NZ275691A

NZ275691A - Di and tripeptides and compositions thereof which inhibit farnesyl transferase

Info

Publication number: NZ275691A
Application number: NZ275691A
Authority: NZ
Inventors: Gary Louis Bolton; Mark Wallace Creswell; John Cooke Hodges; Michael William Wilson
Original assignee: Warner Lambert Co
Priority date: 1993-11-05
Filing date: 1994-10-12
Publication date: 1998-03-25
Also published as: CA2170766A1; JP3597863B2; HU9601193D0; HUT75308A; FI961819A0; AU7976094A; JPH09504547A; EP0730605A1; NO961814D0; KR100362338B1; NO961814L; FI961819A; WO1995012612A1; AU681454B2

Description

New Zealand Paient Spedficaiion for Paient Number £75691 New Zealand No. 275691 International No.

TO BE ENTERED AFTER ACCEPTANCE AMD PUBLICATION Priority dates: 05.11.1993;13.09.1994; Complete Specification Filed: 12.10.1994 Classification^) C07K5/06,078,08,097; A61K38/05.06 Publication date: 25 March 1998 Journal No.: 1426 Title of Invention: Substituted di- and tripeptide inhibitors of protein:farnesyl transferase Name, address and nationality of applicant(s) as in international application form: WARNER-LAMBERT COMPANY, a US company of 201 Tabor Road, Morris Plains, New Jersey 07950, United States of America NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION AVO 95/12612 2756^1 * SUBSTITUTED DI- AND TRIPEPTIDE INHIBITORS OF PROTEIN:FARHESYL TRANSFERASE FIELD OF THE INVENTION The present invention pertains to a number of compounds which can be used in the medicinal field to treat, prophylactically or otherwise, uncontrolled or 10 abnormal proliferation of human tissues. More specifically, the present invention pertains to a number of compounds which act to inhibit the farnesyl transferase enzyme that has been determined to activate ras proteins which in turn activate cellular division 15 and are implicated in cancer and restenosis.

BACKGROUND OF THE INVENTION Ras protein (or p2l) has been examined extensively because mutant forms are found in 20% of most types of human cancer and greater than 50% of colon and pancreatic carcinomas (Gibbs J.B., Cell. 65:1 (1991), Cartwright T., et al., Chimica Qggi, 1£:26 (1992)). 25 These mutant ras proteins are deficient in the capability for feedback regulation that is present in native ras and this deficiency is associated with their oncogenic action since the ability to stimulate normal cell division can not be controlled by the normal 30 endogenous regulatory cofactors. The recent discovery that the transforming activity of mutant ras is critically dependent on post-translational modifications (Gibbs J., et al., Microbiol. Rev.. 5J.:l7l (1S89M has unveiled an important aspect of ras 35 function and identified novel prospects for cancer therapy.

In addition to cancer, there are other conditions of uncontrolled cellular proliferation that may be related to excessive expression and/or function of native ras proteins. Post surgical vascular restenosis 5 is such a condition. The use of various surgical revascularization techniques such as saphenous vein bypass grafting, endarterectomy and transluminal coronary angioplasty is often accompanied by complications due to uncontrolled growth of neointimal 10 tissue, known as restenosis. The biochemical causes of restenosis are poorly understood and numerous growth factors and protooncogenes have been implicated (Naftilan A.J., et al., Hypertension. 13:706 (1989) and J. Clin. Invest.. 83.: 1419; Gibbons G.H. , et al., 15 Hypertension. 14:358 (1989); Satoh T., et al., Mollec.

Cell. Biol.. 1^:3706 (1993)). The fact that ras proteins are known to be involved in cell division processes makes them a candidate for intervention in many situations where cells are dividing uncontrol-20 lably. In direct analogy to the inhibition of mutant ras related cancer, blockade of ras dependant processes has the potential to reduce or eliminate the inappropriate tissue proliferation associated with restenosis, particularly in those instances where 25 normal ras expression and/or function is exaggerated by growth stimulatory factors.

Ras functioning is dependent upon the modification of the proteins in order to associate with the inner face of plasma membranes. Unlike other membrane-30 associated proteins, ras proteins lack conventional transmembrane or hydrophobic sequences and are initially synthesized in a cytosol soluble form. Ras protein membrane association is triggered by a series of posttranslational processing steps that are signaled 35 by a carboxyl terminal amino acid consensus sequence that is recognized by protein:faraesyl transferase.

This consensus sequence consists of a cysteine residue located four amino acids from the carboxyl terminus, followed by two lipophilic amino acids and the C-terminal residue. The sulfhydryl group of the 5 cysteine residue is alkylated by farnesyl pyrophosphate in a reaction that is catalyzed by protein:farnesyl transferase. Following prenylation, the C-terminal three amino acids are cleaved by an endoprotease and the newly exposed alpha-carboxyl group of the 10 prenylated cysteine is methylated by a methyl transferase. The enzymatic processing of ras proteins that begins with farnesylation enables the protein to associate with the cell membrane. Mutational analysis of oncogenic ras proteins indicate that these 15 posttranslational modifications are essential for transforming activity. Replacement of the consensus sequence cysteine residue with other amino acids gives a ras protein that is no longer farnesylated, fails to migrate to the cell membrane and lacks the ability to 20 stimulate cell proliferation (Hancock J.F., et al., Cell. 57:: 1617 (1989); Schafer W.R., et al., Science. 245:379 (1989); Casey P.J., Proc. Natl. Acad. Sci. USA. 86:8323 (1989)).

Recently, protein:farnesyl transferases (PFTs, 25 also referred to as farnesyl:protein transferases) have been identified and a specific PFT from rat brain was purified to homogeneity (Reiss Y., et al., Bioch. Soc. Trans.. 2£:487-88 (1992)). The enzyme was characterized as a heterodimer composed of one alpha-subunit 30 (49 kDa) and one beta-subvm.it (46 kDa) , both of which are required for catalytic activity. High level expression of mammalian PFT in a baculovirus system and purification of the recombinant enzyme in active form has also been accomplished (Chen W.-J., et al., 35 J. Biol. Chem.. 268:9675 (1993)).

In light of the foregoing, the discovery that the function of oncogenic ras proteins is critically dependent on their posttranslational processing provides a means of cancer chemotherapy through 5 inhibition of the processing enzymes. The identification and isolation of a protein:farnesyl transferase that catalyzes the addition of a farnesyl group to ras proteins provides a promising target for such intervention. Recently it has been determined 10 that prototypical inhibitors of PFT can inhibit ras processing and reverse cancerous morphology in tumor cell models (Kohl N.E., et al., Science. 260:1934 (1993); James G.L., et al., Science. 260:1937 (1993); Garcia A.M., et al., J. Biol. Chem.. 268:18415 (1993)). 15 Thus, it is possible to prevent or delay the onset of cellular proliferation in cancers that exhibit mutant ras proteins by blocking PFT. By analogous logic, inhibition of PFT would provide a potential means for controlling cellular proliferation associated with 20 restenosis, especially in those cases wherein the expression and/or function of native ras is overs timiilated.

PCT Application W091/16340 discloses cysteine containing tetrapeptide inhibitors of PFT of the 25 formula CAAX.

European Patent Application 0461869 discloses cysteine containing tetrapeptide inhibitors of PFT of the formula Cys-Aaa1-Aaa2-Xaa.

European Patent Application 0520823 discloses 30 cysteine containing tetrapeptide inhibitors of PFT of the formula Cys-Xaa1-dXaa2-Xaa3.

European Patent Application 0523873 discloses cysteine containing tetrapeptide inhibitors of PFT of the formula Cys-Xaa1-Xaa2-Xaa3.

European Patent Application 0528486 discloses cysteine containing tetrapeptide amides inhibitors of PFT of the formula Cys-Xaa1-Xaa2-Xaa3-NRR1.

European Patent Application 0535730 discloses pseudotetrapeptide inhibitors of PFT of the following two formulas: x r2 0 h'nrv^n'VsV^N'^Y .zh oh hs' y r x r 0 (), ^Y^rVY hs' y r European Patent Application 0535731 (US 5,238,922) discloses esters of pseudotetrapeptide inhibitors of 20 PFT of the formula: h1™V^n'\'SV1S''IY0R! ;h hs y r" European Patent Application 0482539 discloses tachykinin antagonists of the formula: r"" r3 y X 0 r' wo 95/12612 YHT& 9 European Patent Application 0457195 discloses endothelin antagonists of the formula: R-A R" X CO-N i? R X CO—N I R X. 8*. f 1998 bf N.w island US 4,022,759 discloses tripeptide antagonists of 10 luteinizing hormone releasing factor of the formula A-R-^-Tyr (benzyl) -Ser (benzyl) -R2, wherein one of the definitions of R2 is His (benzyl) .

Compounds disclosed in the above references do not disclose or suggest a novel combination of structural 15 variations found in the present invention described hereinafter. All cited references are hereby incorporated by reference.

SUMMARY OF THE INVENTION Accordingly, the present invention is a substituted di- or tripeptide compound of Formula I: wo 95/12612 275"f91 wherein: n = 1 or 2; A . COR3, C02R3, CONHR3, CSR3, C(S)OR3, C(S)NHR3, CF3S02, aryl-S02, or alkyl-S02, wherein R3 is 5 alkyl, (CH2)m-cycloalkyl, (CH2)m-aryl, (CH2)m-heteroaryl, or (CH2)m0-alkyl, and m « 0, 1, 2, or 3; R = independently H or Me; S 6j § E I V E P Y = independently H or Me *Pop9rty 0Wcfe Z = independently H or Me; 1 2 FEB 1998 R1 = H, CO-aryl,. <CH2)m-aryl, 0(CH2)m-cycloalky^NewZofl|and 0(CH2)m-aryl, or O (CK2)m-heteroaryl, wherein m is as defined above and R1 is located at either the meta or para position; X = one to four substituents, including H, alkyl, CF3, F, CI, Br, I, HO, MeO, N02, NH2, N(Me)2, 0P03H2, or CH2P03H2; R2 = NR(CH2)nC02R3, NR(CH2)nC0NHR3, NR(CH2)nR3, NR (CH2) n+10R4, NR(CH2)n+1SR4, NRCH(COR5) (CH2)n-heteroaryl, NRCH(C0R5) (CH2)nOR3, NRCH (COR5) (CH2)nSR3, or n N—R .3 wherein R, R3, and n are as defined above, R4 « H 25 or R3, and R5 «= OH, NH2, OR3, or NHR3; an optical isomer, diastereomer, or a pharmaceutically acceptable salt thereof; wherein the following compounds are excluded: Z-His-Phe-Gly-OMe, Z-His-Phe-Trp-OEt, and Z-His-Phe(N02)-Gly-OMe.

The present invention is also directed to the use of a compound of Formula I, or a pharmaceutically acceptable salt therefrom, to inhibit the activity of a protein:farnesyl 35 transferase enzyme, in the manufacture of a medicament for treating tissue proliferative diseases.

A further embodiment of the present invention is the use of a compound of Formula I in the manufacture of a medicament for the treatment of cancer.

PCT/U S94/11553 27 569 1 A still further embodiment of the present invention is the use of a compound of Formula I in the manufacture of a medicament for the treatment oof restenosis. fnt ft % 6 BI V A still further embodiment of the present *l§" Ut invention is a pharmaceutical composition for ' 2 jggy administering an effective amount of a compound of ofjvow Formula I in unit dosage for.n in the treatment methods mentioned above.

A final embodiment of the present invention pertains to methods for the preparation of compounds of Formula I by solid phase synthesis, solution phase synthesis, and simultaneous multiple syntheses using a multiple simultaneous synthesis apparatus.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In the compounds of Formula I, the term "alkyl" 20 means a straight or branched hydrocarbon radical having from 1 to 6 carbon atoms and includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, and the like.

The term "cycloalkyl" means a saturated 25 hydrocarbon ring which contains from 3 to 10 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl, and the like, unsubstitilted or substituted by an alkyl or aryl group.

The term "aryl" means an aromatic ring which is a 30 phenyl, 5-fluorenyl, l-naphthyl or 2-naphthyl group, unsubstituted or substituted by l to 3 substituents selected from alkyl, 0-alkyl and S-alkyl, 0-aryl, OH, SH, F, CI, Br, I, CF3, N02, NH2, NHCH3, N(CH3)2> NHCO-alkyl, (CH2)mC02H, (CH2) BC02-alkyl, (CH2)mS03H, 35 (CH2)mP03H2, (CH2)mP03(alkyl)2, (CH2)mS02NH2, and WO 95/12612 PCT/US94/11553 (CH2)inS02NH-alkyl wherein alkyl is defined as above and m = 0, 1, 2, or 3.

The term "heteroaryl" means a heteroaromatic ring which is a 2- or 3-thienyl, 2- or 3-furanyl, 2- or 5 3-pyrrolyl, 2-, 3- or 4-pyridyl, 2-, 3-, 4-, 5-, 6-, or 7-indolyl group, unsubstituted or with l or 2 substituents from the group of substituents described above for aryl.

The following table provides a list of 10 abbreviations and definitions thereof used in the present invention.

TABLE OF ABBREVIATIONS Abbreviation* Amino Acid Ala Alanine Arg Arginine Asn Asparagine Asp Aspartic acid Cys Cysteine Glu Glutamic acid Gin Glutamine Gly Glycine His Histidine lie Isoleucine Leu Leucine Lys Lysine Met Methionine Phe Phenylalanine Pro Proline Ser Serine Thr Threonine Trp Tryptophan If the optical activity of the amino acid is other than L(S), the amino acid or abbreviation is preceded by the appropriate configuration D(R) or DL(RS).

FCT/US94/11553 Abbreviation* Tyr Val Amino Acid (continued) Tyrosine Valine Abbreviation* Aaa-C02R Aaa-CONHR 3 Hyp 4 Hyp Hey Nva Nle Orn Bal Abu Ahe Acp Aoc Apn Bpa Chx Cit Modified and Unusual Amino Acid An amino acid ester, for examples: Gly-C02Bn is Glycine, benzyl ester; Ser(OBn)-C02Me is 0-Benzyl-serine, methyl ester.

An amino acid amide, for examples: Gly-CONHBn is Glycine, N-benzyl amide; Ser(OBn)-CONHEt is O-Benzyl-serine, N-ethyl amide; Tyr (OBn)-CONHCH2CH2OBn is O-Benzyl-tyrosine, N- (2-(phenylmethoxy)ethyl) amide. 3-Hydroxyproline 4-Hydroxyproline Homo cysteine Norvaline Norleucine Ornithine Beta-alanine (or 3-aminopropionic acid) 4-Aminobutyric acid 7-Aminoheptanoic acid 6-Aminocaproic acid 8-Aminooctanoic acid -Aminopentanoic acid (4-Benzoylphenyl)alanine 3-Cyclohexylalanine (or Hexahydrophenylalanine) Citrulline If the optical activity of the amino acid is other than L(S) , the amino acid or abbreviation is preceded by the appropriate configuration D(R) or DL(RS). ■11- Abbreviation* His(1-Me) His(Tr) homoPhe homoTyr homoTyr(OBn) 1-Nal 2-Nal Pen Phe(3-OBn) Phe(4-Ph) Pgl Pyr Ser(OBn) Thr(OBn) Tic Tyr(OMe) Tyr(OEt) Tyr(OBn) (a-Me)Tyr(OBn) (N-Me)Tyr(OBn) Trp(For) Modified and Unusual Amino Acid (continued.) 1-Methyl-histidine (or N(r)-Methyl-histidine) 1-Triphenylmethyl-histidine (or N(r)-Trityl-histidine) 2-Amino-4-phenylbutanoic acid (or Homophenylalanine) 2-Amino-4-(4-hydroxyphenyl)butanoic acid (or Homotyrosine) 2-Amino-4-[4-(phenylmethoxy)phenyl]-butanoic acid (or O-Benzyl-homotyrosiue) 3-(1'-Naphthyl)alanine 3-(2'-Naphthyl)alanine Penicillamine (3 -Benzyloxyphenyl)alanine 3-(1,1'Biphen-4-yl)alanine (or 4-Phenyl-phenylalanine) Phenylglycine 2-Amino-3-(3-pyridyl)-propanoic acid (or 3-Pyridylalanine) 0-Benzyl-serine 0-Benzyl-threonine 1,2,3,4-Tetrahydro-3-isoquinoline-carboxylic acid O- Methyl-tyros ine 0-Ethyl- tyrosine O-Benzyl-tyros ine 2-Amino-3- (4-benzyloxyphenyl)-2-methyl-propionic acid (or a-Met'nyl-0-benzyl- tyros ine) N-Methyl-0-benzyl- tyrosine Nin-Formyltryptophan If the optical activity of the the amino acid or abbreviation configuration D(R) or DL(RS). amino acid is other than L(S), is preceded by the appropriate Abbreviation Maa Mba Mpa Mercapto Acids Mercaptoacetic acid 4-Mercaptobutyric acid 3-Mercaptopropionic acid Abbreviation Ac Ada Adoc 10 Bn MeBn Cbz 2-Br-Cbz 2-CI-Cbz 15 Bom Boc Dnp For Fmoc 20 N02 TMS Tos Tr Protecting Group Acetyl l-Adamantyl acetic acid Adamantyloxycarbonyl Benzyl 4-Methylbenzyl Benzyloxycarbonyl or tho-Bromobenzvloxycarbonyl ortho-Chlorobenzyloxycarbonyl Benzy1oxymethy1 tertiary Butyloxycarbonyl 2,4-Dinitrophenyl Formyl 9 -Fluorenylmethy1oxycarbonyl Nitro Trimethylsilyl 4-Toluenesulfonyl (tosyl) Triphenylmethyl (trityl) Abbreviation HOAc CF3S02H DCM DCC DIC DIEA DMAP DMF EDAC EtOAc Solvents and Reagents Acetic acid Trifluoromethanesulfonic acid Dichloromethane N,N'-Dicyclohexylcarbodiimide N,N' -Diisopropylcarbodiimide N, N-Diisopropylethylamine 4 -Dimethylaminopyridine N,N' -Dimethylformamide N-Ethyl-N' -Dimethylaininopropyl- carbodiimide Ethyl acetate Abbreviation Et20 HC1 HP HOBT KOH MeCN MeOH NHOS NMP iPrOH TBAF TFA Solvents and Reagents Diethyl ether Hydrochloric acid Hydrofluoric acid l-Hydroxybenzotriazole Potassium hydroxide Acetonitrile Methanol N-Hydroxysuccinimide N- Me thylpyrrolidone iso-Propanol Tetra n-Butylammonium Fluoride Trifluoroacetic acid Abbreviation HMP Resin MBHA Resin PAM Resin 2-Cl-Tr Resin NH2-Rink Resin Solid Phase Peptide Synthesis Resins 4-(Hydroxymethyl)-phenoxymethyl-polystyrene resin Methylbenzhydrylamine resin 4-(Hydroxymethyl)-phenylacetamidomethyl-polystyrene resin 2-Chlorotrityl-polystyrene resin 4-(amino-(2',4'-dimethoxy-phenyl)methyl)-phenoxymethyl-polystyrene resin Abbreviation FPP PFT DTT BSA Biological Reagents Farnesyl pyrophosphate Protein:farnesyl transferase Dithiothreitol Bovine serum albumin Abbreviation COR3 CONHR3 CSR3 C (S) OR3 C (S) NHR3 Miscellaneoug 0 CR3 0 1 3 CNHR CR" COR- CNHR CH (COR5) (CH2)n0R" CH (COR5) (CH2) n-heteroaryl CH (COR5) (CH2)n0R: (CH2)nCNHR R5 o=c I C- (CH2)n-heteroaryl H R5 0=C I C-(CH2)n0R I H 40 CH (COR5) (CH2) nOR" 0=C I C- (CH2)_SRJ I H 45 CO(4-Bn-piperazin-l-yl) 0 / \ C—N N—CH2Ph Preferred compounds of Formula I consist of compounds of Formula II below: II wherein: A' R Y Z R1 = C02R3, CONHR3, C(S)NHR3, or aryl-S02, wherein R3 is alkyl, (CH2)m-cycloalkyl, (CH2)m-aryl, (C^Jjn-heteroaryl, and m = 0, 1, 2, or 3; = independently H or Me; = independently H or Me; = independently H or Me; = (CH2)m-aryl, 0(CH2)m-aryl, 0P03H2, or CH2P03H2> wherein m is as defined above; = NR(CH2)2OR4, NR(CH2)2SR4, NRCH (COR5 )CH20R3, NRCH (COR5) CH2SR3, or , N—CH-Ph wherein R, R3, and n are ?.s defined above, R4 = H or R3, and R5 = OH, NH2, OR3, or NHR3; an optical isomer, diastereomer, or a pharmaceutica'iiy acceptable salt thereof.

Other preferred compounds of the present invention are those of Formula I as defined above wherein A is C02R3 or CONHR3; or as defined above in Formula I wherein at least one of Y and Z is Me; or as defined above in Formula I wherein R2 is (CH2)20R4 or 35 CH (COR5) CH2OR3; or as defined above in Formula I O 95/12612 wherein A is CONHR3, R2 is (CH2)2OR4, and at least one of Y and Z is Me.

The most preferred compounds of Formula I include the following: 5 Cbz-His-Tyr(OBn)-Ser(OBn)-C02Me; Cbz-His-Tyr(OBn)-Ser(OBn)-CONH2; Cbz-His-Tyr(OBn)-Ser(OBn)-CONHEt; Cbz-His-Tyr(OBn)-Ser(OBn); Cbz-His-Tyr(OBn)-D-Ser(OBn)-C02Me; 10 Cbz-D-His-Tyr(OBn)-Ser(Cta)-CONH2; Cbz-D-His-Tyr(OEn)-Ser(OBn)-CONHEt; Cbz-D-His-Tyr(OBn)-Ser(OBn)-C02Me; Cbz-D-His-Tyr(OBn)-Ser(OBn); Cbz-His(1-Me)-Tyr(OBn)-Ser(OBn)-C02Me; 15 Cbz-His(1-Me)-Tyr(OBn)-Ser(OBn)-CONH2; Cbz-His(l-Me)-Tyr(OBn)-Ser(OBn)-CONHEt; Cbz-His(1-Me)-Tyr(OBn)-Ser(OBn); Cbz-D-His(l-Me)-Tyr(OBn)-Ser(OBn)-C02Me; Cbz-D-His(l-Me)-Tyr(OBn)-Ser(OBn)-CONH2; 20 Cbz-D-His(l-Me)-Tyr(OBn)-Ser(OBn)-CONHEt; Cbz-D-His(l-Me)-Tyr(OBn)-Ser(OBn); Cbz-His-(a-Me)Tyr(OBn)-Ser(OBn)-C02Me; Cbz-His-(a-Me)Tyr(OBn)-Ser(OBn)-CONH2; Cbz-His- (a-Me) Tyr (OBn) -Ser (OBn) -CONHEt; 25 Cbz-His-(a-Me)Tyr(OBn)-Ser(OBn); Cbz-His-D-(a-Me)Tyr(OBn)-Ser(OBn)-C02Me? Cbz-His-D-(a-Me)Tyr(OBn)-Ser(OBn)-CONH2; Cbz-His-D-(a-Me)Tyr(OBn)-Ser(OBn)-CONHEt; Cbz-His-D-(a-Me)Tyr(OBn)-Ser(OBn); 30 Cbz-D-His-homoTyr(OBn)-Ser(OBn)-C02Me; Cbz-His-Phe(4-Ph)-Ser(OBn)-C02Me; Cbz-D-His-Phe(4-Ph)-Ser(OBn)-C02Me; Cbz-His -Tyr(OBn)-Pyr-C02Me ; Cbz-D-His-Tyr(OBn)-Pyr-C02Me; 35 Cbz-His-Tyr (OBn) -CONHCH2CH2OBn; Cbz-D-His - Tyr (OBn) - CONHCH2CH2OBn; \VO 95/12612 Cbz-His- (N-Me)Tyr (OBn) -CONHCH2CH2OBn; Cbz-D-His-(N-Me)Tyr(OBn)-CONHCH2CH2OBn; Cbz-His-Tyr(OBn)-CONH (CH2) 2Ph; Cbz-D-His-Tyr(OBn)-CONH (CH2) 2Ph; 5 .Cbz-His-Tyr (OBn)-Gly-C02Bn; Cbz-D-His-Tyr(OBn)-Gly-C02Bn; Cbz-His-Tyr(OBn)-Gly-CONHBn; Cbz-D-His-Tyr(OBn)-Gly-CONHBn; BnNHCO-His-Tyr(OBn)-Ser(OBn)-C02Me; 10 BnNHCO-His-Tyr(OBn)-Ser(OBn)-CONH2; BnNHCO-His-Tyr(OBn)-Ser(OBn)-CONHEt; BnNHCO-His-Tyr(OBn)-Ser(OBn); BnNHCO-His-Tyr (OBn) -C0NHCH2CH20Bn; BnNHCO -His- Tyr (OBn) - CONHCH2 CH2 CH2 OPh ; 15 BnNHCO-D-His-Tyr(OBn)-Ser(OBn)-C02Me; BnNHCO-D-His-Tyr(OBn)-Ser(OBn)-CONH2; BnNHCO-D-His-Tyr(OBn)-Ser(OBn)-CONHEt; BnNHCO-D-His-Tyr(OBn)-Ser(OBn); BnNHCO-D-His-Tyr (OBn) -CONHCH2CH2OBn; 2 0 BnNHCO - D - Hi s - Ty r (OBn) - CONHCH2 CH2 CH2OPh ; Cbz-His-Tyr(OBn)-CON(Me)CH2CH20Bn; (4 -EtOPh)NHCO-D-His-Tyr(OBn)-CONH(CH2) 3OPh ; PhCH2CO-D-His-Tyr (OBn) -CONH(CH2) 3- (2-MeOPh) ; (4-PhOPh)NHCO-D-His-Tyr (OBn) -COHN(CH2) 2Ph; and 25 (4-MePh)S02-D-His-Tyr(OBn)-CO (4-Bn- piperazin-l-yl) .

GENERAL METHODS FOR THE PREPARATION, EVALUATION 30 AND USE OF COMPOUNDS OF FORMULA I The compounds of Formula I may be prepared by solid phase peptide synthesis on a peptide synthesizer, for example, an Applied Biosystems 431A peptide 35 synthesizer using activated esters or anhydrides of Boc or Fmcc protected amino acids, acid chlorides, -ir- isocyanates, isothiocyanates, etc., on PAM, MBHA, or NH2-Rink resins with solution phase modifications to the carboxyl terminus as appropriate. Methodology for the solid phase synthesis of peptides is widely known 5 to those skilled in the art thereof (see, for example: J.M. Stewart and J.D. Young in Solid Phase Peptide Synthesis; Pierce Chemical Co.; Rockford, IL (1984); Fields G.B. and Noble R.L., Int. J. Peptide Protein Res.. 1^:161-214 (1990)).

Additionally, the compounds of Formula I may also be prepared by conventional solution peptide synthesis, substituting amines, acid chlorides, isocyanates, etc, for amino acid derivatives where appropriate. Methods for solution phase synthesis of peptides are widely 15 known to those skilled in the art (see, for example, M. Bodanszky, Principles of Peptide Synthesis. Springer-Verlag (1984)).

Finally, the compounds of Formula I may be prepared by simultaneous multiple solid phase syntheses 20 using an apparatus described by S. H. DeWitt, et al., Proc. Natl. Acad. Sci. USA. ££:6909 (1993), and referred to by the trademark, Diversomer™, both trademark and apparatus being owned in whole by the Warner-Lambert Company. The multiple solid phase 25 synthesis apparatus is currently the subject of now abandoned US Serial 07/958,383 filed October 8, 1992 and pending continuation-in-part US Serial 08/012,557 filed February 2, 1993.

For example (Scheme I below), Fmoc-D-His-Tyr(OBn)-30 C02-CH2CH2Si (CH3) 3 is linked to 2-Cl-Tr resin using a sterically hindered amine such as DIEA as an HC1 scavenger, the Fmoc protecting group is removed with piperidine, the resulting free amino terminus is acylated with a series of isocyanates, isothiocyanates, 35 activated esters, acid chlorides and the like, the TMS-ethyl ester is cleaved with TBAF, the resulting free carboxy terminus is activated with a carbodiimide reagent such as EDAC, DCC, or DIC, the activated carboxyl group is reacted with alcohols such as HOBT, NHOS, or pentachlorophenol to give an activated ester, 5 the activated ester is reacted with a series of amines and the resulting array of compounds of Formula I is cleaved from the resin by with hot HOAc or by treatment with TFA at room temperature.

For all three synthetic methods described above 10 appropriate consideration is given to protection and deprotection of reactive functional groups and to the sequence of synthetic steps. Knowledge of the use of common protecting groups and strategy for the assembly of complex organic molecules are within the usual realm 15 of expertise of a practitioner of the art of organic chemistry (see, for example: T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Chemistry. John Wiley and Sons (1991); E.J. Corey and X.-M. Cheng, The Logic of Chemical Synthesis. John Wiley and Sons 20 (1989)).

The homogeneity and composition of the resulting compounds is verified by reverse phase-high pressure liquid chromatography (RP-HPLC), capillary electrophoresis, thin layer chromatography (TLC), 25 proton nuclear magnetic resonance spectrometry (NMR), amino acid analysis, fast atom bombardment mass spectrometry (FAB-MS) and electrospray mass spectrometry (ES-MS).

SCHEME I: Multiple Simultaneous Synthesis Method Resin Resin 1. Piperidine 2. Eight acylating agents FmocNH ^r »>AC BnO H ,N„ R = 0 N ^ OCH,CHjTMS BnO 1. TBAF 2. EDAC, HOBT 3. Five amines Resin "■"G-'SrV^ BnO 4 0 Compounds of Formula I BnO WO 95/.2612 PCTrtJS94/11553 The compounds of Formula I are capable of further forming both pharmaceutically acceptable acid addition and/or base salts. All of these forms are within the scope of the present invention.

Pharmaceutically acceptable acid addition salts of the compounds of Formula I include salts derived from nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, hydrofluoric, phosphorous, and the like, as well as the 10 salts derived from nontoxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Such salts thus include 15 sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, trifluoroacetate, propionate, caprylate, isobutyrate, oxalate, malonate, 20 succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, and the like. 25 Also contemplated are salts of amino acids such as arginate and the like and gluconate, galacturonate, n-meth.yl glucamine (see, for example, Berge S.M., et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science. 6£:1-19 (1977)). 30 The acid addition salts of said basic compounds are prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt in the conventional manner. Preferably a compound of Formula I can be converted to an acidic salt by 35 treating with an aqueous solution of the desired acid, such that the resulting pH is less than 4. The 95/12612 solution can be passed through a C18 cartridge to absorb the compound, washed with copious amounts of water, the compound eluted with a polar organic solvent such as, for example, methanol, acetonitrile, and the 5 like, and isolated by concentrating under reduced pressure followed by lyophilization. The free base form may be regenerated by contacting the salt form with a base and isolating the free base in the conventional manner or as above. The free base forms 10 differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free base for purposes of the present invention.

Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable 20 amines.are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge S.M., et al., "Pharmaceutical Salts", Journal of Pharmaceutical 25 Science. ££:1"19 (1977)).

The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. Preferably, a 30 compound of Formula I can be converted to a base salt by treating with an aqueous solution of the desired base, such that the resulting pH is greater than 9. The solution can be passed through a C18 cartridge to absorb the compound, washed with copious amounts of 35 water, the compound eluted with a polar organic solvent such as, for example, methanol, acetonitrile and the like, and isolated by concentrating under reduced pressure followed by lyophilization. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the 5 conventional manner or as above. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present 10 invention.

Certain of the compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms, including hydrated forms, are 15 equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain of the compounds of the present invention possess one or more chiral centers and each center may exist in the R(D) or S(L) configuration. The present 20 invention includes all enantiomeric and epimeric forms as well as the appropriate mixtures thereof.

The PFT inhibitory activity of compounds of Formula I was assayed in 30 mM potassium phosphate buffer, pH 7.4, containing 7 mM DTT, l.'2 mM MgCl2, 25 0.1 mM leupeptin, 0.1 mM pepstatin and 0.2 mM phenylmethylsulfonyl fluoride. Assays were performed in 96 well plates (Wallec) and employed solutions composed of varying concentrations of a compound of Formula I in 100% DMSO. Upon addition of both 30 substrates, radiolabeled farnesyl pyrophosphate ([1-3H], specific activity 15-30 Ci/mmol, final concentration 0.12 jtM) and (biotinyl)-Ahe-Tyr-Lys-Cys-Val-Ile-Met peptide (final concentration 0.1 fiM) , the enzyme reaction was started by addition of 40-fold 35 purified rat brain farnesyl protein transferase. After incubation at 37°C for 30 minutes, the reaction was terminated by diluting the reaction 2.5-fold with a stop buffer containing 1.5 M magnesium acetate, 0.2 M H3P04, 0.5% BSA, and strepavidin beads (Amersham) at a concentration of 1.3 mg/mL. After allowing the plate 5 to settle for 30 minutes at room temperature, radioactivity was quantitated on a microBeta counter (model 1450, Wallec). Compounds of Formula I show IC50 values of 0.5 nM to 80 /iM (see data table) in this assay and are thus valuable inhibitors of protein: 10 farnesyl transferase enzyme which may be used in the medical treatment of tissue proliferative diseases, including cancer and restenosis.

IC50 Values for Selected Compounds of 15 Formula I Against PFT Example Number IC50 (/iM) l 4.4 4 1.0 (3) 2 .1 (4) 7.3 (23) 0.64 (27) (28) 0.73 (30) 73 (31) 0.76 (35) 66 (36) 1.9 (46) 1.0 (49) 2.9 (40) 0.75 (52) 1.6 (56) 1.1 (59) (60) 1.4 IC50 Values for Selected Compounds of Formula I Against PFT Example Number IC50 (fiM) (61) 7.2 (62) 1.5 (63) 1.0 (64) 1.7 (69) 0.48 (79) 3.0 (80) 1.6 6 0.42 7 0.26 8 0.074 9 0.27 o • H O 11 0.17 12 0.028 13 0.083 16 0.60 17 0.039 18 0.82 19 0.31 21 0.31 22 0.37 23 1.9 IC50 Values for Selected Formula I Against PFT Compounds of (cont'd) Example Number IC50 (/iM) 24 1.0 3.7 28 11 29 3.0 The compounds of the present invention can be prepared and administered in a wide variety of oral, rectal and parenteral dosage forms. Thus, the compounds of the present invention can be administered 15 by injection, that is, intravenously, intramuscularly, intracutaneously, subcutaneously, intraduodenally, or intraperitoneally. Also, the compounds of the present invention can be administered by inhalation, for example, intranasally. Additionally, the compounds of 20 the present invention can be administered transdermally. It will be obvious to those skilled in the art that the following dosage forms may comprise as the active component, either a compound of Formula I or a corresponding pharmaceutically acceptable salt of a 25 compound of Formula I.

For preparing pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers can be either solid or liquid.

Solid form preparations include powders, tablets, 30 pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.

PCT/U S94/11553 In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component.

In tablets, the active component is mixed with the 5 carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

The powders and tablets preferably contain from 5 or 10 to about 70 percent of the active compound. 10 Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and th® like. The term "preparation" is 15 intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, 20 cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa 25 butter, it first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogenous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water propylene glycol solutions. For parenteral injection liquid preparations can be formulated in solution in aqueous polyethylene glycol solution. 35 Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorant9, flavors, stabilizing and thickening agents as desired.

Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component 5 in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.

Also included are solid form preparations which 0 are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, 5 stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is 0 subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or !5 ampoules. Also, the unit dosage form can be a capsules, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

The quantity of active component in a unit dose 10 preparation may be varied or adjusted from 0.1 mg to 100 mg preferably 0.5 mg to 100 mg according to the particular application and the potency of the active component. The composition can, if desired, also contain other compatible therapeutic agents. !5 In therapeutic use as inhibitors of PFT, the compounds utilized in the pharmaceutical methods of this invention are administered at the initial dosage of about 0.01 mg/kg to about 20 mg/kg daily. A daily dose range of about 0.01 mg/kg to about 10 mg/kg is preferred. The dosages, however, may be varied 5 depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with 10 smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in 15 portions during the day, if desired.

The following nonlimiting examples illustrate the inventors' preferred methods for preparing the compounds of the invention. For added clarity, complex chemical name? describing compounds of Formula I are 20 followed by structural abbrev;ations, which are shown in braces, where?.: the structural elements are as defined in the T .le of Abbreviations above.

EXAMPLE 1 Nq.- TN- TN- f (Phenvlmethoxv) carbonvll -L-histidvll - 0-(phenvlmethvl)-L-tvrosvll-0-(phenylmethvl)-L-serinamide (Cbz-Hjg-Tvr (OBn)-Ser(OBn)-CONH=) Using an ABI model 431A solid phase peptide synthesizer, Fmoc-NH2-Rink resin (0.25 mmol scale) was 30 treated with 20% piperidine in NMP to afford NH2-Rink resin. Sequential coupling of Fmoc-protected Ser(OBn) and Tyr (OBn) (DCC and HOBT in NMP) and Fmoc deprotection (20% piperidine in NMP) reactions were run using a 4-fold excess of reagents in the coupling steps 35 and traditional resin washing cycles to afford Tyr(OBn)-Ser(OBn)-CONR2-Rink resin. This dipeptide resin was transferred to an uninstruxnented reaction vessel and treated with a 4-fold excess of Cbz-His, DCC, and HOBT in DMF, shaking overnight at room temperature. After removal of excess reagents, the 5 resulting substituted tripeptide was cleaved from the resin by treatment with 50% TFA in DCM at room temperature for 2.5 hours. Evaporation of solvents and purification by reversed phase chromatography (C18-column, eluted with a 20-70% gradient of MeCN in 10 water (both solvents acidified with 0.1% TFA) afforded Cbz-His-Tyr (OBn)-Ser (OBn)-C0NH2 as its TFA salt upon lyophilization. ES-MS: 719 (m+1).

Using analogous methods the following most preferred compounds of Formula I with carboxamides at 15 the C-terminus may be prepared: Cbz-D-His-Tyr(OBn)-Ser(OBn)-CONH2; Cbz-His (l-Me) -Tyr (OBn) -Ser (OBn) -CONH2; Cbz-D-His(l-Me)-Tyr (OBn)-Ser(OBn)-CONH2; Cbz-His-DL-(a-Me) Tyr(OBn)-Ser(OBn)-CONH2; 20 BnNHCO-His-Tyr (OBn)-Ser (OBn)-CONH2; and BnNHCO-D-His-Tyr (OBn) -Ser (OBn) -CONH2.

EXAMPLE 2 N- FN- fN- T (Phenvlmethoxv) carbonyll -L-histidvll -25 0- (phenvlmethvl) -L-tyrosvll -O- (phenvlmethyl) -L-serine ICbz-His-Tvr(OBn)-Ser(OBn)) Beginning with PAM resin or HMP resin, Fmoc-Ser(OBn), Fmoc-Tyr(OBn) , and Cbz-His are sequentially coupled using the deprotection and 30 coupling conditions described in Example 1. Cleavage from the resin is accomplished by treatment with CF3S02H for the PAM supported tripeptide or with 50% TFA in DCM for the HMP supported tripeptide. Chromatography as in Example 1 provides 35 Cbz-His-Tyr (OBn)-Ser (OBn) as its TFA salt upon lyophilization. See also Example 7 for a solution phase method.

Using analogous methods the following most preferred compounds of Formula I with a free carboxyl 5 terminus may be prepared: Cbz-D-His-Tyr(OBn)-Ser(OBn); Cbz-His(l-Me)-Tyr(OBn)-Ser(OBn); Cbz-D-His(l-Me)-Tyr(OBn)-Ser(OBn); Cbz-His-DL- (a-Me) Tyr (OBn)-Ser (OBn) ; 0 BnNHCO-His-Tyr(OBn)-Ser(OBn); and BnNHCO-D-His-Tyr(OBn)-Ser(OBn).

EXAMPLE 3 Solid phase supported N- TN-T(9H-Fluoren-9-5 ylmethoxy)carbonyll -L-histidvll -0-(phenylmethyl)-L-tyrosine. 2-trimethylsilylethyl ester fFmoc-His (2-Cl-Tr Resin) -Tvr (OBn) -CO^CH^CH^TMS) Step 1: Boc-Tvr(OBn) -COoCH^CH^TMS 2-Trimethylsilyl ethanol (2.6 g, 22.6 mmol) was 0 added to a premixed solution of EDAC (4.3 g, 22.6 mmol), DMAP (0.5 g), and Boc-Tyr(OBn)-OH (7.0 g, 18.8 mmol) in dry THF (25 mL) . .The resulting mixture was stirred for 18 hours at room temperature. The solution was diluted with 1:1 Et0Ac:Et20 (40 mL), 15 washed with saturated aqueous NaHC03 (2 x 10 mL) and with saturated aqueous NaCl (2 x 10 mL), dried (MgS04) , filtered and concentrated in vacuo to provide an oil which was further purified by flash chromatography (Si02, EtOAc:hexane eluent) to give the pure TMS-ethyl 10 ester as an oil; XH NMR (HCDC13): 5 0.04 (s, 9H) , 1.43 (s, 9H), 3.03 (m, 2H), 4.22 (m, 2H), 4.51 (m; 1H), 4.95 (m, 1H), 5.05 (s, 2H), 6.85-7.48 (m, 9H). step 2: Tvr(OBn)-CQ2CH,CH?TMS Eighty percent TFA in CH2C12 (20 mL, v/v) was added to an ice-cooled solution of Boc-Tyr(OBn)-C02CH2CH2TMS (14.8 g, 31.4 mmol) in CH2C12 (40 mL) . The resulting mixture was stirred for 1.0 minute before concentrating in vacuo. The procedure was repeated once more, and the resulting residue was diluted with CH2C12 and saturated aqueous NaHC03. The resulting mixture was filtered through celite. The organic layer was then separated, washed with saturated aqueous NaCl, and dried (MgS04) . Filtration and concentration in vacuo provided an oil which was further purified by flash chromatography (Si02, CHCl3:MeOH eluent) to give the desired product; NMR (HCDC13): b 0.06 (s, 9H), 1.68 (br s, 2H), 2.85-3.03 (m, 2H) , 3.67 (m, 1H), 4.22 (m, 2H), 5.05 (s, 2H), 6.91-7.45 (m, 9H).

Step 3: Fmoc-His (Tr) -Tyr (OBn) -CO.CH^CH.TMS To a solution of HOBT (2.6 g, 19.3 mmol) in DMF (10 mL) was added Fmoc-His(Tr) ( 10.0 g, 16.1 mmol) followed by EDAC (3.7 g, 19.3 mmol). The mixture was stirred at room temperature for 20 minutes before adding a solution of Tyr (OBn) -C02CH2CH2TMS (from Step 2 above, 5.8 g, 16.1 mmol) in DMF (10 mL) . The mixture was stirred overnight at room temperature before partitioning between a mixture of water and 1:1 Et20:EtOAc (50 mL) . The layers were separated, and the organic phase was washed with saturated aqueous NaCl (4 x 20 mL) and dried (MgS04). Filtration and concentration in vacuo provided an oil which was further purified by flash chromatography (Si02, CHCl3:MeOH eluent) to give the protected His-Tyr dipeptide; FAB-MS 974 (m+1).

Step 4: Fmoc-Hig-Tvr(OBn)-Q^CH^CH^TMS Fmoc-His(Tr)-Tyr(OBn)-C02CH2CH2TMS (from Step 3 above, 5.0 g, 5.1 mmol) was treated with pyridineoHCl (1.0 g) in MeOH (20 mL). The mixture was allowed to 5 stir 8 hours at 65'C. The solution was concentrated in vacuo, and the residue was dissolved in CH2C12, washed with H20 (lx), saturated aqueous NaHC03 (2x), and dried (MgS04). Filtration and concentration in vacuo provided an oil which was further purified by 10 flash chromatography (Si02, CHCl3:MeOH eluent) to give Fmoc-His-Tyr (OBn)-C02CH2CH2STMS as a white solid; FAB-MS 72.1 (m+1) .

Step 5: Fmoc-His (2-Cl-Tr Resin) -Tyr(OBn) -CO.-.CH^CH^TMS 15 To a suspension of Fmoc-His-Tyr (OBn) - C02CH2CH2TMS (from Step 4 above, 5.3 g, 7.3 mmol) in CHC13 (20 mL) was added 2-chloroltrityl chloride resin (Novabiochem) (7.1 g) followed by DIEA (0.96 g, 7.4 mmol). The resulting mixture was subjected to brief sonication to 20 disperse the resin and then agitated on a shaker for 2.0 hours. The modified resin was collected by filtration, washed with DMF (2x), MeOH (2x), CHC12 (2x), and dried in vacuo for 18 hours to yield 10.5 g (loading corresponds to approximately 1 mmol/g resin).

EXAMPLE 4 N-r3-Phenoxvpropy11-0-(phenvlmethvl)-Na- TN-T T(phenylmethvl)amino!carbonyll-L-histidyll-L-tvrosinamide (BnNHCO-His-Tvr (OBn) -CONH (CH,.) 3OPh) 30 Fmoc-His (2-CI-Tr-Resir.) -Tyr (OBn) -C02CH2CH2TMS (from Example 3 above, 2.0 g) was suspended in 20% piperidine in DMF. The resulting suspension was subjected to sonication for 10 minutes and then agitated by shaking for 30 minutes. The resin was 35 filtered and washed with DMF (3x). The resin was again subjected to the same reaction conditions for an additional 20 minutes. The resin was filtered and washed with DMF (4x) and CHC13 (3x) to provide His (2-Cl-Tr-Resin)-Tyr (OBn)-C02CH2CH2TMS which was suspended in DCM (10 mL), agitated by shaking for 5 30 minutes, treated with benzyl isocyanate (1.1 g, 8.0 mmol), and agitated for an additional 30 minutes. The resin was filtered, washed with DCM (3x), resuspended in DCM, and the benzyl isocyanate treatment was repeated. The resin was filtered and washed with 10 DMF (2x) and CHC13 (5x) to give BnNHCO-His (2-Cl-Tr- Resin)-Tyr (OBn)-C02CH2CH2TMS which was ne^t suspended in a mixture of 4:3 dioxane:MeOH (14 mL) and treated with 1.0 M TBAF in THF (2.0 mL, 2.0 mmol) . The suspension was agitated by shaking for 18 hours, 15 filtered, washed sequentially with a 2:1 mixture of dioxane and 10% citric acid (3x 10 mL) , dioxane:MeOH (3x 10 mL), dioxane (3x 10 mL), and CHC13 (3x 10 mL) to provide BnNHCO-His(2-Cl-Tr-Resin)-Tyr(OBn). The BnNHCO-His (2-CI-Tr-Resin)-Tyr (OBn) was suspended in DMF 20 (10 mL) and treated with a carbodiimide coupling reagent such as DIC (0.2 g, 1.6 mmol) and HOBT (0.22 g, 1.6 mmol). The resulting mixture was stirred 30 minutes and 3-phenoxypropylamine (0.24 g, 1.6 mmol) was added. The resulting mixture was shaken 18 hours 25 before filtering the resin and washing with DMF (3x) and CHC13 (3x) * The resin was suspended in DMF (10 mL) and the carbodiimide/H0BT/3-phenoxypropylamine coupling reaction was repeated. After 18 hours, the resin was filtered and washed with 10 mL each of MeOH (2x) , DCM 30 (3x) , DMF (2x) , MeOH (2x), and CHC13 (2x) to give BnNHCO-His (2-Cl-Tr-Resin) -Tyr (OBn) -CONH (CH2) 3OPh. The highly substiuted dipeptide was cleaved from the resin by treatment with 40% TFA in DCM, shaking for 1 hour at room temperature. The supernate, containing the free 35 dipeptide, was filtered away from the resin and the resin was washed with DCM (6x) . The combined supernate and washings were concentrated in vacuo to provide BnNHCO-His-Tyr (OBn)-CONH (CH2)3OPh-TFA. The product was partitioned between water and DCM, and both layers were treated dropwise with saturated aqueous NaHC03 until the aqueous layer remained basic. The layers were separated, and the organic phase was washed with saturated aqueous NaCl and dried (MgS04). Filtration and concentration yielded BnNHCO-His-Tyr(OBn)-CONH(CH2)3OPh; ES-MS 675 (m+1).

EXAMPLE 5 Multiple. Simultaneous Solid Phase Synthesis The method described in Example 4 may be employed in simultaneous multiple syntheses using the Diversomer 15 apparatus described by S.H. DeWitt, et al., Proc. Natl.

Acad. Sci. USA. <L&:6909 (1993). Fmoc-D-His(2-C1-Tr Resin)-Tyr(OBn)-C02iPr, prepared according to from Example 3 by substituting Fmoc-D-His(Tr) for Fmoc-His (Tr) in Step 3, (100-200 mg) is placed in each of 20 40 gas dispersion tubes, and the tubes are placed in the Diversomer apparatus. The sequential deprotection and coupling reactions described in Example 4 are followed, employing the following acylating agents and amines in various combinations: Acylating agents Amines 1) benzyl isocyanate 1) 3-phenoxypropylamine 2) p-toluenesulfonyl chloride 2) 2 -(phenylmethoxy) ethyl amine 3) cyclohexyl isocyanate 3) 2-[(phenylmethyl)-30 thiol -ethylamine 4) phenyl isocyanate 4) 4 -phenylbutylainine ) i-propyl isocyanate 5) 3-(2-methoxyphenyl)- propylamine 6) n-butyl isocyanate 6) 1-benzyl piperazine 35 7) 4 -chlorophenyl isocyanate 7) o-benzyl-hydroxylamine 8) l-napthyl isocyanate 8) methionine methyl eBter 9) 3-methoxypropyl isocyanate 9) benzylamine ) 4-ethoxyphenyl isocyanate 10) 2-phenylethylamine 11) 2-phenethyl isocyante 12} 3-phenylpropionyl chloride 14) pi. enyl acetyl chloride ) 4-phenoxyphenyl isocyanate 16) benzyl chloroformate 17) (trans)-2-phenylcyclopropyl isocyanate 18) 1-adamantyl chloroformate 2. 3. 4. . 7. 8. 9. . 11. 12. 13. 14. . 16. 17. 18. 19. . 21. 22. 23. 24. . 26. 27.

ES-MS 677 (m+1) BS-MS 661 (m+1) BS-MS 639 (m+1) BS-MS 665 (m) BS-MS 666 (m+1) ES-MS 712 (m+1) Array 1. Following cleavage from the resin and work-up as described in Example 4, the following substituted 10 dipeptides (1-40) of Formula I are prepared: 1. PhNHCO-D-His-Tyr (OBn) -CONHCH2CH2OBn PhNHCO-D-His-Tyr (OBn) -CONHCH2CH2SBn PhNHCO-D-His-Tyr (OBn) -C0NHCH2CH2CH20Ph BuNHCO-D-His-Tyr (OBn) -CONH (CH2)4Ph 15 5. BuNHCO-D-His-Tyr (OBn) -CO(4-Bn-piperazin- l-yl) (4-MePh) SOj-D-His-Tyr (OBn) -C0NHCH2CH2Ph (4-MePh)S02-D-His-Tyr(0Bn) -C0NHCH2CH2SBn CF3S02-D-His-Tyr (OBn) -CONH-Met-COjMe 20 9. CF3S02-D-His-Tyr(0Bn)-CONHCH2CH2CH2-(2-MeO-Ph) CF3S02-D-His-Tyr (OBn) -CO (4 -Bn-piperazin-1 -yl) BnNHCO - D - Hi s - Tyr (OBn) C0NHCH2CH2Ph MeO (CH2) 3NHCO-D-His-Tyr (OBn) CONHOBn MeO (CH2) 3NHCO-D-His -X/r (OBn) CONHCH2CH2CH2OPh MeO (CH2)3NHCO-D-His-Tyr (OBn) CONH (CH2)3-(2-MeO-Ph) BnNHCO-D-His-Tyr(OBn)CONHCH2Ph (4 -CIPh) NHCO-D-His-Tyr (OBn) CONHCH2CH2OBn 1 - Napthyl - NHCO -D - His - Tyr (OBn) CONHOBn 1 - Nap thy 1 - NHCO - D - H i s - Tyr (OBn) CONH - Met - CO^Me (4-ClPh) NHCO-D-His-Tyr (OBn) CONH (CH2) 4Ph (4-CIPh)NHCO-D-His-Tyr(OBn)CONHCH2Ph BnOCO-D-His-Tyr (OBn) CONHCH2CH2OBn 1 - adamantyl - OCO - D - Hi s - Tyr (OBn) CONHCH2CH2SBn 35 23. BnOCO - D - Hi s - Tyr (OBn) CONHCH2CH2CH2OPh 1-adamantyl-OCO-D-His-Tyr (OBn) CONHCH2CH2CH2CH2Ph BnOCO-D-His-Tyr (OBn) CO (4 -Bn-piperazin-1-yl) 40 26. PhCH2CO-D-His-Tyr(OBn)CONHCH2CH2Ph PhCH2CH2NHCO - D - Hi s - Tyr (OBn) CONHCH2CH2SBn ES-MS 631 (m+1) ES-MS 695 (m+1) ES-MS 683 (m+1) BS-MS 723 (m+1) BS-MS 693 (m+1) ES-MS 651 (m+1) ES-MS 676 (m+1) ES-MS 676 (m+1) BS-MS 700 (m) BS-MS 630 (m+1) ES-MS 70S (m+1) 28.

PhCH2CH2NHCO-D-His-Tyr (OBn) CONH-Met- C02Me ES' •MS 701 (nw-1) 29.

PhCH2CH2NHCO-D-His-Tyr (OBn) CONH (C<::)3- (2-MeO-Ph) • ES' •MS 703 (m+1) .

PhCH2CO-D-His-Tyr(OBn)CO(4-Bn-piperazin- 1-yl) ES ■MS 684 (m) 31. (t-2-Ph-c-propyl)-NHCO-D-His- Tyr (OBn) CONHCH2CH2Ph ES -MS 671 (m+l) 32. (t-2-Ph-c-propyl)-NHCO-D-His- Tyr (OBn)CONHOBn ES -MS 673 (m+1) 33. c-hexyl-NHCO-D-His-Tyr(OBn) CONH (CH2) 3OPh ES -MS 666 (m+1) 34 . c - hexy 1 - NHCO - D - Hi s - Tyr (OBn) CONH (CH2) 3 - (2-MeO-Ph) ES -MS 681 (m+1) . c-hexyl-NHCO-D-His-Tyr(OBn)CONHCH2Ph ES -MS 623 (m+1) 36.

PhCH2CH2CO - D - His - Tyr (OBn) CONHCH2CH2OBn ES -MS 674 (m+1) 37.

PhCH2CH2CO-D-His -Tyr (OBn) CONHOBn ES -MS 646 (m+1) 38.

(CH3)2CHNHCO-D-His-Tyr (OBn) CONH Met- C02Me ES -MS 639 (m+1) 39 .

PhCH2CH2CO-D-His-Tyr (OBn) CONH (CH2)3Ph ES -MS 672 (m+1) 40.

(CH3) 2CHNHCO - D - Hi s - Tyr (OBn) CONHCH2Ph ES -MS 583 (m+1) Array 2. Following cleavage from the resin and work-up as described in Example 4, the following substituted dipeptides (41-80) of Formula I are prepared: 41. n-BuNHCO-D-His-Tyr (OBn) -CONHCH2CH2OBn ES' -MS 641 (m+1) 42. n-BuNHCO-D-His-Tyr (OBn) -CONHCH2CH2SBn ES- -MS 657 (m+1) 43. n-BuNHCO-D-His-Tyr (OBn) -CONHCH2CH2CH2OPh ES' -MS 641 (m+i) 44 .

PhNHCO-D-His-Tyr (OBn) -CONHCH,CH2CH2CH2Ph ES -MS 659 (m+1) 45.

PhNHCO-D-His-Tyr (OBn) -C0(4-Bn-piperazin-l-yl) ES-MS in CD (m) 46. (4 -PhOPh) NHCO-D-His-Tyr (OBn) -CONHCH2CH2Ph ES -MS 723 (m+1) 47 . (4 - PhOPh) NHCO - D - Hi s - Tyr (OBn) - CONHCH2CH2SBn ES -MS 769 (m+l) 48. (4-MePh) SOj-D-His-Tyr (OBn) -CONH-Met-CO^e ES -MS 708 (m+1) 49. (4-MePh) S02-D-His-Tyr (OBn) -CONHCH2CH2CH2-(2-MeO-Ph) 50. (4-MePh)S02-.D-His-Tyr(OBn) -CO(4-Bn-piperazin- l-yl) ES -MS 720 (m) 51.

MeO (CH2) 3NHCO-D-His -Tyr (OBn) CONHCH2CH2Ph 52.

BnNHCO-D-His-Tyr(OBn)CONHOBn ES -MS 647 (m+1) 53.

BnNHCO-D-His-Tyr (OBn) CONHCH2CH2CH2OPh ES -MS 675 (m+l) 55. 56. 57.

S8. 59. 60. 61. 62. 63. 64. 65. 66 67 68 69 70 71 72 73 74 75 76 77 78 79 PCT/U S94/11553 ■38- BnNHCO-D-His-Tyr(OBn)CONH(CH2) 3-(2-MeO-Ph) MeO (CHj) 3NHCO-D-His-Tyr (OBn) CONHCH2Ph 1 - nap thyl - NHCO - D - Hi s - Tyr (OBn) CONHCH2CH2OBn (4-CIPh)NHCO-D-HiB-Tyr(OBn)CONHOBn (4-CIPh)NHCO-D-His-Tyr(OBn)CONH-Met-COjjMe 1-napthyl-NHCO-D-His-Tyr (OBn) CONHCH2CH2CH2CH2Ph 1-napthy1-NHCO-D-Hi s-Tyr(OBn)C0NHCH2Ph (4-EtOPh)NHCO-D-His-Tyr (OBn) CONHCH2CH2OBn BnOCO-D-His - Tyr (OBn) CONHCH2CH2SBn (4-BtOPh)NHCO-His-Tyr (OBn) CONHCH2CH2CH2OPh BnOCO-D-His -Tyr (OBn) CONHCH2CH2CH2CH2Ph (4-EtOPh)NHCO-D-His-Tyr(OBn)CO(4-Bn-piperazin-l-yl) PhCH2CO-D-His-Tyr (OBn) CONHCH2CH2Ph PhCH2CO - D - Hi s - Tyr (OBn) CONHCH2CH2SBn PhCH2CO-D-His-Tyr(OBn)CONH-Met-COjMe PhCH2CO-D-His-Tyr (OBn) CONH (CH2) 3-(2-MeO-Ph) PhCH2CH2NHCO - D - Hi s - Tyr (OBn) CO (4 - Bn -piperazin-l-yl) c-hexyl-NHCO-D-His-Tyr (OBn) CONHCH2CH2Ph c-hexyl -NHCO-D-His-Tyr (OBn) CONHOBn (t-2-Ph-c-propyl)-NHCO-D-His-Tyr (OBn) CONH (CH2) 3OPh (t-2-Ph-c-propyl)-NHCO-D-His-Tyr (OBn)CONH(CH2)3- (2-MeO-Ph) (t-2-Ph-c-propyl)-NHCO-D-His-Tyr (OBn) CONHCH2Ph (CH3) 2CHNHCO - D - Hi s-Tyr (OBn) CONHCH2CH2OBn (CH3) 2C"TICO-D-His-Tyr (OBn) CONHOBn PhCH2CH2 vJO - D - Hi s - Tyr (OBn) CONH - Me t - O^Me (CH3) 2CHNHCO - D - Hi s-Tyr (OBn) CONHCH2CH2CH2CH2Ph PhCH2CH2CO-D - His - Tyr (OBn) CONHCH2Ph ES-MS 689 (m+1) ES-MS 711 (m+1) BS-MS 707 (m+1) ES-MS 709 (m+1) ES-MS 705 (m+1) ES-MS 705 (m+1) ES-MS 729 (m) ES-MS 659 (m+1) ES-MS 676 (m+1) ES-MS 674 (m+1) ES-MS 713 (m) ES-MS 637 (m+1) ES-MS 639 (m+1) ES-MS 701 (m+1) ES-MS 657 (m+1) BS-MS 627 (m+1) ES-MS 599 (m+1) ES-MS 686 (m+1) ES-MS 625 (m+1) ES-MS 630 (m+1) ^ wo 95/12612 -39-EXAMPLE 6 N- TN- fN-r(Phenvlmethoxv)carbonvll-L-histidvll-O- (phenvlmethvl) -L-t-.vrosvll -0- (phenvlmethvl) -L-serine. methvl ester {Cbz-His-Tvr(OBn)-Ser(OBn)-C02Me) 5 Step 1: Boc-Tvr(OBn)-Ser(OBn)-CO.Me To a solution of Boc-Tyr(OBn) (1.88 g, 6.50 mmol) in EtOAc (30 mL) at 0°C was added HOBT hydrate (1.19 g, 7.80 mmol) followed by DCC (1.61 g, 7.80 mmol). A solution of Ser(OBn)-C02Me-TFA (2.1 g, 6.50 mmol) in 10 EtOAc (20 mL) was added followed by Et3N (1.09 mL, 7.80 mmol). The mixture was allowed to warm to room temperature and stirred overnight. The mixture was filtered, diluted with EtOAc, and washed twice with saturated aqueous NaHC03, brine, dried over MgS04, and 15 concentrated. Flash chromatography (40% EtOAc/hexane) gave 2.67 g (73%) of the title compound as a white solid, mp 81-84°C.

Step 2: Tyr(OBn)-Ser(OBn)-CQ2Me-TFA 20 Boc-Tyr(OBn)-Ser(OBn)-C02Me (from Step l above, 2.64 g, 4.69 mmol) was dissolved in CH2C12 (15 mL), cooled to 0°C and TFA (5 mL) was added. The solution was warmed to room temperature and stirred for 4 hours. The solution was concentrated, taken up in CH2C12 and 25 reconcentrated twice. The resulting oil was triturated with ether to provide 2.7 g of the title compound as a white solid.

Step 3: Cbz-His-Tvr(OBn)-Ser(OBn)-CQ2Me To a solution of Cbz-His (1.00 g, 3.47 mmol) in DMF (15 mL) at 0°C was added HOBT (0.64 g, 4.16 mmol) and DCC (0.86 g, 4.16 mmol). Tyr(OBn)-Ser(OBn)-C02Me'TFA (from Step 2 above, 2.0 g, 3.47 mmol) was added followed by Et3N (0.58 mL, 4.16 mmol). The 35 mixture was allowed to warm to room temperature and stirred overnight. The mixture was filtered and the PCT7US94/11553 filtrate was diluted with CHC13, washed twice with saturated aqueous NaHC03, brine, dried over MgS04, and concentrated. Flash chromatography (2-5% MeOH/CHCl3) gave 2.14 g of the title compound as a white solid, 5 mp 175- 176°C; FAB-MS 734 (m+1) ; Anal. Calc. for C41H43N508: C, 67.11? H, 5.91; N, 9.54; Found: C, 66.96; H, 6.01; N, 9.41.

EXAMPLE 7 N-fN- TN-f(Phenvlmethoxv)carbonvll-L-histidyll-O-(phenvlmethyl)-L-tvrosvll-0-(phenvlmethvl)-L-serine ICbz-His-Tyr(OBn)-Ser(OBn)} To a solution of Cbz-His-Tyr(OBn)-Ser(OBn)-C02Me 15 (from Example 6 above, 2.02 g, 2.75 mmol) in THF (50 mL) and MeOH (15 mL) at 0°C was added 0.1N LiOH (30.3 mL, 3.03 mmol). The solution was stirred for 6 hours at 0°C, then concentrated. Water was added and the pH was adjusted to 4-5 with IN HC1. The mixture 20 was filtered, and the solid was collected and dried to provide 1.55 g (78%) of the title compound as a white solid, mp 187-192°C; ES-MS 720 (m+1); Anal. Calc. for C40H41N5O8-1.5H20: C, 64.33; H, 5.94; N, 9.38; Found: C, 64.29; H, 5.73; N, 9.15.

EXAMPLE 8 N- TN- fN- r (Phenvlmethoxv) carbonyll -D-histidyll -O-(phenvlmethyl)-L-tyrosvll-0-(phenylmethvl)-L-serine. 30 methvl ester {Cbz-D-His-Tvr(OBn)-Ser(OBn)-CO-.Me) Step 1: Tvr(OBn)-Ser(OBn)-CO-.Me-HCl A solution of Boc-Tyr(OBn)-Ser(OBn)-C02Me (from Example 6, Step 1 above, 9.90 g, 17.6 mmol) in EtOAc was cooled to 0°C. Anhydrous HC1 gas was bubbled 35 through the cold solution for 5 minutes. The solution was allowed to warm to room temperature and stirred overnight. The solution was concentrated, taken up in EtOAc and reconcentrated to provide 8.75 g of the title compound as a foam; CI-MS 463 (m+1) .

Step 2: Cbz-D-His(Tr)-Tvr(OBn)-Ser(OBn)-CQ2Me According to Example 6, Step 3, by substituting Cbz-D-His(Tr) for Cbz-His and Tyr(OBn)-Ser(OBn)-C02Me*HC1 for Tyr(OBn)-Ser(OBn)-C02Me-TFA, the title compound was obtained as a white solid, mp 78-88°C; 10 FAB-MS 976 (m+1).

Step 3: Cbz-D-His-Tyr(OBn)-Ser(OBn)-CO^Me A solution of Cbz-D-His(Tr)-Tyr(OBn)-Ser(OBn)-C02Me (from Step 2 above, 0.27 g, 0.28 mmol) in 15 H0Ac:H20 (4:1, 2 mL) was stirred at 80°C for 5 minutes, then cooled to room temperature. The solution was partitioned between EtOAc and saturated aqueous NaHC03. The organic layer was washed with brine, dried (MgS04), and concentrated. Flash chromatography (2-5% 20 MeOH:CHCl3) yielded 0.10 g of the title compound as a foam; FAB-MS 734 (m+I) .

EXAMPLE 9 N- fN- fN-r(Phenylmethoxv)carbonyll-L-histidyll-0-25 (phenylmethyl)-L-tvrosvll-O-(phenvlmethyl)-D-serine. methvl ester {Cbz-His-Tvr(OBn)-D-Ser(OBn)-CO^Me) According to Example 6, by substituting D-Ser (OBn)-C02Me-TFA for Ser (OBn)-C02Me • TFA in Step 1, the title compound was obtained, mp 168-170°C; 30 FAB-MS 734 (m+1). -42-EXAMPLE 10 N- for-Methvl-N- fN- f (phenvlmethoxy)carbonyll -L-histidyll -0- (phenvlmethvl)-DL-tyrosvll -0-(phenvlmethvl)-L-serine. methyl ester (Cbz-His-DL- (o;-Mg) Tyr (OBn) -Ser (OBn)-CO^Me} According to Example 6, by substituting Boc-DL-(a-Me) Tyr (OBn) for Boc-Tyr(OBn) in Step 1, the title compound was obtained; FAB-MS 748 (m+1).

EXAMPLE 11 N-Ethvl-Ng- fN- fN- T (phenvlmethoxy)carbonyll -L-histidyll -0-(phenylmethyl)-L-tvrosvll -O-(phenvlmethyl)-L-serinamide {Cbz-His-Tvr(OBn)-Ser(OBn)-CONHEt) According to Example 6, by substituting Boc-Tyr(OBn)-Ser(OBn)-CONHEt for Boc-Tyr(OBn)-Ser (OBn)-C02Me in Step 2, the title compound was obtained, mp 182-188°C; FAB-MS 747 (m+1).

EXAMPLE 12 N-Ethvl-Na.- fN- fN- f (Phenvlmethoxy) carbonvll -D-histidyll -O-(phenvlmethyl)-L-tvrosvll -O-(phenylmethyl)-L-serinamide {Cbz-D-His-Tyr(OBn)-Ser(OBn)-CONHEt} According to Example 6, by substituting Boc-Tyr (OBn) -Ser (OBn) -CONHEt for Boc-Tyr(OBn)-Ser(OBn)-C02Me in Step 2 and Cbz-D-His for Cbz-His in Step 3, the title compound was obtained, mp 193-196°C; ES-MS 747 (m+1)- EXAMPLE 13 N-fN-fl-Methvl-N-f(phenvlmethoxv)carbonvll 1-L-histidyll- C-(phenylmethyl)-L-tvrosvll -0-(phenylmethyl)-L-serine. methvl ester {Cbz-His(l-Me)-Tvr(OBn)-Ser(OBn)-CO^Me) Step 1: Cbz-His(l-Me) Benzyl chloroformate (0.24 mL, 1.7 mmol) was added dropwise to a slurry of 1-methyl-L-histidine (0.25 g, 1.5 mmol) in THF (5 mL) and saturated aqueous NaHC03 (5 mL) at 0°C. The mixture was allowed to warm to room temperature and stirred overnight. The mixture was concentrated and diluted with H20, washed with ether, 5 and the pH adjusted to 6-7 with IN HCl. The mixture was concentrated, then diluted with CHC13 (150 mL) and MeOH (15 mL), and stirred for 1 hour. The mixture was dried (MgS04) and concentrated to provide 0.48 g of the title compound which was used without further 0 purification.

Step 2: Cbz-His(l-Me)-Tvr(OBn)-Ser(OBn)-CO^Me To a slurry of Cbz-His (l-Me) (from Step 1 above, 0.36 g, 1.2 mmol), Tyr (OBn)-Ser (OBn)-C02Me*HCl (from 5 Example 8, Step. 1 above, 0.60 g, 1.2 mmol), DCC (0.30 g, 1.4 mmol), and HOBT (0.19 g, 1.4 mmol) in CH2C12 was added Et3N (0.17 mL, 1.2 mmol) and the mixture was stirred overnight at room temperature. The mixture was diluted with CHC13, washed with saturated 0 aqueous NaHC03, brine, dried (MgS04), and concentrated. Flash chromatography (1% MeOH:CHCl3) provided the title compound as a white solid, mp 161.5-163,5°C; FAB-MS 748 (m+1).

EXAMPLE 14 N- fN- fl-Methvl-N- f (phenylmethoxv) carbonvll -D-histidvll -O- (phenvlmethvl) -L-tvrosvll -0- (phenylmethyl) -L-serine. methvl ester (Cbz-D-His (l-Me) -Tvr (OBn) -Ser (OBn) -CQ2Me} According to Example 13, by substituting 1-methyl-10 D-histidine for 1-methyl-L-histidine, the title compound was obtained; FAB-MS 748 (m+1) . -44-EXAMPLE 15 N- rL-2-Amino-N- fN- T(Phenvlmethoxv)carbonvll-D-histidvll-4-T4- (phenvlmethoxy)phenyl!butanoyll-0-(phenvlmethvl-L-serine, methyl ester (Cbz-D-His-homoTyr(OBn) - Ser (OBn) -CQ2Me) According to Example 6, by substituting Boc-homoTyr(OBn) for Boc-Tyr(OBn) in Step 1, and substituting Cbz-D-His for Cbz-His in Step 3, the title compound was obtained; ES-MS 748 (m+1).

EXAMPLE 16 N- T4-Phenyl-N-TN-f(phenylmethoxy)carbonvll-L-histidyll-L-phenylalanvil -0-(phenylmethyl)-L-serine. methyl ester I Cbz-His-Phe(4-Ph)-Ser(OBn)-C02Mel 15 According to Example 6, by substituting Boc- Phe(4-Ph) for Boc-Tyr(OBn) in Step 1, the title compound was obtained, mp 184-187°C; FAB-MS 704 (m+1).

EXAMPLE 17 N- fO- (Phenylmethyl) -N[N- r f (Phenvlmethvl)amino! - carbonvll - L-histidyll -L-tyrosy]!-O-(phenylmethyl)-serine, methyl ester (BnNHCO-His-Tvr(OBn)-Ser(OBn) -CQ2Mei Step 1: Fmoc-His(Tr)-Tyr(OBn)-Ser(OBn)-CQ2Me 25 According to Example 13, Step 2, by substituting Fmoc-His (Tr) for Cbz-His (l-Me) , the title compound was obtainedi mp 82-92°C.

Step 2: His(Tr) -Tyr(OBn)-Ser(OBn)-C02Me 30 Piperidine (4.0 mL) was added to a slurry of Fmoc-His (Tr)-Tyr (OBn)-Ser (OBn)-C02Me (from Step 1 above, 1.85 g, 1.74 mmol) in CH2C12 (20 mL) . The solution was stirred for 2 hours at room temperature, then concentrated. The residue was taken up in EtOAc 35 (150 mL), washed with water (3 x 50 mL), dried (MgS04), and concentrated. The resulting oil was triturated with Et20/hexane. Flash chromatography of the residue (2* MeOH/CHCl3) gave 1.03 g of the title compound as a foam, mp 61.5-70°C; ES-MS 843 (m+1). step 3: BnNHCO-His (Tr) -Tvr (OBn) -Ser (OBn) -CO.Me Benzyl isocyanate (0.053 mL, 0.43 ETmol) was added in one portion to a solution of His (Tr)-Tyr (OBn)-Ser (OBn)-C02Me (from Step 2 above, 0.33 g, 0.39 mmol) in EtOAc (5 mL) . The resulting slurry was stirred for 0 3 hours at room temperature, then concentrated to yield the title compound (0.4 g) , which was used without further purification. step 4: BnNHCO-His-Tvr(OBn)-Ser(OBn)-CO^Me 5 According to Example 8, by substituting BnNHCO- His (Tr) -Tyr (OBn) -Ser (OBn) -C02Me for Cbz-His(Tr)-Tyr(OBn)-Ser(OBn)-C02Me, the title compound was obtained, mp 196.5-199°C; ES-MS 733 (m+1). 0 EXAMPLE 18 N- fN- fN- (l-0xo-3-phenvlpropvl)-L-histidyll-O-(phenvlmethvl) - L-tvrosvll -0- (phenvlmethvl) -L-serine. methyl ester IPhCH2CHnCQ-His-Tvr(OBn)-Ser(OBn)-CO?Me} Step 1: PhCH* CH.. CO - His (Tr) -Tvr (OBn) -Ser (OBn) -CQ2Me 5 To a cooled (0°C) solution of His(Tr)-Tyr(OBn)- Ser (OBn)-C02Me (from Example 17, Step 2 above, 0.33 g, 0.39 mmol) in THF (5 mL) was added Et3N (0.06 mL, 0.43 mmol) followed by phenylpropionyl chloride (0.064 mL, 0.43 mmol). The resulting slurry was 0 brought to room temperature and stirred overnight. The mixture was partitioned between EtOAc and saturated aqueous NaHC03. The organic layer was washed with brine, dried (MgS04) , and concentrated to yield the title compound as a solid which was used without 5 further purification.

Step 2 : PhCHoCHnCO-His-Tvr (OBn) -Ser (OBn) -CQ2Me According to Example 8, Step 3, by substituting PhCH2 CH2 CO - His (Tr) -Tyr (OBn) -Ser (OBn) -C02Me for Cbz-D-His (Tr)-Tyr (OBn)-Ser (OBn)-C02Me, the title compound was 5 obtained, mp 193-196.5°C; ES-MS 732 (m+1).

EXAMPLE 19 Nffl- fN- f (Phenylmethoxy) carbonvll -L-histidvll -0- (phenvlmethvl) -N- f2- (phenvlmethoxv) ethvll -10 L-tvroginamide (Cbz-His-Tvr (OBn) -CONHCH2CH2OBn) Step 1: Cbz-His-Tvr(OBn)-CQ2Me According to Example 6, Step 3, by substituting Tyr (OBn)-C02Me'TFA for Tyr (OBn) -Ser (OBn)-C02Me-TFA, the title compound was obtained as a white powder, 15 mp 145-148°C; CI-MS 557 (m+1) .

Step 2: Cbz-His-Tvr(OBn) According to Example 7, by substituting Cbz-His-Tyr (OBn) -C02Me for Cbz-His-Tyr(OBn)-Ser(OBn)-C02Me, the 20 title compound was obtained as a white powder, mp 79-92°C; CI-MS 543 (m+1).

Step 3 : Cbz-His -Tvr (OBn) -CONHCH2CH2OBn To a solution of Cbz-His-Tyr (OBn) (froir. Step 2 25 above, 0.43 g, 0.79 mmol) in DMF (4 mL) at 0°C was added HOBT (0.15 g, 0.95 mmol) and DCC (0.20 g, 0.95 mmol). A solution of 2-(phenylmethoxy)ethylamine (0.12 g, 0.79 mmol) in DMF (1 mL) was then added. The mixture was allowed to warm to room temperature and 30 stirred overnight. The mixture was filtered, diluted with CHC13, washed twice with saturated aqueous NiHC03, washed with brine, dried over MgS04, and concentrated. Flash chromatography (3-5% MeOH/CHCl3) afforded 0.34 g (63%) of the title compound as a white solid, 35 mp 136-150°C; FAB-MS 676 (m+1); Anal. Calc. for C39H41N5Oe: C, 69.32; H, 6.12; N, 10.36; Found: C, 69.43; H, 6.24; N, 10.45.

EXAMPLE 20 Na- FN-f(Phenvlmethoxv)carbonvll-D-histidvll-N-f2 - (phenvlmethoxv) ethvll -0- (phenylmethyl) -L-tvrosinamide (Cbz-D-His-Tyr (OBn) -CONHC^CH^OBn} According to Example 6, by substituting 2-(phenylmethoxy ) e thy lamine for Ser(OBn)-TFA and omitting Et3N in Step 1 and by substituting Cbz-D-His for Cbz-His in Step 3, the title compound was prepared, mp 161-165°C; FAB-MS 676 (m+1).

EXAMPLE 21 Ng- FN-Methvl-Nf(phenvlmethoxy)carbonvll-D-histidyll -N-f2- (phenylmethoxv)ethvll-O-(phenvlmethvl)-L-tvrosinamide 'Cbz-D-His- (N-Me) Tyr (OBn) -CQNHCH^CH-.OBn) According to Example 20, by substituting Boc-(N-Me)Tyr(OBn) for Boc-Tyr(OBn), the title compound was obtained, mp 64-78°C; ES-MS 690 (m+1).

EXAMPLE 27 Na-fa-Methyl-N-FN-f (phenvlmethoxv)carbonyll -D-histidyll-N-F2-(phenvlmethoxv)ethyl!-0-(phenylmethyl) -L- tyr ?sinamide I Cbz-D-His- (o?-Me) Tvr (OBn) -CONHCH^CHoOBn) According to Example 20, by substituting Boc-(a-Me)Tyr(OBn) for Boc-Tyr(OBn), the title compound was obtained, mp 66-78°C; ES-MS 690 (m+1).

WO 95/12612 PCT/US94/11553 EXAMPLE 23 N- (2-Phenvlethvl)-Na- fN-f(phenvlmethoxv)carbonvll -L-histidvll -0-(phenvlmethvl)-L-tvrosinamide (Cbz-His-Tvr (OBn) -CONHCH-XHoPhl 5 According to Example 19, Step 3, by substituting 2-phenylethylamine for 2-(phenylmethoxy)ethylamine, the title compound was obtained as a white solid, mp 188-189.5°C; FAB-MS 646 (m+1).

EXAMPLE 24 N- fN- fN- T (Phenvlmethoxv) carbonyl7 - L-histidvll -O-(phenylmethyl)-l-tvrosvll-3- (3-pvridinvl)-L-alanine. methvl ester {Cbz-His-Tyr (OBn)-Pvr-COnMe) According to Example 19, Step 3, by substituting 15 Pyr-C02Me for 2-(phenylmethoxy) ethylamine, the title compound was obtained as a white solid, mp 180-182.5°C (dec); FAB-MS 705 (m+1).

EXAMPLE 25 (S.R)-N-f2-(4-Benzyloxv-phenyl)-1- (3-phenoxv- propvlcarbamoyl)-ethvll-2-f3-(4-ethoxv-phenyl) -ureidol-3-(3H-imidazol-4-vl)-propionamide / (4-EtOPh) NHCO-D-His-Tyr (OBn) -CONH(CH=) 3OPhl Step 1. Boc-Tvr (OBn) -CONH(CH:,) 30Ph 25 2-(Phenylmethoxy) ethylamine (0.81 g, 5.4 mmol) was added to a premixed solution of EDAC (1.2 g, 6.5 mmol), HOBT (0.87 g, 6.5 mmol), and Boc-Tyr(OBn)-OH (2.0 g, 5.4 mmol) in dry DMF (15 mL) . The resulting mixture was stirred for 18 hours at room temperature. The 30 solution was diluted with 1:1 Et0Ac:Et20 (40 mL), washed with saturated aqueous NaCl (4 x 10 mL) , dried (MgS04), filtered, and concentrated in vacuo to provide a solid which was further purified by trituration with hexane to give the pure product, mp 145-146'C.

PCT71JS94/11553 Step 2. Tvr (OBn) - CONH (CHg) 3OPh Dry HCl gas was bubbled into an ice cold solution of Doc-Tyr (OBn) - CONH (CH2) 3OPh (from Step 1 ?J?ove, 2.0 g, 3.9 mmol) in MeOH (15 mL) for 4 minutes. The 5 resulting mixture was stirred for 1 hour at O'C and then allowed to warm to room temperature and stir 1 hour. The solution was concentrated in vacuo to provide a solid which was triturated with ether to provide Tyr (OBn) -CONH (CH2) 30Ph-HCl? CI-MS 405 (m+1). 10 The title compour"! was suspended in CHC13, cooled in an ice bath, and NH3 gas was bubbled through the mixture for 2 minutes. The NH4C1 was filtered off, and the supernate was concentrated in vacuo to yield the free Lase of the title compound which was used in the next 15 step without further purification.

Step 3 . Fmoc-D-His (Tr) -Tvr (OBn) - CONH (CH=) 3OPh To a solution of HOBT (0.48 g, 3.5 mmol) in DMF (10 mL) was added Fmoc-D-His (Tr)-C02H (2.0 g, 3.2 mmol) 20 followed by EDAC (0.67 g, 3.5 mmol). The mixture was stirred at room temperature for 20 minutes before adding a solution of Tyr (OBn)-CONH (CH2) 3 OPh (from Step 2 above, 1.4 g, 3.2 mmol) in DMF (10 mL) . The mixture was stirred overnight at room temperature 25 before partitioning between a mixture of water (20 mL) and 1:1 Et20:EtOAc (50 mL) . The layers were separated, and the organic phase was washed with saturated aqueous NaCl (4x 20 mL) and dried (MgS04) . Filtration and concentration in vacuo provided an oil which was 30 further purified by flash chromatography (Si02, CHC13: MeOH eluent) to give the protected His-Tyr dipeptide> FAB-MS 1006 (m).

Step 4. D-His (Tr) -Tvr (OBn)-CONH(CH=) 3OPh 35 Fmoc-D-His (Tr)-Tyr (OBn)-CONH(CH2)30Ph (from Step 3 above, 1.0 g, 0.99 mmol) in CH2C12 (5 mL) was treated with piperidine (0.18 g, 2.1 mmol). The resulting mixture was stirred 2 hours before concentrating in vacuo and purifying the resulting oil by flash chromatography (Si02, CHCl3:MeOH eluent) to give 5 (4-EtOPh)NHCO-D-His(Tr) -Tyr (OBn) -CONH(CH2) 3OPh; ES-MS 784 (m+1).

Step 5 . (4 - EtOPh) NHCO-D-His (Tr) -Tvr (OBn) -CONH (CHO OPh D-His(Tr)-Tyr(OBn)-CONH(CH2)3OPh (from Step 4 10 above, 0.55 g, 0.7 mmol) in CH2C12 (5 mL) was treated with 4-ethoxyphenyl isocyanate (0.1 g, 0.7 mmol). The resulting mixture was stirred l hour at room temperature. Concentrated in vacuo. The resulting oil was purified by flash chromatography (Si02, CHCl3:MeOH 15 eluent) to give (4-EtOPh)NHCO-D-His(Tr)-Tyr(OBn)-CONH (CH2) 3OPh; ES-MS 947 (m+1).

Step 6. (4-EtOPh) NHCO-D-His -Tvr (OBn) -CONH(CH2) 3OPh (4-EtOPh)NHCO-D-His (Tr) -Tyr (OBn) -CONH(CH2) 3OPh 20 (from Step 5 above, 0.5 g, 0.52 mmol) in MeOH (5 mL) was treated with Pyridine-HCl (catalytic). The resulting mixture was stirred at 65*C for 6 hours. Concentrated in vacuo to obtain an oil which was purified by by flash chromatography (Si02, CHCl3:MeOH 25 eluent) to give (4-EtO-Ph) NHCO-D-His-Tyr (OBn) -CONH (CH2) 3OPh, mp 185-187 *C; ES-MS 705 (m+1).

EXAMPLE 26 (S.R)-N-(2-(4-Benzvloxv-phenvl)-1-T3-(2-methoxv-30 phenyl) -propylcarbamoyl 1 -ethvl \ - 3 - (3H - inK ■iazol-4-vl) -2-phenvlacetvlamino-propionamide (PhCH2CO-D-His-Tvr(OBn)- CONH(CH.)3(2-MeOPh)) Step 1. Boc-Tvr (OBn) -CONH(CH2)3 (2-MeOPh) According to Example 25, Step 1, by substituting 35 3- (2-methoxyphenyl) propyl amine for 2-(phenylmethoxy)- ethylamine, the title compound was obtained as a white solid, rnp 125-126.5'C.

Step 2. Tvr (OBn) -CONH(CH2)3 (2-MeOPh) 5 According to Example 25, step 2, by substituting Boc-Tyr(OBn)-CONH(CH2)3(2-MeOPh) for Boc-Tyr(OBn)-CONH (CH2) 3OPh, the title compound was obtained as a white solid; CI-MS 419 (m+1) . 0 Step 3. Fmoc-D-His (Tr) -Tvr(OBn) - CONH (CH..) 3 (2-MeOPh) According to Example 25, Step 3, by substituting Tyr(OBn)-CONH(CH2)3(2-MeOPh) for Tyr(OBn)-CONH (CH2) 3OPh, the title compound was obtained as a foam; ES-MS 1020 (m).

Step 4. D-His(Tr)-Tvr(OBn)-CONH(CH2)3(2-MeOPh) According to Example 25, Step 4, by substituting Fmoc-D-His (Tr)-Tyr (OBn)-CONH(CH2)3 (2-MeOPh) for Fmoc-D-His (Tr)-Tyr (OBn)-CONH (CH2) 3OPh, the title compound was 0 obtained as a white foam; ES-MS 798 (m+1).

Step 5. PhCH-.CO-D-His(Tr)-Tvr(OBn)-CONH(CH2)3(2-MeOPh) To a solution of D-His(Tr)-Tyr(OBn)-CONH(CH2)3 (2-MeOPh) (0.4 g, 0.5 mmol) in CH2C12 (5 mL) :5 was added N-methyl morpholine (0.05 g, 0.5 mmol) followed by phenyl acetyl chloride (0-08 g, 0.5 rnmol) . The resulting mixture was stirred 2 hours at room temperature. Diluted with DCM and washed with saturated aqueous NaHC03, saturated aqueous NaCl, and 10 dried (MgS04). Purified by flash chromatography (Si02, CHC13:MeOH.eluent). The title compound was obtained as a foam; FAB-MS 916 (m).

Step 6. PhCH3CO-D-His-Tvr(OBn)-CONH(CH2)3(2-MeOPh) According to Example 25, Step 6, by substituting PhCH2CO-D-His (Tr) -Tyr (OBn) -CONH(CH2) 3 (2-MeOPh) for O 95/12612 (4-EtOPh) NHCO-D-His (Tr) -Tyr (OBn) -CONH (CH2) 3OPh, the title compound was obtained as a white foam; ES-MS 674 (m+1) .

EXAMPLE 27 (S.R)-N-f2-(4-Benzvloxv-Phenyl)-1-phenethvlcarbamoyl-ethvll -3-(3H-imidazol-4-vl)-2-T3-(4-phenoxv-phenyl)-ureidol-propionamide ((4-PhOPh)NHCO-D-His-Tvr(OBn)-CONH(CH2UPh) Step 1. Boc-Tvr(OBn)CONH(CH2) 2Ph According to Example 25, Step 1, by substituting phenethylamine for 2-(phenylmethoxy)ethylamine, the title compound was obtained as a white solid; CI-MS 475 (m+1) .

Step 2. Tvr (OBn) - CONH (CH2) 2Ph According to Example 25, Step 2, by substituting BocTyr(OBn)-CONH(CH2)2Ph for BocTyr (OBn)-CONH (CH2) 30Ph, the title compound was obtained as a white solid; 20 CI-MS 375 (m+1) .

Step 3 . Fmoc-D-His (Tr) -Tvr (OBn) -CONH (CH2) 2Ph According to Example 25, Step 3, by substituting Tyr (OBn)-CONH (CH2)2Ph for Tyr (OBn)-CONH (CH2) 3 OPh, the 25 title compound was obtained as a foam; ES-MS 977 (m+1).

Step 4. Fmoc-D-His-Tvr(OBn)-CONH(CH2)=Ph Fmoc-D-His (Tr)-Tyr (OBn)-CONH(CH2)2Ph (1.2 g, 1.6 mmol) in MeOH (5 juL) was treated with Pyridine-HCl 30 (catalytic). The resulting mixture was stirred at 65'C overnight and concentrated in vacuo to obtain an oil which was purified by by flash chromatography (Si02, CHCl3:MeOH eluent) to give a white solid; ES-MS 734 (m+1) . wo 95/12612 0 Step 5. (4 -PhOPh)NHCO-D-His-Tvr(OBn)-CONH(CH2)2Ph Fmoc-D-His-Tyr(OBn)-CONH(CH2) 2Ph (0.6 g, 0.8 mmol) in CH2C12 (5 mL) was treated with piperdine (0.14 g, 1.6 mmol). The resulting mixture was stirred 2 hours 5 before concentrating in vacuo and purifying the resulting by flash chromatography (Si02, CHCl3:MeOH eluent) to give D-His-Tyr (OBn)-CONH (CH2) 2Ph. The foam was dissolved in CH2C12 (5 mL) and treated with 4-phenoxyphenyl isocyanate (0.05 g, 0.23 mmol). The 10 resulting mixture was stirred 1 hour at room temperature, concentrated in vacuo, and purified the resulting oil by flash chromatography (Si02, CHCl3:MeOH eluent) to obtain (4-PhOPh)NHCO-D-His-Tyr(OBn)-CONH (CH2) 2Ph as a foam; ES-MS 723 (m+1).

EXAMPLE 28 (S.R)-N-[1-(4-Benzyloxv-benzvl)-2-(4-benzyl-piperazin-1-yl)-2-oxo-ethvn-3-(3H-imidazol-4-yl)-2-(toluene-4-sulfonvlamino)-propionamide {(4-MePh)SQ2-D-His-20 Tvr(OBn)-CO(4-Bn-piperazin-l-vl)•HCl) Step 1. Boc-Tvr(OBn)-CO(4-Bn-piperazin-l-yl) According to Example 25, Step 1, by substituting 1-benzylpiperizine for 2-(phenylmethoxy)ethylamine, the 25 title compound was obtained as a white solid; CX-MS 530 (m+1).

Step 2. Tyr(OBn)-CO(4-Bn-pjperazin-l-yl) According to Example 25, Step 2, by substituting 30 Boc-Tyr(OBn)-CO(4-Bn-piperazin-l-yl) for Boc-Tyr(OBn)- CONH (CH2) 3OPh, the title compound was obtained as a white solid; CI-MS 430 (m+1).

AVO 95/12612 step 3. Fmoc-D-His (Tr) -Tvr (OBn) -CO (4-Bn-pjperazin- i-yj-) According to Example 25, Step 3, by substituting Tyr (OBn)-CO (4-Bn-piperazin-l-yl) for Tyr(OBn)-5 CONH (CH2) 3OPh, the title compound was obtained as a foam; ES-MS 1032 (m+1) .

Step 4. (4-MePh) SQ2-D-His(Tr) -Tvr (OBn) -CO(4-Bn-pjperazin-l-vl) Fmoc-D-His-Tyr (OBn) -CONH(CH2) 3OPh, (0.7 g, 0.69 mmol) in CH2C12 (5 mL) was treated with piperidine (0.14 g, 1.6 mmol). The resulting mixture was stirred 2 hours before concentrating in vacuo and purifying the resulting oil by flash chromatography (Si02, CHCl3:MeOH 15 eluent) to give D-His-Tyr (OBn)-CONH(CH2) 2Ph. The foam was dissolved in CH2C12 (5 mL) and treated with pyridine (0.05 g, 0.63 mmol) , followed by 4-toluenesulfonyl chloride (0.12 g, 0.63 mmol). The resulting mixture was stirred 3 hours at room 20 temperature, concentrated in vacuo, and purified the resulting oil by flash chromatography (Si02, CHCl3:MeOH eluent) to obtain (4-MePh) S02-D-His (Tr)-Tyr (OBn)-CO (4-Bn-piperazin-l-yl) ; ES-MS 963 (m) .

Step 5. (4-MePh) S02-D-His-Tvr (OBn) -CO (4-Bn-oiperazin- 1-vl)•HCl (4-MePh) S02-D-His (Tr) -Tyr (OBn) -CO (4-Bn-piperazin-l-yl) (0.21 g, 0.22 mmol) was treated with 80% aqueous HCl (3 mL) and heated to 80'C for 5 minutes. The 30 mixture was cooled and diluted with water (5 mL) . The solid was filtered off, and the supernate was concentrated in vacuo to provide an oil. The oil was dissolved in water (15 mL) , frozen, and lyophilized to provide (4-MePh)S02-D-His-Tyr (OBn) -CO (4-Bn-piperazin-35 l-yl) *HC1; ES-MS 720 (m) .

Claims

WO 95/12612* m EXAMPLE 29 Na- fN- f (Phenvlmethoxv) carbonvll -L-histidvll -0-(phenvlmethvl) -N-methvl-N- T2- (phenvlmethoxv) ethvll -L-tyrosinamide {Cbz-His-Tvr (OBn) -CON(Me) CH2CH20Bn) 5 According to Example 19, Step 3, by substituting N-methyl-N- [2- (phenylmethoxy) ethyl] amine for 2-(phenylmtthoxy) ethylamine, the title compound was prepared; FAB-MS 690 (m+1). 10 The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the 15 invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope. PCT/US94/11553 -55- 0 WO 95/12612 -56-CLAIMS

1. A compound of the Formula I: PCT/uSy4/fl55 10 15 20 25 30 wherein: n A R Y Z X » 1 or 2; « COR3, C02R3, CONHR3, CSR3, C(S)OR3, C(S)NHR3, where.ln R3 is (CH2)m-aryl, CF3S0.j, aryl-S02, or alkyl-S02, alkyl, (CH2)m-cycloalkyl, (CH2)m-heteroaryl, or (CH2)mO-alkyl, and m « 0, 1, 2, or 3; independently H or Me; independently H or Me; independently H or Me; H, CO-aryl, (CH2)m-aryl, 0 (CH2)m-cycloalkyl, 0 (CH2)m-aryl, or 0(CH2)m-heteroaryl wherein m is as defined above and R1 is located at either the meta or para position; one to four substituents, including H, alkyl, CF3, F, CI, Br, I, HO, MeO, N02, NH2, N(Me)2, 0PO3H2, or CH2P03H2; and NR(CH2)nC02R3, NR(CH2)nCONHR3, NR(CH2)nR3, NR(CH2)n+1SR4, WO 95/12612 PCT/US94/11553 35 40 -57- NRCH (COR5) (CH2)n-heteroaryl, NRCH (COR5) (CH2) n0R3, NRCH (COR5) (CH2) nSR3 f 275691 N—R3 wherein R, R3, and n are or as defined above, R4 = H or R3, and R5 » OH, NH2, OR3, or NHR3; an optical isomer, diastereomer, or a pharmaceutically acceptable salt thereof; wherein the following compounds are exclud roperty Off,Ce Z-His-Phe-Gly-OMe, Z-His-Phe-Trp-OEt, and Z-His-Phe(NO2)-Gly-OMe. '2 FEB 1998 Zealand

A compound according to Claim 1 which is a compound of Formula II: 10 15 20 25 II wherein: A' - C02R3, CONHR3, C(S)NHR3, cr aryl-S02, wherein R3 is alkyl, (CH2)m-cycloAlkyl, (CH2)m-aryl, (CH2)m-heteroaryl, and m « 0, 1, 2 or 3; R <= independently H or Me; Y = independently H or Me; Z » independently H or Me; R1' = {CH2)nj-aryl, 0(CH2)m-aryl, OP03H2, or CH2P03H2, wherein m is as defined above; R2' ® NR(CH2)20R4, NR(CH2)2SR4, NRCH (COR5) CH20R3, or NRCH (COR5) CH2SR3, wherein R3 and n are as defined above, R4 « H or R3, and R5 « OH, NH2, OR3, or NHR3; an optical isomer, diastereomer, or a pharmaceutically acceptable salt thereof. WO 95/12612 PCT/US94/11553 27 5691

3. A compound according to Claim 1 wherein A is C02R3 or CONHR3.

4. A compound according to Claim 1 wherein at least one of Y and Z is Me.

5 . A compound according to Claim l wherein R2 iff g C E I V E 0 NH (CH2) 2OR4 or NHCH (COR5) CH2OR3. fntellaotual Pronarty Offle# f 2 1998

6. A compound according to Claim l wherein A is.-:, , . . % ? 3 of New Zealand CONHR 7 R is NH(CH2)2OR4, and at least one of V and Z is Me.

7. A compound according to Claim l selected from the group consisting of: Cbz-His-Tyr (OBn) -Ser (OBn) -C02Me; Cbz-His-Tyr (OBn) -Ser (OBn) -CONH2; 5 Cbz-His-Tyr (OBn)-Ser (OBn)-CONHEt; and Cbz-His-Tyr(OBn)-Ser(OBn).

8. A compound according to Claim 1 selected from the group consisting of: Cbz-His-Tyr (OBn) -D-Ser (OBn) -C02Me; Cbz-D-His-Tyr (OBn) - Ser (OBn) -CONH2; 5 Cbz-D-His-Tyr (OBn) -Ser(OBn) -CONHEt; Cbz-D-His-Tyr (OBn) -Ser(OBn) -C02Me; and Cbz-D-His-Tyr (OBn) -Ser (OBn) .

9. A compound according to claim 1 selected from the group consisting of: Cbz-His (l-Me) -Tyr (OBn) -Ser (OBn) -COoMe; Cbz-His (l-Me) -Tyr (OBn) -Ser (OBn) -CONH2; 5 Cbz-His (l-Me) -Tyr (OBn)-Ser (OBn) -CONHEt; Cbz-His (l-Me)-Tyr (OBn)-Ser (OBn) ; Cbz-D-His (l-Me)-Tyr (OBn) -Ser (OBn) -C02Me; Cbz-D-His (l-Me)-Tyr (OBn) -Ser (OBn) -CONH2; #, WO 95/12612 PCT/l'S94/l 1553 -59- Cbz-D-His(l-Me)-Tyr(OBn)-Ser(OBn)-CONHEt; and 10 Cbz-D-His(l-Me)-Tyr(OBn)-Ser(OBn).

10. A compound according to claim 1 selected from the group consisting of: Cbz-His-(a-Me)Tyr(OBn)-Ser(OBn)-C02Me; Cbz-His-(a-Me)Tyr(OBn)-Ser(OBn)-CONH2 ? 5 Cbz-His-(a-Me)Tyr\OBn)-Ser(OBn)-CONHEt; Cbz-His-(a-Me)Tyr(OBn)-Ser(OBn); Cbz-His-D-(a-Me)Tyr(OBn)-Ser(OBn)-C02Me; Cbz-His-D-(a-Me)Tyr(OBn)-Ser(OBn)-CONH2; Cbz-His-D-(a-Me)Tyr(OBn)-Ser(OBn)-CONHEt; and 10 Cbz-His-D-(a-Me)Tyr(OBn)-Ser(OBn).

11. A compound according to Claim 1 selected from the group consisting of: Cbz-D-His-homoTyr(OBn)-Ser(OBn)-C02Me; Cbz-His-Phe(4-Ph)-Ser(OBn)-C02Me; 5 Cbz-D-His-Phe (4-Ph)-Ser(OBn)-C02Me; Cbz-His-Tyr(OBn)-Pyr-C02Me; and Cbz-D-His-Tyr (OBn) -Pyr-C02Me.

12. A compound according to Claim 1 selected from the group consisting of: Cbz-His-Tyr (OBn)-CONHCH2CH2OBn; Cbz-D-His-Tyr (OBn) -CONHCH2CH2OBn; 5 Cbz-His- (N-Me) Tyr (OBn) -CONHCH2CH2OBn; Cbz-D-His- (N-Me) Tyr (OBn) -CONHCH2CH2OBn; Cbz-His-Tyr (OBn) -C0NH(CH2) 2Ph; and Cbz-D-His-Tyr (OBn) -CONH (CH2) 2Ph.

13. A compound according to Claim 1 selected from the group consisting of: Cbz-His-Tyr (OBn) -Gly-C02Bn; Cbz-D-His-Tyr (OBn) -Gly-C02Bn; WO 95/12612 PCT/US94/11553 •60- 275 6 Cbz-His-Tyr (OBn)-Gly-CONHBn; and Cbz-D-His-Tyr(OBn)-Gly-CONHBn.

14. A compound according to Claim 1 selected from the group consisting of: BnNHCO-His - Tyr (OBn) - Ser (OBn) - C02Me; BnNHCO-His-Tyr (OBn) -Ser (OBn) -CONH2; BnNHCO-His-Tyr (OBn) -Ser (OBn) -CONHEt; BnNHCO-His-Tyr (OBn) -Ser (OBn) ; ' ^ FEB 1998 BnNHCO-His-Tyr (OBn)-CONHCH2CH2OBn; and of NevvZoafand BnNHCO-His-Tyr (OBn) -CONHCH2CH2CH2OPh.

15. A compound according to Claim 1 selected from the group consisting of: BnNHCO-D-His-Tyr (OBn) -Ser (OBn) -C02Me; BnNHCO-D-His-Tyr (OBn) -Ser (OBn) -CONH2; BnNHCO-D-His-Tyr (OBn) -Ser (OBn) -CONHEt; BnNHCO-D-His-Tyr (OBn) -Ser (OBn) ; BnNHCO-D-His - Tyr (OBn) - CONHCH2CH2OBn; and BnNHCO - D- His - Tyr (OBn) -CONHCH2CH2CH2OPh.

16. A compoiind according to Claim 1 selected from the group consisting of: Cbz-His-Tyr (OBn) -CON(Me) CH2CH20Bn; (4 - EtOPh) NHCO - D - Hi s - Tyr (OBn) - CONH (CH2) 3OPh ; PhCH2CO-D-His-Tyr (OBn) -CONH(CH2)3- (2-MeOPh) ; (4-PhOPh) NHCO-D-His-Tyr (OBn) -C0HN (CH2) 2Ph; and (4-MePh) S02-D-His-Tyr (OBn) -CO(4-Bn-piperazin-l-yl) .

17. The use of a compound according to claim 1 in the manufacture of a medicament for treating tissue proliferative diseases. ^ WO 95/12612 PCT/US94/11553 61- 27 569 ^

18. A pharmaceutical composition adapted for administration as an antiproliferative agent comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient, diluent or carrier.

19. The use of a compound according to claim 1 in the manufacture of a medicament for treating cancer.

20. A pharmaceutical composition adapted for administration as an anticancer agent comprising a therapeutically effective amount of a compound according to Claim l in admixture with a pharmaceutically acceptable excipient, diluent or carrier.

21. The use of a compoiind according to claim 1 in the manufacture of a medicament for treating restenosis.

22. A pharmaceutical composition adapted for administration as a restenosis inhibiting agent comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient, diluent, or carrier.

23. A process for the preparation of organic compounds according to Claim l, or a pharmaceutically acceptable salt thereof, comprising the steps of employing solid phase support technology and 5 sequentially coupling building blocks utilizing a WO 95/12612 PCT/US94/11553 27569 1 -62- solid phase peptide synthesizer, cleaving the organic compound from the solid phase support and subsequently optionally modifying the C-terminus of the organic compound in solution phase to 10 afford a compound or a pharmaceutically acceptable salt thereof of Formula I.

24. A process for the preparation of compounds according to Claim 1, or a pharmaceutically acceptable salt thereof, comprising the steps of employing solution phase technology and 5 sequentially coupling building blocks to afford a compound or a pharmaceutically acceptable salt thereof of Formula I. . . ' lnd?aftU0l Property Office

25. A process for the preparation of compounds according to Claim l, or a pharmaceutically 1 2 FEB acceptable salt thereof, comprising simultaneo^^^ ^land synthesis of compounds of Formula I in a multiple 5 simultaneous synthesis apparatus, using a D- or L- histidine containing dipeptide derivative that is supported on 2-chlorotrityl resin with sequential deprotection and acylation of the N-terminus followed by sequential deprotection of the carboxy 10 terminus, carboxyl activation and condensation with a series of amines, followed by cleavage from the solid support.

26. A compound as claimed in claim 1 as specifically set forth herein.

27. A process for the preparation of a compound of claim 1 substantially as herein described with reference to any one of the Examples.

28. A pharmaceutical composition containing a compound of claim 1 substantially as herein described with reference to any example thereof. END OF CLAIMS