}phenoxy}-acetamidomethyl-copoly(styrene-l% divinylbenzene) resin. The dried amino acid-solid support typically showed a leucine content of 0.6 to 0.8 mmol/g for the product, as determined by deprotection 5 of an aliquot, followed by picric acid titration, B.F. Gisin, Anal.

Chim. Acta, 58, 248 (1972). The latter amino acid-solid support was used to build up the required sequence of units (i.e. amino acid residues, derived amino acid residues) of the desired peptide by solid phase methodology. Two molar equivalents (per mole of the 10 amino acid-solid support) of the appropriate amino acid residues were coupled serially to the solid support system using BOP (2 molar equivalents), or BOP (2 molar equivalents)/HOBt (1 molar equivalent), in the presence of N-methylmorpholine (6 molar equivalents) in dry DMF. Completion of coupling was verified by 15 a negative ninhydrin test, E. Kaiser et al., Anal Biochem., 34> 595 (1979). Double coupling was used when necessary.

Cleavage of the protected peptide from the solid support was accomplished by irradiation at 330 nm in EtOH/DMF (1:4) at 0°C under an argon atmosphere for 6 to 18 h. Protective groups (Bzl), 20 if present, were removed from the cleavage product by hydrogenolysis over 5% or 10% Pd/C or 20% Pd(OH)2/C by standard procedures (cf. example 1). Purification of the final product was performed by reversed-phase HPLC to better than 95% homogeneity using 0.06% aqueous TFA/acetonitrile gradients.

More specifically exemplified, the protected peptide, (CH3)2CHCH2CO-Ile-Asp(pyrrolidino)-Asp(OBzl)-Leu-OH was assembled by the preceding procedure on a BHA photoresin using BOP/HOBt as the coupling agent, followed by cleavage of the resulting protected peptide resin by photolysis under argon at -5°C 30 for 6 h. DMF:EtOH(4:l) was used as the photolysis medium. 234 373 27 Deprotection of the cleavage product was effected by hydrogeno-lysis using 5% Pd/C as catalyst. Purification of the product was done by HPLC, the product being dissolved in O.IN aqueous NH4OH solution and the solution adjusted to pH6 with O.IN 5 aqueous AcOH. Whatman Partisil • 100DS-3 C-18 column (2.2 X 50cm2), 10 micron particle size, was used. Elution was done with a gradient of acetonitrile and 0.06% aqueous TFA. Pure fractions (determined by analytical HPLC) were pooled and lyophilized to give (CH3)2CHCH7CO-lle-Asp(pyrrolidino)-Asp-Leu-OH. MS: 612 10 (M + H)+.

The procedure of example 7 was used to prepare the peptides listed in the table of example 8. Commercially available Boc-amino acids were used. Unnatural amino acids were used in their Boc protected form; they were either commercially available, 15 readily prepared from commercially available corresponding amino acids by reaction with di-tertiary-butyl carbonate, or prepared by standard methods.

With reference to the preparation of peptides of formula 1 wherein R2 is lower alkyl or phenyl(lower)alkyl, the required N-20 alkylated Boc amino acids can be prepared by standard N-alkylation of corresponding Boc-amino acids. For example, Boc-N-Me-Asp-(NEt^-OH was obtained by reacting Boc-Asp^E^-OH of example 2 with 2.5 molar equivalents of methy] iodide and 2.1 molar equivalents of potassium hydride in THF at 0°C for 18h to 25 give a mixture of Boc-N-Me-Asp^Et^-OH and its corresponding methyl ester. The mixture was esterified fully (diazomethane) and then saponified (NaOH/H20/dioxane) to yield the desired compound.

Example 8 23 4 3 7 28 Inhibition of Heroes Simplex Virus rHSV. type 1) Ribonucleotide Reductase a) Prepargtign of Ensyme HSV-1 ribonucleotide reductase (partially purified) was obtained from quiescent BHK-21/C13 cells infected with strain F HSV-1 virus at 10 plaque forming units/cell as described by E.A. Cohen et al., J. Gen. Virol., 66, 733 (1985). b) Assay and Results for Exemplified Peptides By following the procedure described by P. Gaudreau et al., J. Biol, Chem., 262. 12413 (1987), the assay results listed in the following table were obtained. The assay result for each peptide is expressed as the concentration of the peptide producing 50% of the maximal inhibition (IC^,) of enzyme activity. The number of units of the enzyme preparation used in each assay was constant, based on the specific activity of the enzyme preparation. The results are relative to the activity obtained in control experiments without peptide and represent the mean of four assays that varied less than 10% with each other.

O ' t < 'j t t i 29 TABLE Peptide (CH3)2CHCH2CO-Ile-Asp-(pyrrolidino)-Asp-Leu-OH 2-Biphenylylcarbonyl-Ile-Asp(morpholino)-Asp-Leu-OH 2-Biphenylylcarbonyl-Ile-Asp(pyrrolidino)-Asp-Leu-OH FAB/MS (M+H)+ 612 724 708 (MM) 2.7 7.2 3.6 [(d,l)-2-(3-Phenyl-l-oxo-propyl)cyclohexanecar-bonyl]-Ile-Asp(NEt2)-Asp-Leu-OH 1 2-Biphenylcarbonyl-Ile-Asp-(pyrrolidino)-Asp-NHCH-[CH2CH(CH3)2]-5-1 H-tetra-zole 2 [(d,l) la,2a,3B,6B-3,6-Dime-thyl-2-(phenylethoxycar-bonyl)cyclohexanecarbonyl]-Ile-Asp(pyrrolidino)-Asp-Leu-OH3 (CH3)2CHCH2CH2CO-Ile-Asp(pyrrolidino)-Asp-Leu-OH (CH3)2CHCH2CH2CO-Ue-Asp(l-methylheptyloxy)-Asp-Leu-OH (C2H5)2CHCO-Tbg-Asp(N Me-N-octyl)-Asp(cyBu)-Leu- OH (C2H5)2CHCO-Tbg-Asp(pyrrolidino)-Asp(diEt)-Leu-OH 772 1.4 and 34 732 814 626 707 4 738 682 13.8 0.5 and 2.3 3.0 >10 0.2 (C2H5)2CHCO-Tbg-Asp(l-methylheptyloxy)-Asp(cyPn)-Leu-OH (C2H8)2CHCO-Thr(OBz)-Asp(pyrrolidino)-Asp-Leu-OH (C2H5)2CHCO-Tbg-Asp(pyrrolidino)-Asp(cyPn)-(L-leucinol) (C2H8)2CHCO-Tbg-Asp(l-methylheptyloxy)-Asp(cyBu )-Leu-OH (C2H5)CHCO-Tbg-Asp(pyrrolidino)-Asp-NH-CH(cyclohexylmethyl)CH2 OH (C2H8)2CHCO-Tbg-Asp(pyrrolidino)-Asp-(L-isoleucinol) (C2Hs)2CHCO-Thr(OMe)-Asp(pyrrolidino)-Asp-NHCH2CH2C(CH3)3 (C2H5)2CHCO-Cpg-Asp(pyrrolidino)-Asp-NHCH2CH2C(CH3)3 (C2H5)sCHCO-Tbg-Asp(pyrrolidino)-Asp-NHCHaCHjCCCHJa (C2H5)2CHCO-Phg-Asp( pyrrolidine )-Asp-NHCHjCHjCCCHgJj (C2H5)2CHCO-NHCH(2-thienylmethyl)CO-Asp(pyrrolidino)-Asp-NHCH2CH2C(CH3)3 (C2H8)8CHCO-Tbg- 234373 31 Asp(pyrrolidino)-Asp(Bu)-Leu-OH (C2H5)2CHCO-Tbg- 6334 1 0 As p(pyrrolidino)-Asp(diMe)-NHCHjCHaCCCHaJa (C2H5)2CHCO-Tbg- 6654 1.9 Asp(pyrrolidino)- Asp*[CSNH]Leu-OH [(S)-(GH3)2CHCH(OH)CO]. 628 2.8 Tbg-Asp(pyrrolidino)-Asp-Leu-OH (C2H5)2CHCO-Tbg- 6764 0.14,9.1 Asp(pyrrolidino)-15 Asp(diMe)-Leu-OH5 [ d , 1 - P h ( C H 2 ) 4 - 744 1.3 CH[CH(CH3)2]CO]-Ile-Asp(pyrrolidino)-Asp-Leu-OH [trans-PhCH2CH2CH=CH- 8746 0.46,0.77 CH[CH(CH3)2]CO]-Ile-Asp-(pynolidino)-Asp-Leu-OH s (2,6-Dimethylcyclohexyl)- 6S84 2.1 c a r b o n y 1 • I I e • 25 Asp(pyrrolidino)-Asp-Leu- OH (2,2,3,3-Tetramethyl- 651 1.4 cyclopropyl)carbonyl-Ile-Asp(pyrrolidino)-Asp-Leu-30 OH 1. By incorporating the product of example 4 into the procedure of example 6, this peptide was obtained as a mixture of diastereoisomers. The diastereoisomers were separated by reversed 23 4 3 7 J 32 phase HPLC using a Waters model 590 programmable solvent delivery module (Millipore Corporation, Milford, MA, USA) and a Whatman Partisil • 10 ODS-3, C-18 column (2.2X50 cm2), 10 micron particle size. Elution was done with a gradient of aceto-nitrile and 0.06% aqueous TFA. The above IC^'s for the diastereoisomers are listed in the order that they are eluted. 234 373 33 2. The tetrazole residue or unit for this peptide was derived from Boc-Leu-NHa in this manner: Boc-Leu-NHa was converted to the corresponding nitrile derivative by treatment with p-toluenesulfonyl chloride in CHaCla in the presence of excess pyridine and a catalytic amount of 4-dimethylaminopyridine (Fieser and Fieser, "Reagents for Organic Synthesis", John Wiley and Sons, Inc., New York, NY, USA, 1967, vol 1, p 1183). The nitrile derivative then was mixed with tributyl tin azide, J.G.A. Luijten et al., Rec. Trav., &1, 202 (1962), to give a tetrazole tin derivative. JO The latter was treated with HC1 gas in EtaO to afford the desired tetrazole residue as a hydrochloride salt (cf. K. Sisido et al., Journal of Organometallic Chemistry, 13, 337 (1971). The tetrazole derivative, or hyd.ochloride salt, was used as such for the coupling with an activated amino acid of the appropriate tripeptide inter-15 mediate, i.e. 2-biphenylylcarbonyl-Ile-Asp(pyrrolidino)-Asp(OBzl)- OH, prepared according to the procedure of example 6. 3. By incorporating the product of example 5 into the procedure of example 6, this peptide was obtained as a mixture of diastereoisomers. The diastereoisomers were separated by HPLC (Waters Delta Prep 3000 •, Millipore Corporation, Milford, MA, USA) by dissolving the mixture of diastereoisomers in 0.6% TFA/HaO and adjusting the pH of the solution to 6.5 using NH«OH and AcOH. Elution was done with a gradient of acetonitrile and 0.06% aqueous TFA. The above ICM's for the diastereoisomers are 25 listed in the order that they are eluted. 4. MS (M+Na)+. 34 . Product obtained as a mixture of diastereoisomers. The isomers were separated by HPLC in the manner described in footnote 1. 6. MS (M + Cs)+ Other examples of peptides of formula 1 are: PhCH2CH2CO-Thr-N-Me-Asp(NEt2)-Asp-Leu-OH [2-(2/-carboxy)biphenylyl]carbonyl-Thr-Asp(NMe8)-Asp-Leu-OH octanoyl-Ue-Asp(octyloxy)-Asp-Leu-NH2 [6-methyl-2-(l-methylethyl)-l,4-dioxoheptyl]-Ue-Asp(pyrrolidino)-Asp-Leu-OH (CH3)2CHCHaCH2CO-NHCH( eye 1 oh exylm ethyl )-CO-Asp(tridecyloxy)-Asp-Leu-OH [3-methyl-2-(l-methylethyl)-l-oxobutyl]-Ue-Asp(pyrrolidino)-Asp-Leu-OH PhCH2CH2CO-Ile-N-Me-Asp(pyrrolidino)-Asp-Leu-OH (CH3)2CHCH2CH2CO-Ile-N-Me-Asp(octyloxy)-Asp-Leu-OH (CH3)2CHCH2CH2CO-Ile-Asp( pyrrolidino)-Asp-NHCH[CH2CH(CH3)2]CH2COOH la, 2B-2-methylcyclohexylcarbonyl-Ile-Asp(pyrrolidino)-Asp-Leu-OH 2-biphenylcarbonyl-Ile-Asp(pyrrolidino)-Aspv»(CSNH]Leu-OH (CH3)aCHCH2CH2CO-Thr-Asp{NEt2)-Asp-NHCH[CH2CH(CH3)2]-CH2CHaCOOH PhCH2CH2CO-Ue-N-Me-Asp(N-Me-N-octyl)-Asp-Leu-OH PhCH2CH2CO-Ile-N-Me-Asp(cis-3-octenyloxy)-Asp-Leu-OH (C2Hs)2CHCO-Tbg-Asp(pyrrolidino)-Asp(Me)-Leu-OH 3<5

Claims (20)

WHAT WE CLAIM IS:-CLA1MS!

1. A peptide of formula 1 X-NH-CHR1-C(W1)-NR2-CH[CH2C(0)-Y1j-C(W2)-NH-CH[CR3(R4)-COOH]-C(W3)-NH-CHR5-Z i wherein X is (C^Jalkanoyl; (C,.10)alkanoyl monosubstituted with halo, hydroxy or lower alkoxy; (C110)alkoxycarbonyl; benzoyl; benzoyl monosubstituted or disubstituted with a substituent selected from halo, hydroxy, lower alkyl, lower alkoxy, phenyl, 2-carboxy-phenyl or benzyl; 2,2-diphenylacetyl; phenyl(C2.10)alkanoyl; phenyl(C2.10)alkanoyl monosubstituted or disubstituted on the aromatic portion thereof with a substituent selected from halo, hydroxy, lower alkyl, lower alkoxy or phenyl; phenyl(C3.,0) alkenoyl; (lower cycloalkyl)carbonyl; (lower cycloalkyl)carbonyl substituted with one to four substituents selected from halo or lower alkyl; cyclohexylcarbonyl substituted at position 2 with lower alkanoyl, phenyl(lower)alkanoyl or phenyl(lower)alkoxycarbonyl; 3,6-dimethyl-2-(phenylethoxycarbonyl)cyclohexylcarbonyl: or a straight or branched chain 1,4-dioxoalkyl containing from five to eleven carbon atoms; R1 is lower alkyl. hydroxy(lower)alkvl, mercapto(lower)alkyl, methoxy(lower)alkyl, methylthio(lower)alkyl, ben/.yloxy(lower)alkyl, henzylthio(lower)alkyl, carboxy(lower)alkyl, low>;r cycloalkyl, (lower cycloalkyl)methyl, phenyl, phenylnieihyl, 2-thienyl or 2-thienylmethyl; R2 is hydrogen, lower alkyl or phenyl(lo\ver)alkyl; R3 and R4 each independently is hydrogen or lower alkyl. or R3 and R4 together with the carbon atom to which they are attadV6tf-f<3rrn R5 is lower alkyl, lower cycloalkyl, or (lower cycloalkyl a lower cycloalkyl; 36 W1, W2, and W3 each independently is oxo or thioxo; Y is a. (C^Jalkoxy, (C^Jalkenyloxy, CH3(0CH2CH2)n-0 wherein n is the integer 1, 2 or 3, lower cycloalkyloxy, lower alkoxy monosubstituted with a lower cycloalkyl, phenoxy, phenoxy monosubstituted with hydroxy, halo, lower alkyl or lower alkoxy, phenyl(lower)alkoxy or phenyl(lower)alkoxy in which the aromatic portion thereof is substituted with hydroxy, halo, lower alkyl or lower alkoxy, or b. NR6R7 wherein R8 is lower alkyl and R7 is lower alkoxy, or c. NR8R7 wherein R6 is hydrogen or lower alkyl and R7 is (C^Jalkyl, lower cycloalkyl, lower alkyl monosubstituted with a lower cycloalkyl; phenyl, phenyl monosubstituted with halo, lower alkyl or lower alkoxy; phenyl(lower)alkyl, phenyl(lower)alkyl in which the aromatic portion thereof is substituted with halo, lower alkyl or lower alkoxy; or (Het)-lower alkyl wherein Het represents a five or six membered heterocyclic radical containing one or two heteroatoms selected from nitrogen, oxygen or sulfur, or d. NRaR7 wherein R6 and R7 together with the nitrogen to which they are attached form a pyrrolidino, piperidino, morpholino, thiomorpholino, piperazino or 4-(lower alkyl)piperazino; and Z is hydrogen; COOH; CH2COOH; CH2CH2COOH; CH2OH; 5-lH-tetrazolyl; COOR8 wherein R8 is lower alkyl; CONR9R10 wherein R9 and R10 each independently is hydror~n or lower alkyl; or CON(R11)OH wherein R11 is hydrogen or lower alkyl; with the provisos that (1) when X is a (C^Jalkanoyl containing one oj/tw^T carbon atoms (i.e. formyl or acetyl) then R2 is lower alkyl or rinenyl lower alkyl, and that (2) when Z is hydrogen then R3 is hydrogen or" 37 lower alkyl and R4 is lower alkyl or R3 or R4 together with the carbon atom to which they are attached form a lower cycloalkyl; or a therapeutically acceptable salt thereof.

2. A peptide of formula 1 as claimed in claim 1 wherein X is (C1.10)alkanoyl; (C^alkanoyl monosubstituted with chloro, fluoro, hydroxy or methoxy; benzoyl monosubstituted with phenyl, 2-carboxyphenylorbenzyl;phenyl(C2.10)alkanoyl;phenyl(C2.10)alkanoyl monosubstituted on the aromatic portion thereof with a substitutent selected from halo, hydroxy, lower alkyl, lower alkoxy or phenyl; phenyl(C3.10)alkenoyl; (lower cycloalkyl)carbonyl; (lower cycloalkyl)carbonyl monosubstituted, disubstituted, trisubstituted or tetrasubstituted with methyl; cyclohexylcarbonyl substituted at position 2 with a phenyl(lower)alkanoyl; la,2a,3B,6B-3,6-dimethyl-2-(phenylethoxycarbonyl)cyclohexanecarbonyl or 6-methyl-2-(l-methylethyl)-l,4-dioxoheptyl; R1 is as defined in claim 1; R2 is hydrogen or lower alkyl; R3 and R4 each independently is hydrogen or lower alkyl or R3 and R4 together with the carbon atom to which they are joined form a lower cycloalkyl; R5 is 1-methylethyl, 1,1-dimethylethyl, 1-methylpropyl, 2-methylpropyl, 2,2-dimethylpropyl, cyclopentyl, cyclopentylmethyl, cyclohexyl or cyclohexylmethyl; W\ W2 and W3 are as defined in claim 1; Y is (C^Jalkoxy, (Gj. 14)alkenyloxy, CH3(0CH2CH2)3-0,lower cycloalkyloxy, lower cyclo-alkylmethoxy, phenyl(lower)alkoxy, NR6R7 wherein R8 is lower alkyl and R7 is lower alkoxy, or NR8R7 wherein R® is hydrogen or lower alkyl and R7 is (C1.14)alkyl, lower cycloalkyl, lower cycloalkylmethyl, phenyl, phenyl monosubstituted with halo, lower alkyl or lower alkoxy, phenyl(lower)alkyl, phenyl(lower)alkyl monosubstituted with halo, lower alkyl or lower alkoxy, (Het)-lower alkyl whereirt Hetis a heterocyclic radical selected from 2-pyrrolyl, 2-pyridin)/l, 4-pyri-dinyl, 2-furyl, 2-isoxazolyl and 2-thiazolyl, or NR6R7 wherein R8 and " 38 R7 together with the nitrogen atom to which they are attached form a pyrrolidino, piperidino or morpholino; and Z is as defined in claim 1; with the provisos that (1) when X is a (C,.10)alkanoyl containing one or two carbon atoms then R2 is methyl, and that (2) when Z is hydrogen then R3 is hydrogen, methyl or ethyl and R4 each is methyl or ethyl, or R3 and R4 together with the carbon atom to which they are attached form a lower cycloalkyl; or a therapeutically acceptable salt thereof.

3. A peptide of formula 1 as claimed in claim 2 wherein X and R5 are as defined in claim 2; R1 is lower alkyl, hydroxy(lower)alkyl, methoxy(low.r)alkyl, benzyloxy(lower)alkyl, lower cycloalkyl, (lower cycloalkyl). nethyl, phenyl, phenylmethyl or 2-thienyl; R2 is hydrogen or methyl; R3 and R4 each independently is hydrogen or lower alkyl or R3 and R4 together with the carbon atom to which they are attached form a lower cycloalkyl; W\ W2 and W3 are oxo; Y is (C,.14)alkoxy, (C3.14)alkenyloxy, CH3(0CH2CH2)3-0, lower cycloalkyloxy, lower cycloalkylmethoxy, phenyl(lower)alkoxy, NR6R7 wherein R6 is lower alkyl and R7 is lower alkoxy, or NR6R7 wherein R6 is hydrogen or lower alkyl and R7 is (C1.14)alkyl, lower cycloalkyl, lower cycloalkylmethyl, phenyl, phenyl(lower)alkyl o» pyridinyl(lower alkyl), or NR6R7 wherein R6 and R7 together with the nitrogen to which they are attached form a pyrrolidino, piperidino or morpholino; and Z is hydrogen, COOH, CH2COOH, CH2OH, 5-lH-tetrazolyl, CONR9R10 wherein R9 and R10 each independently is hydrogen or lower alkyl, or CON(R11)OH wherein R11 is hydrogen or methyl; or a therapeutically acceptable salt thereof.

4. A peptide of formula 1 as claimed in claim 3 wherein X is 2- ethylbutanoyl, 3-methylbutanoyl, 4-methylpentanoyl, octanoyl, f- hydroxy-3-methylbutanoyl, 2-biphenylylcarbonyl, phenylace phenylpropionyl, 2-(l-methylethyl)-6-phenylhexanoyl, 2 methylethyl)-6-phenyl-3-hexenoyl, cyclopropylcarbonyl, 2, 39 tetramethylcyclopropylcarbonyl, cyclohexylcarbonyl, 2-methylcyclohexylcarbonyl, 2,6-dimethylcyclohexylcarbonyl, 2-(3-phenyl-l-oxopropyl)cyclohexanecarbonyl or la,2a,3B,6B-3,6-dimethyl-2-(phenylethoxycarbonyl)cyclohexylcarbonyl; R' is lower alkyl, hydroxymethyl, 1-hydroxyethyl, 1-benzyloxyethyl, cyclopentyl, cyclohexyl, cyclohexylmethyl, phenyl, phenylmethyl or 2-thienyl; R2 is hydrogen or methyl; R3 and R4 each independently is hydrogen or lower alkyl or R3 and R4 together with the carbon atom to which they are attached form a lower cycloalkyl; R5 is 1-methylpropyl, 2-methylpropyl, 2,2-dimethylpropyl or cyclohexylmethyl, W1, W2 and W3 are oxo, Y is hexyloxy, 1-methylheptyloxy, octyloxy, decyloxy, trans-3-heptenyloxy, cis-3-octenyloxy, CH3(0CH2CH2)3-0, cyclopen-tyloxy, cyclohexyloxy, cyclohexy'.methoxy, phenylpropoxy, N(Me)OMe,ethylamino,phenylamino, phenylethylamino,N-methyl-N-phenylethylamino, 2-pyridinylethyl, N,N-dimethylamino, N,N-diethylamino, N,N-diisopropylamino, N-methyl-N-octylamino, pyrrolidino, piperidino or morpholino; and Z is hydrogen, COOH, CH2COOH, 5-lH-tetrazolyl, CH2OH or CONR9R10 wherein RB and R10 each independently is hydrogen, methyl, ethyl or propyl; or a therapeutically acceptable salt thereof.

5. A peptide as claimed in claim 4 wherein X is 2-ethylbutanoyl, R1 is 1,1-dimethylethyl or 1-ethylpropyl, R2 is hydrogen, R3, R4, R5, W1, W2 and W3 are as defined in claim 4 and Z is hydrogen, COOH or CH2OH; or a therapeutically acceptable salt thereof.

6. A peptide of claim 1 selected from: (CH3)2CHCH2CO-IIe-Asp(pyrrolidino)-Asp-Leu-OH 2-Biphenylylcarbonyl-Ile-Asp(morpholino)-Asp-Leu-OH 2-Biphenylylcarbonyl-Ile-Asp(pyrrolidino)-Asp-Leu-OH 40 d,l-2-(3-Phenyl-l-oxopropyl)cyclohexanecarbonyl-Ile-Asp(NEt2)-Asp-Leu-OH 2-Biphenylcarbonyl-IIe-Asp(pyrrolidino)-Asp-NHCH-[CH2CH(CH3)2]-5-lH-tetrazole [ la,2a,3B,6B-3,6-Dimethyl-2-(phenylethoxycarbonyl)-cyclohexanecarbonyl]-Ile-Asp(pyrrolidino)-Asp-Leu-OH (CH3)2CHCH2CH2CO-Ile-Asp(pyrrolidino)-Asp-Leu-OH (CH/^HCH2CH2CO-Ile-Asp(l-methylheptyloxy)Asp-Leu-OH (C2H5)2CHCO-Tbg-Asp(N-Me-N-octyl)-Asp(cyBu)-Leu-OH (C2H5)2CHCO-Tbg-Asp(pyrrolidino)-Asp(diEt)-Leu-OH (C2H5)2CHCO-Tbg-Asp(l-methylheptyloxy)-Asp(cyPn)-Leu-OH (C2H5)2CHCO-Thr(OBz)-Asp(pyrrolidino)-Asp-Leu-OH (C2H5)2CHCO-Tbg-Asp(pyrrolidino)-Asp(cyPn)-(L-leucinol) (C2H5)2CHCO-Tbg-Asp(l-methylheptyloxy)-Asp(cyBu)-Leu-OH (C2H5)CHCO-Tbg-Asp(pyrrolidino)-Asp-NHCH(cyclohexyl-methyl)CH2OH (C2H5)2CHCO-Tbg-Asp(pyrrolidino)-Asp-(L-isoleucinol) (C2H5)2CHCO-Thr(OMe)-Asp(pyrrolidino)-Asp-NHCH2CH2C(CH3)3 (C2H5)2CHCO-Cpg-Asp(pyrrolidino)-Asp-NHCH2CH2C(CH3)3 (C2H5)2CHCO-Tbg-Asp(pyrrolidino)-Asp-NHCH2CH2C(CH)3 (C2H5)2CHCO-Phg-Asp(pyrrolidino)-Asp-NHCH2CH2C(CH3)3 (C2H5)2CHCO-NHCH(2-thienylmethyl)CO-Asp(pyrrolidino)-Asp-NHCH2CH2C(CH3)3 (C2H5)2CHCO-Tbg-Asp(pyrrolidino)-Asp(Bu)-Leu-OH A" (C2H5)2CHCO-Tbg-Asp(pyrrolidino)-Asp(diMe)-NHCH2CH2C(CH3)3 (C2H,)2CHCO-Tbg-Asp(pyrrolidino)-Asp4»[CSNH]Leu-OH 41 [(S)-(CH3)2CHCH(OH)CO]-Tbg-Asp(pyrrolidino)-Asp-Leu-OH (C2H5)2CHCO-Tbg-Asp(pyrrolidino)-Asp(diMe)-Leu-OH [d,l-Ph(CH2)4CH[CH(CH3)2]CO]-De-Asp(pyrrolidino)-Asp-Leu-OH [trans-PhCH2CH2CH=CH-CH[CH(CH3)2]CO]-Ile-Asp(pyrrolidino)-Asp-Leu-OH (2,6-Dimethylcyclohexyl)-carbonyl-Ile-Asp(pyrrolidino)-Asp-Leu-OH or (2,2,3, 3-Tetramethyl-cyclopropyl)carbonyl-Ile-Asp(pyrrolidino)-Asp-Leu-OH. or a therapeutically acceptable salt thereof.

7. A peptide as claimed in claim 1 substantially as herein described and as illustrated with reference to the Examples.

8. A pharmaceutical or cosmetic composition comprising a peptide as defmed in any one of claims 1-7, or a therapeutically acceptable salt thereof, together with at least one pharmaceutically or veterinarily acceptable carrier or excipient.

9. A pharmaceutical or cosmetic composition comprising a peptide as defined in any one of claims 1-7, or a therapeutically acceptable salt thereof, and a physiologically acceptable carrier suitable for topical application.

10. A composition as claimed in claim 8 or claim 9 substantially as herein described.

11. A method of treating, or prophylaxis of, a herpes viral infection in a non-human mammal comprising administering to said mammal an effective amount of a peptide as defined in any one of claims 1-7, or a therapeutically acceptable salt thereof. 42

12. A method of claim 11 wherein the herpes viral infection is a herpes simplex viral infection.

13. An invitro method of inhibiting the replication of herpes virus comprising contacting the virus with a herpes viral ribonucleotide reductase inhibiting amount of peptide as defined in any one of claims 1-7, or a therapeutically acceptable salt thereof.

14. A method of inhibiting the replication of herpes virus in a non-human mammal comprising contacting the virus with a herpes viral ribonucleotide reductase inhibiting amount of peptide as defined in any 10 one of claims 1-7, or a therapeutically acceptable salt thereof.

15. A method as claimed in any one of claims 11-14 substantially as herein described.

16. Use of a peptide as defined in any one of claims 1-7, or a therapeutically acceptable salt thereof, for the manufacture of a IS pharmaceutical or cosmetic composition for the treatment of herpes virus infections.

17. Use of a peptide as defined in any one of claims 1-7, or a therapeutically acceptable salt thereof, for the manufacture of a composition for use in inhibiting the replication of herpes virus. 20

18. Use of a peptide as claimed in claim 16 or claim 17 substantially as herein described.

19. A process for preparing a peptide as claimed in any one of claims 1-7, or a therapeutically acceptable salt thereof, comprising: a) stepwise coupling, in the order of the sequence of the peptide, of 2S the amino acid or derived amino acid residues, or fragments of the peptide, in which i) reactive side chain groups of the residue or fragments are protected with suitable protective groups to prevent chemical reactions from occurring at that site until the protective group is ultimately removed after the completion of the stepwise coupling; ii) an a-amino group of a coupling reactant is protected by an a-amino protective group while the free carboxy group of that reactant couples with the free a-amino group of the second reactant; the a-amino protective group being one which can be selectively removed to allow the subsequent coupling step to take place at that a-amino group; and iii) the C-terminal carboxyl of the amino acid residue of the amino acid residue or peptide fragment, which is to become the C-terminal function of the protected peptide, if present, is protected with a suitable protective group which will prevent chemical reaction occurring at that site until after the desired amino acid sequence for the peptide has been assembled; and b) at the completion of the coupling, eliminating any protecting groups and, if required, effecting standard transformations to obtain the peptide of claim 1; and if desired, converting the peptide into a therapeutically acceptable salt.

20. A process as claimed in claim 19 substantially as herein described and as illustrated with reference to the Examples. BIQ-MEGA/BOEHRINGER 1NGELHEIM RESEARCH INC end of claims