AU715632B2 - Conjugates useful in the treatment of prostate cancer - Google Patents

Description

WO 98/10651 PCT/US97/16087 -1- TITLE OF THE INVENTION CONJUGATES USEFUL IN THE TREATMENT OF PROSTATE CANCER BACKGROUND OF THE INVENTION In 1994 cancer of the prostate gland is expected to be diagnosed in 200,000 men in the U.S. and 38,000 American males will die from this disease (Gamick, M.B. (1994). The Dilemmas of Prostate Cancer. Scientific American, April:72-81). Thus, prostate cancer is the most frequently diagnosed malignancy (other than that of the skin) in U.S. men and the second leading cause of cancer-related deaths (behind lung cancer) in that group.

Prostate specific Antigen (PSA) is a single chain 33 kDa glycoprotein that is produced almost exclusively by the human prostate epithelium and occurs at levels of 0.5 to 2.0 mg/ml in human seminal fluid (Nadji, Taber, Castro, et al. (1981) Cancer 48:1229; Papsidero, Kuriyama, Wang, et al. (1981). JNCI 66:37; Qui, Young, Bihartz, et al. (1990), J. Urol.

144:1550; Wang, Valenzuela, Murphy, et al. (1979).

Invest. Urol. 17:159). The single carbohydrate unit is attached at asparagine residue number 45 and accounts for 2 to 3 kDa of the total molecular mass. PSA is a protease with chymotrypsin-like specificity (Christensson, Laurell, Lilja, H. (1990). Eur. J. Biochem.

194:755-763). It has been shown that PSA is mainly responsible for dissolution of the gel structure formed at ejaculation by proteolysis of the major proteins in the sperm entrapping gel, Semenogelin I and Semenogelin II, and fibronectin (Lilja, H. (1985). J. Clin. Invest.

76:1899; Lilja, Oldbring, Rannevik, et al. (1987). J. Clin.

Invest. 80:281; McGee, Herr, J.C. (1988). Biol. Reprod. 39:499).

The PSA mediated proteolysis of the gel-forming proteins generates several soluble Semenogelin I and Semenogelin II fragments and soluble fibronectin fragments with liquefaction of the ejaculate and release of progressively motile spermatoza (Lilja, Laurell, C.B. (1984).

Scand. J. Clin. Lab. Invest. 44:447; McGee, Herr, J.C. (1987).

WO 98/10651 PCT/US97/16087 -2- Biol. Reprod. 37:431). Furthermore, PSA may proteolytically degrade IGFBP-3 (insulin-like growth factor binding protein 3) allowing IGF to stimulate specifically the growth of PSA secreting cells (Cohen et al., (1992) J. Clin. Endo. Meta. 75:1046-1053).

PSA complexed to alpha 1 antichymotrypsin is the predominant molecular form of serum PSA and may account for up to of the detected serum PSA (Christensson, Bjirk, Nilsson, et al. (1993). J. Urol. 150:100-105; Lilja, Christensson, A., Dahl6n, U. (1991). Clin. Chem. 37:1618-1625; Stenman, U.H., Leinoven, Alfthan, et al. (1991). Cancer Res. 51:222-226). The prostatic tissue (normal, benign hyperplastic, or malignant tissue) is implicated to predominantly release the mature, enzymatically active form of PSA, as this form is required for complex formation with alpha 1 antichymotrypsin (Mast, Enghild, Pizzo, et al.

(1991). Biochemistry 30:1723-1730; Perlmutter, Glover, G.I., Rivetna, et al. (1990). Proc. Natl. Acad. Sci. USA 87:3753-3757).

Therefore, in the microenvironment of prostatic PSA secreting cells the PSA is believed to be processed and secreted in its mature enzymatically active form not complexed to any inhibitory molecule. PSA also forms stable complexes with alpha 2 macroglobulin, but as this results in encapsulation of PSA and complete loss of the PSA epitopes, the in vivo significance of this complex formation is unclear. A free, noncomplexed form of PSA constitutes a minor fraction of the serum PSA (Christensson, Bjirk, Nilsson, et al. (1993). J. Urol.

150:100-105; Lilja, Christensson, Dahl6n, U. (1991). Clin.

Chem. 37:1618-1625). The size of this form of serum PSA is similar to that of PSA in seminal fluid (Lilja, Christensson, Dahl6n, U.

(1991). Clin. Chem. 37:1618-1625) but it is yet unknown as to whether the free form of serum PSA may be a zymogen; an internally cleaved, inactive form of mature PSA; or PSA manifesting enzyme activity.

However, it seems unlikely that the free form of serum PSA manifests enzyme activity, since there is considerable (100 to 1000 fold) molar excess of both unreacted alpha 1 antichymotrypsin and alpha 2 macroglobulin in serum as compared with the detected serum levels of WO 98/10651 PCT/US97/16087 -3the free 33 kDa form of PSA (Christensson, Bjork, Nilsson, O., et al. (1993). J. Urol. 150:100-105; Lilja, Christensson, A., Dahl6n, U. (1991). Clin. Chem. 37:1618-1625).

Serum measurements of PSA are useful for monitoring the treatment of adenocarcinoma of the prostate (Duffy, M.S. (1989). Ann.

Clin. Biochem. 26:379-387; Brawer, M.K. and Lange, P.H. (1989).

Urol. Suppl. 5:11-16; Hara, M. and Kimura, H. (1989). J. Lab. Clin.

Med. 113:541-548), although above normal serum concentrations of PSA have also been reported in benign prostatic hyperplasia and subsequent to surgical trauma of the prostate (Lilja, Christensson, Dahl6n, U. (1991). Clin. Chem. 37:1618-1625). Prostate metastases are also known to secrete immunologically reactive PSA since serum PSA is detectable at high levels in prostatectomized patients showing widespread metatstatic prostate cancer (Ford, Butcher, Masters, et al. (1985). Brit. J. Urology 57:50-55).

Therefore, a cytotoxic compound that could be activated by the proteolytic activity of PSA should be prostate cell specific as well as specific for PSA secreting prostate metastases.

It is the object of this invention to provide a novel anticancer composition useful for the treatment of prostate cancer which comprises oligopeptides having solubility augmenting oligopeptide blocking groups in conjugation with a cytotoxic agent.

Another object of this invention is to provide a method of treating prostate cancer which comprises administration of the novel anti-cancer composition.

SUMMARY OF THE INVENTION Chemical conjugates which comprise oligopeptides, having amino acid sequences that are selectively proteolytically cleaved by free prostate specific antigen (PSA), hydrophilic oligopeptide blocking groups and known cytotoxic agents are disclosed. Such conjugates are useful in the treatment of prostatic cancer and benign prostatic hypertrophy (BPH).

WO 98/10651 PCT/US97/16087 -4- DETAILED DESCRIPTION OF THE INVENTION The instant invention relates to novel anti-cancer compositions useful for the treatment of prostate cancer. Such compositions comprise the oligopeptides covalently bonded directly, or through a chemical linker, to a cytotoxic agent. The oligopeptides are chosen from oligomers that are selectively recognized by the free prostate specific antigen (PSA) and are capable of being proteolytically cleaved by the enzymatic activity of the free prostate specific antigen.

Such a combination of an oligopeptide and cytotoxic agent may be termed a conjugate.

The conjugates of the instant invention are further characterized by having a hydrophilic blocking group at the N-terminus of the oligopeptide which contributes to the aqueous solubility of the conjugate. Examples of such hydrophilic blocking groups include but are not limited to hydroxylated and polyhydroxylated alkanoyl moieties and alkanoyl moieties that incorporate ether functionalities.

Ideally, the cytotoxic activity of the cytotoxic agent is greatly reduced or absent when the oligopeptide containing the PSA proteolytic cleavage site is bonded directly, or through a chemical linker, to the cytotoxic agent and is intact. Also ideally, the cytotoxic activity of the cytotoxic agent increases significantly or returns to the activity of the unmodified cytotoxic agent upon proteolytic cleavage of the attached oligopeptide at the cleavage site.

Furthermore, it is preferred that the oligopeptide is selected from oligopeptides that are not cleaved or are cleaved at a much slower rate in the presence of non-PSA proteolytic enzymes when compared to the cleavage of the oligopeptides in the presence of free enzymatically active PSA.

For the reasons above, it is desireable for the oligopeptide to comprise a short peptide sequence, preferably less than ten amino acids. Most preferably the oligopeptide comprises seven or fewer amino acids. Because the conjugate preferably comprises a short amino acid sequence, the solubility of the conjugate may be influenced to a greater extent by the generally hydrophobic character of the cytotoxic WO 98/10651 PCT/US97/16087 agent component. Therefore, the hydrophilic blocking groups of the instant conjugates are selected to offset or diminish such a hydrophobic contribution by the cytotoxic agent.

While it is not necessary for practicing this aspect of the invention, a preferred embodiment of this invention is a conjugate wherein the oligopeptide, and the chemical linker if present, are detached from the cytotoxic agent by the proteolytic activity of the free PSA and any other native proteolytic enzymes present in the tissue proximity, thereby releasing unmodified cytotoxic agent into the physiological environment at the place of proteolytic cleavage.

Pharmaceutically acceptable salts of the conjugates are also included.

It is understood that the oligopeptide that is conjugated to the cytotoxic agent, whether through a direct covalent bond or through a chemical linker, does not need to be the oligopeptide that has the greatest recognition by free PSA and is most readily proteolytically cleaved by free PSA. Thus, the oligopeptide that is selected for incorporation in such an anti-cancer composition will be chosen both for its selective, proteolytic cleavage by free PSA and for the cytotoxic activity of the cytotoxic agent-proteolytic residue conjugate (or, in what is felt to be an ideal situation, the unmodified cytotoxic agent) which results from such a cleavage. The term "selective" as used in connection with the proteolytic PSA cleavage means a greater rate of cleavage of an oligopeptide component of the instant invention by free PSA relative to cleavage of an oligopeptide which comprises a random sequence of amino acids. Therefore, oligopeptide component of the instant invention is a prefered substrate of free PSA. The term "selective" also indicates that the oligopeptide is proteolytically cleaved by free PSA between two specific amino acids in the oligopeptide.

The oligopeptide components of the instant invention are selectively recognized by the free prostate specific antigen (PSA) and are capable of being proteolytically cleaved by the enzymatic activity of the free prostate specific antigen. Such oligopeptides comprise an oligomer selected from: WO 98/10651 WO 9810651PCT/US97/16087 -6a) b) c) d) AsnLysIleSerTyrGlnlSer LyslleSerTyrGInjSer AsnLyslleSerTyrTyrjSer AsnLysAlaSerTyrG miSer (SEQ.LD.NO.: 1), (SEQ.LD.NO.: 2), (SEQ.ID.NO.: 3), (SEQ.ID.NO.: 4), e) SerTyrGinjSerSer f) LysTyrGinjSerSer g) hArgTyrGlnlSerSei h) hArgChaGlnjSerSei i) TyrGiniSerSer j) TyrGlnlSerLeu k) TyrGinjSerNie 1) ChgGlnjSerLeu m) ChgGlnjSerNle (SEQ.ID.NO.: (SEQ.LD.NO.: 6); (SEQ.LD.NO.: 7); r (SEQ.ID.NO.: 8); (SEQ.LD.NO.: 9); (SEQ.ID.NO.: (SEQ.ID.NO.: 11); (SEQ.ID.NO.: 12); (SEQ.ID.NO.: 13); wherein hArg is homoarginine, Cha is cyclohexylalanine and Chg is cyclohexyiglycine.

In an embodiment of the instant invention, the oligopeptide comprises an oligomer that is selected from: a) AsnLys~leSerTyrGlniSerSer (SEQ.ID.NO.: 14), WO 98/10651 WO 9810651PCT/US97116087 b)

C)

d) e) f) g) h) 1) j) k) m) AsnLyslleSerTyrGlnlSerAla AlaAsnLys LieSerTyrTyri Ser AlaAsnLysAlaSerTyrGlnlSer SerTyrGlnlSerSerThr

(SI

SerTyrGIniSerSerSer

(S[

LysTyrGlnjSerSerSer

(SI

hArgTyrGlnjSerSerSer SerTyrGlnlSerSerLeu

(SI

SerTyrGlnlSerLeu

(SEQ.

SerChgGlnjSerLeu

(SEQ

hArgChgGlnjSerLeu

(SE(

hArgTyrGlnlSerLeu

(SE

(SEQ.ID.NO.: (SEQ.LD.NO.: 16), (SEQ.ID.NO.: 17), EQ. ID. NO.: 18), ~Q.ID.NO.: 19), EQ.ID.NO.: SEQ.ID.NO.: 21), ~Q.LD.NO.: 22); ID.NO.: 23); .ID.NO.: 24); 25); and ~Q.LD.NO.: 26).

In a more preferred embodiment of the instant invention, the oligopeptide comprises an oligomer selected from: GlyGluAsnGlyVaIG~nLysAspVaISerGnArgSerlieTyriSerGInThrGlu (SEQ.ID.NO.: 27), AlaSerTyrGlnlSerSerLeu (SEQ.LD.NO.: 28); SerhArgChgGlnjSerLeu (SEQ.ID.NO.: 29); WO 98/10651 WO 9810651PCTIUS97/16087 -8 hArgSerSerTyrGlnjSerNle (SEQ.ID.NO.: hArgAlaSerChgGlnjSerLeu (SEQ.LD.NO.: 31); hArgSerSerTyrGlnjSerLeu (SEQ.LD.NO.: 32); hArgSerSerChglSerLeu (SEQ.LD.NO.: 33); SerhArgChgGlnlSerLeu (SEQ.LD.NO.: 34); hArgTyrGlnlSerLeu (SEQ.ID.NO.: hArgSerSerChgGlnlSerLeu (SEQ.ID.NO.: 36); SerhArgTyrGlnjSerLeu (SEQ.ID.NO.: 37); SerSerTyrGlnjSerLeu (SEQ.ID.NO.: 38); SerSerSerChgGlnjSerLeu (SEQ.ID.NO.: 39); 3PAL-.SerSerChgG~nISerLeu (SEQ.ID.NO.: SerSerChgGlnjSerLeu (SEQ.LD.NO.: 41); SerSerSerChgGlnlSer(dLeu) (SEQ.ID.NO.: 42); SerSerSerChgGlnjSerVal (SEQ.ID.NO.: 43); ProSerSerChgGlnjSerVal (SEQ.ID.NO.: 44); GlySerSerChgGlnjSerLeu (SEQ.LD.NO.: hSerSerSerChgG in SerLeu (SQDNO:4) (SEQ.ID.NO.: 46); WO 98/10651 PCT/US97/16087 -9hArgSerSerChgGlnjSerNle (SEQ.ID.NO.: 47); hArgTyrGln|SerSerSerLeu (SEQ.ID.NO.: LysTyrGlnjSerSerSerLeu (SEQ.ID.NO.: 56); SerTyrGlnlSerSerSerLeu (SEQ.ID.NO.: 57); SerSerChgGln-Ser(dLeu) (SEQ.ID.NO.: 58); and 3PAL-SerSerChgGln-Ser(dLeu) (SEQ.ID.NO.: 59); and AlaSerChgGln-SerLeu (SEQ.ID.NO.: The phrase "oligomers that comprise an amino acid sequence" as used hereinabove, and elsewhere in the Detailed Description of the Invention, describes oligomers of from about 3 to about 100 amino acids residues which include in their amino acid sequence the specific amino acid sequence decribed and which are therefore proteolytically cleaved within the amino acid sequence described by free PSA. Preferably, the oligomer is from 5 to amino acid residues. Thus, for example, the following oligomer: hArgSerAlaChgGlnjSerLeu (SEQ.ID.NO.: 48); comprises the amino acid sequence: ChgGlnjSerLeu (SEQ.ID.NO.: 12); and would therefore come within the instant invention. It is understood that such oligomers do not include semenogelin I and semenogelin II.

A person of ordinary skill in the peptide chemistry art would readily appreciate that certain amino acids in a biologically active oligopeptide may be replaced by other homologous, isosteric and/or isoelectronic amino acids wherein the biological activity of the original oligopeptide has been conserved in the modified oligopeptide. Certain WO 98/10651 PCT/US97/16087 unnatural and modified natural amino acids may also be utilized to replace the corresponding natural amino acid in the oligopeptides of the instant invention. Thus, for example, tyrosine may be replaced by 3-iodotyrosine, 2-methyltyrosine, 3-fluorotyrosine, 3-methyltyrosine and the like. Further for example, lysine may be replaced with N'-(2-imidazolyl)lysine and the like. The following list of amino acid replacements is meant to be illustrative and is not limiting: Original Amino Acid Ala Arg Asn Asp Glu Gin Gly Ile Leu Lys Met Omithine Phe Ser Thr Trp Tyr Val Replacement Amino Acid(s) Gly Lys, Omithine Gln Glu Asp Asn Ala Val, Leu, Met, Nie Ile, Val, Met, Nle Arg, Omithine Leu, Ile, Nle, Val Lys, Arg Tyr, Trp Thr Ser Phe, Tyr Phe, Trp Leu, Ile, Met, Nie Thus, for example, the following oligopeptides may be synthesized by techniques well known to persons of ordinary skill in the art and would be expected to be proteolytically cleaved by free PSA: AsnArgIleSerTyrGlnjSer (SEQ.ID.NO.: 49) AsnLysValSerTyrGlnjSer (SEQ.ID.NO.: AsnLysMetSerTyrGlnjSerSer (SEQ.ID.NO.: 51) WO 98/10651 PCT/US97/16087 11 AsnLysLeuSerTyrGln ISerSer AsnLysIleSerTyrGlnlSer GlnLysIleSerTyrGlnlSerSer (SEQ.ID.NO.: 52) (SEQ.ID.NO.: 53) (SEQ.ID.NO.: 54).

The inclusion of the symbol within an amino acid sequence indicates the point within that sequence where the oligopeptide is proteolytically cleaved by free PSA.

The compounds of the present invention may have asymmetric centers and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers, including optical isomers, being included in the present invention. Unless otherwise specified, named amino acids are understood to have the natural "L" stereoconfiguration The following abbreviations are utilized in the specification and figures to denote the indicated amino acids and moieties: hR or hArg: hY or hTyr: Cha: Amf:

DPL:

(imidazolyl)K: Me2PO3-Y: O-Me-Y:

TIC:

DAP:

TFA:

AA:

3PAL homoarginine homotyrosine cyclohexylalanine 4-aminomethylphenylalanine 2-(4,6-dimethylpyrimidinyl)lysine N'-(2-imidazolyl)lysine O-dimethylphosphotyrosine O-methyltyrosine tetrahydro-3-isoquinoline carboxylic acid 1,3-diaminopropane trifluoroacetic acid acetic acid 3-pyridyl-alanine The conjugates of the instant invention comprise oligomers wherein the N-terminus amino acid is modified with a hydrophilic blocking group. Such blocking groups are chosen based upon the presence of hydrophilic functionality. The presence of the hydrophilic WO 98/10651 PCT/US97/16087 12 functionality distinquishes the instant conjugates from conjugates previously disclosed that also had N-terminus blocking groups. Such blocking of the terminal amino group may also reduce or eliminate the enzymatic degradation of such peptidyl therapeutic agents by the action of exogenous amino peptidases which are present in the blood plasma of warm blooded animals. Blocking groups that increase the hydrophilicity of the conjugates and therefore increase the aqueous solubility of the conjugates include but are not limited to hydroylated alkanoyl, polyhydroxylated alkanoyl, polyethylene glycol, glycosylates, sugars and crown ethers.

Preferably the blocking group is selected from

HO,

R

1

R

2

H

3 C 0 q P o wherein: R1 a) b) and R 2 are independently selected from: hydrogen, unsubstituted or substituted aryl, unsubstituted or substituted heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, halogen, Cl-C6 perfluoroalkyl, R 12 0-,

R

3

C(O)NR

3

(R

3

R

3 2N-C(NR 3

R

4 S(O)mNH, CN, N02, R 3 N3, -N(R 3 or R 4 0C(O)NR 3 unsubstituted C1-C6 alkyl, substituted C -C6 alkyl wherein the substituent on the substituted C1-C6 alkyl is selected from unsubstituted or WO 98/10651 PCT/US97/16087 13 substituted aryl, unsubstituted or substituted heterocyclic, C3-C10 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, R 3 0-,

R

4 S(O)mNH, R 3

C(O)NR

3

(R

3

R

3 2N-C(NR 3 CN, R 3 N3, -N(R 3 and R 4 0C(O)-NR 3 or R I and R 2 are combined to form (CH2)s wherein one of the carbon atoms is optionally replaced by a moiety selected from: 0, S(O)m, NH and -N(COR 4

R

3 is selected from: hydrogen, aryl, substituted aryl. heterocycle, substituted heterocycle, Cl-C6 alkyl and C3-C10 cycloalkyl;

R

4 is selected from: aryl, substituted aryl, heterocycle, substituted heterocycle, CI-C6 alkyl and C3-C10 cycloalkyl; mis 0, 1 or 2; nis 1, 2, 3 or 4; p is zero or an integer between 1 and 100; and q is 0 or 1, provided that if p is zero, q is 1; and s is 3, 4 or The conjugates of the present invention may have asymmetric centers and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers, including optical isomers, being included in the present invention. When any variable aryl, heterocycle, R 3 etc.) occurs more than one time in any constituent, its definition on each occurence is independent of every other occurence. For example, HO(CRIR 2 represents HOCH2CH2-, HOCH2CH(OH)-, HOCH(CH3)CH(OH)-, etc. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

As used herein, "alkyl" and the alkyl portion of aralkyl and similar terms, is intended to include both branched and straight-chain WO 98/10651 PCT/US97/16087 14saturated aliphatic hydrocarbon groups having the specified number of carbon atoms; "alkoxy" represents an alkyl group of indicated number of carbon atoms attached through an oxygen bridge.

As used herein, "cycloalkyl" is intended to include nonaromatic cyclic hydrocarbon groups having the specified number of carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

"Alkenyl" groups include those groups having the specified number of carbon atoms and having one or several double bonds.

Examples of alkenyl groups include vinyl, allyl, isopropenyl, pentenyl, hexenyl, heptenyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, 1-propenyl, 2-butenyl, 2-methyl-2-butenyl, isoprenyl, faresyl, geranyl, geranylgeranyl and the like.

"Alkynyl" groups include those groups having the specified number of carbon atoms and having one triple bonds. Examples of alkynyl groups include acetylene, 2-butynyl, 2-pentynyl, 3-pentynyl and the like.

"Halogen" or "halo" as used herein means fluoro, chloro, bromo and iodo.

As used herein, "aryl," and the aryl portion of aralkyl and aroyl, is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 members in each ring, wherein at least one ring is aromatic.

Examples of such aryl elements include phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl.

The term heterocycle or heterocyclic, as used herein, represents a stable 5- to 7-membered monocyclic or stable 8- to 11-membered bicyclic heterocyclic ring which is either saturated or unsaturated, and which consists of carbon atoms and from one to four heteroatoms selected from the group consisting of N, 0, and S, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure. Examples of such heterocyclic elements WO 98/10651 WO 9810651PCTIUS97/16087 15 include, but are not limited to, azepinyl, benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinno linyl, dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, furyl, imidazolidinyl, imidazolinyl, imidazolyl, indolinyl, indolyl, isochromanyl, i soindolinyl, isoquino linyl, isothiazolidinyl, isothiazolyl, isothiazolidinyl, morpholinyl, naphthyridinyl, oxadiazo lyl, 2.-oxoazepinyl, oxazolyl, 2-oxopiperazinyl, 2-oxopiperdinyl, 2-oxopyrrolidinyl, piperidyl, piperazinyl, pyridyl, pyrazinyl, pyrazo lidinyl, pyrazo lyl, pyridazinyl, pyrimidinyl, pyrrolidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiazolyl, thiazolinyl, thienofuryl, thienothienyl, and thienyl.

As used herein in the terms "substituted CI _g alkyl", "substituted aryl" and "substituted heterocycle" include moieties containing from 1 to 3 substituents in addition to the point of attachment to the rest of the compound. Such additional substituents are selected from F, Cl, Br, CF3, NH2, N(C I-C6 alkyl)2, N02, CN, (ClI-C6 alkyl)O-, -OH, (CI-C6 alkyl)S(O)m-, (CI-C6 alkyl)C(O)NH-, H2N-C(NIH)-, (C1-C6 alkyl)C(O)-, (CI-C6 alkyl)OC(O)-, N3, (CI-C6 alkyl)OC(O)NH- and CI-C20 alkyl.

When R I and R 2 are combined to form (CH2)s the cyclic moieties and heteroatom -containing cyclic moieties so defined include, but are not limited to: WO 98/10651 PCT/US97/16087 -16-

H

o0 H

O

COR

4 As used herein, the term "PEG" represents certain polyethylene glycol containing substituents having the designated number of ethyleneoxy subunits. Thus the term PEG(2) represents H3C- O 0- 0 and the term PEG(6) represents H3C-o//O O O O O O- As used herein, the term "(d)(2,3-dihydroxypropionyl)" represents the following structure:

OH

HO

0 As used herein, the term "(2R,3S) 2,3,4trihydroxybutanoyl" represents the following structure:

OH

HO

HO

WO 98/10651 PCT/US97/16087 17- Because the conjugates of the invention can be used for modifying a given biological response, cytotoxic agent is not to be construed as limited to classical chemical therapeutic agents. For example, the cytotoxic agent may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, cQ-interferon, pinterferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-l interleukin-2 interleukin-6 granulocyte macrophage colony stimulating factor granulocyte colony stimulating factor or other growth factors.

The preferred cytotoxic agents include, in general, alkylating agents, antiproliferative agents, tubulin binding agents and the like. Preferred classes of cytotoxic agents include, for example, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the taxanes, the pteridine family of drugs, diynenes and the podophyllotoxins. Particularly useful members of those classes include, for example, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, dichloro-methotrexate, mitomycin C, porfiromycin, 6-mercaptopurine, cytosine arabinoside, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine, taxol and the like. Other useful cytotoxic agents include estramustine, cisplatin and cyclophosphamide. One skilled in the art may make chemical modifications to the desired cytotoxic agent in order to make reactions of that compound more convenient for purposes of preparing conjugates of the invention.

A highly preferred group of cytotoxic agents for the present invention include drugs of the following formulae: WO 98/10651 WO 9810651PCTfUS97/16087 18 THE METHOTREXATE GROUP OF FORMULA( F:

H

2 N N N) 7 R 8

COR

9 N N 12 CONHCHCH 2

CH

2

CO

2

H

R

8 in which

R

12 is amino or hydroxy;

R

7 is hydrogen or methyl;

R

8 is hydrogen, fluoro, chioro, bromo or iodo;

R

9 is hydroxy or a moiety which completes a salt of the carboxylic acid; THE MITOMYCIN GROUP OF FORMULA

H

2

N

H

3

C

H

2 000NH 2 (2) in which RIO is hydrogen or methyl; WO 98/10651 WO 9810651PCTIUS97/16087 19 THE BLEOMYCIN GROUP OF FORMULA (3)

CONH

2

NH

2

CONH

2 0 N N HO CH3H 0 N R 1 1

H

2 N l 0 0 N NHI NHN N

CH

3 N 0 I H, N OH 3 H HO OH 3

O

HO

'N

HO

OH 0 OH

H

0

OH

CONH

2 in which R I is hydroxy, amino, C1I-C3 alkylamino, di(CI-C3 alkyl)amino, C4-C6 polymethylene amino,

NH

11

-NHOH

2

CH

2

CH

2

S-CH

3 or -NHCH 2

CH

2

CH

2

CH

2

NH-C-NH

2

OH

3 MELPHALAN OF FORMULA H0 2

-CH-CH

2 N(CH 2

CH

2

CI)

2

NH

2 WO 98/10651 WO 9810651PCT/US97/16087 20 6-MERCAPTOPURINE OF FORMULA SH

H

N

N

N

N

A CYTOSINE ARABIINOSIDE OF FORMULA

NH

2

HOH

2

C,

(6) THE PODOPHYLLOTOXINS OF FORMULA(7:

HC

(7) WO 98/10651 PCTIUS97/16087 -21 in which

R

13 is

R

14 is or a phosphate hydrogen or methyl; methyl or thienyl; salt thereof; THE VINCA ALKALOID GROUP OF DRUGS OF FORMULA

H

CH

3

O'

CO

2

CH

3 in which R1 5 is H, CH3 or CHO; when R 17 and R 18 are taken singly;

R

1 8 is H, and one of R 1 6 and R 17 is ethyl and the other is H or OH; when R 17 and R 1 8 are taken together with the carbons to which they are attached, they form an oxirane ring in which case R 1 6 is ethyl;

R

1 9 is hydrogen, (C -C3 alkyl)-CO, or chlorosubstituted (C1-C3 alkyl)-CO; WO 98/10651 WO 9810651PCTfUS97/16087 22 DIFLUORONUCLEOSIDES OF FORMULA R 1 0 C H 2 0H F OH in which R 2 1 is a base of one of the formnulae: 0 0 N 2

NH

2 0 HN

N\>

IN

N

NH

2 N

N

N

'N

R

23 N N CH-CHR 2 4 in which

R

22 is hydrogen, methyl, bromo,

R

2 3 is -OH or -NH-2; fluoro, chioro or iodo; R24 is R 4 ishydrogen, bromo, chioro or iodo; WO 98/10651 WO 9810651PCT/US97/16087 23 -I THE ANTHRACYCLINES ANTIBIOTICS OF FORMULA wherein Ra is -CH3, -CH2OH, -CH2OCO(CH2)3CH3, or -CH2OCOCH(0C2H5)2; Rb is -OCH3, -OH or -H; RC is -NH2, -NHCOCF3, 4-morpholinyl, 3-cyano-4morpholinyl, I -piperidinyl, 4-methoxy- 1-piperidinyl, benzylamine, dibenzy lamine, cyanomethylamnine, or 1 -cyano-2-methoxyethyl amine; is -OH -OTHP or and

R

6 is -OH or -H provided that

R

6 is not -OH when R 5 is -OH or -OTHP.

WO 98/10651 PCT/US97/16087 -24- ESTRAMUSTINE (11)

(CICH

2

CH

2 2 N00 (11) CYCLOPHOSPHAMIDE (12) O, pN(CH 2

CH

2

CI)

2 P- 0

NH

12 The most highly preferred drugs are the anthracycline antiobiotic agents of Formula described previously. One skilled in the art understands that this structural formula includes compounds which are drugs, or are derivatives of drugs, which have acquired in the art different generic or trivial names. Table 1, which follows, represents a number of anthracycline drugs and their generic or trivial names and which are especially preferred for use in the present invention.

Table 1 Rc (11) Compound daunorubicina doxorubicinb detorubicin carrninomyc in idarubicin epirubicin esomubicin

THP

AD-32 Ra CH3

CHOH

CH2OCOCH(0C2H 5 2 CH3 CH3 CH20H CH2OH CH20H CH2OCO(CH2)3CH3 Rb OCH3 OCH3 OCH3

OH

H

OCH3 OCH3 OCH3 OCH3

RC

NH2 NH2 NH12 NH-2 NH2 NH2 NH2 NH2 NHCOCF3

OH

OH

OH

OH

OH

H

OTHP

H

OH

H

H

a,'daunomycin" is an alternative name for daunortibicin b"adriamycin" is an alternative name for doxorubicin I WO 98/10651 PCT/US97/16087 -26- Of the compounds shown in Table 1, the most highly preferred cytotoxic agents are doxorubicin, vinblastine and desacetylvinblastine. Doxorubicin (also referred to herein as "DOX") is that anthracycline of Formula (10) in which R a is -CH20H, Rc is -OCH3,

R

4 is -NH2, R 5 is -OH, and R 6 is -H.

The blocked oligopeptide-cytotoxic agent conjugate of the instant invention wherein the cytotoxic agent is the preferred cytotoxic agent doxorubicin may be described by the general formula I below: 0 OH 0

SOH

O OH O

CH

3 0 NH 0 OH oligopeptide R C-terminus N-terminus wherein: oligopeptide is an oligopeptide which is selectively recognized by the free prostate specific antigen (PSA) and is capable of being proteolytically cleaved by the enzymatic activity of the free prostate specific antigen, and wherein the C-terminus carbonyl is covalently bound to the amine of doxorubicin and the N-terminus amine is covalently bound to the carbonyl of the blocking group; R is selected from WO 98/10651 PCT/US97/16087 -27a)

HO

R

1

R

2 b)

H

3 C 0 p

R

1 and R 2 are independently selected from: hydrogen. OH, CI-C6 alkyl, C1-C6 alkoxy, CI-C6 aralkyl and aryl; n is 1, 2, 3 or 4; p is zero or an integer between 1 and 100; q is 0 or 1, provided that if p is zero, q is 1; or the pharmaceutically acceptable salt thereof.

In a preferred embodiment of the oligopeptide-cytotoxic agent conjugate: R is selected from a)

HO.

WO 98/10651 PCT/US97/16087 28 L' n' R' R 2

H

3 a R a RI and R 2 are independently selected from: hydrogen, CI -C6 alkyl and n is n' is p is q is aryl; 1, 2, 3 or 4; 0, 1, 2 or 3; zero or an integer between I and 14; 0 or 1, provided that if p is zero, q is 1; or the pharmaceutically acceptable salt thereof.

The following compounds are specific examples of the oligopeptide-cytotoxic agent conjugate of the instant invention: WO 98/10651 WO 9810651PCT/US97/16087 29

-CH

2 0H

"OH

CH

3 0

OH

3 OH x wherein X is: 0 HO,, SerSerSerChgGInSerLeu+~ 0 HO__-kSerSerChgGlnserLeu (SEQ.ID.NO.: 61), (SEQ.ID.NO.: 62), 0 'I"SerSerSerChgGnSerLeu- (SEQ.ID.NO.: 63), or the pharmaceutically acceptable salt thereof.

Further examples of conjugates of an oligopeptide and doxorubicin wherein the N-terminus of the oligopeptide is blocked by a hydrophilic moiety and the C-terminus of the oligopeptide is attached to the doxorubicin at the 3'-amine are as follows: WO 98/10651 WO 9810651PCTIUS97/16087 30 2-hydroxyacetyl-hArgSerSerTyrGln-SerNIe-DOX (3Y) (SEQ.ID.NO.: 64) 2-hydroxyacetyl-hArgSerSerChgGln-SerNle-DOX

(SEQ.LD.NO.:

2-hydroxyacetyl-SerhArgChgGln-SerLeu-DOX (SEQ.ID.NO.: 66) 2-hydroxyacetyl-hArgSerSerChgGln-SerLeu-Dox

(SEQ.LD.NO.:

67) 2-hydroxyacetyl-hArgAlaSerChgGln-SerLeu-DOX

(SEQ.ID.NO.:

68) 2,3 -dihydroxypropionyl-SerhArgChgGln-SerLeu-DOX (SEQ.ID.NO.: 69) 2,3-dihydroxypropionyl-SerhArgChgGn-SerLeu.DOX (SEQ.LD.NO.: PEG(2)-SerhArgChgGln-SerLeu-DOX (SEQ.IID.NO.: 71) PEG(2)-hArgChgGln-SerLeu-DOX (SEQ.ID.NO.: 72) (2R,3S) 2,3,4-trihydroxybutanoyl-hArgChgGln-SerLeu-DOX (SEQ.ID.NO.: 73) PEG (2)-SerhArgTyrGlIn-SerLeu-DOX(3') (SEQ.ID.NO.: 74) PEG(2)-hArgTyrGln-SerSerSerLeu-DOX (SEQ.ID.NO.: PEG (2)-LysTyrGln-SerSerSerLeu-DO X (SEQ.ID.NO.: 76) 2-hydroxyacetyl-hArgSerSerTyrGln-SerLeu-DOX

(SEQ.ID.NO.:

77) 2 ,3-dihydroxypropionyl)hArgSerSerChgGlnSerLeu..DOX (SEQ.ID.NO.: 78) PEG(2)-hArgSerSerChgGln-SerLeu-DOX (SEQ.ID.NO.: 79) 2-hydroxyacetyl-SerTyrGln-SerSerSerLeu.DOX

(SEQ.ID.NO.:

PI3G( 16)-SerhArgTyrGln-SerLeu-DOX (SEQ.ID.NO.: 81) (2R ,3S) 2,3 ,4-trihydroxybutanoyl-SerhArgChgGln-SerLeu..Dox (SEQ.LD.NO.: 82) PEG (2)-SerhArgTy-GlIn- SerLeu-DO X (SEQ.LD.NO.: 83) 2 3 -dihydroxypropionyl)-hArgSerSerChgGn-.SerLeu..DOX(3') (SEQ.ID.NO.: 84) WO 98/10651 WO 9810651PCT/US97/16087 31 (1)(2,3-dihydroxypropionyl)SerSerSerChgGln-Ser(dLeu)-DOX (SEQ.ID.NO.: (d)(2,3-dihydroxypropionyl)SerSerSerChgGln-SerLeu-DOX (SEQ.ID.NO.: 86) (1)(2,3-dihydroxypropionyl)SerSerSerChgGln-SerLeu-DOX

(Y)

(SEQ.ID.NO.: 87) (l)(2,3-dihydroxypropionyl)SerSerChgG ln-Ser(dLeu)-DOX (SEQ.ID.NO.: 88) (d)(2,3-dihydroxypropionyl)SerSerChgGln-SerLeu-DOX (SEQ.ID.NO.: 89) PEG (2)-SerSerChgGIn -Ser(dLeu)-D OX (SEQ.ID.NO.: PEG(2)SerSerChgGln-SerLeu-DOX (SEQ.ID.NO.: 91) PEG (2)-SerSerSerChgGlIn-S er(dLeu)-DOX (SEQ.LD.NO.: 92) (2,3-dihydroxypropionyl)-3PAL-SerSerChgGln-Ser(dLeu)-DOX (SEQ.ID.NO.: 93) (d)(2,3-dihydroxypropionyl)-3PAL-SerSerChgGln-SerLeu-DOX (SEQ.ID.NO.: 94) (1)(2,3-dihydroxypropionyl)-SerSerChgGln-SerLeu-DOX (SEQ.LD.NO.: (2,3-dihydroxypropionyl)-hSerSerSerChgGln-SerLeu-DOX (SEQ.LD.NO.: 96) PEG(2)-AlaSerChgGIn-SerLeu-DOX (SEQ.ID.NO.: 97) PEG erSerChgG In-SerLeu-DOX (3Y) (SEQ.LD.NO.: 98) PEG(6)-SerSerSerChgG ln-SerLeu-DOX (SEQ.ID 99) PEC(6)-AlaSerChgGln-SerLeu-DOX (SEQ.LD. NO.: 100) PEG(4)-3PALSerSerChgGln-SerLeu-DOX (SEQ.ID.NO.: 101) or the pharmaceutically acceptable salt thereof.

The oligopeptide-cytotoxic agent conjugate of the instant invention wherein the cytotoxic agent is the preferred cytotoxic agent vinbiastine or desacetylvinbiastine may be described by the general formula 11 below: WO 98/10651 PCT/US97/16087 32-

CH

2

CH

3

'OR

1 9 oligopeptide R wherein: oligopeptide is an oligopeptide which is specifically recognized by the free prostate specific antigen (PSA) and is capable of being proteolytically cleaved by the enzymatic activity of the free prostate specific antigen; XL is NH -(CH2)r NH R is selected from a) HO,/ 0

H

3 C O0 .oj \p WO 98/10651 PCTIUS97/16087 33 Ri and R 2 are independently selected from: hydrogen, OH, C1-C6 alkyl, CI-C6 alkoxy, CI-C6 aralkyl and aryl;

R

19 is hydrogen, (C1-C3 alkyl)-CO, or chlorosubstituted (Cl-C3 alkyl)-CO; n is 1, 2, 3 or 4; p is zero or an integer between 1 and 100; q is 0 or 1, provided that if p is zero, q is 1; r is 1, 2, 3, 4 or or the pharmaceutically acceptable salt thereof.

The another embodiment of the oligopeptide-cytotoxic agent conjugate of the instant invention wherein the cytotoxic agent is the preferred cytotoxic agent vinblastine or desacetylvinblastine may be described by the general formula III below:

OH

Et

H

CH

2

CH

3 CO oligopeptide NRdRe III N-terminus C-terminus wherein: WO 98/10651 PCT/US97/16087 -34oligopeptide is an oligopeptide which is specifically recognized by the free prostate specific antigen (PSA) and is capable of being proteolytically cleaved by the enzymatic activity of the free prostate specific antigen, Rd and Re are independently selected from: hydrogen, Cl-C6-alkyl, -C -C6-alkyl-OH, -C I-C6-alkyl-di-OH, -C -C6-alkyl-tri-OH and

H

3 C 0.

provided that at least one Rd and Re are not hydrogen or Cl-C6-alkyl, or Rd and Re are combined to form a -CH2CH20CH2CH2- diradical; R1 9 is hydrogen, (C -C3 alkyl)-CO, or chlorosubstituted (Cl-C3 alkyl)-CO; p is zero or an integer between 1 and 100; q is 0 or 1, provided that if p is zero, q is 1; The following compounds are specific examples of the oligopeptide-desacetylvinblastine conjugate of the instant invention: WO 98/10651 WO 9810651PCT/US97/16087 35

OH

N

1

H

N 00CH I C0CH H F

CH

2

CH

3

*OH

0 HO,) (SEQ.ID.NO.: 61), 0 OH SerSerSerChgGtn-SerLeu-

N

H

(SEO.ID.NO.: 102), WO 98/10651 PCT/US97/16087 -36or the pharmaceutically acceptable salt thereof.

The oligopeptides, peptide subunits and peptide derivatives (also termed "peptides") of the present invention can be synthesized from their constituent amino acids by conventional peptide synthesis techniques, preferably by solid-phase technology. The peptides are then purified by reverse-phase high performance liquid chromatography

(HPLC).

Standard methods of peptide synthesis are disclosed, for example, in the following works: Schroeder et al., "The Peptides", Vol. I, Academic Press 1965; Bodansky et al., "Peptide Synthesis", Interscience Publishers, 1966; McOmie "Protective Groups in Organic Chemistry", Plenum Press, 1973; Barany et al., "The Peptides: Analysis, Synthesis, Biology" 2, Chapter 1, Academic Press, 1980, and Stewart et al., "Solid Phase Peptide Synthesis", Second Edition, Pierce Chemical Company, 1984. The teachings of these works are hereby incorporated by reference.

The pharmaceutically acceptable salts of the compounds of this invention include the conventional non-toxic salts of the compounds of this invention as formed, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like: and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenyl-acetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the like.

The conjugates of the instant invention which comprise the oligopeptide containing the PSA cleavage site and a cytotoxic agent may similarly be synthesized by techniques well known in the medicinal chemistry art. For example, a free amine moiety on the cytotoxic agent may be covalently attached to the oligopeptide at the carboxyl terminus WO 98/10651 PCT/US97/16087 -37such that an amide bond is formed. Similarly, an amide bond may be formed by covalently coupling an amine moiety of the oligopeptide and a carboxyl moiety of the cytotoxic agent. For these purposes a reagent such as 2-(1H-benzotriazol-1-yl)-1,3,3-tetramethyluronium hexafluorophosphate (known as HBTU) and 1 -hyroxybenzotriazole hydrate (known as HOBT), dicyclohexyl- carbodiimide (DCC), N-ethyl-N-(3-dimethylaminopropyl)- carbodiimide (EDC), diphenylphosphorylazide (DPPA), benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate (BOP) and the like, used in combination or singularly, may be utilized.

Furthermore, the instant conjugate may be formed by a non-peptidyl bond between the PSA cleavage site and a cytotoxic agent.

For example, the cytotoxic agent may be covalently attached to the carboxyl terminus of the oligopeptide via a hydroxyl moiety on the cytotoxic agent, thereby forming an ester linkage. For this purpose a reagent such as a combination of HBTU and HOBT, a combination of BOP and imidazole, a combination of DCC and DMAP, and the like may be utilized. The carboxylic acid may also be activated by forming the nitro-phenyl ester or the like and reacted in the presence of DBU (1,8-diazabicyclo[5,4,0]undec-7-ene.

The instant conjugate may also be formed by attachment of the oligopeptide to the cytotoxic agent via a linker unit. Such linker units include, for example, a biscarbonyl alkyl diradical whereby an amine moiety on the cytotoxic agent is connected with the linker unit to form an amide bond and the amino terminus of the oligopeptide is connected with the other end of the linker unit also forming an amide bond. Conversely, a diaminoalkyl diradical linker unit, whereby a carbonyl moiety on the cyctotoxic agent is covalently attacted to one of the amines of the linker unit while the other amine of the linker unit is covalently attached to the C terminus of the oligopeptide, may also be uselful. Other such linker units which are stable to the physiological environment when not in the presence of free PSA, but are cleavable upon the cleavage of the PSA proteolytic cleavage site, are also envisioned. Furthermore, linker units may be utilized that, upon WO 98/10651 PCT/US97/16087 -38 cleavage of the PSA proteolytic cleavage site, remain attached to the cytotoxic agent but do not significantly decrease the cytotoxic activity of such a post-cleavage cytotoxic agent derivative when compared with an unmodified cytotoxic agent.

One skilled in the art understands that in the synthesis of compounds of the invention, one may need to protect various reactive functionalities on the starting compounds and intermediates while a desired reaction is carried out on other portions of the molecule. After the desired reactions are complete, or at any desired time, normally such protecting groups will be removed by, for example, hydrolytic or hydrogenolytic means. Such protection and deprotection steps are conventional in organic chemistry. One skilled in the art is referred to Protective Groups in Organic Chemistry, McOmie, ed., Plenum Press, NY, NY (1973); and, Protective Groups in Organic Synthesis, Greene, ed., John Wiley Sons, NY, NY (1981) for the teaching of protective groups which may be useful in the preparation of compounds of the present invention.

By way of example only, useful amino-protecting groups may include, for example, Cl-C10 alkanoyl groups such as formyl, acetyl, dichloroacetyl, propionyl, hexanoyl, 3,3-diethylhexanoyl, y-chlorobutryl, and the like; CI-C10 alkoxycarbonyl and C5-C15 aryloxycarbonyl groups such as tert-butoxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl, 4-nitrobenzyloxycarbonyl, fluorenylmethyloxycarbonyl and cinnamoyloxycarbonyl; halo- (Cl-C10)-alkoxycarbonyl such as 2,2,2-trichloroethoxycarbonyl; and arylalkyl and alkenyl group such as benzyl, phenethyl, allyl, trityl, and the like. Other commonly used amino-protecting groups are those in the form of enamines prepared with 3-keto-esters such as methyl or ethyl acetoacetate.

Useful carboxy-protecting groups may include, for example, CI-C10 alkyl groups such as methyl, tert-butyl, decyl; haloalkyl such as 2,2,2-trichloroethyl, and 2-iodoethyl; C5-C15 arylalkyl such as benzyl, 4-methoxybenzyl, 4-nitrobenzyl, triphenylmethyl, diphenyl-methyl; CI-C10 alkanoyloxymethyl such as acetoxy- WO 98/10651 PCT/US97/16087 -39methyl, propionoxymethyl and the like; and groups such as phenacyl, 4-halophenacyl, allyl, dimethylallyl, tri-(C -C3 alkyl)silyl, such as trimethylsilyl, P-p-toluenesulfonylethyl, P-p-nitrophenyl-thioethyl, 2,4,6-trimethylbenzyl, P-methylthioethyl, phthalimidomethyl, 2,4dinitro-phenylsulphenyl, 2-nitrobenzhydryl and related groups.

Similarly, useful hydroxy protecting groups may include, for example, the formyl group, the chloroacetyl group, the benzyl group, the benzhydryl group, the trityl group, the 4-nitrobenzyl group, the trimethylsilyl group, the phenacyl group, the tert-butyl group, the methoxymethyl group, the tetrahydropyranyl group, and the like.

With respect to the preferred embodiment of an oligopeptide combined with the anthracycline antibiotic doxorubicin, the following Reaction Schemes illustrate the synthsis of the conjugates of the instant invention.

WO 98/10651 WO 9810651PCT/US97/16087 40 REACTION SCHEME I

CH

2 0H

CH

3 0

CH

2 0H

OH

dox

CH

3

O

CH

3

NH

ofigopepticle -G

CH

3 0

CH

2 O oligopeptidle -jG

H

G is a hydrophilic N-terminus 3 blocking group WO 99/10651 WO 9810651PCT/US97/16087 41 REACTION SCHEME il

CH

2 0H

CH

3 0

NH

2

CH

2 0H

"OH

11

OH

CH

3

O

CH

3 NH-protect

OH

HN- otigopeptide TG

CH

2 0H 14

CH

3 0

OH

WO 98/10651 WO 9810651PCT/US97/16087 42 REACTION SCHEME IlI

ICH

2 0H

CH

3 0 0 Y(CH 2 2 C0 2

H

OH

NH

2 oligopeptide tG'

CH

3 0

CH

2 0H 15 G' is a hydrophilic C-terminal moiety

CH

3

NH-

2

OH

WO 98/10651 WO 9810651PCTIUS97/16087 43 REACTION SCHEME IV

CH

3 0

OH

3

NH

2

H

2

NHN

7 oligopeptide TjG 0 OH Hm oligopeptideTjG

CH

3 0

OH

WO 98/10651 WO 9810651PCT/US97/16087 44 REACTION SCHEME V

-CH

3

"OH

Br 2 CC1 4

CH

2 Br

CH

3 0

OH

HS--

H

2 N NTo~igopepide -G 0

HI

H

2

N

H

2 N N- 1 o~figopeptide-7G 0

HI

CH

3 0 Reaction Scheme VI illustrates preparation of conjugates of the oligopeptides of the instant invention and the ymnca alkaloid cytotoxic agent vinbiastine wherein the attachment of vinbiastine is at WO 98/10651 PCT/US97/16087 the C-terminus of the oligopeptide. The use of the 1,3-diaminopropane linker is illustrative only; other spacer units between the carbonyl of vinblastine and the C-terminus of the oligopeptide are also envisioned.

Furthermore, Scheme VI illustrates a synthesis of conjugates wherein the C-4-position hydroxy moiety is reacetylated following the addition of the linker unit. Applicants have discovered that the desacetyl vinblastine conjugate is also efficacious and may be prepared by eliminating the steps shown in Reaction Scheme VI of protecting the primary amine of the linker and reacting the intermediate with acetic anhydride, followed by deprotection of the amine. Conjugation of the oligopeptide at other positions and functional groups of vinblastine may be readily accomplished by one of ordinary skill in the art and is also expected to provide compounds useful in the treatment of prostate cancer.

WO 98/10651 WO 9810651PCTIUS97/16087 46 REACTION SCHEME VI

OH

*H 1. H 2 N -0H 2 0H 2 0H 2

-NH

2 2. BOO-Cl

CH

3

O

OH

3

CH

3 0 OH

OH

H3 2 NH -BOO

AC

2 0, pyridilne .aq. HCI 0H 3 0~ N 0H

OH

3 w NH 2 WO 98/10651 PCT/US97/16087 -47- REACTION SCHEME VI (Continued) G- oligopeptide

'OCOCH

3 oligopeptide -G H H

H

The oligopeptide-cytotoxic agent conjugates of the invention are administered to the patient in the form of a pharmaceutical composition which comprises a conjugate of of the instant invention and a pharmaceutically acceptable carrier, excipient or diluent therefor.

As used, "pharmaceutically acceptable" refers to those agents which are useful in the treatment or diagnosis of a warm-blooded animal including, for example, a human, equine, procine, bovine, murine, canine, feline, or other mammal, as well as an avian or other warmblooded animal. The preferred mode of administration is parenterally, particularly by the intravenous, intramuscular, subcutaneous, WO 98/10651 PCT/US97/16087 -48intraperitoneal, or intralymphatic route. Such formulatiops can be prepared using carriers, diluents or excipients familiar to one skilled in the art. In this regard, See, e.g. Remington's Pharmaceutical Sciences, 16th ed., 1980, Mack Publishing Company, edited by Osol et al. Such compositions may include proteins, such as serum proteins, for example, human serum albumin, buffers or buffering substances such as phosphates, other salts, or electrolytes, and the like. Suitable diluents may include, for example, sterile water, isotonic saline, dilute aqueous dextrose, a polyhydric alcohol or mixtures of such alcohols, for example, glycerin, propylene glycol, polyethylene glycol and the like.

The compositions may contain preservatives such as phenethyl alcohol, methyl and propyl parabens, thimerosal, and the like. If desired, the composition can include about 0.05 to about .20 percent by weight of an antioxidant such as sodium metabisulfite or sodium bisulfite.

As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specific amounts, as well as any product which results, directly or indirectly, from combination of the specific ingredients in the specified amounts.

For intravenous administration, the composition preferably will be prepared so that the amount administered to the patient will be from about .01 to about 1 g of the conjugate. Preferably, the amount administered will be in the range of about .2 g to about 1 g of the conjugate. The conjugates of the invention are effective over a wide dosage range depending on factors such as the disease state to be treated or the biological effect to be modified, the manner in which the conjugate is administered, the age, weight and condition of the patient as well as other factors to be determined by the treating physician. Thus, the amount administered to any given patient must be determined on an individual basis.

One skilled in the art will appreciate that although specific reagents and reaction conditions are outlined in the following examples, modification can be made which are meant to be encompassed by the spirit and scope of the invention. The following preparations and examples, therefore, are provided to further illustrate the invention, WO 98/10651 PCT/US97/16087 -49and are not limiting.

EXAMPLES

EXAMPLE 1 Preparation of Oligopeptides which Comprise the PSA Mediated Cleavage Site Blocked oligopeptides were prepared by solid-phase synthesis, using a double coupling protocol for the introduction of amino acids on the Applied Biosystems model 430A automated peptide synthesizer. Deprotection and removal of the oligopeptide from the resin support were achieved by treatment with liquid hydrofluoric acid. The oligopeptides were purified by preparative high pressure liquid chromatography on reverse phase C18 silica columns using an aqueous 0.1% trifluoroacetic acid/acetonitrile gradient. Identity and homogeneity of the oligopeptides were confirmed by amino acid composition analysis, high pressure liquid chromatography, and fast atom bombardment mass spectral analysis. The oligopeptides that were prepared by this method are shown in Table 2.

WO 98/10651 WO 9810651PCTIUTS97/16087 50 TABLE 2 SEQ.ID.NO. PEPTIDE PEPTIDE-DOX CONJUGATE Time to 50% Substrate_ Cleavage by ____orkSMPiA_[j 1 03 Ac-ANKASYQ-SL-acid 135 1 04 Ac-ANKASYQ-SL-acid 220 1 05 Ac-hR(CHA)Q-SNIe-acid 200 (PS) 1 06 Ac-ShRYQ-SNIe-acid 25 (PS) 1 07 Ac-ShRChgQ-SNIe-acid

INSOLUBLE

1 08 Ac-hRSSYQ-SNIe-acid 2 5 (PS) 1 09 AchRSSChgQ-SL-acid 120(45-) 6 6 2-hydroxyacetyl-ShRChgQ-SL-acid 120(30*) 11 0 Ac-hRSSYQ-SNie-acid 25 (PS) 64 2-hydroxyacetyl-hRSSYQ-SNie-acici ill Ac-hRASChgQ-SL-acid 6 8 2-hydroxyacetyl-hRASChgQ-SL-acid 64 2-hydroxyacetyl-hRSSYQ-SL-acid 35 (PP) 6 7 2-hydroxyacetyl-hRSSChgSL-acid

(PP)

6 9 2,3-dihydroxypropionylShflChgQ-SL-acid 7 0 2(S)-2,3-dihydroxy pro pionylS hRCl-igQSL-acid 11 2 2-hydroxyacetylhRYQ-SL-acid 1 29 ShRChgQ-SL -acid 4 HOUR=8% 7 1 PEG(2)-S-hRChgQ-SL-acid 11 3 PEG(1)-ShRChgQ-SL-acid 120* 7 8 2 2,3 -di hydroxypropio nly- hRSSC hg Q-S L-acid 7 9 PEG(2)-hRSSChgQ-SL-acid 74 PEG(2)-ShRYQ-SL-acid 11 4 PEG(1)-hRSSChgQ-SL-acid 115 PEG(1)-ShRYQ-SL-acid 11 6 PEG(1 5)-ShRYQ-SL-acid 81 PEG(1 6)-ShRYQ-SL-acid 11 7 PEG(1 7)-ShRYQ-SL-acid 8 2 (2R,3S) 2,3,4-trihydroxybutanoyl-ShRChgQ-SL-acid 8 3 2,3-dihydroxypropionyl-h RSSChgQ-SL-acid 11 8 PEG(2)-SSYQ-SL-acid 150 11 9 PEG(14)ShRYQ-SL-acid 1 20 PEG(18)ShRYQ-SL-acid WO 98/10651 WO 9810651PCT/US97/16087 51 TABLE 2 (continued) SEQ.ID.NO. PEPTIDE I PEPTIDE-DOX CONJUGATE Time to 50% Substrate- Cleavage by _or PQ[E~flin 1 21 PEG(19)ShRYQ-SL-acid 94 (d)2,3-dihydroxypropionyl-3PAL-SSChgQSL-acid 6 3 PEG(2)SSSChgQ-SL-acid 150 1 01 PEG(2)-3PAL-SSChgQ-SL-acid 8 7 (I)2,3-dihydroxypropionyl-SSSChgQ-S L-acid 6 1 (OH-Ac)SSSChgQ-SL-acid 120 1 22 (1)2,3-dihydroxypropionyl-SSChigQ-SL-acid 1 8 7 (I)2,3-dihydroxypropionyi-SSSChgQ-SL-acid 11 0 1 23 (1)2,3-dihydroxypropionyl-3PAL-SSChgQ-SL-acid 1 24 2,3-dihydroxypropionyl-SSSChgQ-SL-acid 120 1 25 2,3-dihydroxypropionyl-ShRYQ-SL-acid 6 2 2-hydroxyacetyl-SSChgQ-SL-acid 180 1 25 2,3-dihyd roxypropio nyl-S hRYQ-SL- acid 1 01 PEG(4)-1-PAL-SSChgQ-SL-acid 1 26 Ac-SSSChgQ-S V-acid 1 27 Ac-PSSChgQ-SV-acid 1 28 2,3-dihydroxypropionyl-GSSChgQ- SL- acid 160 9 6 2,3-dihydroxypropionyl-hSSSChgQ--SL-acid 160 EXAMPLE 2 Assessment of the Recognition of Oligopeptides by Free PSA The oligopeptides prepared as described in Example I were individually dissolved in PSA digestion buffer (.12 mM tris(hydroxymethyl)-aminomethane pH8.0, 25 mM4 NaCI, 0.5 mM CaCI2) and the solution added to PSA at a molar ration of 100 to 1.

The reaction is quenched after various reaction times by the addition of trifluoroacetic acid (TFA) to a final 1I% (volume/volume). The quenched reaction was analyzed by HPLC on a reversed-phase C 18 column using an aqueous 0. 1 %TFA/acetonitrile gradient. The results of the assessment are shown in Table 2. Table 2 shows the amount of time (in minutes) required for 50% cleavage of the noted oligopeptides with enzymatically active free PSA. Oligopeptides containing free amine WO 98/10651 PCT/US97/16087 -52moieties (ie. comprising hArg, Om, Lys and or 3PAL) were tested as TFA salts. All other oligopeptides were tested as neutral compounds.

EXAMPLE 3 Preparation of N-(2-Hydroxyacetyl)-Ser-Ser-Ser-Chg-Gln-Ser-Leu-Dox (3-3) Step A: 2-HO-Ac-Ser(Bzl)-Ser(Bzl)-Ser(Bzl)-Chg-Gln-Ser-Leu- PAM Resin Starting with 0.5 mmol (0.67g) Boc-Leu-PAM resin (Applied Biosystems Inc. ABI), the protected peptide was synthesized on a 430A ABI peptide synthesizer. The protocol used a 4 fold excess (2 mmol) of each of the following protected amino acids: Boc-Ser(OBzl), Boc-Gln, Boc-Chg. Coupling was achieved using DCC and HOBT activation in methyl-2-pyrrolidinone. Removal of the Boc group was performed using 50% TFA in methylene chloride and the TFA salt neutralized with diisopropylethylamine. 2-Hydroxyacetic acid was used for the introduction of the N terminal blocking group, which was also carried out on the peptide synthesizer. At the completion of the synthesis, the peptide resin was dried to provide the title resinpeptide conjugate.

Step B: 2-HO-Ac-Ser-Ser-Ser-Chg-Ser-Leu-OH The protected peptide resin 1.2 g, was treated with HF (15 ml) for lhr at 0°C in the presence of anisole (1.5 ml). After evaporation of the HF, the residue was washed with ether 3 times, and extracted with 20% HOAc. The crude peptide products from the HF-cleavage after lyophilization were purified by preparatory HPLC on a Delta-Pak C18 column with 0.1 trifluoroacetic acid -aqueous acetonitrile solvent systems using 100-70% 0.1%TFA-H20, linear gradient. Fractions containing product of at least 99% (HPLC) purity were combined to provide the title blocked peptide.

ft WO 98/10651 PCT/US97/16087 -53

FABMS:

Peptide Content:

HPLC:

804.85 1.03NMOle/mg.

99% pure @214, retention times= 11.16 min, (Vydac C18, gradient of 95%A/B to 50%A/B over 30 min, A=0.1%TFA-H20, B=0.1%TFA-CH3CN) Step C: 2 -HO-Ac-Ser-Ser-Ser-Chg-Ser-Leu-Dox (3-3) A solution of 241 mg (0.30 mmol) of OH-Ac-Ser-Ser-Ser- Chg-Gln-Leu-OH in 3.0 ml anhyd. N-methyl pyrrolidine (NMP) (or DMF), 46 mg (0.30 mmol) of HOBT, 63 mg (0.33 mmol) of EDC 46 mg (0.09 mmol) of doxorubicin was added and pH was adjusted with diisopropylethylamine (DIEA) to pH 8.5. The solution was stirred at 0 0 C for 1 lhrs., and then reaction was quenched by The organic solvent was removed under reduced pressure and the residue was diluted with 15ml of water, and purified by preparative HPLC using a NH4Ac (4g/4L)-CH3CN gradient, ie. 95-50%A, 60min. Lyophilization of pure fractions gave a red powder. The red powder was dissolved in distil.

filtered, and lyophilized to provide the title conjugate ES+ NH4+ Peptide Content:

HPLC:

1347.61 541.72 NMOle/mg.

99% pure @214, retention times= 20.8 min, (Vydac C18, gradient of 95%A/B to 50%A/B over 30 min, A=0.1%TFA-H20, B=0.1%TFA, CH3CN) EXAMPLE 4 Preparation of 2 -(2-methoxyethoxy)ethoxy }acetyl]-Ser-Ser-Ser- Chg-Gln-Ser-Leu-Dox The title conjugate was prepared in the manner described in Example 3, but substituting 2 2 2 -methoxyethoxy)ethoxy} acetic acid for 2 -hydroxyacetic acid in Step A.

WO 98/10651 PCT/US97/16087 54- ES+ NH4+ Peptide Content:

HPLC:

1450.72 534.36 NMOle/mg.

99% pure @214, retention times= 21.99 min, (Vydac Cl8, gradient of 95%A/B to 50%A/B over 30 min, A=0.1%TFA-H20, B=0.1%TFA, CH3CN) EXAMPLE Preparation of N-2(R)-2,3-dihydroxypropionyl-Ser-Ser-Ser-Chg-Gln- Ser-Leu-Dox (5-3) Step A: N-2(R)-2,3-dihydroxypropionyl-Ser(Bzl)-Ser(Bzl)- Ser(Bzl)-Chg-Gln-Ser-Leu-PAM Resin Starting with 0.5 mmol (0.67g) Boc-Leu-PAM resin, the protected peptide was synthesized on a 430A ABI peptide synthesizer. The protocol used a 4 fold excess (2 mmol) of each of the following protected amino acids: Boc-Ser(OBzl), Boc-Gln and Boc-Chg.

Coupling was achieved using DCC and HOBT activation in methyl- 2-pyrrolidinone. Removal of the Boc group was performed using 50% TFA in methylene chloride and the TFA salt neutralized with diisopropylethylamine. D-Glyceric acid, which was converted from D-Glyceric acid calcium salt, was used for the introduction of the N terminal blocking group. At the completion of the synthesis, the peptide resin was dried to provide the title resin-peptide conjugate.

Step B: in Example 5-1 for the N-2(R)-2,3-dihydroxypropionyl-Ser-Ser-Ser-Chg-Gln-Ser- Leu-Dox (5-3) The title conjugate was prepared in the manner described 3, Steps B and C, but substituting the resin peptide conjugate resin-peptide conjugate used in Example 3, Step B.

ES+ NH4+ Peptide Content: 1377.55 620.85 NMOle/mg.

WO 98/10651 PCT/US97/16087

HPLC:

55 99% pure @214, retention times= 20.71 min, (Vydac C18, gradient of 95%A/B to 50%A/B over 30 min, A=0.1%TFA-H20, B=0.1%TFA, CH3CN) EXAMPLE 6 N-2(S)-2,3-dihydroxypropionyl-Ser-Ser-Ser-Chg-Gln- Preparation of Ser-Leu-Dox The title conjugate was prepared in the manner described in Example 5, but substituting L-glyceric acid for D-glyceric acid in Step A.

ES+ NH4 Peptide Content:

HPLC:

1377.62 641.59 NMOle/mg.

99% pure @214, retention times= 20.57 min, C18, gradient of 95%A/B to 50%A/B over 30 A=0.1%TFA-H20, B=0.1%TFA, CH3CN) (Vydac min, Table 3 shows other blocked peptide-doxorubicin conjugates that were prepared by the procedures described in Examples 3-6, but utilizing the appropriate amino acid residues and blocking group acylation.

WO 98/10651 WO 9810651PCT/US97/16087 56 TABLE 3 PEPTIDE PEPTIDE-DOX CONJUGATE Time to 50% Substrate IDNOCleavage by PSA (Min) 6 4 2-hydroxyacetyl-HomoRSSYQ-SNie-DOX 60 66 2-hydroxyacetyl-SHomoRChgQ-SL-DOX 6 7 2-hydroxyacetyl-HomoRSSChgQ-SL-DOX 12 6 8 2-hydroxyacetyl-HomoRASChgQ-SL-DOX 6 9 (d)2,3-dihydroxypropiony-SHomoRChgo-SL-DOX 7 0 (1)2,3-dihydroxypropionyl-SHomoRChgQ-SL-DOX 71 PEG(2)-SHomoRChgQ-SL-DOX 72 PEG(2)-HomoRCl-gQ-SL-DOX 4 HOUR =12% 7 3 (2R,3S) 2,3,4-trihydroxybutanoyl -Homo RC hgQ-S L- DOX 4 HOUR =0% 74 PEG(2)-SHomoRYQ-SL-DOX(3') PEG(2) -Homo RYQ-SSSL- DOX 4 HOUR 40% (PS) 76 PEG(2)-KYQ-SSSL-DOX 4 HOUR 20% (PS) 77 2-hydroxyacetyl-Homo RSSYQ-SL- DOX 16 (PS) 7 8 ()2,3-dihydroxypropionylHomoRSSChgOsL-DOX 12 79 PEG(2)-HomoRSSChgQ-SL-DOX 11 8 0 2-hydroxyacetyl-SYQ-SSSL-DOX

(PS)

8 1 PEG(16)-SHomoRYQ-SL-DOX 8 2 (2R,3S) 2,3,4-trihydroxybutanoyl-SHomoRChgQ-SL-DOX 83 PEG(2)-SHomoRYQ-SL-DOX 8 4 (d)2,3-dihydroxypropionyl-HomoRSSChgQSL-DOX(3') 12 (l)2,3-dihydroxypropionylSSSChgQ-S(dL)-DOX 180 8 6 (d)2,3-dihydroxypropioniySSSChgQ-SL-DOX 87 (l)2,3-dihydroxypropionylSSSChgQ-SL-DOX 88 (l)2,3-dihydroxypropionylSSChgo-S(dL)-DOX 3 HOUR 22% 89 (d)2,3-dihydroxypropionylSSChgQ-SL-DOX 120 91 PEG(2)SSChgQ-SL-DOX 92 PEG(2)-SSSChgQ-S(dL)-DOX 3 HOURS 46% 63 PEG (2)-SSSChgQ-SL- DOX 94 (d)2,3-dihydroxypropionyl-3PALSSChgQ-SL-DOX (3').AcOH 12 (PS) (1)2 ,3-dihydroxypropionyl-SSChgQ-SL- DOX 61 2-hydroxyacetyi-SSSChgQ-SL-DOX 96 2,3-dihydroxypropiony-HomoSSSChg-SLDOX 97 PEG(2)-ASChgQ-SL-DOX S98 PEG(6)-ASChgQ-SL-DOX 160 6 2 1 2-hydroxyacetyl-SSChgO-SL-DOX WO 98/10651 PCT/US97/16087 -57- EXAMPLE 7 Assessment of the Recognition of Oligopeptide-Doxorubicin Conjugates by Free PSA The conjugates prepared as described in Examples 3-6 were individually dissolved in PSA digestion buffer (50 mM tris(hydroxymethyl)-aminomethane pH7.4, 140 mM NaCI) and the solution added to PSA at a molar ration of 100 to 1. The reaction is quenched after various reaction times by the addition of trifluoroacetic acid (TFA) to a final 1% (volume/volume). The quenched reaction was analyzed by HPLC on a reversed-phase C18 column using an aqueous 0.1%TFA/acetonitrile gradient. The results of the assessment are shown in Table 3. Table 3 shows the amount of time (in minutes) required for cleavage of the noted oligopeptide-cytotoxic agent conjugates with enzymatically active free PSA. If no salt is indicated for the conjugate, the free conjugate was tested. An alternative PSA digestion buffer (12 mM tris(hydroxymethyl)-aminomethane pH8.0, 25 mM NaC1, mM CaCl2) was utilized in the assessment of the 2-hydroxyacetylhArgSerSerTyrGln-SerNle-DOX (SEQ.ID.NO.: 30) conjugate.

EXAMPLE 8 In vitro Assay of Cvtotoxicitv of Peptidvl Derivatives of Doxorubicin The cytotoxicities of the cleaveable oligopeptidedoxorubicin conjugates, prepared as described in Examples 3-6, against a line of cells which is known to be killed by unmodified doxorubicin was assessed with an Alamar Blue assay. Specifically, cell cultures of LNCap prostate tumor cells or DuPRO cells in 96 well plates was diluted with medium containing various concentrations of a given conjugate (final plate well volume of 200il). The cells were incubated for 3 days at 37 0 C, 2 0l of Alamar Blue is added to the assay well. The cells were further incubated and the assay plates were read on a EL-310 ELISA reader at the dual wavelengths of 570 and 600 nm at 4 and 7 hours after addition of Alamar Blue. Relative percentage viability at the WO 98/1065 1 PCTIUS97/16087 58 -I various concentration of conjugate tested was then calculated versus control (no conjugate) cultures. Results of this assay are shown in Table 4. If no salt is indicated, the free conjugate was tested.

TABLE 4 SE, PEPTIDE I-PEPTIDE-DOX CONJUGATE LNCaP Cell Kill in I NQ. 72 HRS, 48 HIRS 6 4 2-hydroxyacetyl-hRSSYQ-SNle-DOX (3T) 3.6 (DuPRO 100) 66 2-hydroxyacetyl-ShRChgQ-SL-DOX 5.1 (DuPRO 100) 6 7 2-hydroxyacetyl-hRSSChgQ-SL-DOX 5.5 (DuPRO 100) 6 8 2-hydroxyacetyl-hRASChgQ-SL-DOX 7.9 (DuPRO 100) (PS) 69 (d)2,3-dihydroxypropionyl..ShRChgQ-SL-DOX 5.8 (DuPRO>I 100) n=2 7 0 (I)2,3-dihydroxypropionyl-ShRChgQ-SL-DOX 9.4 (Du PRO 100) n 2 7 1 PEG(2)-ShRChgQ-SL-DOX 8.1 (DuPRO 100) 72 PEG(2)-IiRChgQ-SL-DOX

INSOLUBLE

73 (2R,3S) 2,3,4-trihydroxybutanoyi-hRChgQ-SL-DOX

PS

74 PEG(2)-ShRYQ-SL-DOX(3') 4.5 (DuPRO 100) PEG(2)-hRYQ-SSSL-DOX (3T) 14 (DuPRO 100) (PS) 76 PEG(2)-KYQ-SSSL-DOX 12.8 (DuPRO 100) (PS) 77 2-hydroxyacetyl-hRSSYQ-SL-DOX 13.6 (DuPRO 100) (PS) 78 (I)2,3-dihydroxypropionylhRSSChgQSL-DOX 7.5 (DuPRO 100) 79 PEG(2)-hRSSChgQ-SL-DOX 5.7 (DuPRO 100) 8 0 2-hydroxyacetyl-SYQ-SSSL-DOX 18.8 (Du PRO 50) (PS) 8 1 PEG(1 6)-ShRYQ-SL-DOX 45 (DuPRO 100) 82 (2R,3S) 2,3,4-trihydroxybutanoyi-ShRChgQ-SL-DOX 14.1 (DuPRO 100) 83 PEG(2)-ShRYQ-SL-DOX (3T) 34 (Du PRO 100) n=2 84 (d)2,3-dihydroxypropionyl-hRSSChgQSL-DOX(3') 7.7 (DuPRO >100) n 2 (l)2.3-dihydroxypropionylSSSohgQ-S(dL)-DOX 91 (DuPRO 100) 86 (d)2,3-dihydroxypropionylSSSChgQ-SL-DOX 5.8 (DuPRO 100) n 3 87 (1)2,3-dihydroxypropionyISSSChgQ-SL-DOX 5.5 (DuPRO 100) 88 (1)2,3-dihydroxypropionylSSChgQ-S(dL)-DOX 100 (DuPRO 100) 89(d)2,3-dihydroxypropionylSSChgQ-SL-Dox 9.1 (DuPRO 100) 91 PEG(2)SSChgQ-SL-DOX 8.8 (DuPRO 100) 63 PEG(2)-SSSChgQ-SL-DOX 10 (DuPRO 100) n=2 94 (d)2,3-dihydroxypropionyl-3PAL-SS~hgQ-SL-DOX 5.5 (DuPRO 100) (1)2,3-dihydroxypropionyl-SSChgQ-SL-DOX 13 (DuPRO 100) n 2 61 2-hYdroxYacetyl-SSSChgQ-SL-DOX 7.2 (DuPRO 100) n 3 96 2,3-dihydroxypropionyl-hSSSChgQ-SL-DOX 5.1 (DuPRO 97 PEG(2)-ASChgQ-SL-DOX 5.6 (DuPRO 100) n=2 98 PEG(6)-ASChgQ-SL-DOX 12 (DuPRO 100) 622-hydroxyacetyl-SS~hgQ-SL-DOX 4.8 (DuPRO 100) WO 98/10651 PCT/US97/16087 -59- EXAMPLE 9 In vivo Efficacy of Peptidvl -Cytotoxic Agent Conjugates LNCaP.FGC or DuPRO-1 cells are trypsinized, resuspended in the growth medium and centifuged for 6 mins. at 200xg. The cells are resuspended in serum-free a-MEM and counted.

The appropriate volume of this solution containing the desired number of cells is then transferred to a conical centrifuge tube, centrifuged as before and resuspended in the appropriate volume of a cold 1:1 mixture of a-MEM-Matrigel. The suspension is kept on ice until the animals are inoculated.

Harlan Sprague Dawley male nude mice (10-12 weeks old) are restrained without anesthesia and are inoculated with 0.5 mL of cell suspension on the left flank by subcutaneous injection using a 22G needle. Mice are either given approximately 5x10 5 DuPRO cells or 1.5x10 7 LNCaP.FGC cells.

Following inoculation with the tumor cells the mice are treated under one of two protocols: Protocol A: One day after cell inoculation the animals are dosed with a 0.1-0.5 mL volume of test conjugate, doxorubicin or vehicle control (sterile water). Dosages of the conjugate and doxorubicin are initially the maximum non-lethal amount, but may be subsequently titrated lower. Identical doses are administered at 24 hour intervals for 5 days.

After 10 days, blood samples are removed from the mice and the serum level of PSA is determined. Similar serum PSA levels are determined at 5-10 day intervals. At the end of 5.5 weeks the mice are sacrificed and weights of any tumors present are measured and serum PSA again determined.The animals' weights are determined at the beginning and end of the assay.

n I WO 98/10651 PCT/US97/16087 Protocol B: Ten days after cell inoculation,blood samples are removed from the animals and serum levels of PSA are determined. Animals are then grouped according to their PSA serum levels. At 14-15 days after cell inoculation, the animals are dosed with a 0.1-0.5 mL volume of test conjugate, doxorubicin or vehicle control (sterile water). Dosages of the conjugate and doxorubicin are initially the maximum non-lethal amount, but may be subsequently titrated lower. Identical doses are administered at 24 hour intervals for 5 days. Serum PSA levels are determined at 5-10 day intervals. At the end of 5.5 weeks the mice are sacrificed, weights of any tumors present are measured and serum PSA again determined. The animals' weights are determined at the beginning and end of the assay.

EXAMPLE In vitro determination of proteolytic cleavage of conjugates by endogenous non-PSA proteases Step A: Preparation of proteolytic tissue extracts All procedures are carried out at 4 0 C. Appropriate animals are sacrificed and the relevant tissues are isolated and stored in liquid nitrogen. The frozen tissue is pulverized using a mortar and pestle and the pulverized tissue is transfered to a Potter-Elvejeh homogenizer and 2 volumes of Buffer A (50 mM Tris containing 1.15% KC1, pH 7.5) are added. The tissue is then disrupted with 20 strokes using first a lose fitting and then a tight fitting pestle. The homogenate is centrifuged at 10,000 x g in a swinging bucket rotor (HB4-5), the pellet is discarded and the re-supernatant centrifuged at 100,000 x g (Ti 70). The supernatant (cytosol) is saved.

The pellet is respuspended in Buffer B (10 mM EDTA containing 1.15% KCI, pH 7.5) using the same volume used in step

J>

WO 98/10651 PCT/US97/16087 -61 as used above with Buffer A. The suspension is homogenized in a dounce homogenizer and the solution centrifuged at 100,000x g. The supematant is discarded and the pellet resuspended in Buffer C (10 mM potassium phosphate buffer containing0.25 M sucrose, pH using 1/2 the volume used above, and homogenized with a dounce homogenizer.

Protein content of the two solutions (cytosol and membrane) is determine using the Bradford assay. Assay aliquots are then removed and frozen in liquid N2. The aliquots are stored at -70 0

C.

Step B: Proteolvtic cleavage assay For each time point, 20 microgram of peptide-doxorubicin conjugate and 150 micrograms of tissue protein, prepared as described in Step A and as determined by Bradford in reaction buffer are placed in solution of final volume of 200 microliters in buffer (50 mM TRIS, 140 mM NaCI, pH Assay reactions are run for 0, 30, 60, 120, and 180 minutes and are then quenched with 9 microliters of 0.1 M ZnCl2 and immediately placed in boiling water for 90 seconds. Reaction products are analyzed by HPLC using a VYDAC C 18 15 cm column in water acetonitrile to 50% acetonitrile over 30 minutes).

WO 98/10651 PCT/US97/16087 -62- SEQUENCE LISTING GENERAL INFORMATION APPLICANT: FENG, DONG-MEI GARSKY, VICTOR, M.

JONES, RAYMOND, E.

OLIFF, ALLEN, I.

WAI, JENNY, M.

(ii) TITLE OF THE INVENTION: CONJUGATES USEFUL IN THE

TREATMENT

OF PROSTATE CANCER (iii) NUMBER OF SEQUENCES: 128 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Merck Co., Inc.

STREET: P.O. Box 2000, 126 E. Lincoln Ave.

CITY: Rahway STATE: NJ COUNTRY: USA ZIP: 07065-0900 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette COMPUTER: IBM Compatible OPERATING SYSTEM: DOS SOFTWARE: FastSEQ for Windows Version (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 60,026,015 FILING DATE: 09-DEC-1996 (viii) ATTORNEY/AGENT INFORMATION: NAME: Muthard, David A REGISTRATION NUMBER: 35,297 REFERENCE/DOCKET NUMBER: 19784Y (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: 908-594-3903 TELEFAX: 908-594-4720

TELEX:

WO 98/10651 PCT/US97/16087 63 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Asn Lys Ile Ser Tyr Gin Ser 1 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Lys Ile Ser Tyr Gin Ser 1 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 40 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Asn Lys Ile Ser Tyr Tyr Ser 1 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: WO 98/10651 PCT/US97/16087 64 Asn Lys Ala Ser Tyr Gin Ser 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Tyr Gin Ser Ser 1 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Lys Tyr Gin Ser Ser 1 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Xaa Tyr Gin Ser Ser 1 INFORMATION FOR SEQ ID NO:8: WO 98/10651 PCT/US97/16087 SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Xaa Cys Gln Ser Ser 1 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Tyr Gin Ser Ser 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Tyr Gln Ser Leu 1 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear WO 98/10651 PCT/US97/16087 -66- (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Norleucine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Tyr Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid STRANDEDNESS: single 40 TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: Cyclohexylglycine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Norleucine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Xaa Gin Ser Leu 1 WO 98/10651 PCT/US97/16087 -67- INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Asn Lys Ile Ser Tyr Gln Ser Ser 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Asn Lys Ile Ser Tyr Gln Ser Ala 1 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Ala Asn Lys Ile Ser Tyr Tyr Ser 1 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide xi) SEQUENCE DESCRIPTION: SEQ ID N:17: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: WO 98/10651 PCT/US97/16087 -68- Ala Asn Lys Ala Ser Tyr Gin Ser 1 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Ser Tyr Gin Ser Ser Thr 1 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Ser Tyr Gln Ser Ser Ser 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Lys Tyr Gln Ser Ser Ser 1 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear WO 98/10651 PCT/US97/16087 69 (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Xaa Tyr Gin Ser Ser Ser 1 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Ser Tyr Gin Ser Ser Leu 1 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Ser Tyr Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 2...2 WO 98/10651 PCT/US97/16087 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: Homoarginine NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID Xaa Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Xaa Tyr Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid WO 98/10651 PCT/US97/16087 -71 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Gly Glu Asn Gly Val Gin Lys Asp Val Ser Gin Arg Ser Ile Tyr Ser 1 5 10 Gln Thr Glu INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single 20 TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: Ala Ser Tyr Gin Ser Ser Leu 1 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: Homoarginine NAME/KEY: Other LOCATION: 3...3 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Ser Xaa Xaa Gln Ser Leu 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid WO 98/10651 PCT/US97/16087 -72- STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: Homoarginine NAME/KEY: Other LOCATION: 7...7 OTHER INFORMATION: Norleucine (xi) SEQUENCE DESCRIPTION: SEQ ID Xaa Ser Ser Tyr Gln Ser Leu 1 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: Homoarginine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: Xaa Ala Ser Xaa Gln Ser Leu 1 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 WO 98/10651 PCT/US97/16087 -73- OTHER INFORMATION: Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: Xaa Ser Ser Tyr Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: Homoarginine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: Xaa Ser Ser Xaa Ser Leu 1 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: Homoarginine NAME/KEY: Other LOCATION: 3...3 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: Ser Xaa Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID WO 98/10651 PCT/US97/16087 -74- SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID Xaa Tyr Gln Ser Leu 1 INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: Homoarginine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: Xaa Ser Ser Xaa Gln Ser Leu 1 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 2...2 WO 98/10651 PCT/US97/16087 OTHER INFORMATION: Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: Ser Xaa Tyr Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Ser Ser Tyr Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Ser Ser Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: 3-Pyridylalanine WO 98/10651 PCT/US97/16087 -76- NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID Xaa Ser Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 3...3 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: Ser Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 3...3 OTHER INFORMATION: Cyclohexylglycine NAME/KEY: Other LOCATION: 7...7 OTHER INFORMATION: Leucine with Unnatural Stereoconfiguration (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: Ser Ser Ser Xaa Gln Ser Xaa 1 WO 98/10651 PCT/US97/16087 -77- INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: Ser Ser Ser Xaa Gin Ser Val 1 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: Pro Ser Ser Xaa Gin Ser Val 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine xi) SEQUENCE DESCRIPTION: SE ID (xi) SEQUENCE DESCRIPTION: SEQ ID f WO 98/10651 PCT/US97/16087 78 Gly Ser Ser Xaa Gin Ser Leu C 1 INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Homoserine Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: Ser Ser Xaa Gin Ser Leu INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: Homoarginine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: NAME/KEY: Other LOCATION: 7...7 OTHER INFORMATION: Cyclohexylglycine Norleucine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: Xaa Ser Ser Xaa Gin Ser Leu 1 WO 98/10651 PCT/US97/16087 -79- INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: Homoarginine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: Xaa Ser Ala Xaa Gln Ser Leu 1 INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: Asn Arg Ile Ser Tyr Gln Ser 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Asn Lys Val Ser Tyr Gln Ser 1 WO 98/10651 PCT/US97/16087 80 INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: Asn Lys Met Ser Tyr Gln Ser Ser 1 INFORMATION FOR SEQ ID NO:52: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: Asn Lys Leu Ser Tyr Gln Ser Ser 1 INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: Asn Lys Ile Ser Tyr Gln Ser 1 INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: WO 98/10651 PCT/US97/16087 81 Gln Lys Ile Ser Tyr Gin Ser Ser 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID Xaa Tyr Gin Ser Ser Ser Leu 1 INFORMATION FOR SEQ ID NO:56: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: Lys Tyr Gin Ser Ser Ser Leu 1 INFORMATION FOR SEQ ID NO:57: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: Ser Tyr Gin Ser Ser Ser Leu 1 INFORMATION FOR SEQ ID NO:58: 1 WO 98/10651 PCT/US97/16087 82- SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 3...3 OTHER INFORMATION: Cyclohexylglycine NAME/KEY: Other LOCATION: 6...6 OTHER INFORMATION: Leucine with Unnatural Stereoconfiguration (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: Ser Ser Xaa Gln Ser Xaa 1 INFORMATION FOR SEQ ID NO:59: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Pyridylalanine Cyclohexylglycine NAME/KEY: Other LOCATION: 7...7 OTHER INFORMATION: Stereoconfiguration Leucine with Unnatural (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: Xaa Ser Ser Xaa Gln Ser Xaa 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids WO 98/10651 PCT/US97/16087 -83 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 3...3 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID Ala Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:61: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(2-Hydroxyacetyl)Serine NAME/KEY: Other LOCATION: 4...4 3 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: Xaa Ser Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:62: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(2-Hydroxyacetyl)Serine NAME/KEY: Other WO 98/10651 PCT/US97/16087 -84- LOCATION: 3...3 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: Xaa Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:63: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(PEG-2)Serine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: Xaa Ser Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:64: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(2-Hydroxyacetyl)Homoarginine NAME/KEY: Other LOCATION: 7...7 OTHER INFORMATION: Norleucine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: Xaa Ser Ser Tyr Gin Ser Leu 1 WO 98/10651 PCT/US97/16087 85 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(2-Hydroxyacetyl)Homoarginine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: NAME/KEY: Other LOCATION: 7...7 OTHER INFORMATION: Cyclohexylglycine Norleucine (xi) SEQUENCE DESCRIPTION: SEQ ID Xaa Ser Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:66: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(2-Hydroxyac NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: Homoarginine NAME/KEY: Other LOCATION: 3...3 OTHER INFORMATION: Cyclohexylglyc (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: Xaa Xaa Xaa Gin Ser Leu etyl)Serine ine 1 WO 98/10651 PCT/US97/16087 -86 INFORMATION FOR SEQ ID NO:67: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(2-Hydroxyacetyl)Homoarginine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: Xaa Ser Ser Xaa Gln Ser Leu 1 INFORMATION FOR SEQ ID NO:68: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(2-Hydroxyacetyl)Homoarginine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: Xaa Ala Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:69: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear WO 98/10651 PCT/US97/16087 87- (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-((d)-2,3-Dihydroxypropionyl)Serine NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: Homoarginine NAME/KEY: Other LOCATION: 3...3 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: Xaa Xaa Gin Ser Leu INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-((1)-2,3-Dihydroxypropionyl)Serine NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: NAME/KEY: Other LOCATION: 3...3 OTHER INFORMATION: Homoarginine Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID Xaa Xaa Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:71: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear WO 98/10651 PCT/US97/16087 88 (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: NAME/KEY: Other LOCATION: 3...3 OTHER INFORMATION: N-(PEG-2)Serine Homoarginine Cyclohexylglycine Xaa 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: Xaa Xaa Gin Ser Leu INFORMATION FOR SEQ ID NO:72: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: N-(PEG-2)Homoarginine Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: Xaa Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:73: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other WO 98/10651 PCT/US97/16087 89 LOCATION: 1...1 OTHER INFORMATION: Trihydroxybutanoyl)Homoarginine NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: N-((2R,3S)-2,3,4- Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: Xaa Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:74: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: N-(PEG-2)Serine Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: Xaa Xaa Tyr Gin Ser Leu 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(PEG-2)Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID Xaa Tyr Gln Ser Ser Ser Leu WO 98/10651 PCT/US97/16087 INFORMATION FOR SEQ ID NO:76: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid -TRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(PEG-2)Lysine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: Xaa Tyr Gln Ser Ser Ser Leu 1 INFORMATION FOR SEQ ID NO:77: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(2-Hydroxyacetyl)Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: Xaa Ser Ser Tyr Gln Ser Leu 1 INFORMATION FOR SEQ ID NO:78: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: Dihydroxypropionyl)Homoarginine WO 98/10651 PCT/US97/16087 -91 NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: Xaa Ser Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:79: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(PEG-2)Homoarginine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: Xaa Ser Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(2-Hydroxyacetyl)Serine (xi) SEQUENCE DESCRIPTION: SEQ ID Xaa Tyr Gin Ser Ser Ser Leu 1 INFORMATION FOR SEQ ID NO:81: WO 98/10651 PCT/US97/16087 -92- SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(PEG-16)Serine NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: Xaa Xaa Tyr Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:82: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-((2R,3S)-2,3,4- Trihydroxybutanoyl)Serine NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: Homoarginine NAME/KEY: Other LOCATION: 3...3 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: Xaa Xaa Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:83: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid WO 98/10651 PCT/US97/16087 -93- STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(PEG-2)Serine NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: Xaa Xaa Tyr Gln Ser Leu 1 INFORMATION FOR SEQ ID NO:84: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: Dihydroxypropionyl)Homoarginine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: Xaa Ser Ser Xaa Gln Ser Leu 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other WO 98/10651 PCT/US97/16087 -94- LOCATION: 1...1 OTHER INFORMATION: N-((1)-2,3-Dihydroxypropionyl)Serine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine NAME/KEY: Other LOCATION: 7...7 OTHER INFORMATION: Leucine with Unnatural Stereoconfiguration (xi) SEQUENCE DESCRIPTION: SEQ ID Xaa Ser Ser Xaa Gin Ser Xaa 1 INFORMATION FOR SEQ ID NO:86: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-((d)-2,3-Dihydroxypropionyl)Serine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: Xaa Ser Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:87: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-((1)-2,3-Dihydroxypropionyl)Serine

I

WO 98/10651 PCT/US97/16087 NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: Xaa Ser Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:88: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: NAME/KEY: Other LOCATION: 3...3 OTHER INFORMATION: NAME/KEY: Other LOCATION: 6...6 OTHER INFORMATION: Stereoconfiguration N-((1)-2,3-Dihydroxypropionyl)Serine Cyclohexylglycine Leucine with Unnatural (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: Xaa Ser Xaa Gin Ser Xaa 1 INFORMATION FOR SEQ ID NO:89: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-((d)-2,3-Dihydroxypropionyl)Serine NAME/KEY: Other LOCATION: 3...3 OTHER INFORMATION: Cyclohexylglycine WO 98/10651 PCT/US97/16087 -96- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89: Xaa Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(PEG-2)Serine NAME/KEY: Other LOCATION: 3...3 OTHER INFORMATION: Cyclohexylglycine NAME/KEY: Other LOCATION: 6...6 OTHER INFORMATION: Leucine with Unnatural Stereoconfiguration (xi) SEQUENCE DESCRIPTION: SEQ ID Xaa Ser Xaa Gin Ser Xaa 1 INFORMATION FOR SEQ ID NO:91: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(PEG-2)Serine S NAME/KEY: Other LOCATION: 3...3 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: WO 98/10651 PCT/US97/16087 -97 Xaa Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:92: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: NAME/KEY: Other LOCATION: 7...7 OTHER INFORMATION: Stereoconfiguration N-(PEG-2)Serine Cyclohexylglycine Leucine with Unnatural (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92: Xaa Ser Ser Xaa Gln Ser Xaa 1 INFORMATION FOR SEQ ID NO:93: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(2,3-Dihydroxypropionyl)-3- Pyridylalanine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine NAME/KEY: Other LOCATION: 7...7 OTHER INFORMATION: Leucine with Unnatural Stereoconfiguration WO 98/10651 PCT/US97/16087 -98- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93: Xaa Ser Ser Xaa Gin Ser Xaa 1 INFORMATION FOR SEQ ID NO:94: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-((d)-2,3-Dihydroxypropionyl)-3- Pyridylalanine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: Xaa Ser Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-((1)-2,3-Dihydroxypropionyl)Serine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine S50 (xi) SEQUENCE DESCRIPTION: SEQ ID Xaa Ser Xaa Gin Ser Leu 1 WO 98/10651 PCT/US97/16087 -99- INFORMATION FOR SEQ ID NO:96: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(2,3-Dihydroxypropionyl)Homoserine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96: Xaa Ser Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:97: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(PEG-2)Alanine NAME/KEY: Other LOCATION: 3...3 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97: Xaa Ser Xaa Gln Ser Leu 1 INFORMATION FOR SEQ ID NO:98: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear WO 98/10651 PCT/US97/16087 100- (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: NAME/KEY: Other LOCATION: 3...3 OTHER INFORMATION: N-(PEG-6)Serine Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98: Xaa 1 Ser Xaa Gin Ser Leu INFORMATION FOR SEQ ID NO:99: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: N-(PEG-6)Serine Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99: Xaa Ser Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:100: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(PEG-6)Alanine NAME/KEY: Other WO 98/10651 PCT/US97/16087 101 LOCATION: 3...3 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100: Xaa Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:101: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: N-(PEG-4)-3-Pyridylalanine Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101: Xaa Ser Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:102: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine NAME/KEY: Other LOCATION: 7...7 OTHER INFORMATION: Leucine-2-Hydroxyethylamine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102: Ser Ser Ser Xaa Gin Ser Xaa 1 s WO 98/10651 PCT/US97/16087 -102- INFORMATION FOR SEQ ID NO:103: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-Acetylalanine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103: Xaa Arg Lys Ala Ser Tyr Gln Ser Leu 1 INFORMATION FOR SEQ ID NO:104: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-Acetylalanine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104: Xaa Arg Lys Ala Ser Tyr Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:105: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-Acetylhomoarginine NAME/KEY: ther NAME/KEY: Other WO 98/10651 PCT/US97/16087 103- LOCATION: 2...2 OTHER INFORMATION: NAME/KEY: Other LOCATION: OTHER INFORMATION: Cyclohexylalanine Norleucine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105: Xaa Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:106: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: NAME/KEY: Other LOCATION: 6...6 OTHER INFORMATION: N-Acetylserine Homoarginine Norleucine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106: Xaa Xaa Tyr Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:107: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: Homoarginine NAME/KEY: Other WO 98/10651 PCT/US97/16087 104- LOCATION: 3...3 OTHER INFORMATION: Cyclohexylglycine NAME/KEY: Other LOCATION: 6...6 OTHER INFORMATION: Norleucine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107: Ser Xaa Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:108: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: OTHER INFORMATION: N-Acetylhomoarginine NAME/KEY: Other LOCATION: 7...7 OTHER INFORMATION: Norleucine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108: Xaa Ser Ser Tyr Gin Ser Leu 1 s INFORMATION FOR SEQ ID NO:109: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-Acetylhomoarginine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109: WO 98/10651 PCT/US97/16087 105- Xaa Ser Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:110: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: NAME/KEY: Other LOCATION: 7...7 OTHER INFORMATION: N-Acetylhomoarginine Norleucine Xaa 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110: Ser Ser Tyr Gin Ser Leu INFORMATION FOR SEQ ID NO:111: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: N-Acetylhomoarginine Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111: Xaa Ala Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:112: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids WO 98/10651 PCT/US97/16087 106 TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(2-Hydroxyacetyl)Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112: Xaa Tyr Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:113: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(PEG-1)Serine NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: Homoarginine NAME/KEY: Other LOCATION: 3...3 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113: Xaa Xaa Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:114: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other NAME/KEY: Other WO 98/10651 PCT/US97/16087 107 LOCATION: 1...1 OTHER INFORMATION: NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: N-(PEG-1)Homoarginine Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114: Xaa 1 Ser Ser Xaa Gin Ser Leu INFORMATION FOR SEQ ID NO:115: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(PEG-1)Serine NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115: Xaa 1 Xaa Tyr Gin Ser Leu INFORMATION FOR SEQ ID NO:116: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: i...1 OTHER INFORMATION: NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116: WO 98/10651 PCT/US97/16087 108 Xaa Xaa Tyr Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:117: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(PEG-17)Serine NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117: Xaa Xaa Tyr Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:118: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(PEG-2)Serine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118: Xaa Ser Tyr Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:119: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear WO 98/10651 PCT/US97/16087 109- (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(PEG-14)Serine NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119: Xaa Xaa Tyr Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:120: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(PEG-18)Serine NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120: Xaa Xaa Tyr Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:121: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(PEG-19)Serine NAME/KEY: Other WO 98/10651 PCT/US97/16087 -110- LOCATION: 2...2 OTHER INFORMATION: Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121: Xaa Xaa Tyr Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:122: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-((1)-2,3-Dihydroxypropionyl)Serine NAME/KEY: Other LOCATION: 3...3 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:122: Xaa Ser Xaa Gin Ser Leu 1 INFORMATION FOR SEQ ID NO:123: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-((1)-2,3-Dihydroxypropionyl)-3- Pyridylalanine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:123: Xaa Ser Ser Xaa Gin Ser Leu 1 WO 98/10651 PCT/US97/16087 111 INFORMATION FOR SEQ ID NO:124: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid (C)-STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(2,3-Dihydroxypropionyl)Serine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124: Xaa Ser Ser Xaa Gln Ser Leu 1 INFORMATION FOR SEQ ID NO:125: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(2,3-Dihydroxypropionyl)Serine NAME/KEY: Other LOCATION: 2...2 OTHER INFORMATION: Homoarginine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125: Xaa Xaa Tyr Gln Ser Leu 1 INFORMATION FOR SEQ ID NO:126: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear WO 98/10651 PCT/US97/16087 -112- (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-acetylserine NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126: Xaa Ser Ser Xaa Gin Ser Val 1 INFORMATION FOR SEQ ID NO:127: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-Acetylproline NAME/KEY: Other LOCATION: 4...4 OTHER INFORMATION: Cyclohexylglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127: Xaa Ser Ser Xaa Gin Ser Val 1 s INFORMATION FOR SEQ ID NO:128: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Other LOCATION: 1...1 OTHER INFORMATION: N-(2,3-Dihydroxypropionyl)Glycine WO 98/10651 PCT/US97/16087 113 NAME/KEY: Other LOCATION: .4 OTHER INFORMATION: Cyclohexyiglycine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i28: Xaa Ser Ser Xaa Gin Ser Leu 1

Claims (11)

1. A conjugate which is useful for the treatment of prostate cancer which comprises a cytotoxic agent attached to a oligopeptide, wherein the oligopeptide comprises a sequence of amino acids that is selectively proteolytically cleaved by free prostate specific antigen, wherein the means of attachment is a covalent bond or through a chemical linker and wherein the point of attachment on the oligopeptide is at the C-terminus, and which further comprises a hydrophilic blocking group at the N-terminus of the oligopeptide, or the pharmaceutically acceptable salt thereof.

2. The conjugate according to Claim 1 wherein the cytotoxic agent is a member of a class of cytotoxic agents selected from the following classes: a) anthracycline family of drugs, b) the vinca alkaloid drugs, c) the mitomycins, d) the bleomycins, e) the cytotoxic nucleosides, f) the pteridine family of drugs, g) diynenes, h) estramustine, i) cyclophosphamide, j) the taxanes and k) the podophyllotoxins, or the pharmaceutically acceptable salt thereof.

3. The conjugate according to Claim 2 wherein the cytotoxic agent is selected from the following cytotoxic agents: a) doxorubicin, b) carminomycin, WO 98/10651 PCT/US97/16087

115- c) daunorubicin, d) aminopterin, e) methotrexate, f) methopterin, g) dichloro-methotrexate, h) mitomycin C, i) porfiromycin, j) k) 6-mercaptopurine, 1) cytosine arabinoside, m) podophyllotoxin, n) etoposide, o) etoposide phosphate, p) melphalan, q) vinblastine, r) vincristine, s) leurosidine, t) vindesine, u) estramustine, v) cisplatin, w) cyclophosphamide, x) taxol, and y) leurosine, or the pharmaceutically acceptable salt thereof. 4. The conjugate according to Claim 2 wherein the cytotoxic agent is selected from doxorubicin and vinblastine or a cytotoxic derivative thereof. The conjugate according to Claim 2 wherein the cytotoxic agent is doxorubicin or a cytotoxic derivative thereof. WO 98/10651 WO 9810651PCTIUS97/16087

116- 6. The conjugate according to Claim 1 wherein the oligopeptide comprises an oligomer selected from: a) AsnLyslleSerTyrGlnlSer (SEQ.ID.NO.: 1), b) LyslleSerTyrGiniSer (SEQ.JD.NO.: 2), c) AsnLyslleSerTyrTyrJSer (SEQ.ID.NO.: 3), d) AsnLysAlaSerTyrGlnlSer (SEQ.LD.NO.: 4), e) SerTyrGlnJSerSer (SEQ.ID.NO.: f) LysTyrGlnJSerSer (SEQ.LD.NO.: 6); g) hArgTyrG~nJSerSer (SEQ.ID.NO.: 7); h) hArgChaGlnfSerSer (SEQ.ID.NO.: 8); i) TyrGiniSerSer (SEQ.LD.NO.: 9); j) TyrGlnlSerLeu (SEQ.LD.NO.: k) TyrGIniSerNie (SEQ.ID.NO.: 11); I) ChgGlnfSerLeu (SEQ.ID.NO.: 12); and mn) ChgGlnISerNle (SEQ.ID.NO.: 13). 7. The conjugate according to Claim 1 wherein the oligopeptide comprises an oligomer selected from: a) AsnLys~leSerTyrGlnJSerSer (SEQ.ID.NO.: 14), WO 98/10651 WO 9810651PCTIUS97/16087 117 b) C) d) e) f) g) hi) i) j) k) 1) AsnLysleSerTyrGlnlSerAla AlaAsnLysleSerTyrTyrlSer AlaAsnLysAlaSerTyrGlnlSer SerTyrGlnJSerSerThr (S SerTyrGiniSerSerSer (SI LysTyrGInJSerSerSer (S hArgTyrGlnJSerSerSer SerTyrGlnlSerSerLeu (SI SerTyrGlnlSerLeu (SEQ. SerChgGlnJSerLeu (SEQ hArgChgGlnlSerLeu (SE hArgTyrGlnlSerLeu (S (SEQ.LD.NO.: (SEQ.ID.NO.: 16), (SEQ.LD.NO.: 17), EQ. ID. NO.: 18), EQ.ID.NO.: 2), EQ.D.NO.: 2), BQ.D.NO.: 2) .ID.NO.: 2); 24); Q.LD.NO.: 25); and EQ.LD.NO.: 26). 8. The conjugate according to Claim 1 wherein the oligopeptide comprises an oligomer selected from: G lyGluAsnGlyValGlnLysAsp ValSerGlnArgSerIeTyrISerG lnThrGlu (SEQ.LD.NO.: 27), AlaSerTyrGlnJSerSerLeu (SEQ.ID.NO.: 28); SerhArgChgGlnISerLeu (SEQ.LD.NO.: 29); I WO 98/10651 PCTIUS97/16087 hArgSerSerTyrGlnjSerNle (SEQ.ID.NO.: hArgAlaSerChgGlnlSerLeu (SEQ.ID.NO.: 31); hArgSerSerT-yrGlnjSerLeu (SEQ.ID.NO.: 32); hArgSerSerChglSerLeu (SEQ.ID.NO.: 33); SerhArgChgGlnlSerLeu (SEQ.ID.NO.: 34); hArgTyrGlnlSerLeu (SEQ.ID.NO.: hArgSerSerChgGlnjSerLeu (SEQ.ID.NO.: 36); SerhArgTyrGlnlSerLeu (SEQ.LD.NO.: 37); SerSerTyrGlnlSerLeu (SEQ.LD.NO.: 38); SerSerSerChgGlnlSerLeu (SEQ.ID.NO.: 39); 3PAL-SerSerChgGlnISerLeu (SEQ.LD.NO.: SerSerChgG~njSerLeu (SEQ.LD.NO.: 41); SerSerSerChgGlnlSer(dLeu) (SEQ.ID.NO.: 42); SerSerSerChgGlnlSerVal (SEQ.LD.NO.: 43); ProSerSerChgGlnlSerVal (SEQ.LD.NO.: 44); GlySerSerChgGlnlSerLeu (SEQ.LD.NO.: hSerSerSerChgGlnjSerLeu (SEQ.ID.NO.: 46); I I WO 98/10651 PCTIUS97/16087

119- hArgSerSerChgGlnJSerNle (SEQ.ID.NO.: 47); hArgTyrGlnlSerSerSerLeu (SEQ.ID.NO.: LysTyrGlnlSerSerSerLeu (SEQ.LD.NO.: 56); SerTyrGlnlSerSerSerLeu (SEQ.LD.NO.: 57); SerSerChgGln-Ser(dLeu) (SEQ.ID.NO.: 58); and 3PAL-SerSerChgGln-Ser(dLeu) (SEQ.LD.NO.: 59); and AlaSerChgGln-SerLeu (SEQ.ID.NO.: 9. The conjugate according to Claim I wherein the hydrophilic blocking group is selected from: a) 0 HO and n R 1 R 2 b) H 3 C 0 q p 0 wherein: R I and R 2 are independently selected from: a) hydrogen, b) unsubstituted or substituted aryl, unsubstituted or substituted heterocycle, C3-C1O cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, halogen, C1I-C6 perfluoroalkyl, R 1 2 0-, A WO 98/10651 PCT/US97/16087

120- R 3 C(O)NR 3 (R 3 R 3 2N-C(NR 3 R 4 S(O)mNH, CN, N02, R 3 N3, -N(R 3 or R 4 0C(O)NR 3 c) unsubstituted C -C6 alkyl, d) substituted C1-C6 alkyl wherein the substituent on the substituted CI-C6 alkyl is selected from unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic, C3-C10 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, R 3 0-, R 4 S(O)mNH, R 3 C(O)NR 3 (R 3 R 3 2N-C(NR 3 CN, R 3 N3, -N(R 3 and R 4 0C(O)-NR 3 or R and R 2 are combined to form (CH2)s wherein one of the carbon atoms is optionally replaced by a moiety selected from: 0, S(O)m, NH and -N(CORIO)-; R 3 is selected from: hydrogen, aryl, substituted aryl, heterocycle, substituted heterocycle, Cl-C6 alkyl and C3-C10 cycloalkyl; R 4 is selected from: aryl, substituted aryl, heterocycle, substituted heterocycle, C -C6 alkyl and C3-C10 cycloalkyl; mis 0,1 or 2; n is 1, 2, 3 or 4; p is zero or an integer between 1 and 100; and q is 0 or 1, provided that if p is zero, q is 1; and s is 3, 4 or A conjugate which is useful for the treatment of prostate cancer of the formula I: .1 WO 98/10651 PCT/US97/16087 121 S .CH, OH SOH CH 3 NH 0 OH oligopeptide R wherein: oligopeptide is an oligopeptide which is selectively recognized by the free prostate specific antigen (PSA) and is capable of being proteolytically cleaved by the enzymatic activity of the free prostate specific antigen, and wherein the C-terminus carbonyl is covalently bound to the amine of doxorubicin and the N-terminus amine is covalently bound to the carbonyl of the blocking group; R is selected from a) HO R 1 R 2 S Op H3C WO 98/10651 PCT/US97/16087

122- RI and R 2 are independently selected from: hydrogen, OH, CI-C6 alkyl, CI-C6 alkoxy, C1-C6 aralkyl and aryl; nis 1, 2, 3 or 4; p is zero or an integer between 1 and 100; q is 0 or 1, provided that if p is zero, q is 1; or the pharmaceutically acceptable salt thereof. 11. The conjugate according to Claim 10 wherein: R is selected from a) HO R 1 R 2 b) OH HO R 1 R 2 c) OH HO R 1 R 2 WO 98/10651 WO 9810651PCTIUS97/16087 123 b) H 3 0 R I and R 2 are independently selected from: hydrogen, C I -C6 alkyl and aryl; n is 1, 2, 3or 4; n' is 0, 1, 2or 3; p is zero or an integer between 1 and 14; q is 0 or 1, provided that if p is zero, q is 1; or the pharmaceutically acceptable salt thereof. 12. The conjugate according to Claim 10 wherein: oligopeptide is an oligomer that comprises an amino acid sequence selected from: a) AsnLys~leSerTyrGlnlSer (SEQ.ID.NO.: 1), b) LyslleSerTyrGinJSer (SEQ.LD.NO.: 2), c) AsnLys~leSerTyrTyrlSer (SEQ.ID.NO.: 3), d) AsnLysAlaSerTyrGlnlSer (SEQ.ID.NO.: 4), e) SerTyrGiniSerSer (SEQ.LD.NO.: f) LysTyrGinJSerSer (SEQ.ID.NO.: 6); g) hArgTyrGlnlSerSer (SEQ.ID.NO.: 7); h) hArgChaGlnlSerSer (SEQ.LD.NO.: 8); WO 98/10651 WO 9810651PCT/US97/16087 124 i) TyrGiniSerSer (SEQ.ID.NO.: 9); j) TyrGlnlSerLeu (SEQ.JD.NO.: k) TyrGiniSerNle (SEQ.ID.NO.: 11); 1) ChgGlnlSerLeu (SEQ.LD.NO.: 12); m) ChgGlnISerNle (SEQ.ID.NO.: 13); or an optical isomer or pharmaceutically acceptable salt thereof. 13. The conjugate according to Claim 10 wherein: oligopeptide is an oligomer that comprises an amino acid sequence selected from: GlyGluAsnGlyValGnLysAspValSerGInArgSerleTyrISerGlnThrGlu (SEQ.ID.NO.: 27), AlaSerTyrGlnJSerSerLeu (SEQ.ID.NO.: 28); SerhArgChgGlnlSerLeu (SEQ.ID.NO.: 29); hArgSerSerTyrGlnlSerNle (SEQ.ID.NO.: hArgAlaSerChgGlnlSerLeu (SEQ.ID.NO.: 31); hArgSerSerTyrGlnJSerLeu (SEQ.LD.NO.: 32); hArgSerSerChgJSerLeu (SEQ.LD.NO.: 33); SerhArgChgGlnISerLeu (SEQ.ID.NO.: 34); WO 98/10651 WO 9810651PCTIUS97/16087 125 hArgTyrGlnlSerLeu (SEQ.LD.NO.: hArgSerSerChgGlnlSerLeu (SEQ.LD.NO.: 36); SerhArgTyrG~njSerLeu (SEQ.ID.NO.: 37); SerSerTyrG~njSerLeu (SEQ.LD.NO.: 38); SerSerSerChgGlnlSerLeu (SEQ.ID.NO.: 39); 3PAL-SerSerChgGlnlSerLeu (SEQ.ID.NO.: SerSerChgGlnlSerLeu (SEQ.LD.NO.: 41); SerSerSerChgGlnjSer(dLeu) (SEQ.LD.NO.: 42); SerSerSerChgGlnISerVal (SEQ.LD.NO.: 43); ProSerSerChgGlnISerVal (SEQ.ID.NO.: 44); GlySerSerChgGlnjSerLeu (SEQ.LD.NO.: hSerSerSerChgGlnjSerLeu (SEQ.ID.NO.: 46); hArgSerSerChgGlnlSerNle (SEQ.LD.NO.: 47); hArgTyrGlnlSerSerSerLeu (SEQ.LD.NO.: LysTyrGlnlSerSerSerLeu (SEQ.ID.NO.: 56); SerTyrGlnlSerSerSerLeu (SEQ.ID.NO.: 57); (SEQ.ID.NO.: 59); and WO 98/10651 WO 9810651PCT/US97/16087 126 3PAL-SerSerChgGln-Ser(dLeu) (SEQ. LD.NO.: 59); and AlaSerChgGln-SerLeu (SEQ.ID.NO.: or an optical isomer or pharmaceutically acceptable salt thereof. 14. The conjugate according to Claimn 10 which is selected from: OH -CH 2 OH "'OH CH 3 O OH 3 r3 NH OH x wherein X is: 0 HO,_I SerSerSerChgGInSerLeu-+ (SEQ.ID.NO.: 61), 0 H, ,,SerSerChgGInSerLeu- (SEQ).ID.NO.: 62), 0 H3C' SerSerSerChgGlnSerLeu- (SEQ.ID.NO.: 63), or an optical isomer or pharmnaceutically acceptable salt thereof. WO 98/10651 WO 9810651PCT/US97/16087 127 The conjugate according to Claim 10 which is selected from: 2-hydroxyacetyl-hArgSerSerTyrG In-SerN le-DOX (SEQ.ID. NO.: 64) 2-hydroxyacetyl-hArgSerSerChgG In-SerNie-DOX (SEQ.ID.NO.: 2-hydroxyacetyl-SerhArgChgGln-SerLeu-DOX (SEQ.ID.NO.: 66) 2-hydroxyacetyl-hArgSerSerChgGln-SerLeu-DOX (SEQ.ID.NO.: 67) 2-hydroxyacetyl-hArgAlaSerChgGln-SerLeu-DOX (SEQ.ID.NO.: 68) 2,3-dihydroxypropionyl-SerhArgChgGln-SerLeu-DOX (SEQ.ID.NO.: 69) 2,3-dihydroxypropionyl-SerhArgChg-Gln-SerLeu-DOX (SEQ.ID.NO.: PEG(2)-SerhArgChgGln-SerLeu-DOX (SEQ.ID.NO.: 71) PEG (2)-hArgChgGlIn-SerLeu-DOX (SEQ.ID.NO.: 72) (2R,3S) 2,3,4-trihydroxybutanoyl-hArgChgGln-SerLeu-DOX (SEQ.ID.NO.: 73) PEG (2)-SerhArgTyrG In -SerLeu-DOX(3') (SEQ.LD.NO.: 74) PEG (2)-hArgTyrGIn -SerSerSerLeu-DO X (SEQ.ID.NO.: PEG(2)-LysTyrGln-SerSerSerLeu-DOX (SEQ.ID.NO.: 76) 2-hydroxyacety I-hArgSerSerTyrGln-SerLeu-DOX (SEQ.LD. NO.: 77) ,3-dihydroxypropionyl)hArgSerSerChgGlnSerLeu-DOX (SEQ.LD.NO.: 78) PEG(2)-hArgSerSerChgGln-SerLeu-DOX (SEQ.ID.NO.: 79) 2-hydroxyacety 1-SerTyrG ln-SerSerSerLeu-DOX (3Y) (SEQ. ID.NO.: PEG(I1 6)-SerhArgTyrGln-SerLeu -DOX (3Y) (SEQ.ID.NO.: 81) (2R,3S) 2,3,4-trihydroxybutanoyl-SerhArgChgGln-SerLeu-DOX (SEQ.ID.NO.: 82) WO 98/10651 WO 9810651PCTIUS97/16087

128-1 PEG (2)-SerhArgTyrGln-SerLeu-DOX (3Y) (SEQ.LD.NO.: 83) -dihydroxypropionyl)-hArgSerSerChgGln-SerLeu-DOX(3') (SEQ.ID.NO.: 84) (1)(2,3-dihydroxypropionyl)SerSerSerChgGln-Ser(dLeu)-DOX (SEQ.ID.NO.: -dihydroxypropionyl)SerSerSerChgGln-SerLeu-DOX (SEQ.ID.NO.: 86) -dihydroxypropionyl)SerSerSerChgGln-SerLeu-DOX (SEQ.ID.NO.: 87) (I)(2,3-dihydroxypropionyl)SerSerChgGln-Ser(dLeu)-DOX (3Y) (SEQ.ID.NO.: 88) (d)(2,3-dihydroxypropionyl)SerSerChgGln-SerLeu-DOX (SEQ.LD.NO.: 89) PEG (2)SerSerChgGln-S erLeu -DOX (3Y) (SEQ.ID.NO.: 91) (d)(2,3-dihydroxypropionyl)-3PAL-SerSerChgGln-SerLeu-DOX (3Y) (SEQ.ID.NO.: 94) (1)(2,3-dihydroxypropionyl)-SerSerChgGln-SerLeu-DOX (SEQ.ID.NO.: (2,3 -dihydroxypropionyl)-hSerSerSerChgG ln-SerLeu-DOX (SEQ.ID.NO.: 96) PEG(2)-AlaSerChgGln-SerLeu-DOX (SEQ.LD.NO.: 97) PEG(6)-SerSerChgGln-SerLeu-DOX (SEQ.LD.NO.: 98) PEG -SerS erSerChgGlIn-SerL-eu -DOX (SEQ.LD.NO.: 99) PEG(6)-AlaSerChgGln-SerLeu-DOX (3Y) (SEQ.LD.NO.: 100) PEG PALS erSerChgGlIn-SerLeu-DOX (SEQ.LD.NO.: 101) or an optical isomer or pharmaceutically acceptable salt thereof. 16. The conjugate according to Claim 1 of the formula 11: WO 98/10651 PCT/US97/16087

129- OH N N s Et N '1H II N CO2CH 3 H N '""CH2CH3 N OR 19 C H, OH CH 3 II XL oligopeptide R wherein: oligopeptide is an oligopeptide which is specifically recognized by the free prostate specific antigen (PSA) and is capable of being proteolytically cleaved by the enzymatic activity of the free prostate specific antigen and wherein the point of attachment of the oligopeptide to XL is at the C-terminus; XL is NH (CH2)r NH R is selected from a) R' R 2 WO 98/10651 PCT/US97/16087 -130- ,C O RI and R2 n is p is qis are independently selected from: hydrogen, OH, CI-C6 alkyl, CI-C6 alkoxy, C1-C6 aralkyl and aryl; 1, 2, 3 or 4; zero or an integer between 1 and 100; 0 or 1, provided that if p is zero, q is 1; r is 1, 2, 3, 4 or or a pharmaceutically acceptable salt thereof. WO 98/10651 WO 9810651PCTIUS97/16087 131 17. The conjugate according to Claim 16 which is: OH 6H 3 O 0 NH 0 HOJ _"SerSerSerChgGln-SerLeu-- NH (SEQ.ID.NO.: 61), or a pharmaceutically acceptable salt or optical isomer thereof. WO 98/10651 PCT/US97/16087

132- 18. A conjugate of the formula III: 'OR 19 CH CO oligopeptide NRdRe III N-terminus C-terminus wherein: oligopeptide is an oligopeptide which is specifically recognized by the free prostate specific antigen (PSA) and is capable of being proteolytically cleaved by the enzymatic activity of the free prostate specific antigen, Rd and Re are independently selected from: hydrogen, C1-C6-alkyl, -Cl-C6-alkyl-OH, -Cl-C6-alkyl-di-OH, -Cl-C6-alkyl-tri- OH and H 3 C- '0) provided that at least one Rd and Re are not hydrogen or Cl-C6-alkyl, or Rd and R e are combined to form a -CH2CH20CH2CH2- diradical; 133 p is zero or an integer between 1 and 100; q is 0 or 1, provided that if p is zero, q is 1; or a pharmaceutically acceptable salt thereof. 19. The conjugate according to claim 18 which is: OH Et N H a"<N CO2CH 3 *N H S f p'CH2CH3 N OH OH \CH 3 0 OH 0000 SerSerSerChgGln-SerLeu- N H (SEQ. ID. NO.: 102), or a pharmaceutically acceptable salt or optical isomer thereof. 20. A conjugate which is useful for the treatment of prostate cancer which comprises a cytotoxic agent attached to an oligopeptide wherein the oligopeptide is 10 substantially as hereinbefore described with reference to any one of the oligopeptides listed in Table 2 herein. 21. A conjugate which is substantially as hereinbefore described with reference to the title conjugate of any one of Examples 3 to 6. 22. A process for preparing a conjugate substantially as hereinbefore described with reference to any one of Examples 3 to 6. 23. A pharmaceutical composition comprising a pharmaceutical carrier, and dispersed therein, a therapeutically effective amount of a compound of any one of claims 1 to 21. 24. A method for treating prostate cancer in a mammal, which method comprises administering to the mammal a therapeutically effective amount of a conjugate of any one of claims 1 to 21 or of a composition of claim 23. A conjugate of any one of claims 1 to 21 or a composition of claim 23 when used for treating prostate cancer in a mammal. 26. Use of a conjugate of any one of claims 1 to 21 or a composition of claim 23 3. s in the manufacture of a medicament for treating prostate cancer in a mammal. [n:\libaa]01563:TAB 27. A method for treating benign prostatic hyperplasia in a mammal which method comprises administering to the mammal a therapeutically effective amount of a conjugate of any one of claims 1 to 21 or of a composition of claim 23. 28. A conjugate of any one of claims 1 to 21 or a composition of claim 23 when used for treating benign prostatic hyperplasia in a mammal. 29. Use of a conjugate of any one of claims 1 to 21 or a composition of claim 23 in the manufacture of a medicament for treating benign prostatic hyperplasia in a mammal. A pharmaceutical composition made by combining a compound of any one of claims 1 to 21 and a pharmaceutically acceptable carrier. 31. A process for making a pharmaceutical composition comprising combining a compound of any of claims 1 to 21 and a pharmaceutically acceptable carrier. Dated 9 April, 1999 Merck Co., Inc. 15 Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON *O a* *0 [n:\libaa]01563:TAB