CA2264610A1 - Branched peptide linkers - Google Patents

Abstract

Conjugates containing a targeting ligand, such as an antibody, a therapeutically active drug and a branched peptide linker. The branched peptide linker contains two or more amino acid moieties that provide an enzyme cleavage site. The number of drugs capable of being bonded to the branched linkers varies by a factor of two for each generation of branching.

Description

‘IO15202530W0 98/ 19705CA 02264610 1999-03-08PCT/US97/19851BRANCHED PEPTIDE LINKERS ,.,Background of the Invention _Bifunctional compounds which link cytotoxic reagents to antibodies(i.e., "linkers") are known in the art. These compounds have beenparticularly useful in the formation of immunoconjugates directed againsttumor associated antigens. Such immunoconjugates allow the selectivedelivery of toxic drugs to tumor cells. (See e.g., Hermentin and Seller,"lnvestigations With Monoclonal Antibody Drug'Conjugates," Behring lnsti._M_itl_. 82:197-215 (1988); Gallego _e_ta_l_., "Preparation of Four Daunomycin-Monoclonal Antibody 791T/36 Conjugates With Anti—Tumor Activity". Int. J.Cancer 332737-44 (1984); Arnon et al., "|_nmc_> and l;i_ Vivo Efficacy ofConjugates of Daunomycin With Anti—Tumor Antibodies," mmunological_F_ie_v, 62:5-27 (1982).Greenfield e_taL have described the formation of acid-sensitiveimmunoconjugates containing .the acylhydrazide conjugated via anacylhydrazone bond to the 13—keto position of an anthracycline molecule,and conjugation of this anthracycline derivative to an antibody molecule(Greenfield ejal, European Patent Publication EP 0 328 147, publishedAugust 16, 1989, which corresponds to pending U.S. Serial No. 07/270,509,filed November 16, 1988 and U.S. Serial No. 07/155,181, filed February 11,1988, now abandoned). This latter reference also discloses specificthioether-containing linkers and conjugates, including hydrazone thioethercontainig immunoconjugates.Kaneko §IE1_'- (U.S. Serial No. 07/522,996, filed May 14, 1990,which is equivalent to European Patent Publication, EP A O 457 250,published November 21, 1991) have also described the formation ofconjugates containing anthracycline antibiotics attached to a bifunctionallinker by an acylhydrazone bond at the C-13 position of an anthracyclinemolecule. In their invention the linkers contain a reactive pyridinyldithio- oran ortho—nitrophenyldithio— group, by which the linker reacts with a suitablegroup attached to a cell reactive ligand, to form the completed conjugate.1015202530W0 98/ 19705CA 02264610 1999-03-08PCT/US97/19851Conjugates which rely on simple acid hydrolysis may release the‘drug prematurely. Accordingly, it would be desirable to have conjugates thatrelease active drug in a more site-specific fashion. European PatentPublication 941075012 discloses lysosomal enzymes-cleavable antitumordrug conjugates which are selectively activatible at the site of the tumor.However, one of the problems with prior art immunoconjugates is therelatively low ratio of drug to targeting ligand (e.g., immunoglobulin)achievable. It would be highly desirable to have immunoconjugates,activatible at the tumor site, which provide a higher ratio of drug to targetingHgand.Summarv of the inventionThe present invention provides novel branched peptide linkers.The novel linkers are used to prepare novel drug/linker molecules andbiologically active conjugates composed of a targeting ligand, atherapeutically active drug, and a branched peptide linker. The novelconjugates are selectively activatible at the site of of a selected target cellpopulation recognized by the targeting ligand.As used hereinthe term "drug/linker" or "linker/drug" refers to thebranched peptide linker molecule coupled to two or more therapeuticallyactive drug molecules, and the term "conjugate" refers to the drug/linkermolecule coupled to the targeting ligand.The branched peptide linker contains a protein peptide spacer andmay also contain a self-immolating spacer which spaces the protein peptidesequence and the drug. The linkers of the invention are branched so thatmore than one drug molecule per linker are coupled to the targeting ligand.The number of drugs attached to each linker varies by a factor of 2 for eachgeneration of branching. Thus, the number of drug molecules per moleculeof linker can be 2, 4, 8, 16, 32, 64, etc. The factor of branching can beexpressed mathematically as 2“, wherein n is a positive integer. Thus, asingly branched linker will have a first generation of branching or 21, i.e.,contains a potential of two drug molecules per linker. A doubly branched1015202530W0 98/ 19705CA 02264610 1999-03-08PCT/US97/ 19851linker will have a second generation of branching or 22, i.e., contains a __potential of four drug molecules per linker.As n rises from n=1, there is a tendency for the solubility of theimmunoconjugate to diminish. Solubility can be enhanced by using morewater-soluble peptides, or addition of a water-solubilizing moiety such aspolyethylene glycol or charged species, e.g., B—alanine, to the drug in such away that it is released from the drug by either the low pH or the enzymes ofthe Iiposomal milieuThe present invention is directed to a branched peptide linker forlinking a thiol group derived from a targeting ligand to two or more drugmoieties which comprises a compound having a terminus containing a thiolacceptor for binding to a thiol group (also called a sulfhydryl group) derivedfrom a targeting ligand, at least one point of branching which is a polyvalentatom, such as a carbon atom or a nitrogen atom, allowing for a level ofbranching of 2“, wherein n is a positive integer, at least two amino acidmoieties per branch providing at least one enzymatic site per branch, and atleast two other termini containing groups capable of forming covalent bondswith chemically reactive functional groups derived from a drug moiety. It ispreferred that n is 1, 2, 3, or 4; more preferably 1, 2, or 3; and most preferably1 or 2. it is also preferred that the targeting ligand is an antibody or fragmentthereof.As used in the preceeding paragraph, the phrase "thiol groupderived from the targeting ligand" means that the thiol group is alreadypresent on the targeting ligand or that the targeting ligand is chemicallymodified to contain a thiol group, which modification optionally includes athiol spacer group between the targeting ligand and the thiol group.Likewise, the phrase "chemically reactive functional group derived from adrug rnoiety" means that the chemically reactive functional group is alreadypresent on the drug or the drug is chemically modified to contain suchchemically reactive functional group. Such chemically reactive functionalgroups are groups that are capable of forming covalent bonds with a linkerterminus. Examples of such chemically reactive functional groups include1015202530W0 98/19705 ’ -CA 02264610 1999-03-08PCT/US97/19851primary or secondary amino, hydroxyl, sulfhydroxyl, carboxyl, aldehyde,ketone, and the like.Also provided by the invention are intermediates for preparing thelinkers, drug/linkers, and/or conjugates; and a method for treating or 9‘preventing a selected disease state which comprises administering to apatient a conjugate of the invention.The selected target cell population recognized by the targetingligand is preferably a tumor.An aspect of the invention provides tumor-specific conjugateswhich are highly selective substrates for drug—activating enzymatic cleavageby one or more tumor—associated enzymes.A further aspect of the invention provides tumor-specific drugconjugates wherein the activating enzyme is one which is present in thetumor in sufficient amounts to generate cytotoxic levels of free drug in thevicinity of the tumor.Another aspect of the invention provides tumor-specific drugconjugates which are stable to adventitious proteases in blood.A still further aspect of the present invention provides a tumor-specific conjugate in accordance with the preceding aspects, which isconsiderably less toxic than the activated drug.lncanother aspect the present invention provides methods fordelivering the conjugates to target cells in which a modification in biologicalprocess is desired, such as in the treatment of diseases such as cancer.The present invention also provides a method for delivering to thesite of tumor cells in a warm-blooded animal an active antitumor drug byadministering to said warm-blooded animal the conjugate according to thisinvention.The above and other aspects of the present invention are achievedby derivatizing a drug (e.g., an an antitumor agent) linked to a ligand througha peptide linker, made up of a protein peptide sequence and a self~immolating spacer, at a reactive site appropriate for inhibiting thepharmacological activity of the antitumor agent to thereby convert theantitumor agent into a pharmacologically inactive peptidyl derivativeconjugate. The peptide linker hasat least two amino acid residue-4-1015202530WO 98/19705CA 02264610 1999-03-08PCT/US97/ 19851sequences specifically tailored so as to render the peptidyl derivativesselective substrates for drug—activating enzymatic cleavage by one or morelysosomal proteases, such as cathepsin B, C, D, or L. The enzymaticcleavage reaction will remove the peptide linker moiety from the drug‘conjugate and effect release of the antitumor agents in pharmacologicallyactive form selectively at the tumor site. _ln comparison with ligand-druglinkers which rely on simple acid hydrolysis for drug release this new methodprovides significantly less systemic toxicity due to premature linkerhydrolysis in the blood, consequently a greater amount of the drug isdelivered to the tumor site, and the method results in a longer storage lifeand simplified handling conditions for the conjugate.The conjugates of the present invention show significantly lesssystemic toxicity than biparte conjugates and free drug. The conjugates ofthe invention retain both specificity and therapeutic drug activity for thetreatment of a selected target cell population. They may be used in apharmaceutical composition, such as one comprising a pharmaceuticallyeffective amount of a compound of Formula (Ill) below, associated with apharmaceutically acceptable carrier, diluent or excipient.The present invention is directed to a linker molecule of the formulaif / (CH2)b'X O)A‘Wc’(CH2)a'(Q)p‘(C)d'E\(CH2)b"Xwherein sA is a thiol acceptor;W is a bridging moiety;0 _is an integer of O to 1;a is an integer of 2 to 12;Q is O, NH, or N-lower alkyl;p is an integer of O or 1;dis an integer of O or 1;E is a polyvalent atom;each b is an integer of 1 to 10;each X is of the formula101520W0 98/ 19705CA 02264610 1999-03-08PCT/US97/19851-CO-Y-Zm-GnwhereinY is two amino acid residues in the L form;Z is one or two amino acid residues;m is an integer of O or 1;G is a self-immolative spacer; andn is a integer of O or 1; provided that when G is 0 then -Y-Zmis aia-Ieu-ala—|eu or gly-phe-leu-gly;or each X is of the formulaoH / (CH2)b-X‘-(CH )a'(Q) -(C) —E2 P d \<cH2>b-X‘.wherein each X1 is of the formula-CO-Y-Zm-Gn;and wherein Y, Z, O, E, G, m, d, p, a, b and n are as definedabove;or each X1 is of the formulaif / (CH2)b'X2-CH2-<o>p—<c>d'E\(CH2)b’X2 ;wherein each X2 is of the formula-CO—Y-Zm-Gn;and wherein Y, Z, G, Q, E, m, d, p, a, b and n are as definedabove;or each X2 is of the formulaii’ / (CH2)b'X3-CH2-<0) ‘(C)d"Ep \<cH2>b-X3 .9wherein each X3 is of the formula-CO-Y-Zm-Gr};CA 02264610 1999-03-08W0 98/ 19705 PCT/US97/ 19851and wherein Y, Z, G, O, E. m, cl, p, a, b and n are as definedabove;or each X3 is of the formula0H (CH2)b'X4- H _ _ _C (Q) (C) E/2 P d \<cH2>b-X4;5 wherein each X4 is of the formula-CO-Y-Zm—Gn;and wherein Y, Z, G. Q, E, m, d, p, a, b and n are as definedabove.The present invention is also directed to a drug/linker molecule of10 the formulawhereinA is a thiol acceptor;W is a bridging moiety;15 c is an integer of O to 1;a is an integer of 2 to 12;Q is O, NH, or N-lower alkyl;p is an integer of O or 1;d is an integer of 0 or 1;20 E is a polyvalent atom;each b is an integer of 1 to 10;each X is of the formula-CO-Y-Zm—GnDwherein25 Y is two amino acid residues in the L form;Z is one or two amino acid residues;m is an integer of O or 1;G is a self-immolative spacer;-7-CA 02264610 1999-03-08W0 98/ 19705 PCTIUS97/19851n is a integer of Clor 1; provided that when G is O then,—Y-Zn,-is ala-leu-ala-leu“or gly-phe-leu-gly; andD is a Drug moiety having a backbone and at least onechemically reactivefffunctional group pendant thereto ‘5 chemicallyreacted to the self-immolative spacer or terminal amino acidresidue to form a covalent bond, said functional group selectedfrom the group consisting of a primary or secondary amine,hydroxyl, sulfhydryl, carboxyl, aldehyde or ketone;10 or each X is of the formulafi’ / (CH2)b'X1-(CH2)a-(Q) '(C)d'E p \<cH2>b-X‘.wherein each X1 is of the formula-CO—Y-Zm-Gn-D;wherein Y, Z, G, D, Q, E, m, d, p and n are as defined above;15 or each X1 is of the formula0H / (CH2)b'X2'CH2'(Q)p‘(C)d'E\ (CH2)b_X2 -wherein each X2 is of the formula-CO-Y-Zm-Gn-D;and wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as defined20 above;or each X2 is of the formulaW / (CH2)b'X3‘CH2-(Q)p-(C)d'E\ 3(C H2)b'X ;wherein each X3 is of the formula-CO-Y—Zm—Gn-D;CA 02264610 1999-03-08WO 98/19705 PCT/US97/19851and wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as defined‘10152025formulawhereinabove;or each X3 is of the formula0H /(CH2)b-X4'CH2'(C)d'N\<cH2>b-X“ .and wherein each X4 is of the formula-CO—Y-Zm-Gn-D;and wherein Y, Z, G, D, Q. E, m, d, p, a, b and n are as definedabove.Furthermore, the present invention is directed to conjugate of theOH / (CH2)b'XL- A'Wc‘(CH2)a"(Q)p‘(C)d'E ‘“'>\<cH2>b-X qL is a ligand;q is an integer of 1 to 10;A is a thiol acceptor;W is a bridging moiety;c is an integer from O to 1;a is an integer of 2 to 12;Q is O, NH, or N-lower alkyl;p is an integer of O or 1;d is an integer of 1 or 2;E is a polyvaient atom;each b is an integer of 1 to 10;each X is of the formula—CO-Y-Zm-Gn-DwhereinY is two amino acid residues in the L form‘)Z is one or two amino acid residues;-9-10152025W0 98/1 9705Or-CH2-(Q)p-(C)'CH2'(Q)CA 02264610 1999-03-08PCT/US97/ 19851m is an integer of O or 1;G is a self-immolative spacer; andn is a integer of O or 1; provided that when G is 0 then -Y—Zm- isala-leu—ala-Ieu or gly-phe-Ieu-gly; 9 ‘D is a drug moiety having a backbone and at least onechemically reactive functional group pendant thereto reacted tothe self—immolative spacer to form a covalent bond, saidfuntional group selected from the group consisting of a primaryor secondary amine, hydroxyl, carboxyl, sulfhydryl, aldehyde,ketone;or X is of the formula0H / (CH2)b'X1'(CH2)a'(Q)p'(C)d'E(CHQVXRVwherein each X1 is of the formula-CO-Y-Zm-Gn-D;and wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as definedabove;or each X1 is of the formula0H / (CH2)b'X2d‘E\ 2(CH2)b-X .wherein each X2 is of the formula—CO—Y-Zm-Gn-D;and wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as definedabove;or each X2 is of the formulaH //(Ctbhrxs'(C)d'EP\<cH2>b-X3 .wherein each X3 is of the formula-10-1015202530CA 02264610 1999-03-08W0 98/ 19705 PCT/U S97/ 19851-CO-Y-Zm-Gn-D; , wherein Y, Z, G, D, Q, E, m, d. p, a, b and n are as definedabove;or each X3 is of the formula0H / (CH2)b'X4'CH2'(Q)p'(C)d'E\ (CH2)b'X4 .wherein end X4 is of the formula—CO-Ym-Zm—Gn-D;wherein Y. Z, G, D, Q, E, m, d, p, a, b and n are as definedabove.As used herein the term “lower alkyl" is an alkyl group having 1 to 3carbon atoms. It is preferred that the polyvalent atom is carbon or nitrogen.In one embodiment the drug moiety is an anthracycline antibioticand the targeting ligand is an antibody.in a preferred embodiment the anthracycline is bound to the linkerat an amino sugar group of the anthriacycline. It is preferred that the sugarmoiety is daunosamine. The antibody then is bound, through the linker, tothe anthracycline compound. in an especially preferred embodiment, thislinkage occurs through a reduced disulfide group (i_.g a free sulfhydryl group(-SH)) on an antibody).In a most preferred embodiment the anthracycline drug moiety isdoxorubicin; the thiol acceptor is a Michael Addition acceptor (from which aMichael Addition Adduct is derived), more preferably is a maleimido— group;and the antibody moiety is a chimeric or humanized antibody, and the pointof attachment of the linker to the drug is at the amino group of the sugarmoiety of the drug.The conjugates of the invention retain both specificity and therapeuticdrug activity for the treatment of a selected target cell population. They maybe used in a pharmaceutical composition, such as one comprising apharmaceutically effective amount of a compound of Formula (lll) associatedwith a pharmaceutically acceptable carrier, diluent or excipient.-11-, WO 98/197051015202530CA 02264610 1999-03-08PCT/US97/19851Detailed Description of the InventionThe present invention provides novel drug-linker-ligand conjugatescomposed of a ligand capable ofitargetinga selected cell population, and adrug connected to the ligand by a branched peptide linker. The linkercontains a thiol acceptor such as a Michael Addition accceptor, a bridgingmoiety, a point of branching, and a peptide sequence per each branchcontaining at least two amino acid moieties which provides an enzymaticcleavage site for an enzyme such as cathepsin B, C, D, or L. The branchedpeptide linker may also contain a self-immolating spacer, which spaces thedrug and the protein peptide sequence. it has been discovered that whenthe peptide seqeunce (i.e., Y-Zm) is ala-leu-a|a-leu- or gly-phe-leu-gly, thenthe presence of the self-immolative spacer is optional. The reason that ase|f—immo|ative spacer is not required for these peptide sequences is notentirely understood; however, it may be due to response to differentenzymes.The targeting ligand molecule can be an immunoreactive proteinsuch as an antibody, or fragment thereof, a non-immunoreactive protein, orpeptide ligand such as bombesin or, a binding ligand recognizing a cellassociated receptor such as a lectin, or any protein or peptide thatpossesses a reactive sulfhydryl group (-SH) or can be modified to containsuch a sulfhydryl group. The thiol acceptor carboxylic is linked to the ligandvia a thioether bond, and the drug is linked to the linker via a functionalgroup selected from primary or secondary amine, hydroxyl, sulfhydryl,carboxyl, aldehyde or ketone.For a better understanding of the invention, the drugs, ligands,peptides and spacers will be discussed individually. The synthesis of theconjugates then will be explained.it will be understood that in the following detailed description andappended claims, the abbreviations and nomenclature employed are thosewhich are standard in amino acid and peptide chemistry, and that all theamino acids referred to are in the L-form unless otherwise specified.Some abbreviations used in the present application, unlessotherwise indicated, are as follows:-12-1015202530W0 93/19705 A 'CA 02264610 1999-03-08PCT/US97/19851ACOH: acetic acid; Allocz allyloxy-carbonyl; Boc: t-buty|oxycarbonyl;.,DBU: __diazobicycloundecene; DCC: dicyclohexylcarbodiimide; DCI: directchemical ionization; DCU: dicyclohexylurea; DIEA: diisopropylethylamine;DMAP: 4-dimethylaminopyridine‘,DME: 1,2-dimethoxyethane; DOX: 1doxorubicin; DTT: dithiothreitol; EEDQ: N-ethcxycarbonyl-2-ethoxy-1,2-dihydroquinoline; EtOAc: ethyl acetate; FAB: fast atom bombardment; Fmoc:fluorenylmethoxycarbonyl; GABA: y—aminobutyric acid; HOBt:m N-hydroxybenzotriazole; HRMS: high resolution mass spectroscopy: LDL: lowdensity lipoprotein; MC: 6-maleimidocaproyl; MP: 3-maleimidopropionyl;MPr-BHP: maleimidopropyl—bis—hydroxypropyl; MPr-Mal: maleimidopropy|-malonyl; MEt—|BHE: maleimidoethyl-imino-bis-hydroxyethyl; MMA:mitomycin A, MMC: mitomycin C; Mtr: 4-methoxytrityl; NHS: N-hydroxysuccinimide; NMP: N-methylpyrrolidinone; PABC: p-aminobenzyl-carbamoyl; PAB-OH: p—aminobenzy| alcohol; PNP: p-nitrophenol; TFA:trifluoroacetic acid; THF: tetrahydrofuran.The Peptide Residue (-Y-Zm-)The peptide linker of the present invention contains two, three orfour amino acid residues per branch, together with the self-immolativespacer if present, that provides one or more enzyme cleavage sites. Theamino acid moieties collectively form a peptide sequence.The amino acid residues making up the peptide residues are eachselected, independently, from the group of amino acids, preferably naturallyoccurring amino acids. The naturally occurring amino acids are alanine(Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys),glutamic acid (Glu), glutamine (Gin), glycine (Gly), histidine (His), isoleucine(lie), Ieucine (Leu), lysine (Lys) methionine (Met), ornithine (Om),phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan(Trp), tyrosine (Tyr), and valine (Val). Preferred naturally occuring aminoacids are Ala, Val. Leu, Lys, lle, Met, Phe, Trp, and Pro. Certain non-naturally occuring amino acids also can be part of the peptide residue. Suchnon—naturally occuring amino acids include citrulline (Cit) and protectedamino acids such as naturally occuring amino acids protected with groupssuch as acetyl, formyl, tosyl, nitro andthe like. When specific amino acids-13-1015202530W0 98/ 19705CA 02264610 1999-03-08PCT/U S97/ 19851are indicated herein as part of a peptide sequence, they are in the L formunless specified otherwise. The amino acid residues making up the moiety must be in the L form. The amino acid residue(s) making up the "Z”moiety can be either in the L or Diform. More preferred amino acids includeLys, Lys protected with acetyi or formyl, Arg, Arg arginine protected with tosylor nitro groups, His, Om, Orn protected with acetyl or formyl, Phe, Val, Ala,and Cit. Most preferred are Lys and Cit..The amino acid residue sequence is specifically tailored so that itwill be selectively enzymatically cleaved from the resulting peptidylderivative drug-conjugate by one or more of the tumor-associated proteases.The amino acid residue chain length of each branch of the peptidelinker preferably ranges from that of a dipeptide to that of a tetrapeptide.It is preferred that the first amino acid residue (i.e., the first aminoacid making up read left to right) is a basic amino acid (e.g., Lys or Arg)or has strong hydrogen bonding capability (e.g., Cit). it is preferred that thesecond amino acid residue (i.e., the second amino acid making up “Y”, readleft to right) has a hydropholic side chain (e.g., Phe, Val, Ala, Leu or lie).The following group of exemplary peptide linker groups, are namedin order to illustrate further the conjugates of the present invention:Phe-Lys, Val-Lys, Phe—Phe—Lys, Lys-Phe-Lys, Gly-Phe-Lys, Ala-Lys,Val—Cit, Phe-Cit, Leu-Cit, lle-Cit, Trp-Cit, Phe-Ala, Gly-F’he—Leu-Gly,Ala-Leu-Ala-Leu, Phe-Ng-tosyl-Arg, Phe-Ng-Nitro-Arg, Lys-Lys,Lys—Cit, and Cit-Cit.Specific examples of the preferred embodiment of peptidesequences include Phe-Lys, Val-Lys, Val-Cit, and D-Phe—Phe-Lys.Numerous specific peptide linker molecules suitable for use in thepresent invention can be designed and optimized in their selectivity forenzymatic cleavage by a particular tumor—associated protease. Thepreferred peptide linkers for use in the present invention are those which areoptimized toward the protease, cathepsin B, C, D, and L.-14-1015202530CA 02264610 1999-03-08WO 98/19705 PCT/US97/19851 ("G") . wThe molecules in accordance with the present invention mayemploy an intermediate self-immolative spacer moiety which spaces andcovalently links together the drughmoiety and the protein peptide moiety. Aself-immolative spacer may be defined as a bifunctional chemical moietywhich is capable of covalently linking together two spaced chemical moietiesinto a normally stable tripartate molecule, releasing one of said spacedchemical moieties from the tripartate molecule by means of enzymaticcleavage; and following said enzymatic cleavage, spontaneously cleavingfrom the remainder of the molecule to release the other of said spacedchemical moieties. In accordance with the present invention, the self-immolative spacer is covalently linked at one of its ends to the proteinpeptide moiety and covalently linked at its other end to the chemical reactivesite of the drug moiety whose derivatization inhibits pharmacological activity,so as to space and covalently link together the protein peptide moiety andthe drug moiety into a tripartate molecule which is stable andpharmacologlcally inactive in the absence of the target enzyme, but which isenzymatically cleavable by such target enzyme at the bond covalentlylinking the spacer moiety and the protein peptide moiety to thereby effectrelease of the protein peptide moiety from the tripartate molecule. Suchenzymatic cleavage, in turn, will activate the self-immolating character of thespacer moiety and initiate spontaneous cleavage of the bond covalentlylinking the spacer moiety to the drug moiety, to thereby effect release of thedrug in pharmacologically active form.In the molecules of Formulas I, II, and Ill:G is a self-immolative spacer moiety which spaces and covalently0 links together the drug moiety and the amino acid, in which thespacer is linked to the drug moiety via the T moiety (as used in thefollowing formulas “T” represents a nucleophilic atom which isalready contained in the Drug), and which may be represented bythe structures of Formulae, (IV), (V), (VI), (V|l),or (VIII):-15-CA 02264610 1999-03-08WO 98/19705 7' ‘ PCT/US97/198510Formula (IV)in which T is O, N or S,—HN—R‘—coT5 Formula (V) -in which T is O, N or S, andR1 is C1-C5 alkyl;T--HN cooriFormula (VI)10 (J. Med. Chem., 27: 1447 (1984)in which T is O, N or S, andR2 is H or C1-C5 alkyl;——NH—< >—~\O/\T' 01'Formula (VII)15 in which T is O, N or 8,COT--OCOFormula (Vlll)in which T is O, S or N.20 As used herein "C1-C5 alkyl'’ is meant to include a branched orunbranched hydrocarbon chain having, unless otherwise noted, one to five-15-CA 02264610 1999-03-08WO 98/19705 PCT/US97/19851carbon atoms, including but not limited to methyl, ethyl, isopropyl, r)-propyl, __sec-butyl, isobutyl, n-butyl and the like.A preferred G self—immolative spacer moiety suitable for use in thepresent invention is PABC represented by the Formula (lVa):5H-—N0Formula (|Va)Another preferred G se|f—lmmolative spacer moiety suitable for usein the present invention is GABA represented by the Formula (Va):10 ——HN’“\/”‘coTFormula (Va)Yet another preferred G self-immolative spacer moiety suitable foruse in the present invention is ot,o«.—dimethy| GABA represented by theFormula (Vb):15 __HN/\><COTFormula (Vb)Another preferred G self-immolative spacer moiety suitable for usein the present invention is B,B-dimethyl GABA represented by the Formula(Vo):20-HN/f><:‘coTFormula (V0)The Thiol Acceptor25 In the molecules of Formulas I, II, and III, the thiol acceptor "A" is linkedto the ligand via a sulfur atom derived from the ligand. The thiol acceptorbecomes a thiol adduct after bonding to the ligand through a thiol group via-17-101520W0 98I19705CA 02264610 1999-03-08PCTIUS97/19851a thioester bond. The thiol acceptor can be , for example, an alpha; substitited acetyl group. Such a group has the formulaE.’ -.Y-CH2--C-wherein Y is a leaving group. Examples of leaving groups include Cl,Br, I, mesylate, tosylate, and the like. If the thiol acceptor is an alpha-substituted acetyl group, the thiol adduct after linkage to the ligand forms thebond -8-CH2-Preferably, the thiol acceptor is a Michael Addition acceptor. Arepresentative Michael Addition acceptor of this invention has the formulaAfter linkage the thiol group of the ligand, the Michael Additionacceptor becomes a Michael Addition adduct, such as of the formula AON__wherein L is ligand.The Brid in Grou "W"The bridging group ia a bifunctional chemical moiety which is capableof covalently linking together two spaced chemical moieties into a stabletripartate molecule. Examples of bridging groups are described in S.S.Wong, Chemistrv of Protein Coniudation and Crosslinkinq. CRC Press,Florida, (1991); and GE. Means and RE. Feeney, Bioconludate Chemistry,vol. 1, pp.2-12, (1990), the disclosures of which are incorporated herein byreference. Specifically, the bridging group "W" covalently links the thiolacceptor to a keto moiety. An example of a bridging group has the formula-18-1015202530CA 02264610 1999-03-08WO 98/19705 PCT/US97/19851'”'(CH2)r _—(Z'>g ""{CH2)h: « Wwherein A "f is an integer of O to 10,h is an integer of O to 10,g is‘an integer of O or 1,provided that when g is 0, then f + h is 1 to 10,Z is S, O, NH, S02, phenyl, naphthyl, a polyethylene glycol, acycloaliphatic hydrocarbon ring containing 3to 10 carbon atoms, or a heteroaromatichydrocarbon ring containing 3 to 6 carbonatoms and 1 or 2 heteroatoms selected from O,N, or 8. ~.Preferred cycloaliphatic moieties include icyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and the like. Preferred heteroaromatic moietiesinclude pyridyl, polyethlene glycol (1-20 repeating units), furanyl, pyranyl,pyrimidinyl, pyrazinyl, pyridazinyl, oxazinyl, pyrrolyl, thiazolyl, morpholinyl,and the like.In the bridging group it is preferred that when g is O, f + h is an integerof 2 to 6 preferably 2 to 4 and more preferably 2. When g is 1, it is preferredthatf is 0, 1 or 2, and that h is O, 1 or 2.Preferred bridging groups coupled to thiol acceptors are shown in thePierce Catalog, pp. E-12, E-13, E—14,E-15, E-16, andE—17 (1992).The DrugThe drug conjugates of the present invention are effective for theusual purposes for which the corresponding drugs are effective, and havesuperior efficacy because of the ability, inherent in the ligand, to transport thedrug to the desired cell where it is of particular benefit. Further, because theconjugates of the invention can be used for modifying a given biologicalresponse, the drug moiety is not to be construed as limited to classicalchemical therapeutic agents. For example, the drug moiety may be a proteinor polypeptide possessing a desired biological activity. Such proteins mayinclude, for example, a protein such as tumor necrosis factor.-19-1015202530WO 98/19705CA 02264610 1999-03-08PCT/US97/1985 1The preferred drugs for use in the present invention are cytotoxicdrugs, particularly those which are used for cancer therapy. Such drugsinclude, in general, DNA damaging agents, anti-metabolites, naturalproducts and their analogs. Preferred classes of cytotoxic agents include, forexample, the enzyme inhibitors such as dihydrofolate reductase inhibitors,and thymidylate synthase inhibitors, DNA intercalators, DNA cleavers,topoisomerase inhibitors, the anthracycline family of drugs, the vinca drugs,the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridinefamily of drugs, diynenes, the podophyllotoxins, differentiation inducers, andtaxanes. Particularly useful members of those classes include, for example,methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine,actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A,carminomycin, aminopterin, tallysomycin, podophyllotoxin andpodophyllotoxin derivatives such as etoposide or etoposide phosphate,vinblastine, vincristine, vindesine, taxol, taxotere, retinoic acid, butyric acid,N8-acetyl spermidine, camptothecin, and their analogues.As noted previously, one skilled in the art may make chemicalmodifications to the desired compound in order to make reactions of thatcompound more convenient for purposes of preparing conjugates of theinvenfion.In the conjugate of Formula I,D is a drug moiety having pendant to the backbone thereof achemically reactive functional group my means of which the drugbackbone is bonded to the protein peptide linker, said functionalgroup selected from the group consisting of a primary or secondaryamine, hydroxyl, sulfhydryl, carboxyl, aldehyde or a ketone.Representative of said amino containing drugs are mitomycin-C,mitomycin-A, daunorubicin, doxorubicin, aminopterin, actinomycin,bleomycin, 9-amino camptothecin, N3-acetyl spermidine, 1-(2-chloroethyl)—1,2-dimethanesulfonyl hydraxide, tallysomycin,cytarabine and derivatives thereof.-20-10152025CA 02264610 1999-03-08WO 98/19705 PCT/US97/19851Representative of said alcohol group containing drugs are _,etoposide, camptothecin, taxol, esperamicin, 1,8-dihydroxy-bicyc|o[7.3.1ltrideca-4-9-diene-2,6~diyne-13-one, (U.S. Patent5,198,560), podophyllotoxin, anguidine, vincristine, vinblastine,morpholine-doxorubioin, n-(5,5-diacetoxy-pentyl)doxorubicin, andderivatives thereof.Representative of said sulfhydryl containing drugs are esperamicinand 6-mercaptopurine, and derivatives thereof.Representative of said carboxyl containing drugs are methotrexate,camptothecin (ring-opened form of the lactone), butyric acid,retinoic acid, and derivatives thereof.Representative of said aldehyde and ketone containing drugs areanguidine and anthracyclines such as doxorubicin, and derivativesthereof.A highly preferred group of cytotoxic agents for use as drugs in thepresent invention include drugs of the following formulae:THE MITOMYCIN GROUP OF FORMULA (1):O2 cH2ocoNh;.OCH,N N-R1H300in whichR1 is hydrogen or methyl;R2 is -NH2, -OCH3, -O(CH2)2OH, —NH(CH2)2SS(CH2)2NHAc, -NHCH—C5CH, —NH(CH2)2SS(C5H4)NO2, -O(CH2l2SS(CH2)2OH, -N=CH~NHOCH3, —NH(C5H4)OH, —NH(CH2)2SS(CH2)2NHCO(CH2)2CH(NH2)COOH-21-CA 02264610 1999-03-08W0 98/19705 PCTIUS97/19851O-——-9 .._ /N‘-"'—‘ , €)___ ‘-_NHcH2c< NH(C,H2)2SSCH2\ ___OC,_b/0"‘ , or ¥—.0 CH3 _THE BLEOMYCIN GROUP OF FORMULA (2):CONH2 NH2&OcoNH2/ ‘N CH3 NH2N 0 5 in whichR1 is hydroxy, amino, C1-C3 a|ky|amino.di(C1-C3 alky|)amino, C4-C5 polymethylene amino,NH+ II-—NH(CH2)3S-'-CH3CH3 , ,—NH(CH2)sCHC|'bCIT|H(CFb)sNH(CH2)4NH2 , or10 NH2 O"'NH(CH2)3NH(CH2)4NH2THE METHOTREXATE GROUP OF FORMULA (3):-22-(2)1015CA 02264010 1999-03-08WO 98119705 PCT/US97/19851H2N\r/N N\ 3 IGOR‘ ' " .Nfijihl/J-—C H2—T CONHCHCi-§CH2COOi-Q, _R“ F‘ R8 is)in whichR1 is amino or hydroxy;R2 is hydrogen or methyl;R3 is hydrogen, fluoro, chloro, bromo or iodo;R4 is hydroxy or a moiety which complete a salt of the carboxylicacid.MELPHALAN OF FORMULA (4):NH2(4)6-MERCAPTOPURINE OF FORMULA (5):HSHN/ I >KN(5)-23-CA 02264610 1999-03-08W0 98/ 19705 PCT/US97/19851A CYTOSINE ARABINOSIDE OF FORMULA (6): ’ QNmI ;“NJ\ooHOH2C—/OH OH(5)THE PODOPHYLLOTOXINS OF FORMULA (7):CH3O OCH3OR?(7)10 whereinR2 is hydrogenR1 is hydrogen or-24-CA 02264610 1999-03-08wo 98/19705 Z ‘ PCT/US97/19851whereinR3 is NH2, OH, ocH3, NH(C1—C3 akyl) orN(C1-C3 alkyl)25 R4 is OH, or NH2,R5 is methyl or thienyl,R5 is hydrogen or methyl, or a phosphate salt thereof.As used herein "(C1-C3 aky|)" means a straight or branched carbonchain having from one to three carbon atoms; examples include methyl,10 ethyl, n-propyl and isopropyl.THE VINCA ALKALOID GROUP OF DRUGS OF FORMULA (8): 15 in whichR1 is H, CH3 or CHO;when R2 and R3 are taken singly, R3 is H, and one of R4 and $2 isethyl and the other is H or OH:-25-CA 02264610 1999-03-08W0 98/ 19705 PCT/US97/19851when R2 and R3 are taken together with the carbons to which they.are attached, they form an oxirane ring in which case R4 is ethyl;R5 is hydrogen, (C1-C3 alkyl)-CO, or chlorosubstituted (C1-C2alkyl)-CO.5 As used herein "C1-C3 alkyl" means a straight or branched carbonchain having from one to three carbon atoms; examples include methyl,ethyl, n—propyl and isopropyl.DiFLUORONUCLEOS|DES OF FORMULA (9):10R1 0 C FF OH(9)in which R1 is a base of one of the formulae:R30/ _HN | R2 mi \ N\> 2:: | CH_CHR‘)\ )\ / N O NO T H2N N l |NH2 NH2/ 2 \ NN l R N \>)\ K / NO T N l15 in whichR2 is hydrogen, methyl, bromo, fluoro, chloro, or iodo;R3 is -OH or -NH2;R4 is hydrogen, bromo, chloro, or iodo.-26-10152025CA 02264610 1999-03-08WO 98/19705 PCT/US97/ 19851TAXOLS OF FORMULA (101: , R4COl\_lH whereinR1 is hydroxy;R2 is hydrogen or hydroxy;R2‘ is hydrogen, hydroxy, or acetoxy;R7 is hydrogen or hydroxy;R3 is hydrogen, hydroxy, or acetoxy;R4 is aryl, substituted aryl, C1-5 alkyl, C2-5 alkenyi, C2-5 alkynyl ort-butoxy;R5 is C1-3 alkyl, C2-5 alkenyl, C2-5 alkynyl, or -Z-R5;Z is a direct bond, C1-5 alkyl or C2-5 alkenyl;R6 is aryl, substituted aryl, C3-5 cycloaikyl, thienyl or furyl.As used herein, "alkyl" means a straight or branched saturatedcarbon chain having from one to six carbon atoms; examples include methyl,ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t—buty|, n-pentyl, sec-butyl, isopentyl, and n—hexyl. "A|kenyl" means a straight or branched carbonchain having at least one carbon-carbon double bond, and having from twoto six carbon atoms; examples include ethenyl, propenyl, isopropenyl,butenyl, isobutenyl, pentenyl, and hexenyl. "Alkynyl" means a straight orbranched carbon chain having at least one carbon-carbon triple bond, andfrom two to six carbon atoms; examples include ethynyl, propynyl, butynyl,and hexynyl. "Aryl" means aromatic hydrocarbon having from six to tencarbon atoms; examples include phenyl and naphthyl. "Substituted aryl"means aryl substituted with at least one group selected from C1-5-27-1O1520CA 02264610 1999-03-08W0 98/ 19705 T ’ PCT/US97/19851alkanoyloxy, hydroxy, halogen, C1-5 alkyl, trifluoromethyi, C1-5 alkpxy, aryI,__C2—5 alkenyi, C1-5 alkanoyl, nitro, amino, and amido.ANGUIDINES OF FORMULA T1“: whereinR1 is OH or O;R2 is H or O;Anguidine can be targeted at the C-3, C-4, C-8 or C-15 positions,as an ester or hydrazonePHENYLENE DIAMINE MUSTARD (12):H2N-C5H4-N(CH2CH2C|)2THE ANTHFIACYCLINES ANTIBIOTICS OF FORMULA (13): 101520W0 98/ 19705CA 02264610 1999-03-08PCT/US97/ 19851whereinR1 is —CH3, -CHQOH, -CH2OCO(CH2)3CH3 or —CH2OCOCH(OC2H5)2R2 is -OCH3, —OH or -HR3 is -NH2, -NHCOCF3, 4-morpholinyl, 3—cyano—4-morpholinyl, 1-piperidinyl, 4-methoxy-1-piperidinyl, benzvlamine, dibenzylamine,cyanomethylamine, 1—cyano—2-methoxyethyl amine, or NH—(CH2)4-CH(OAc)2;R4 is —OH, —OTHP, or —H; andR5 is —OH or -H provided that R5 is not —OH when R4 is -OH or -OTHP.One skilled in the art understands that structural Formula (13)includes compounds which are drugs, or are derivatives of drugs, whichhave acquired in the art different generic or trivial names. Table l, whichfollows, represents a number of anthracycline drugs and their generic ortrivial names and which are especially preferred for use in the presentinvenfion.Of the compounds shown in Table I, the most highly preferred drugis Doxorubicin. Doxorubicin (also referred to herein as "DOX") is thatanthracyclin shown in the formula of Table l in which R1 is -CH2OH, R2 is -OCH3, R3 is -NH2, R4 is -OH, and R5 is -H. --29-Euunuuocaac won 25: ouafiufia .3 3 sfizmwmonzaa...PCT/US97/19851CA 02264610 1999-03-08WO 98/19705u .8 £o¢oEm.1nu:_z.. inc no.5 xonea3B so a 3:! nuuo no.8 xoaooafionmuonaoanao/I.\= .6 .\ /.1 nauo aofu xoonacfiozmuoz. nan. Eu. «.8 2 En . auo .H ea. .5: 3.. .3... = = «:2 :80 =c~=u Suuaauonu.8 a £2 nzuo =o~=u 5u3EEm2 so «:2 a man a ouaauucn= :0 «:2 no nzo cue nonuauao2 no «:2 inc :.=~uo.auouo~_.u .:u3.aB3a an u=z nun .=m=o cmuanauuxon= =0 «.5. J50 nan -auuunauozaanmm 2:. -9 . 2 z ...__...._..a -30-1015202530W0 98/ 19705CA 02264610 1999-03-08PCT/US97/19851The most highly preferred drugs are taxol, mitomycin C, and theanthracycline antibiotic agents of Formula (13), described previously.The LigandThe "ligand" includes within its scope any molecule that specificallybinds or reactively associates or complexes with a receptor or otherreceptive moiety associated with a given target cell population. This cellreactive molecule, to which the drug reagent is linked via the linker in theconjugate, can be any molecule that binds to, complexes with or reacts withthe cell population sought to be therapeutically or otherwise biologicallymodified and, which possesses a free reactive sulfhydryl (-SH) group or canbe modified to contain such a sulfhydryl greup. The cell reactive moleculeacts to deliver the therapeutically active drug moiety to the particular targetcell population with which the ligand reacts. Such molecules include, butare not limited to, large molecular weight proteins such as, for example,antibodies, smaller molecular weight proteins, polypeptides or peptideligands, and non-peptidyl ligands.The non-immunoreactive protein, polypeptide, or peptide ligandswhich can be used to form the conjugates of this invention may include, butare not limited to, transferrin, epidermal growth factors ("EGF"), bombesin,gastrin, gastrin-releasing peptide, platelet—derived growth factor, IL-2, IL-6,tumor growth factors (”TGF"), such as TGF-(X and TGF-B, vaccinia growthfactor ("VGF"), insulin and insulin-like growth factors I and ll. Non-peptidylligands may include, for example, carbohydrates, lectins, and apoproteinfrom low density lipoprotein.The immunoreactive ligands comprise in antigen-recognizingimmunoglobulin (also referred to as "antibody"), or an antigen-recognizingfragment thereof. Particularly preferred immunoglcbulins are thoseimmunoglcbulins which can recognize a tumor-associated antigen. Asused, "immunoglobulin" may refer to any recognized class or subclass ofimmunoglobulins such as |gG, lgA, lgM, lgD, or lgE. Preferred are thoseimmunoglcbulins which fall within the IgG class of immunoglobulins. Theimmunoglobuin can be derived from any species. Preferably, however, the-31-1O15202530WO 98/19705CA 02264610 1999-03-08PCT/US97/ 19851immunoglobulin is of human, murine, or rabbit origin. Furthermore, theimmunoglobulin may be polyclonal or monoclonal, preferably monoclonal.As noted, one skilled in the art will appreciate that the inventionalso encompasses the use of antigen recognizing immunoglobulinfragments. Such immunoglobulin fragments may include, for example, theFab‘, F(ab')2, FV or Fab fragments, or other antigen recognizingimmunoglobulin fragments. Such immunoglobulin fragments can beprepared, for example, by proteolytic enzyme digestion, for example, bypepsin or papain digestion, reductive alkylation, or recombinant techniques.The materials and methods for preparing such immunoglobulin fragmentsare well-known to those skilled in the art. See generally, Parham, ;immunology, @, 2895 (1983); Lamoyi _e_1_aL, J. Immunological Methods,E, 235 (1983); Parham, i_d., 5_3, 133 (1982); and Matthew etii, Q, fl, 239(1982).The immunoglobulin can be a "chimeric antibody" as that term isrecognized in the art. Also the immunoglobulin may be a ''bifunctional'' or"hybrid" antibody, that is, an antibody which may have one arm having aspecificity for one antigenic site. such as a tumor associated antigen whilethe other arm recognizes a different target, for example, a hapten which is, orto which is bound, an agent lethal to the antigen-bearing tumor cell.Alternatively, the bifunctional antibody may be one in which each arm hasspecificity for a different epitope of a tumor associated antigen of the cell tobe therapeutically or biologically modified. In any case, the hybridantibodies have a dual specificity, preferably with one or more binding sitesspecific for the hapten of choice or more or more binding sites specific for atarget antigen, for example, an antigen associated with a tumor, an infectiousorganism, or ‘other disease state.Biological bifunctional antibodies are described, for example, inEuropean Patent Publication, EPA O 105 360, to which those skilled in theart are referred. Such hybrid or bifunctional antibodies may be derived, asnoted, either biologically, by cell fusion techniques, or chemically, especiallywith cross-linking agents or disulfide bridge—forming reagents, and may becomprised of whole antibodies and/or fragments thereof. Methods forobtaining such hybrid antibodies are disclosed, for example, in PCT-32-1015202530W0 98/ 19705CA 02264610 1999-03-08PCT/US97/19851Application W083/03679, published October 27, 1983, and publishedEuropean Application EPA O 217 577, published April 8, 1987, both ofwhich are incorporated herein by reference. Particularly preferred ‘bifunctional antibodies are thoseibiologically prepared from a "polydorna" or"quadroma" or which are synthetically prepared with cross—linking agentssuch as bis-(maleimido)-methyl ether ("BMME"), or with other cross-linkingagents familiar to those skilled in the art.In addition the immunoglobulin may be a single chain antibody("SCA"). These may consist of single chain Fv fragments ("scFv") in whichthe variable light ("VL") and variable heavy ("VH") domains are linked by apeptide bridge or by disulfide bonds. Also, the immunoglobulin may consistof single VH domains (dAbs) which possess antigen-binding activity. 1, ’ G. Winter and C. Milstein, _Na_m_re, gfl, 295 (1991); R. Glockshuber gta_l., Biochemistry _2__9, 1362 (1990); and E. 8. Ward Lal.,l_\l_a1mr_e_§_4fl, 544(1989).Especially preferred for use in the present invention are chimericmonoclonal antibodies, preferably those chimeric antibodies havingspecificity toward a tumor associated antigen. As used herein, the term"chimeric antibody" refers to a monoclonal antibody comprising a variableregion, i_.<;, binding region, from one source or species and at least a portionof a constant region derived from a difference source of species, usuallyprepared by recombinant DNA techniques. Chimeric antibodies comprisinga murine variable region and a human constant region are especiallypreferred in certain applications of the invention, particularly human therapy,because such antibodies are readily prepared and may be lessimmunogenic than purely murine monoclonal antibodies. Suchmurine/human chimeric antibodies are the product of expressedimmunoglobulin genes comprising DNA segments encoding murineimmungobulin constant regions. Other forms of chimeric antibodiesencompassed by the invention are those in which the class or subclass hasbeen modified or changed from that of the original antibody. Such "chimeric"Methods forproducing chimeric antibodies involve conventional recombinant DNA andantibodies are also referred to as "class-switched antibodies".-33-1015202530W0 98/ 19705CA 02264610 1999-03-08PCT/US97/ 19851gene transfection techniques now well known in the art. &a_e, _e_._g,,.Morrison,_,_S. L., et al., Proc. Nat'l Acad. Schfl 6851 (1984).Encompassed by the term "chimeric antibody" is the concept of"humanized antibody", that is those antibodies in which the framework for"complementarity determining regions ("CDR") have been modified tocomprise the CDR of an immunoglobulin of different specificitry as comparedto that of the parent immunoglobulin. in a preferred embodiment, a murineCDR is grafted into the framework region of a human antibody to prepare the"humanized antibody". Sis, e_.g_., L. Riechmann <_afi1_L,_l_\l_atgLe3Ci, 323(1988); M. S. Neubergerge’r_al.,hl_;;1tiJi§_1A, 268 (1985). Particularlypreferred CDR‘s correspond to those representing sequences recognizingthe antigens noted above for the chimeric a_nd bifunctional antibodies. Thereader is referred to the teaching of EPA O 239 400 (published September30, 1987), incorporated herein by reference, for its teaching of CDR modifiedantibodies.One skilled in the art will recognize that a bifunctional-chimericantibody can be prepared which would have the benefits of lowerimmunogenicity of the chimeric or humanized antibody, as well as theflexibility, especially for therapeutic treatment, of the bifunctional antibodiesdescribed above. Such bifunctional-chimeric antibodies can besynthesized, for instance, by chemical synthesis using cross—linking agentsand/or recombinant methods of the type described above. in any event, thepresent invention should not be construed as limited in scope by anyparticular method of production of an antibody whether bifunctional,chimeric, bifunctional-chimeric, humanized, or an antigen-recognizingfragment or derivative thereof.In addition, the invention encompasses within its scopeimmunoglobulins (as defined above) or immunoglobulin fragments to whichare fused active proteins, for example, an enzyme of the type disclosed inNeuberger, gal; PCT application, WO86/01533, published March 13, 1986.The disclosure of such products is incorporated herein by reference.As noted, "bifunctional", "fused", "chimeric" (including humanized),and "bifunctional-chimeric" (including humanized) antibody constructionsalso include, within their individual contexts constructions comprising-34-10152025W0 98l19705CA 02264610 1999-03-08PCT/US97/ 19851antigen recognizing fragments. As one skilled in the art will recognize, such__fragments could be prepared by traditional enzymatic cleavage of intactbifunctional, chimeric, humanized, or chimeric-bifunctional antibodies. If,however, intact antibodies are not susceptible to such cleavage, because ofthe nature of the construction involved, the noted constructions can beprepared with immunoglobulin fragments used as the starting materials; or, ifrecombinant techniques are used, the DNA sequences, themselves, can betailored to encode the desired "fragment" which, when expressed, can becombined in vivo or in vitro, by chemical or biological means, to prepare thefinal desired intact immunoglobulin "fragment". It is in this context, therefore,that the term "fragment" is used.Furthermore, as noted above, the immunoglobulin (antibody), orfragment thereof, used in the present invention may be polyclonal ormonoclonal in nature. Monoclonal antibodies are the preferredimmunoglobulins, however. The preparation of such polyclonal ormonoclonal antibodies now is well known to those skilled in the art who, ofcourse, are fully capable of producing useful immunoglobulins which can beused in the invention. gcfi, ggu G. Kohler and C. Milstein, Nature g§_§, 495(1975). In addition, hybridomas and/or monoclonal antibodies which areproduced by such hybridomas and which are useful in the practice of thepresent invention are publicly available from sources such as the AmericanType Culture Collection ("ATCC") 12301 Parklawn Drive, Ftockville,Maryland 20852 or, commercially, for example, from Boehringer—MannheimBiochemicals, P.O. Box 50816, Indianapolis, Indiana 46250.Particularly preferred monoclonal antibodies for use in the presentinvention are those which recognize tumor associated antigens. Suchmonoclonal antibodies, are not to be so limited, however, and may include,for example, the following:-35-W0 98/19705Antigen SiteRecognizedLung TumorsSquamous LungSmall Cell LungCancerColon CancerCarcinoembryonicMelanomaCA 02264610MonoclonalAntibodiesKS1/4534,F8;604A9G1, LuCa2,LuCa3, LuCa4TFS-211.285.1414.95.55NS—3a-22,NS—1 ONS-19-9,NS—33aNS—52a,1 7-1 AMoAb 35 orZCEO259.2.271999-03-03PCT/US97/19851ReferenceN. M. Varki et al., CancerRes. 44:681, 1984.F. Cuttitta et al., in: G. L.Wright (ed) MonoclonalAntibodies and Cancer,Marcel Dekker, Inc., NY., p.161, 1984.Kyoizumi et al., CancerRes., 4523274, 1985.Okabe et al., Cancer Res.,4521930, 1985.G. Rowland et al., CancerImmunol. Immunother, 19:1,1985.Z. Steplewski et al., CancerRes., 4112723, 1981.Acolla, R. S. et al., Proc. Nat.Acad. Sci., (USA), 772563,1980.T. F. Bumol and R. A.Reiseld, Proc. Natl. Acad.SCi., lUSAl, 79:1245, 1982.-35-W0 98/ 19705p97Antigen T65FerritinNeuroblastomaGliomaCA 02264610 1999-03-0896.5T101AntiferrinR24P1 153/3MIN 1UJ13ABF7, GE2, CG12PCT/US97/19851K. E. Hellstrom et al.,Monoclonal Antibodies andCancer, loc. cit. p. 31.Boehringer-MannheimP.O. Box 50816Indianapolis, IN 46250Boehringer-MannheimPO. Box 50816Indianapolis, IN 46250W. G. Dippold et al., Proc.Natl. Acad. Sci. (USA),7726114, 1980.R. H. Kennet and F. Gilbert,Science, 203:1120, 1979.J. T. Kemshead inMonoclonal Antibodies andCancer, loc. cit. p. 49.Goldman et al., Pediatrics,105252, 1984.N. de Tribolet et al., inMonoclonal Antibodies andCancer, loc. cit. p. 81.-37-, WO 98/19705GangliosideLewis YfucosylatedLewis YCA 02264610 1999-03-08L6Chimeric L6BR64BR96, ChimericBR96PCT/US97/19851l. Hellstrom _et_a|_., Proc. Natl. ~.Acad. Sci. (USA), 83:7059(1986); U.S. Patent Nos.4.906562, issued March 6,1990 and 4,935,495, issuedJune 19, 1990.«iiU.S. Serial No. 07/923,244,filed October 27, 1986,equivalent to PCT PatentPublication, WO 88/03145,published May 5, 1988.U.S. Serial No. 07/289,635,filed December 22, 1988,and U.S. Serial No.07/443,696, filed November29, 1989, equivalent toEuropean Patent TPublication, EP A 0 375562, published June 27,1990.U.S. Serial No. 07/374,947,filed June 30, 1989, andU.S. Serial No. 07/544,246,filed June 26, 1990,equivalent to PCT PatentPublication, WO 91/00295,pubhshedJanuary 10, 1991.-38-10CA 02264610 1999-03-08wo 98/19705 PCT/US97/19851Breast Cancer B6.2, B72.3 D. Colcher et al., in _Monoclonal Antibodies andCancer, loc. cit. p. 121.Osteogenic 791T/48, M. J. Embleton, p. 181.Sarcoma 791T/36Leukemia CALL 2 C. T. Teng et al., Lancet,1:01, 1982.anti—idiotypeR.A. Miller et al., N. Engl. J.Med., 306:517, 1982.Ovarian Cancer OC 125 R. C. Bast et al., J. Clin.lnvest., 68:1331, 1981.Prostrate Cancer D83.21, P6.2, J. J. Starling et al., inTurp-27 Monoclonal Antibodies andCancer, loc. cit, p. 253.Renal Cancer A6H, D5D P. H. Lange et al., Surgery,982143, 1985.In the most preferred embodiment, the ligand containing conjugateis derived from chimeric antibody BR96, "ChiBR96", disclosed in U.S. SerialNo. 07/544,246, filed June 26, 1990, and which is equivalent to PCTPublished Application, WO 91/00295, published January 10, 1991.ChiBFt96 is an internalizing murine/human chimeric antibody and is reactive,ad noted, with the fucosylated Lewis Y antigen expressed by humancarcinoma cells such as those derived from breast, lung, colon, and ovariancarcinomas. The hybridoma expressing chimeric BR96 and identified asChiBR96 was deposited on May 23, 1990, under the terms of the BudapestTreaty, with the American Type Culture Collection ("ATCC"), 12301 ParklawnDrive, Rockville, Maryland 20852. Samples of this hybridoma are available-39-1015202530W0 98/19705 9 ‘CA 02264610 1999-03-08PCT/US97/19851under the accession number ATCC 10460. ChiBFt96 is derived, in part, fromits source parent, BR96. The hybridoma expressing BR96 was deposited, onFebruary 21, 1989, at the ATCC, under the terms of the Budapest Treaty andis available under the accessioninumber HB 10036. The desired hybridomais cultured and the resulting antibodies are isolated from the cell culturesupernatant using standard techniques now well known in the art. E <_a_.g,,"Monoclonal Hybridoma Antibodies: Techniques and Applications", Hurell(ed.) (CRC Press, 1982).Thus, as used "immunog|obulin" or "antibody" encompasses withinits meaning all of the immunoglobulin/antibody forms or constructions notedabove.Svnthesis of the Compounds of the InventionThe linkers, drug/linkers, and conjugates of the invention can bemade techniques taught herein, known in the art, or can be made via routineexperimentation using as guidance the techniques taught herein and/orknown in the art. The attachment of the drug to the linker is accomplished byreacting a nucleophilic atom of the drug (0, N or S) to an electrophilic atom(C, S, P) on eitherthe self—immolating spacer or the carboxy terminus of thepeptide. This is illustrated as follows:0 0JKR X ’' D'“9’V —-—-> RJ]\y-Drug+XOR-O/\X + DVU9‘Y ——-—-> R-O y-Drugwhere: X = leaving group such as Cl‘, Br‘, tosylate, N—hydroxysuccinimidey = nucleophilic group such as OH, NH2, SH, NH—|ower alkylR = rest of linker.Linkage to an aldehyde or ketone can be effected by having thealdehyde or ketone in the form of an enol or N—|oweralkyl enamine. It isexpected that on release of the free enamine, it will spontaneously hydrolyzeto an aldehyde or ketone.The following reaction schemes are illustrative preparativetechniques (compound numbers correspond to Example numbers):-40-CA 02264610 1999-03-08WO 98/19705 PCTIUS97/19851Scheme 1 .A-ii\o\HNI. TMS—Cl, DIEA " /(Z O EIQNH '3 OOHFmoc-Lys —~—--——-—--—~cn —‘:_T"’ /(12. DES H1202 HQN E0”. 21/ONH\ o\DCC fit)" OCO 0 ?L N 0%o 3D111‘/\. DMF9° log0 OH OHEL N N B0030, pyridine OJOL N “£0”0 H o 5 H OH O "' ° 'H2N/©/\ Hg5 : /o 0 ‘3Ewnmfifmf2 Hz“0 0,-41-CA 02264610 1999-03-08W0 98/ 19705Scheme 2on flit?OJLN/Y\‘ToH__1x11~1?3_’o3OPCT/US97/19851O 0£5 +CO H o DCC O 0 OHO Q "2" O 27a R=CI-12Ph7b R=CH(CH3 )27c R=CH3IIHI0 R H 0JL JkDME. water: 0 OHNJHCO3 HN OHOLQNH2EEDQ, THF0 0/ 821 R=CH2Ph81) R=CH(CH3)28C R=CH3OH0 H H 0 Q) E NHO 0JLNJ\[(N\:)L 2O H 5 CHQCIZ0 H"o C,91) R=CH(CH3 )39C R=CH3-42-OHR H 0 Q)HZN/'fir"\)‘Ncp/ HHN O0 0/10a R=CH2Ph101; R=CH(CI-I 3)310c R=CH3CA 02264610 1999-03-08W0 98/ 19705 PCT/US97/ 19851S_cheme 3 (Part I)0 HCBZ-CI JL /\/N 0 /© _5_L_,H2N/\/NH2 “"130;-—* | «O 3 \lOf \ AcOH,A/11H0 My Q0 W/E ;<O/“\ N/\/ N”; O \H/ N\/\ O' El';N OH CI ~ 13CH3Clg/CH3OH )<O O12O* J< I. :1“-;hd"d, /H2. Pd—C H3N\/\Fl\'J/ O” nDHIElL lhngfvgjfln C _ §;‘r\:/‘|\r‘\/E0/kHC1. ELOH 14 2 C" 2. TMSCI, DIEA, A 0\O/|< ; 15(:LO/l<O O/ NHS DCC /TSQH, ‘N/Vi . .CH2Cl3 O N\/‘H OH MeCN O N‘/\ - Osu16 T50» j\0 OH O 0Su-43..CA 02264610 1999-03-08W0 98/ 19705 ’ PCT/U S97/ 19851Scheme 3 (Part II) , .T ___MEt-IDP-(0Su)2 17 DME.:-1+ __ MEt-IDP-AA-Lys(Mtr)-PABOH)2 ‘AA-Lys(Mtr)-PABOH 18:) AA=Lys(Mu‘)6b AA=Lys(Mtr) 131’ A/\=Ph°10a AA=Phe 13“ AA=/W10c AA=AlaPNP co -——:—’————‘ MEt-IDP-AA-Lys(Mtr)-PABC-PNP);_ L)Xi:‘——»DIEA. CH2C12 DIEA. DMF19:: AA=Lys(Mt1')191; AA=Phc19¢ AA=AlaC13CHCO3H, zmisoleCHZCIZMEt-IDP-AA-Lys(Mtr)- PA BC-DOX)220a AA=Lys( Mu’)20b AA=Phc20c AA=A]a0 VM J W2- ncI2cHco’221a R=(CH3)4NH3+, n:5211) R=CH3Ph. 11:3216 R=CH(CH_‘;)'_1. n=3 -44- CA 02264610 1999-03-08WO 98/19705Scheme 4 (Part I)r<°J<O + _®/\oJLN/\,NH,. c» m|<_H§(_)LQ§/If’ @OfiNNN\ 3O / K?1; By/fig 0%O/u\0_ fgfig O 0 0H2,Pd-C, +N,\/N o L if C”2C'2______. 0AC0“ KS 2 TMS-Cl. Et;N23 14 McC‘.N,AoJ< 0,, so0 /A . J)0 o 'rsoH (3 L-qu_.yi* 0 + K4 EKb:/\/K70 CH3Cl7,.'s'nl1iC. \ N/\’N{‘ 8 W)o of ’ ' 0 0,, .24 25I}_‘HS. DCC, DMEAA«Lys(M1r)-PAB-OH “N ,4’10a AA=Phe ;L\ 010b AA=Vnl Li JOH2621 R=CH«_;Ph26b R=CH(CH3)2-45- PCT/US97/19851CA 02264610 1999-03-08W0 98l19705 M PCT/US97/ 19851Scheme 4 (Part II) a1 - _ - _ 7T—————> < - - - ‘ ‘ - -M1: [DA AA L M PABOH PNPQCO’ MEt IDA (AA L (Mt )PABC PNP‘ ( Y5‘ tr) )‘DII:‘A.CHgC|2 ‘V’ ' ~ )326-a AA=Phe 27a AA=Phe26b AA=Va1 27b AA=ValDOX.HC1 CIQCHCO 2H, anisoleME .11) - AA-L~ Mt -PABC-DOX ——————_?.DIEA, NMP t A ( 3“ r) )2 CHZCIZ28a AA=Phe281) AA=VaI 2921 R=CH3Ph291) R=CH(CH3)2-45-CA 02264610 1999-03-08WO 98/19705 PCT/U S97/ 19851Scheme 5MEt-IDA-(Phe-Lys(Mtr)-PABC-i’—NP)2 + 7 VMMC27*‘ HOBI. DIEANMP, sievesMEt-IDA-(Phe-Lys(Mtr)-PABC-MMC)230CICI--I3CO2H,z1nis0lc.CH2Cl30 NH,H2N3\O\O N 0Hk /N0H —<oN0“)4 0 _N 4/ H 0 A ’/\/N O OQ \¢° )Lo 0 ”“=0 HN “ZN\-47-CA 02264610 1999-03-08wo 93/19705 PCT/US97/19851Scheme6 , .., __O0 JL OHpyridine, CHQCIQ ° l. diphosgcne, pyridine, DIEA' Z-—-—:——> E O ' oP“°"”"‘°' O c1 O oQ”° ° r“ 2, MEt-IDA-(Phe-Lys(Mtr)-PABOH)20 26a0‘ 0'32CICHQCOQH. anisolcMEt-IDA-(Phe-Lys(Mtr)-PABC-7-Paclitaxel-2'-Mtr)233cHgc13-43-CA 02264610 1999-03-08WO 98/19705 PCT/US97/ 19851Scheme 7 oj’LN,\flN; KHCO3, DMF ©»oJi,.«/x N/\n,o\[< H2‘ MC,CF H 3 ar/\n/°\’< H 0%;/I 0 ACOHo 35 o7<+ /\/\ O _ 0O H,N 0%/7/fig j< L 0&0 <L/1/\(:\N/§:0\K— -:———--—-~ 00 0 2. TMSCI. DIEA, A V36 7<. 37 °W<O TSOH \ ~’\”L““,f°" Phc—Lys(M1r)«PABOH 10:.“{H,C,j o °§/ ° _ DCC,NI-1S,DIEA.DME' ‘ on Tso38I’NP3C()MPr-IDA-(Phe-Lys(Mtr)-PABOH);—];;\———- MPr-IDA-(Phe-Lys-(Mtr)-PABC-PNP)239 CH3C|3 4"D0X'HC] C]'>CHCO'>H anisole—-'—-———*MPr-IDA-(Phe-Lys(Mt')-PABC-DOX) — ~ ‘DIEA, I CH2C[2DMF -49..CA 02264610 1999-03-08 A wo 93/19705 PCT/US97/19851Scheme 8 , ...0 + C 0 H O>L0Jl\/NH, + i LB, >L0JJ\,"‘\/LLoJ<- 0 DMFc| 43 O o' " N/\)LC|\ODIEAo 0 ° xi/\)L OH TSOH N N/\[(0\K\ N 0 ofig"/T cH3c13 ‘ o °§/ °0on 45 7<Phc-Lys(Mtr)«PABOH 7 44DCC, NHS.DIEA, DMEMP-IDA-(Phe-Lys(Mtr)-PABOH) MP-lDA-(Phe-Lys(Mtr)-PABC-PNP)246 CHQCI3 47DOX-HCI MP IDA (Pl L ‘Wu ) PABC DOX) CIQCHCOZH. zmisolcDIEA, - - 1e- ys J r - CH2Cl2DMF 43 -50-CA 02264610 1999-03-08WO 98/19705 ' PCT/U S97/ 19851Scheme 9 (Part I)O O O O O0- JL /\JL NHS V———w“ ©ro : °~ ( ‘ . Hso 51/—CO2HHN O o o O\’C°2” [D/\oJLN’\/‘LN/\n,oH E)/‘O/u\N’\/EN/\rroEj§' H H I-1OH oY| 0 0?) O0 00.N on52 53 )50° 0 H o H @o*Nm~«»«x H,~«/‘*m[r“\/Wr°7<DME ° 0 0 j,(T> 0g o oHN\/\n/o\l< HN\/\n,0W<54 O 55 0_ o0410 N/\}oL : o /« 0 /\)(i HO / H O N/\n/ V/Y \]\ N N/\n/Nx/\n/OK O” W’) 0 0 EIJN \ O0? 0 Oo HN o\/fif HN o0 7< A; w<O 0 H H O/\)L ._1;I:‘A_. fl§=_, oJ“g”wg:;§CHQCIQ O o§/ DCC-51-CA 02264610 1999-03-08WO 98/19705 PCT/US97/ 19851Scheme 9 (Part II) , MP-IDA-(BAla-OSu)2 + Phe-Ly,S.(Mtr)-PABOH DMF59 10a rtPNP2CODIEA, CH2C]2MP-IDA-( BAla-Phe-Lys(Mtr)-PABOH)260DOX-HCIDIEA, DMFMP-IDA-(BAla-Phe-Lys(Mtr)-PABC-PNP)261C13CHCOgH, anisoleCHgC1gMP-IDA-(BAla-Phe-Lys(Mtr)-PABC-DOX)262-52-CA 02264610 1999-03-08WO 98/19705 PCTIUS97/19851Scheme 10“N/\/\oH B0620. >|\ /E CH_qSO3Cl, >L Jok2 ——:. \ __.___.CH2CI2 ° W °” El3N. CH3C13 o N/\/\0S02CHa66 ‘T H 670 J0 A/\oJ\/?Lo’\ >LoJi O I-_LI1_3£z:____. >LoJ1N OHNaH,THF n O O 2. AcOH H/\/Yas 9 69 °”CF3CO3H L,»/\/W\/\oH 0&0 DIE“ E: OHCH 2C1:/H20 CF Co- 0,. TMSCI, CH3CN \ m3 Z70 71o Phe-Lys(MMT)-PABOHphosgcnc/toluene gfl/\/W\/\oJl\c| 10aCH2Cl3 \ O O C. DIEA.CH2C1272 If0 \ N’\/\(o’kPhe-Lys(MMT)-PABOH 0; 0 N020 o\n/Phe-Lys(MMT)-PABOH DIEA. CHZCIZ73 0O&:’\/\f 0)kPhe-Lys(MM'I‘)—PABC-PNP DOX-HCI. DIEA.0 om/Phe-Lys(MMT)-PABC-PNP DMF74 °0\ N/\/\(\OJkPhe-Lys(MMT)-PABC-DOX Cl3CHCOgH. anisoleo om/Phe-Lys(MMT)-PABC-DOX CH2c]275 °0M0JJ\Phe—Lys-1>ABC-Dox-CI2CHc0zH0 OYPl1e-I.ys-PABC-DOX°Cl3CHCO2Ho76-53-CA 02264610 1999-03-08WO 98/19705 PCTfUS97/19851Scheme 11 _ __' 7water, EHN. CH2Cl2HN21HCO3 ~ 78O OH2”/\/\°H Ad we-C1 @><oJLwAc,5o2cH3H77J<>LOj\/&oJ< o/EN O 0 CF3CO2H, anisole ;‘ 0/KNaH. THF H/\/0):: CH QCI2 ‘N/_\/;(§°79 C|aCC0-2 0/9 so 4‘_ O/k 7‘ H(%’o>:o DIEA o CF3C(_)2H_ SEE OTMSCl.CH3CN \ O O CH2C1: \ O H O81 4* 82Phe-Lys(MMT)-PABOH °103 Phe-I4ys(MMT)-PABOHv \“HS” DCC' DME 0 0 he-l,ys(MMT)-PABOH830\n,O 00o2 O \©\N0; €\E: lIe-Lys(MMT)-PABC-PNP0 he-l,.ys(MMT)-PABC—PNPDIEA,CH2Cl284DOX-HCI. DIEA. gl Phe-Lys(MMT)-PABC-DOX\DMF 0 0 he-Lys(MMT)-PABC-DOXss0GECHCOZH’ ‘““5°'3 H he-Lys-PABC—DOX-C17CHCOaHCHQCIQ \ \ ~ ‘0 0 he-Lys-PABC-DOX-Cl2CHCOzHso-54-CA 02264610 1999-03-08WO 98/19705 PCT/US97/19851_Schcme 12 ’ _W_Kék - 0 » °H gon0 0/[L /\/ 0 9F3CO2H \ AOJLN/\/N o I. NHS,DCC \ JL /\/N[j° 2 Kg’ CH:Cl3 H 2. N-.1BH4 ©A° S K‘22 xg -87 as 0"- H _ //)7'" ion0H2.Pd_C QM/V Kfogo DIEA N/\/M‘ phosgene/toluene N/\/N clTo 3 ’ \ \ K‘0 on o O90 91CI8‘)HC1‘ C1430“ — OJMSCI, CHKCN C”2C]'_>0J\Phe-L_vs(MMT)-PAB OHPhe-Lys(MMT)-PABOH0 01021 h V“'5” C”?C'2 ‘ K‘ 7/Phi:-I.vs(MM’l')~I'AB()H92 '50o o o)kl’he-Lys(MMT)=PABC-IENP~’® \g (1 O H0, No,of“DIEA-CH2Cl2 ‘ ‘o \\L\§l’lne—Lys(MM'I‘)-PABC-PNP93fi\l-‘he-Lys(MMT)-PABC-DOXODOX-HC]. DIEA. {L/2/\/NlDMF ‘ ‘o Kl, Phe-Lys(MM’I‘)-PABC-DOX‘)4 0o’fi\Phe-Lys-PABC-DOX'Cl3CHC()3HCIQCHCOQH. anisolc N/\,NCW3‘: ‘ o on/Phe-Lys-PABC-DOX-CIZCHCOZII95The following examples are to illustrate the invention but should not beconstrued as a limitation thereon.-55-1015202530WO 98/19705CA 02264610 1999-03-08PCT/US97/ 19851Example 1 , __Fmoc-Lys(Mtr)A stirred suspension of Fmoc—Lys hydrochloride (23.78 g, 56.42 mmol) in drymethylene chloride (250 mL) under argon at it was treated with trlmethylsilylchloride (15 mL, 2.1 equiv) and DIEA (10.3 mL, 1.05 equiv). The mixturewas heated at reflux for 1 h, during which time it became homogeneous, andthen cooled to 0°C. DIEA (31 mL, 3.1 equiv) was added, followed by p-anisyldiphenylmethyl chloride (18.29 g, 1.05 equiv). The reaction wasstirred at rt for 14 h. The solvent was evaporated and the residue partitionedbetween ethyl acetate and pH 5 buffer (0.05 M biphthalate). The organicphase was washed with more pH 5 buffer, water and brine, dried oversodium sulfate and evaporated to give a pale—ye|low foam (34.71 g, 96%).1H-NMR (CDCI3) 5 1.26 and 1.68 (m, 2H and 4H), 2.45 (m, 2H), 3.71 (s,3H), 4.05-4.40 (m, 4H), 6.81 (d, 2H), 7.15-7.77 (m, 20H). MS (FAB) 641(MH)+, 663 (M+Na)+, 679 (M+K)+. HRMS Calcdz 641.3015. Found:641.3001.Example 2Lys(Mtr)Fmoc-Lys(Mtr) 1 (5.25 g, 8.19 mmol) in 1:1 methylene chloride/acetonitrile(80 mL) at rt was treated with diethylamine (80 mL). After 1.5 h the solventswere evaporated. The residue was flushed with acetonitrile (2 x 50 mL) at60°C, and then triturated with ether (80 mL). The resulting solid wascollected by filtration, washed with ether, and then dissolved as far aspossible in 1:1 methylene chloride/methanol. Some solid byproduct wasremoved by filtration and the filtrate was concentrated invacuo. The resultinglight tan solid was dried in vacuo for 4 h (3.2221 g, 94%). 1H-NMR (DMSO-de) 6 1.34, 1.57 and 1.72 (m, 6H), 2.05 (m, 2H), 3.38 (m, 1H), 3.68 (s, 3H),3.71 (d, 2H), 7.03-7.40 (m, 12H). MS (FAB) 419.2 (MH)+, 441.4 (M+Na)"',457.4 (M+K)'*'. MS (FAB) 419.2 (MH)+, 441.4 (M+Na)+, 457.4 (M+K)+.-55-1015202530WO 98/19705CA 02264610 1999-03-08PCT/U S97/ 19851Example 3 . _Fmoc-Lys(Mtr)-0SuA stirred solution of Fmoc-Lys(Mtr) 2 (5.556? g, 8.672 mmol) and N-V_hydroxysuccinimide (1.0978 g, 1.1 equiv) in DME (100 mL) at 0°C wastreated with 0.5M DCC in methylene chloride (19.1 mL, 1.1 equiv). Themixture was gradually warmed to rt. after 14 h the solid DCU was filtered offand the filtrate evaporated to dryness. The crude active ester was usedwithout further purification. 1H—NMFi (CDCI3) 8 1.52, 1.73 and 1.92 (m, 6H),2.14 (1, 2H), 2.79 (brs, 4H), 3.75 (s, 3H), 4.21 (1, 1H), 4.42 (d, 21-1), 4.71 (m,1H), 5.40 (d, 1H), 6.79 (d, 2H), 7.10-7.76 (m, 20H).Example 4Fmoc-Lys(Mtr)-Lys(Mtr)A stirred solution of Fmoc-Lys(Mtr)-0Su 3 (ca 8.672 mmol) and Lys(Mtr) 2(3.6294 g, 1 equiv) in DMF (70 mL) at rt was treated with DIEA (4.5 mL, 3equiv). After 4 h the mixture was diluted with ethyl acetate (250 mL). Thesolution was washed with pH 4 buffer (0.05M biphthalate) (2x), water andbrine, dried over sodium sulfate and evaporated. the residue was flushedwith methylene chloride (400 mL) to give a pale-yellow foam which wascarried on without further purification. 1H-NMF1 (CDCI3/CD3OD) 6 1.10-1.85 (m, 12H), 2.22 (m, 4H), 3.70 (m, 6H), 4.21 (m, 5H), 6.76 (m, 4H), 7.00-7.75 (m, 32H). MS (ESI) 1041.6 (MH)+.Example 5Fmoc-Lys(Mtr)-Lys(Mtr)-PABOHA stirred solution of Fmoc-Lys(Mtr)-Lys(Mtr) 4 (ca 8.672 mmol) and di-t-butyldicarbonate (2.8390 g, 1.5 equiv) in methylene chloride (100 mL) at rtwas treated with pyridine (0.736 mL, 1.05 equiv). After 20 min p-aminobenzyl alcohol (1.6020 g, 1.5 equiv) was added. Stirring wascontinued for 16 h and then the solvent was evaporated and the residuedried in vacuo for 2 h. The crude product was carried on without furtherpurification. 7H-NMR (CDCI3/CD3OD) 6 1.00-1.75 (m, 12H), 2.09 (m, 4H),3.73 (rn, 6H), 4.14 (m, 3H), 4.39 (m, 2H), 4.58 (m, 2H), 6.76 (m, 4H), 7.05-- _1015202530W0 98/ 19705CA 02264610 1999-03-08PCT/US97/198517.75 (36H). MS (ESI) 1146.6 '(MH)+. Anal. Calcd for C74H75N5O7-"2H2O: .-C-75.17, H-6.73, N-5.92. Found: C-74.89, H—6.37, N-5.75.Example 6Lys(Mtr)-Lys(Mtr)-PABOHFmoc-Lys(Mtr)-Lys(Mtr)-PABOH 5 (ca 8.672 mmol) in acetonitrile (100 mL) atrt was treated with diethylamine (75 mL). After 2 h the solvents wereremoved on the rotovap at 40°C and the residue dried in vacuo for 1 h andthen dissolved as far as possible in methylene chloride (150 mL). The solidwas removed by filtration and the filtrate concentrated in vacuo. The residuewas chromatographed on silica, eluting with 1) 2.5%, 2) 3%, and 3.5%methanol/methylene chloride, to give the productflas a colorless foam(2.8403 g, 35% (4 steps)). 1H—NMR (CDCI3) 5 1.42 and 1.78 (m, 12H), 2.12(brt, 4H), 3.32 (m, 1H), 3.75 (s, 3H), 3.79 (s, 3H), 4.44 (m, 1H), 4.57 (s, 2H),6.77 (ABq, 4H), 7.10-7.70 (m, 28H), 7.91 (d, 1H), 9.09 (s, 1H). MS (ESI)924.5 (MH)+. Anal. Calcd for C59He5N5O5-H20: C—75.21, H—7.17, N—7.43.Found: C-75.37, H—6.96, N—7.24.Example 7aFmoc-Phe-OSuFmoc-Phe (5.1043 g, 13.17 mmol) and NHS (1.592 g, 1.05 equiv) inmethylene chloride (100 mL) at 0°C were treated with DCC (2.854 g, 1.05equiv). The ice bath was allowed to warm to rt and the mixture was stirredfor 14 h. DCU was removed by filtration and the filtrate was evaporated. Theresulting crude product, a colorless glass, was used without furtherpurification.Example 7bFmoc-Val-0Su 7bThis was prepared from Fmoc-Val (7.02 g, 20.7 mmol) as described abovefor 7a.-58-1015202530WO 98/19705CA 02264610 1999-03-08PCT/U S97/ 19851Example 7c . __Fmoc-Ala-OSuThis was prepared from Fmoc-Ala (5.0414 g, 15.31 mmol) as describedabove for 7a. . 9 9Example 8aFmoc-Phe-Lys(Mtr)A suspension of Lys(Mtr) 2 (4.686 g, 11.20 mmol) and NaHCO3 (941.0 mg, 1equiv) in water (100 mL) and DME (50 mL) was treated with a solution ofFmoc—Phe—OSu 7a (11.20 mmol) in DME (50 mL). THF (25 mL) was thenadded to aid solubility. The mixture was stirred at rt for 2 days and then asmuch DME as possible was removed on the rotovap (bath at 30°C), Theresulting gummy suspension was partitioned between ethyl acetate and pH5 buffer. The organic phase was washed with water and brine, dried andevaporated to give a pale yellow foam. This was flushed with methylenechloride (100 mL). TLC showed the product to be satisfactorily pure and itwas carried on without further purification (8.559 g, 97%). 1H—NMR(CDCI3/CD3OD) 5 1.10-1.93 (m, 6H), 2.31 (t, 2H), 3.00 (m, 2H), 3.71 (s, 3H),4.02-4.48 (m, 5H), 6.79 (d, 2H), 7.00-7.75 (m, 25H). MS (FAB) 788.2 (MH)+,810.4 (M+Na)+, 826 (M+K)+. Anal. Calcd for C5oH49N3O5—H2O: C-74.51,H-6.38, N—5.21. Found: C-74.17, H-6.57, N—5.41.Example 8bFmoc-Val-Lys(Mtr)This was prepared from Fmoc-Val-OSu 7b (20.7 mmol) and Lys(Mtr) 2 (9.09g, 1.05 equiv) as described above for 8a (15.28 g, 100%). TH-NMR(CDCI3/CD3OD) 5 0.85 (m, 6H), 1.20 (m, 2H), 1.59 (m, 4H), 1.99 (m, 1H),2.41 (m, 2H), 3.71 (s, 3H), 4.21 (m, 5H), 6.78 (d, 2H), 7.29 (m, 16H), 7.51 (brt,2H), 7.71 (d, 2H). MS (FAB) 740 (MH)+, 762 (M+Na)+. HRMS Calcd:740.3700. Found: 740.3712.-59-10152025WO 98/19705CA 02264610 1999-03-08PCT/US97/ 19851Example 8c , __ _Fmoc-Ala-Lys(Mtr)A solution of Fmoc-Ala-OSu 7c (15.31 mmol) in DME (100 mL) was "added toa stirred solution of Lys(Mtr) 2 (64080 g, 1 equiv) and potassium 9bicarbonate (1.5331 mg, 1 equiv) in 2:1 water/DME (150 mL). After 16 h theDME was removed in vacuo and the resulting suspension was treated withcitric acid (3.22 g, 1.1 equiv) in water (25mL). The mixture was extractedwith ethyl acetate. The organic phase was washed with water (2x) andbrine, dried over sodium sulfate, and concentrated in vacuo, giving a yellowfoam which was carried on without further purification. 7H-NMFI(CDCI3/CD3OD) 8 1.25 (m, 2H), 1.32 (d, 3H), 1.57 (m, 3H), 1.73 (m, 1H),2.41 (brt, 2H), 3.72 (s, 3H), 4.14 (m, 1H), 4.29 (m, 4H), 6.79 (d, 2H), 7.22 (m,16H), 7.38 (d, 4H), 7.52 (m, 2H), 7.72 (d, 2H). MS (FAB) 712.2 (MH)+, 750.2(M+K)+.Example 9aFmoc-Phe-Lys(Mtr)-PAB-OHA stirred solution of Fmoc-Phe—Lys(Mtr) 8a (7.728 g, 9.808 mmol) and p-aminobenzyl alcohol (1.450 g, 1.2 equiv) in methylene chloride (100 mL) atrt was treated with EEDQ (3.640 g, 1.5 equiv). After 20 h the solvent wasevaporated (water bath at 30°C). The solid residue was triturated with ether(200 mL) and the resulting suspension sonicated for 15 min and left to standat it for 2 h. The resulting solid was collected by filtration, washed well withether, and dried in vacuo (7.6140 g, 87%). 1H-NMR (CDCI3/CD3OD) 60.98-1.91 (m, 6H), 2.06 (t, 2H), 2.97 (m, 2H), 3.71 (s, 3H), 4.12 (1, 1H), 4.20-4.41 (m, 4H), 4.59 (s, 2H), 6.72 (d, 2H), 7.00-7.73 (m, 29H). MS (FAB) 891.4(MH)+, 916.7 (M+Na)+, 931 (M+K)+. Anal. Calcd for C57H55N4O5-H20: C-75.14, H-6.42, N-6.15. Found: C-75.25, H-6.02, N-6.49.-60-1015202530W0 98/ 19705CA 02264610 1999-03-08PCT/US97/19851Example 9b , __Fmoc-Val-Lys(Mtr)-PABOH 'This was prepared from Fmoc—Va|-Lys(Mtr) 8b (15.28 g, 20.65 mmol) asdescribed above for 9a (14.24 82%). 1H—NMR (DMSO—d5) 8 0.84 (ABq,6H), 1.15-1.80 (m, 6H), 1.94 (m, 3H), 2.37 (brt, 1H), 3.69 (s, 3H), 3.91 (t, 1H),4.21 (m, 4H), 4.41 (d and m, 3H), 5.10 (t, 1H), 6.79 (d, 2H), 7.29 (m, 18H),7.53 (d, 2H), 7.68 (t, 2H), 7.86 (m, 2H), 8.02 (d, 1H), 9.94 (brs, 1H). IR (KBr)700, 740, 1650, 1695. MS (ESJ) 845.6 (MH)+. Anal Calcd forC53H55N4O5—0.5H2O: C-73.76, H—6.77, N-6.49. Found: C-74.01, H-6.68,N-6.63.Example 9cFmoc-Ala-Lys(Mtr)-PABOHThis was prepared from Fmoc-Ala-Lys(Mtr) 8c (108985 g, 15.31 mmol) asdescribed above for 9a (11.0073 g, 88%). ‘H-NMR (CDCI3/CD3OD) 6 1.29(d and m, 5H), 1.42 (m, 2H), 1.57 (m, 1H), 1.79 (m, 1H), 2.05 (m, 2H), 3.68 (s,3H), 4.11 (m, 2H), 4.30 (m, 3H), 4.51 (s, 2H), 6.69 (d, 2H), 7.00-7.55 (m,22H), 7.66 (d, 2H). MS (ESI) 818.0 (MH)+. Anal Calcd for C51H52N4O5-H20: C-73.36, H-6.52, N-6.71. Found: C-73.02, H—6.72, N—6.54.Example 10aPhe-Lys(Mtr)-PAB-OHFmoc-Phe—Lys(Mtr)-PAB-OH 9a (4.285? g, 4.80 mmol) in methylene chloride(35 mL) at rt was treated with diethylamine (50 mL). The mixture wassonicated briefly and stirred at rt for 4 h. after which time no starting materialwas observed by TLC. The solvents were evaporated and the residue wasflushed with methylene chloride and chromatographed on silica, eluting with1) 2%, 2) 3%, and 3) 4% methanol/methylene chloride, to give the product asa colorless foam (2.230 g, 69%). TH-NMR (CDCI3) 5 1.26-2.00 (m, 6H),2.12 (t, 2H), 2.75 and 3.21 (ABq, each 1H), 3.68 (ABq, 1H), 3.76 (s, 3H), 4.42((1. 1H), 4.66 (brs, 2H), 6.79 (d, 2H), 7.10-7.42 (m, 21H), 7.81 (cl, 1H), 8.71 (S,-61-1015202530CA 02264610 1999-03-08_ W098/197051H). MS (FAB) 693.4 (M+Na)+, 709 (M+K)+. Anal. Calcd for C42H46N404;.PCT/US97/198511/2H20: C-74.20, H-6.97, N-8.24. Found: C—74.28, H-7.00, N-8.34.Example Val-Lys(Mtr)-PABOHA solution of Fmoc-Val-Lys(Mtr)—PABOH 9b (13.94 g, 16.50 mmol) in 5:1methylene chloride/methanol (75 mL) was treated with diethylamine (150mL). The reaction was sonicated for 30 min and then allowed to stand at rtfor 3 h. The solvents were evaporated and the residue flushed withmethylene chloride. The remaining material was chromatographed on silica,eluting with 1) 2%, 2) 3%, 3) 3.5%, and 4) 4% methanol/methylene chloride,to give the product as a colorless foam (6.2510 g, 61%). 1H—NMR(CDCI3/CD3OD) 6 0.80 (d, 3H), 0.97 (d, 3H), 1.48 (m, 4H), 1.70 (m, 1H),1.90 (m, 1H), 2.11 (t, 2H), 2.27 (m, 1H), 3.27 (cl, 1H), 3.76 (s, 3H), 4.53 (q,1H), 4.58 (s, 2H), 6.78 (d, 2H), 7.10-7.50 (m, 16H), 7.95 (d, 1H), 9.11 (s, 1H).IR (KBr) 706, 830, 1034, 1248, 1510, 1648, 3310. MS (ESl) 623.2 (MH)+.HRMS Calcd for C33H47N404: 623.3598. Found: 623.3606. Anal. Calcdfor C33H45N404-H20: C, 71.22, H, 7.55, N, 8.74. Found: C, 71.41, H, 7.51,N, 8.66.Example 10cAla-Lys(Mtr)-PABOHA solution of Fmoc-Ala-Lys(Mtr)-PABOH 9c (11.0073 g, 13.47 mmol) in DMF(50 mL) at rt was treated with diethylamine (25 mL). After 2.5 h the mixturewas concentrated under high vacuum and the residue was flushed withmethylene chloride (2x100 mL) and then toluene (150 mL). The remainingmaterial was chromatographed on silica. eluting with 1) 4:1, 2) 1:1, 3) 121.6and 4) 1:3 methylene chloride/(5:1 ethyl acetate/methanol), to give theproduct as a pale-yellow foam (5.7817 g, 72%). 1H-NMR (CDCI3/CD3OD) 81.32 (d, 3H), 1.25-2.01 (m, 6H), 2.13 (t, 2H), 3.51 (q, 1H), 3.75 (s, 3H), 4.48(q, 1H), 4.60 (brs, 2H), 6.79 (d, 2H), 7.12-7.50 (m, 16H), 7.89 (d, 1H), 8.89(brs, 1H). MS (ESI) 595.2 (MH)+.-62-1015202530WO 98/19705 A ‘CA 02264610 1999-03-08PCT/US97/ 19851Example 11N, N-Bis-CarbobenzyloxyethylenediamineBenzyl chloroformate (40 mL, 280 mmol) was added dropwise over 45. minto a vigorously stirred solution of ethylenediamine (9.4 mL, 0.5 equiv) in 1MNaOH (310 mL, 1 equiv) at 0°C. A heavy white precipitate formed and themixture was stirred for 4 h following addition. The resulting solid productwas collected by filtration, washed with water and then hexane, and then air-dried overnight (46.17 g, 100%). 1H—NMFl (CDCI3) 8 3.32 (s, 4H), 5.10 (s,4H), 5.23 (br, 2H), 7.35 (s, 10H). MS (ESI) 329.3 (MH)+.Example 12N-Carbobenzyloxyethylenediamine hydrochlorideN, N-Bis-Carbobenzyloxyethylenediamine 11 (23.91 g, 72.8 mmol) in glacialacetic acid (100 mL) was treated with 12M HCI (12.1 mL, 2 equiv). Thestirred mixture was heated at reflux for 1 h and then left to stand at rtovernight. A small amount of solid was removed by filtration and the filtratewas diluted with ether (700 mL) and left to stand at rt for 2 h. The resultingwhite solid product was collected by filtration, washed repeatedly with ether,and dried in a vacuum dessicator overnight (9.2440 g, 55%). 1H—NMR(DMSO-d5) 5 2.87 (t, 2H), 3.30 (q, 2H), 5.04 (s, 2H), 7.33 (m, 5H), 7.49 (brt,1H), 8.22 (br, 3H).Example 13N-Carbobenzyloxy-N‘, N'-bis-(t-butoxycarbonylethyl)-ethylenediamineA stirred solution of N-carbobenzyloxyethylenediamine hydrochloride 12(50009 g, 21.68 mmol) in 3:1 methanol/methylene chloride (100 mL) at rtwas treated with t-butyl acrylate (64 mL, 20 equiv) and triethylamine (3.3 mL,1.1 equiv). After 2 days the solvents were removed on the rotovap. Theresidue was dried in vacuo for 30 min and then partitioned between 30%ethyl acetate/ether and 50% sat. NaHCO3. The organic phase was washedwith water and brine, dried over sodium sulfate and evaporated. The-53-1015202530WO 98/19705CA 02264610 1999-03-08PCT/US97/19851residue was chromatographed on silica, eluting with 20°/O ethyl . ..<3acetate/hexane, to give the product as a thick, colorless oil (9.3801 g,'96%).1H-NMR (CDCl3) 5 1.42 (s, 181-l), 2.33 (t, 4H), 2.54 (t, 2H), 2.72 (t, 4H), 3.28(Q. 2H), 5.11 (s, 2H), 5.49 (brt, 1H), 7.33 (m, SH). MS(DCl) 451 (MH)+. Anal.Calcd for C24H38N2O5—0.5 H20: C, 62.72, H, 8.55, N, 6.09. Found: C,62.54, H, 8.27, N, 5.95.Example 14N, N-Bis-(t-butoxycarbonylethyl)ethylenediaminedihydrochlorideA solution of N-carbobenzyloxy-N‘, N‘-bis—t-butoxypropionylethylenediamine13 (18.3640 g, 40.76 mmol) was passed through a bed of Raney nickel in afritted glass funnel. The filtrate was degassed with nitrogen and 10% Pd-C(1 g) and 12M HCl (6.8 mL, 2 equiv) were added. The mixture washydrogenated on a Parr shaker at 50 psi for 16 h and then degassed onceagain with nitrogen. The catalyst was filtered off on a bed of celite. Thefiltrate was concentrated in vacuo and the residue flushed with methylenechloride (2x), giving an off-white foam (158696 g, 100%). 1H-NMR (CDCI3)8 1.48 (s, 18H), 2.97 (brt, 4H), 3.53 (m, 4H), 3.79 (m, 2H), 3.88 (m, 2H), 8.61(br, 3H), 11.24 (br, 1H).Example 15N-Maleoyl-N‘, N'-bis-(t-butoxycarbonylethyl)-ethylenediamineA stirred suspension of N, N~bis-t-butoxypropionylethylenediaminedihydrochloride 14 (5.3363 g, 13.70 mmol) in acetonitrile (30 mL) at 0°Cunder argon was treated with DIEA (4.77 mL, 2 equiv) and maleic anhydride(1.4111 g, 1.05 equiv). The reaction was allowed to warm gradually to rt.After 16 h trimethylsilyl chloride (5.2 mL, 3 equiv) and DIEA (7.2 mL, 3 equiv)were added. The mixture was heated at reflux for 4 h. Upon cooling, thesolvents were removed in vacuo and the residue taken up in ethyl acetate.The solution was washed with sat. NaHCO3, water and brine, dried oversodium sulfate and evaporated. The residue was chromatographed on-54-1015202530W0 98/19705CA 02264610 1999-03-08PCT/US97/ 19851silica, eluting with 2.511 hexane/ethyl acetate, to give the product as .a pale-yellow oil (29986 g, 55%). 1H-NMR (CDCI3) 5 1.42 (s, 18H), 2.30 (t, 4H),i2.61 (t, 2H), 2.73 (t, 4H), 3.59 (t, 2H), 6.68 (s, 2H). MS (ESI) 397.4 (MH.)+.Anal. Calcd for C2oH32N2O5: C-60.59, H-8.13, N—7.07. Found: C—60.65, H-7.98, N-6.88.Example 16N-Maleoyl-N‘, N'-bis-(carboxyethyl)ethy|enediaminep-toluenesulfonic acidN-Maleoyl-N‘, N'-bis-t—butoxypropionylethylenediamine 15 (3.9934 g, 10.07mmol) in methylene chloride (100 mL) at rt was treated with p-toluenesulfonic acid monohydrate (5.75 g, 3 equiv). The reaction was stirredat rt overnight. The solvent was evaporated at rt and the gummy residuedried in vacuo for 3 h. NMR showed complete loss of t-butyl ester signal.The crude product was carried on without further purification. 1H-NMR(DMSO-d5) 5 (contains 3 equiv of p-toluenesulfonic acid) 2.29 (s, 9H), 2.76(m, 4H), 3.39 (m, 6H), 3.81 (brt, 2H), 7.11 (s, 2H), 7.17 (d, 6H), 7.49 (d, 6H),9.27 (br, 1H).Example 17N-Maleoyl-N‘, N‘-bis-(succinimidyloxycarbony|-ethyi)ethy|enediamineA stirred solution of N-maleoyl-N‘, N‘-bis-propionylethylenediamine p-toluenesulfonate 16 (ca 10.07 mmol, containing two extra equiv of p-toluenesulfonic acid), triethylamine (4.4 mL, 3.1 equiv) and N-hydroxysuccinimide (2.5502 g, 2.2 equiv) in acetonitrile (90 mL) at 0°C wastreatedwith 0.5M DCC in methylene chloride (45.3 mL, 2.25 equiv). After 14h the reaction was concentrated in vacuo and the residue partitionedbetween ethyl acetate and water. The aqueous phase was re—extracted withethyl acetate. The combined organic phases were washed with water (2x)and brine, dried over sodium sulfate and evaporated. The residue waschromatographed on silica, eluting with 1:1 ethyl acetate/methylene chloride,-65-CA 02264610 1999-03-08W0 98/ 19705 ‘ PCT/US97/ 19851to give the product as a pa|e—ye|low foam (1.2043 g, 25%, 2 steps): 3H-NMB.(CDCI3) 8 2.72 (t, 4H), 2.84 (brs, 6H), 2.92 (t, 4H), 3.61 (t, 2H), 6.68 (s, 2H),Example 18a5 MEt-IDP-(Lys(Mtr)-Lys(Mtr)-PABOH)2A solution of Lys(Mtr)-Lys(Mtr)-PABOH 6 (2.0903 g, 2.262 mmol) and N-maleoyl-N‘, N'—bis-succinimidyloxypropionylethylenediamine 17 (541.0 mg,0.5 equiv) in DME (30 mL) was stirred for 2 days at rt. The reaction wasconcentrated in vacuo at 30°C and the residuedissolved in ethyl acetate.10 The solution was washed with water (2x) and brine, dried over sodiumsulfate and evaporated to give the product as a colorless glass (2.3623 g,99%). ‘H-NMR (CDCI3/CH3OD) 6 1.10-1.97 (m, 24H), 2.09 (m, 8H), 2.48(m, 2H), 2.63 (m, 4H), 3.45 (m, 2H), 3.72 (s, 6H), 3.74 (s, 6H), 4.31 (m, 2H),4.46 (m, 2H), 4.50 (s, 4H), 6.52 (s, 2H), 6.77 (m, 8H), 7.05-7.52 (m, 56H). MS15 (ESI) 2096.4 (MH)+.-55-1015202530CA 02264610 1999-03-08W0 98/ 19705 PCT/US97/19851Exampel 18b . ..MEt-IDP-(Phe-Lys(Mtr)-PABOH)2A stirred mixture of N-maleoyl-N‘, N'—bis—(carboxyethyl)ethylenediamine p—toluenesulfonic acid 16 (1.0643 g, 1.696 mmol) in DME (50 mL) at 0°C wastreated with triethylamine (0.6 mL, 2.5 equiv), Phe-Lys(Mtr)—PABOH 10a(2.2750 g, 2 equiv), NHS (439.0 mg, 2.25 equiv) and DCC (0.5M inmethylene chloride, 8.5 mL, 2.5 equiv). The reaction was allowed to warmgradually to rt. After 16 h the DCU byproduct was filtered off and the filtratewas concentrated in vacuo. The residue was triturated with ether and theresulting solid was collected by filtration, washed with ether and dried invacuo (2.0494 g, 76%). 1H—NMR (CDCI3/CD3OD) 5 1.20-1.95 (m, 12H),2.07 (brt, 4H), 2.18 (m, 4H), 2.39 (t, 2H), 2.54 (m, 4H), 3.02 (m, 4H), 3.29 (m,2H), 3.71 (s, 6H), 4.41 (m, 2H), 4.56 (brs, 4H), 4.69 (m, 2H), 6.57 (s, 2H), 6.72(d, 4H), 6.97-7.55 (m, 42H), 9.00 (br, 2H). MS(FAB) 1611 (M+Na)+, 1627(M+K)+.Example 18cMEt-lDP-(Ala-Lys(Mtr)-PAB0H)2This was prepared from N—ma|eoyl—N', N'—bis-succinimidyloxy-propionylethylenediamine 17 (. 1H—NMFl (CDCI3/CD3OD) 5 1.20-1.80 (m,15H), 1.95 (m, 4H), 2.13 (m, 4H), 2.39 (m, 2H), 2.55 (m, 4H), 3.31 (m, 2H),3.60 (S, 6H), 4.28 (m, 4H), 4.40 (brs, 4H), 6.50 (5, 2H), 6.61 (d, 4H), 6.90-7.50(m, 32H). MS (ESI) 1437.8 (MH)+. HRMS Calcd for C34H95N1oO12Na:1459.7107. Found: 1459.707.Example 19aMEt-IDP-(Lys(Mtr)-Lys(Mtr)-PABC—PNP)2MEt-lDP-(Lys(Mtr)—Lys(Mtr)-PABOH)2 18a (2.3623 g, 1.127 mmol) and bis-p-nitrophenylcarbonate (1.5424 g, 4.5 equiv) in dry methylene chloride (60mL) were treated with freshly activated 4/\ powdered sieves (8.5 g) and DIEA(0.883 mL, 4.5 equiv). The mixture was stirred at rt for 16 h and then filtered.The filtrate was concentrated in vacuo to give a yellow solid which was-67-1015202530WO 98/19705CA 02264610 1999-03-08PCT/US97/ 19851dissolved as far as possible in ether (200 mL). The suspension wasisonicated and stirred overnight at rt. The resulting white solid was collectedby filtration, washed repeatedly with ether, and dried in vacuo (2.4084 g,488%). 1H—NMR (CDCI3/CH3OD) 5 1.10-1.97 (m, 24H), 2.04 (m, 8H), 2.28(m, 4H), 2.48 (m, 2H), 2.62 (m, 4H), 3.41 (m, 2H), 3.71 (s, 6H), 3.73 (s, 6H),4.36 (m, 2H), 4.42 (m, 2H), 5.14 (s, 4H), 6.51 (s, 2H), 6.73 (m, 8H), 7.00-7.60(m, 60H), 8.20 (d, 4H). Anal. Calcd for C144H143N14O22-2H2O: C-70.23,H-6.22, N-7.96. Found: C-70.11, H-6.22, N-7.96.Example 19bMEt-IDP-(Phe-Lys(Mtr)-PABC-PNP)2This was prepared from MEt-IDP-(Phe-Lys(Mtr)—PABOH)2 18b (517.8 mg,0.326 mmol) as described above for 19a (509.1 mg, 74%). ‘H-NMFI(CDCl3/CD3OD) 6 1.20-1.97 (m, 12H), 2.08 (m, 4H), 2.24 (m, 4H), 2.40 (t,2H), 2.61 (m, 4H), 3.10 (m, 4H), 3.29 (m, 2H), 3.73 (s, 6H), 4.41 (q, 2H), 4.69(q, 2H), 5.22 (s, 4H), 6.58 (s, 2H), 6.77 (d, 4H), 7.00-7.50 (m, 42H), 7.56 (d,4H), 8.22 (d, 4H), 8.74 (brs, 2H).Example 19cMEt-IDP-(Ala-Lys(Mtr)-PABC-PNP)2This was prepared from MEt-lDP—(Ala-Lys(Mtr)-PABOH)2 18c (6.24 g, 4.34mmol) as described above for 19a (6.00 g, 74%). 1H—NMFt (CDCI3/CD3OD)5 1.26 (d, 6H), 1.33 (m, 4H), 1.42 (m, 4H), 1.62 (m, 2H), 1.83 (m, 2H), 2.18(m, 4H), 2.41 (m, 6H), 2.64 (m, 4H), 3.72 (s, 6H), 4.40 (m, 4H), 5.18 (brs, 4H),6.60 (s, 2H), 6.72 (d, 4H), 7.05-7.50 (m, 38H), 7.56 (d, 4H), 8.21 (d, 4H).Example 20aMEt-IDP-(Lys(Mtr)-Lys(Mtr)-PABC-DOX)2A stirred solution of MEt—lDP—(Lys(Mtr)-Lys(Mtr)—PABC-PNP)2 19a (753.0mg, 0.3103 mmol) and DOX hydrochloride (377.9 mg, 2.1 equiv) in DMF (25mL) at rt was treated with DlEA (0.114 mL, 2.1 equiv). After 3 days themixture was poured into ethyl acetate (200 mL) and the solution was washed-53-1015202530WO 98/19705CA 02264610 1999-03-08PCT/US97/ 19851with water (4x) and brine, dried over sodium sulfate, and evaporated to give,an orange glass. This was triturated with ether (200 mL), and the resultingsolid was collected by filtration and then chromatographed on silica, elutingwith 16:1 methylene chloride/methanol, to give the product as an orangesolid (803.8 mg, 80%). 1H-NMR (DMF-d7) 5 1.23 (d, 6H), 1.30-1.90 (m,28H), 2.04 (m, 10H), 2.30 (m, 6H), 2.41 (d, 2H), 2.51 (m, 2H), 2.72 (m, 4H),3.12 (q, 4H), 3.64 (m, 2H), 3.75 (s, 12H), 3.91 (m, 2H), 4.08 (s and m, 8H),4.33 (m, 4H), 4.45 (m, 2H), 4.79 (s, 4H), 4.89 (d, 2H), 4.96 (brs, 4H), 5.12 (m,2H), 5.40 (brs, 2H), 5.60 (s, 2H), 6.72 (d, 2H), 6.87 (q, 8H), 6.92 (s, 2H), 7.10-7.50 (m, 54H), 7.67 (m, 6H), 7.93 (m, 2H), 9.91 (brs, 2H).Example 20bMEt-lDP-(Phe-Lys(Mtr)-PABC-DOX)‘2- ‘This was prepared from MEt-lDP-(Phe—Lys(Mtr)—PABC—PNP)2 19b (340.8mg, 0.161 mmol) as described above for 20a (411.2 mg, 93%). 1H—NMR(CDCI3/CD3OD) 8 1.10-1.65 (m and d, 16H), 2.07 (m, 6H), 2.10-2.45 (m,10H), 2.55 (m, 4H), 3.00 (m, 6H), 3.26 (m and cl, 4H), 3.71 (s, 6H), 3.82 (m,2H), 4.00 (brs, 6H), 4.11 (m, 2H), 4.40 (m,'2H), 4.63 (m, 2H), 4.74 (s, 4H),4.90 (ABq, 4H), 5.13 (brs, 2H), 5.44 (brs, 2H), 5.58 (brd, 2H), 6.60 (s, 2H),6.71 (d, 4H), 6.90-7.55 (m, 44H), 7.72 (t, 2H), 7.94 (d, 2H).Example 20cMEt-IDP-(Ala-Lys(Mtr)-PABC-DOX)2This was prepared from MEt—lDP—(Ala-Lys(Mtr)-PABC-PNP)2 19c (6.00 g,3.39 mmol) as described above for 20a (620 mg, 7%). 1H-NMR(CDCI3/CDSOD) 5 1.17 (m, 16H), 1.29 (m, 4H), 1.40 (m, 4H), 1.72 (m, 7H),1.99 (m, 4H), 2.15 (m, 10H), 2.53 (m, 6H), 3.03 (ABq, 4H), 3.39 (m, 2H), 3.52(brs, 2H), 3.64 (8, 5H), 3.95 (S, 6H), 4.04 (m, 2H), 4.26 (IT), 2H), 4.33 (m, 2H),4.68 (8, 4H), 4.80 (ABq, 4H), 5.10 (brs, 2H), 5.47 (brs, 2H), 5.55 (5, 2H), 5.64(d, 4H), 6.98-7.40 (m, 34H), 7.68 (t, 2H), 7.89 (d, 2H).-59-1015202530W0 98/ 19705CA 02264610 1999-03-08PCT/US97/ 19851Example 21a , .. __MEt—lDP-(Lys-Lys-PABC-DOX)2-5C|2CHCO2HA stirred solution of MEt-lDP-(Lys(Mtr)—Lys(Mtr)-PABC-DOX)2 20a (318.7mg, 0.0985 mmol) in methylene chloride (21 mL) at rt was treated withanisole (3.2 mL, 300 equiv) and dichloroacetic acid (0.244 mL, 30 equiv).After 2.5 h ethyl acetate (50 mL) was added. The mixture was stored at 4°Cfor 2 h. The resulting solid was collected by filtration and washed repeatedlywith ethyl acetate followed by ether, and then air-dried (274.5 mg, 99.8 °/o).1H-NMR (DMF-d7) 8 1.23 (cl, 6H), 1.54 (m, 8H), 1.75 (m, 18H), 2.01 (m, 2H),2.22 (q, 2H), 2.40 (m, 6H), 3.07 (m, 8H), 3.89 (m, 2H), 4.06 (s, 6H), 4.34 (m,4H), 4.52 (m, 2H), 4.79 (s, 4H), 4.96 (s, 4H), 5.13 (brs, 2H), 5.39 (s, 2H), 5.72(m, 2H), 6.30 (s, 5H), 6.77 (d, 2H), 6.99 (s, 2H), 7.30 (d, 4H), 7.71 (m, 6H),7.93 (m, 2H), 8.45 (m, BH). M8 (ESI) 2147.6 (MH)+.Example 21bMEt-lDP-(Phe-Lys-PABC-DOX)2-3C|2CHCO2HThis was prepared from MEt-lDP-(Phe-Lys(Mtr)-PABC-DOX)2 20b (280.0mg, 0.103 mmol) as described above for 21a (237.1 mg, 90%). 1H-NMR(CDCI3/CD3OD) 5 1.20 (d, 6H), 1.37 (m, 4H), 1.70 (m, 10H), 2.06 (m, 2H),2.29 (m ,6H), 2.61 (m, 2H), 2.82 (m, 8H), 3.00 (m, 4H), 3.16 (d, 2H), 3.75 (m,2H), 3.99 (s, 6H), 4.07 (m, 2H), 4.44 (m, 4H), 4.70 (s, 4H), 4.89 (ABq, 4H),5.16 (brs, 2H), 5.40 (brs, 2H), 5.85 (s, 3H), 6.63 (s, 2H), 7.08 (m,. 14H), 7.39(d, 2H), 7.69 (t, 2H), 7.90 (d, 2H). MS (ESI) 1092.8 (MH2)2+. 2184.8 (MH)+.. Example 21cMEt-IDP-(Ala-Lys-PABC-DOX)2-3C|2CHCO2HThis was prepared from MEt-lDP-(A|a—Lys(Mtr)-PABC-DOX)2 20c (569 mg,0.248 mmol) as described above for 21a (455 mg, 80%). 1H—NMR(CDCI3/CD3OD) 5 1.17 (d and m, 8H), 1.22 (brd, 6H), 1.38 (m, 4H), 1.65 (m,10H), 2.02 (d, 2H), 2.23 (d, 2H), 2.41 (m, 4H), 2.82 (m, 6H), 2.99 (m, 4H),3.10 (d, 2H), 3.54 (m and brs, 4H), 3.93 (s, 6H), 4.05 (m, 2H), 4.19 (m, 2H),4.40 (m, 2H), 4.68 (brs, 4H), 4.82 (_ABq, 4H), 5.10 (brs, 2H), 5.38 (brs, 2H),-70-1015202530WO 98/19705CA 02264610 1999-03-08PCT/U S97/ 198515.79 (s, 3H), 6.61 (S, 2H), 7.09 (d, 4H), 7.29 (m, 2H), 7.40 (d, 4H), 7.67 (t, 2H)_,_7.82 (d, 21-1). MS (ESI) 2032.8 (MH)+. MS (ESI) 1016.6 (iviH2)2+, 2032.4(MH)+.Example 22N-Carbobenzyloxy-N‘, N‘-bis-(t-butoxycarbonylmethyl)-ethylenediamineA stirred mixture of N—carbobenzyloxyethylenediamine hydrochloride 12(13.95 g, 60.48 mmol) and potassium bicarbonate (24.22 g, 4 equiv) in DMF(100 mL) at 0°C was treated with t-butyl bromoacetate (22 mL, 2.25 equiv),dropwise over 10 min. The mixture was allowed to warm to rt. Stirring wascontinued for 12 h and then the mixture was partitioned between 3:1ether/ethyl acetate and 50% saturated sodium bicarbonate. The organicphase was washed with water (3x), brine, dried over sodium sulfate andevaporated to give a thick oil. This was flushed with heptane several timesat 60°C to remove excess t-butyl bromoacetate and then chromatographedon silica, eluting with 1) hexane, 2) 5% ethyl acetate/hexane, and 3) 25%ethyl acetate/hexane, to give the product as a thick, colorless oil (18.53 g,73%). 1H-NMR (CDCI3) 5 1.43 (s, 9H), 2.83 (t, 2H), 3.22 (q, 2H), 3.41 (s,4H), 5.12 (s, 2H), 6.04 (brt, 1H), 7.33 (m, 5H). MS (ESI) 423.4 (MH)+.Example 23N, N-Bis-(t-butoxycarbonylmethyl)ethylenediaminedihydrochlorideA solution of N-carbobenzy|oxy—N‘, N‘-bis-(t-butoxyacetyl)ethylenediamine22 (15.02 g, 35.54 mmol) in ethanol (75 mL) was filtered through a bed aRaney nickel. Acetic acid (6.1 mL, 3 equiv) was added and the mixture washydrogenated as described above for 4. the crude product was carried onwithout further purification.-71-1015202530CA 02264610 1999-03-08W0 98/19705 PCT/US97/19851Example 24 , .. __N-Maleoyl-N’, N '-bis-(t-butoxycarbonylmethyl)-ethylenediamine 'N, N-Bis-(t-butoxyacety|)ethylenediamine dihydrochloride 23 (ca. 35.594mmol) in CHCI3 (150 mL) at rt was treated with maleic anhydride (3.83 g, 1.1equiv). The mixture was stirred for 14 h and then the solvent wasevaporated. The residue was flushed with CHCI3 (200 mL) and thendissolved in acetonitrile (200 mL) under argon. The stirred mixture wascooled to 0°C and treated with trimethylsilyl chloride (18 mL, 4 equiv) andtriethylamine (20 mL, 4 equiv). The ice bath was removed and the mixturewas heated at reflux for 2.5 h. The solvents were removed under reducedpressure and the residue partitioned between ether and 30% sat sodiumbicarbonate. The organic phase was washed with water, brine, dried andevaporated. The residue was chromatographed on silica, eluting with 2.5:1hexane/ethyl acetate, to give the product as a waxy solid (8.25 g, 63%, 3steps). 1H-NMR (CDCI3) 6 1.42 (s, 9H), 2.91 (t, 2H), 3.41 (s, 4H). 0.62 (t,2H), 6.68 (s, 2H). MS (DCI) 369 (MH)+.Example 25N-Maleoyl-N’, N'-bis-(carboxymethyl)ethylenediamineop-toluenesulfonic acidA stirred solution of N—maleoyl—N', N'-bis-(t-butoxycarbony|methyl)-ethylenediamine 24 (2.6718 g, 7.25 mmol) in methylene chloride (70 mL) atrt was treated with p-toluenesulfonic acid monohydrate (4.1400 g, 3 equiv).After 36 h the resulting white solid was collected by filtration, washedrepeatedly with methylene chloride and ether, and then dried in vacuo. NMRshowed 1.4-1.5 equiv of p-toluenesulfonic acid per equiv of product (3.7531g, 100%). 1H-NMR (DMSO-d5) 6 2.29 (s, 3.9H), 3.39 (t, 2H), 3.77 (t, 2H),4.07 (s, 4H), 7.08 (s, 2H), 7.12 (d, 2.6H), 7.49 (d, 2.6H). MS (DCI) 257(MH)+. Anal. Calcd for C1oH13N205-1.3C7H703S-2H20: C—44.48, H-5.10,N-5.43. Found: C-44.51, H-4.96, N—5.20.-72-1015202530W0 98/ 19705CA 02264610 1999-03-08PCT/US97/19851Example 26a , ., __MEt-lDA-(Phe-Lys(Mtr)-PABOH)2A stirred solution of Phe-Lys(Mtr)-PABOH 10a (2.5179 g, 3.753 mmol), N-maleoyl-N‘, N'-bis—(carboxymethyl)ethylenediamine - p—to|uenesu|fonic acid25 (804.0 mg, 0.5 equiv) and N-hydroxysuccinimide (475.2 mg, 1 equiv) inDME (150 mL) at 0°C was treated with DIEA (0.33 mL, 0.5 equiv) and 0.5MDCC in methylene chloride (9.0 mL, 1.2 equiv). After 6 h DCU was removedby filtration and the filtrate was concentrated in vacuo. The residue waspartitioned between ethyl acetate and water. The organic phase waswashed with water (2x) and brine, dried over sodium sulfate, andconcentrated in vacuo. The residue was dissolved as far as possible inmethylene chloride (100 mL) and then the mixture was diluted with ether(100 mL). After being left to stand overnight the resulting solid was collectedby filtration and air-dried (2.6295 g, 90%). ‘H-NMR (CDCI3) 6 1.05-1.98(m, 12H), 2.09 (brt, 4H), 2.51 (brt, 2H), 2.91 (brs, 4H), 3.03 (m, 4H), 3.39 (brt,2H), 3.72 (s, 6H), 4.34 (q, 2H). 4.52 (s, 4H), 4.71 (q, 2H), 6.55 (s, 2H), 6.78 (d,4H), 7.03-7.50 (m, 34H), 8.72 (brs, 2H). MS (FAB) 1583.9 (M+Na)+, 1600.9(M+K)"'.Example 26bMEt-IDA-(Val-Lys(Mtr)-PABOH)2This was prepared from N-maleoyl-N’, N'-bis-(carboxymethy|)ethyIenediamine-p-toluenesulfonic acid 25 (500 mg, 1.16mmol) and Val-Lys(Mtr)-PABOH 10b (1.44 g, 2 equiv) as described abovefor 26a (1.68 g, 95%). 1H-NMR (CDCI3/CD3OD) 5 0.90 (t, 12H), 1.10-1.88(m, 14H), 2.10 (rn, 4H), 2.71 (bn, 2H), 3.24 (brs, 4H), 3.46 (m, 2H), 3.77 (s,6H), 4.25 (d, 2H), 4.42 (t, 2H), 4.58 (s, 4H), 6.63 (s, 2H), 6.79 (d, 4H), 7.05-7.55 (m, 32H). IR (KBr) 698, 1510, 1638, 1708. MS (ESI) 1466.0 (MH)+.Anal. Calcd for C85H1ooN1oO12-2H2O: C—68.78, H-6.98 N-9.33. Found: C-68.87, H—7.54, N-9.48.-73-. W0 98/ 197051015202530CA 02264610 1999-03-08PCT/US97/ 19851Example 27a , ., __MEt-lDA-(Phe-Lys(Mtr)-PABC-PNP)2A stirred suspension of MEt-lDA-(Phe-Lys(Mtr)-PABOH)2 26a (471.5 mg,0.319 mmol) and bis-p-nitrophenylcarbonate (971.0 mg, 10 equiv) inmethylene chloride (25 mL) at rt under argon was treated with DIEA (0.611mL, 11 equiv). After 16 h the solvent was removed in vacuo and the residuedried in vacuo for 3 h. The residue was dissolved in methylene chloride (15mL) and to this was added ether (30 mL) with slow stirring. The resultingsuspension was left to stand overnight. The solid was collected by filtration,washed with 2:1 ether/methylene chloride and dried in vacuo (411.5 mg,68%). 1H-NMR (CDCI3) 8 1.30 (m, 4H), 1.45 (m, 4H), 1.62 (m, 2H), 1.89 (m,2H), 2.10 (t, 4H), 2.58 (brt, 2H), 3.00 (brs, 4H), 3.06 (m, 4H), 3.31 (t, 2H), 3.73(s, 6H), 4.38 (q, 2H), 4.68 (q, 2H), 5.21 (s, 4H), 6.53 (s, 2H), 6.77 (cl, 4H),7.03-7.38 (m, 34H), 7.41 (d, 8H), 7.57 (d, 4H), 8.22 (cl, 4H), 8.79 (brs, 2H).MS (ESl) 1893.9 (MH)+.Example 27bMEt-lDA-(Val-Lys(Mtr)-PABC-PNP)2This was prepared from MEt-IDA-(Val-Lys(Mtr)-PABOH)2 26b (300 mg,0.205 mmol) as described above for 27a (347.7 mg, 94°/0). 1H—NMR(CDCI3) 5 0.88 (t, 12H), 1.25-1.75 (m, 12H), 1.93 (m, 2H), 2.07 (m, 4H), 2.71(m, 2H), 3.21 (brs, 4H), 3.55 (m, 2H), 4.26 (m, 2H), 4.34 (m. 2H), 5.19 (s, 4H),6.59 (s, 2H), 6.76 (d, 4H), 7.10-7.40 (m, 32H), 7.59 (m, 4H), 8.21 (d, 4H), 8.65(br, 2H).Example 28aMEt-lDA-(Phe-Lys(Mtr)-PABC-DOX)2A solution of MEt-IDA—(Phe-Lys(Mtr)-PABC—PNP)2 27a (410.3 mg, 0.217mmol) and doxorubicin hydrochloride (283.0 mg, 2.25 equiv) in NMP (10mL) at rt was treated with DIEA (0.094 mL, 2.5 equiv). After 48 h the mixturewas poured into ethyl acetate. The solution was washed with water (3x) andevaporated in vacuo. In each case some orange solid, that was shown by-74-1015202530CA 02264610 1999-03-08wo 98/1970S " - PCT/US97/19851TLC to contain product, was formed. This was collected by filtration and __combined later with the organic-soluble components beforechromatography. The combined materials were chromatographed on silica,eluting with 1) 6:4 , and 2) 1:1 methylene chloride/(521 ethyl 2acetate/methanol), to give the product as an orange solid (351.8 mg, 60%).1H-NMR (CDCl3/CH3OD) 5 1.22 (brd, 10H), 1.40 (m, 4H), 1.51 (m, 4H),1.75 (m, 6H), 2.07 (m, 4H), 2.20 (ABq, 4H), 2.50 (brt, 2H), 2.93 (brs, 4H), 2.99(m, 4H), 3.23 (brs, 4H), 3.57 (m, 2H), 3.68 (s. 6H), 3.75 (m, 2H), 3.97 (s, 6H),4.06 (m, 2H), 4.29 (m, 2H), 4.56 (m, 2H), 4.71 (s‘, 4H), 4.81 (ABq, 4H), 5.16(brs, 2H), 5.42 (brs, 2H), 6.55 (s, 2H), 6.71 (d, 4H), 6.98-7.50 (m, 44H), 7.71(t, 2H), 7.94 (d, 2H), 8.69 (br, 2H). MS (ESI) 2702.4 (MH)"".Example 28bMEt-IDA-(Val-Lys(Mtr)-PABC-DOX)2This was prepared from MEt-IDA-(Val-Lys(Mtr)-PABC-PNP)2 27b (6.351 g,3.536 mmol) as described above for 28a (2.79 g, 30%). 1H—NMFl (DMSO-d5) 5 0.76 (ABq, 12H), 1.10 (d, 6H), 1.20-1.60 (m, 12H), 1.88 (m, 8H), 2.13(q, 4H), 2.33 (m, 2H), 2.64 (m, 2H), 2.93 (m, 4H), 3.21 (brs, 4H), 3.64 (s, 6H),3.70 (m, 2H), 3.91 (s, 6H), 4.15 (m, 4H), 4.26 (m, 2H), 4.55 (d, 4H), 4.70 (d,2H), 4.88 (m, 4H), 5.19 (brs, 2H), 5.45 (s, 2H), 6.75 (d, 4H), 6.86 (s, 2H), 7.10(m, 4H), 7.18 (m, 18H), 7.30 (d, 6H), 7.49 (m, 4H), 7.55 (m, 2H), 7.82 (m, 4H),8.04 (m, 2H), 9.94 (br, 2H). MS (ESI) 2608.0 (MH)+.Example 29aMEt-IDA-(Phe-Lys-PABC-DOX)2~2Cl2CHCO2HA stirred solution of MEt-IDA-(Phe-Lys(Mtr)-PABC-DOX)2 28a (192.0 mg,0.071 mmol) in methylene chloride (15 mL), at rt was treated with anisole(1.54 mL, 200 equiv) and dichloroacetic acid (0.117 mL, 20 equiv). After 1hethyl acetate (15 mL) was added. The resulting solid was collected byfiltration, washed with ethyl acetate and ether and dried in vacuo (181.0 mg,100%). TH-NMR (CDCl3/CH3OD) 8 1.19 (d, 6H), 1.33 (m, 4H), 1.52 (m,6H), 1.74 (m, 6H), 2.14 (ABq, 4H), 2.49 (brt, 2H), 2.93 (m, 12H), 3.25 (brs,-75-CA 02264610 1999-03-08W0 98/ 19705 PCT/US97/198514H), 3.51 (m, 2H), 3.77 (m, 2H), 3.90 (3, 6H), 4.04 (m, 2H), 4.32 (m,.2H), 4.6; -1015202530(s,_4H), 4.32 (m, 4H), 5.05 (brs, 2H), 5.33 (brs, 21-1), 5.32 (brs, 2H), 6.63 (s,2H), 7.01 (m, 14H), 7.32 (m, 6H), 7.63 (1, 211), 7.79 (cl, 211). MS (ESI) 1073.3(MH2)2+, 2156.3 (MH)+.Example 29bMEt-IDA-(Val-Lys-PABC-DOX)2-2Cl2CHCO2HThis was prepared from MEt—IDA-(Val-Lys(Mtr)—PABC—DOX)2 28b (232 mg,0.0891 mmol) as described above for 29a (171 mg, 78%). 1H-NMR(DMSO-d5) 5 0.73 (ABq, 1211), 1.10 (d, 6H), 1.15-1.70 (m, 1211), 1.73 (m,2H), 1.92 (m, 2H), 2.11 (m, 4H), 2.72 (m, 4H), 2.94 (m, 4H), 3.68 (m, 2H), 3.94(s, 6H), 4.15 (m, 4H), 4.26 (m, 2H), 4.55 (brs, 41-1), 4.66 (brs, 211), 4.36 (m,4H), 5.19 (brs, 211), 5.45 (s, 2H), 5.91 (s, 2H), 6.32 (d, 2H), 6.93 (s, 2H), 7.21(d, 4H), 7.51 (d, 4H), 7.53 (m, 21-1), 7.76 (m, 41-1), 7.34 (m, 411), 3.19 (m, 211),10.00 (brs, 6H). IR (KBr) 1648, 1709.‘ MS (ESI) 1030.9 (1v11—12)+2, 2060.3(MH)+.Example 30MEt-IDA-(Phe-Lys(Mtr)-PABC—MMC)2A stirred mixture of MEt-|DA—(Phe—Lys(Mtr)-PABC-PNP)2 27a (183.8 mg,0.0971 mmol), mitomycin C (73.1 mg, 2.25 equiv), N-hydroxybenzotriazolemonohydrate (131.3 mg, 10 equiv) and freshly activated 4A powderedsieves (0.5 g) in NMP (6 mL) under argon at rt was treated with DIEA (0.17mL, 10 equiv). After 40 h the mixture was filtered and the filtrate diluted withethyl acetate (80 mL). The solution was washed with water (4x), brine, driedover sodium sulfate and evaporated. The residue was chromatographed onsilica, eluting with 2:1 methylene chloride/(521 ethyl acetate/methanol), togive the product as a light purple solid (201.7 mg, 91%). 1H-NMR (CDCI3)5 1.27 (m, 4H), 1.42 (m, 4H), 1.60 (m, 2H), 1.69 (s, 6H), 1.33 (m, 2H), 2.06(brt, 4H), 2.57 (m, 2H), 3.00 (m, 4H), 3.07 (brs, 4H), 3.13 (s, 6H), 3.28 (m, 4H),3.41 (d, 2H), 3.59 (ABq, 2H), 3.72 (5, 6H), 4.20 (t, 2H), 4.41 (d and m, 3H),4.66 (m, 2H), 4.88 (d, 4H), 5.10 (d, 2H), 5.39 (br, 6H), 6.49 (s, 2H), 6.72 (d,-76..1015202530WO 98/19705CA 02264610 1999-03-08PCT/U S97/ 198514H), 6.90-7.55 (m, 42H), 7.62 (br, 2H), 9.07 (br, 2H). MS (ESI) 1142.0(MH2)2+, 2282.0 (MH)+..fExample 31MEt-IDA-(Phe-Lys-PABC-MMC)2-2C|2CHCO2HA solution of MEt-IDA-(Phe-Lys(Mtr)-PABC-MMC)2 30 (134.6 mg, 0.0590mmol) in methylene chloride (3 mL) at rt was treated with anisole (1.28 mL,200 equiv) and 1M chloroacetic acid in methylene chloride (1.18 mL, 20equiv). After 3.5 h ethyl acetate (30 mL0 was added. the resulting purplesolid was collected by filtration, washed with ethyl acetate and air-dried (97.8mg, 82%). The product appeared to decompose in solution to one productby HPLC. ‘H-NMR (CDCI3/CH3OD) 8 1.33 (m, 4H), 1.63 (s and M, 12H),1.77 (m, 2H), 2.52 (m, 2H), 2.82 (m, 6H), 3.03 (m, 2H), 3.10 (s and brs, 10H),3.27 (m, 2H), 3.53 (ABq, 2H), 3.96 (brs, 4H), 4.11 (m, 2H), 4.35 (d and m,3H), 4.53 (m, 2H), 4.78 (ABq, 2H), 4.94 (m, 4H), 6.62 (s, 2H), 7.10 (m, 10H),7.19 (d, 4H), 7.41 (d, 4H). MS (FAB) 1738.3 (M+Na)+, 1776.4 (M+K)+.Example 322'-Methoxytrityl-PaclitaxelA stirred solution of paclitaxel (0.51 g, 0.597 mmol) and p—methoxytritylchloride (4.63 g, 25 equiv) in methylene chloride (14 mL) under nitrogen at rtwas treated with pyridine (1.23 mL, 25 equiv). After 16 h at rt the solvent wasevaporated and the residue dissolved in ethyl acetate. The solution waswashed with cold pH 5 buffer (2x100mL), water and brine, dried andevaporated. The residue was chromatographed on silica, eluting with 3%methanol/methylene chloride, to give the product as a white solid (482 mg,72%). "TH NMR(CDCl3)51.11 (s, 3H), 1.17 (s, 3H), 1.55 (s, 3H), 1.67 (s,3H), 1.90 and 2.54 (m, 2H), 2.26 (s, 3H), 2.51 (s, 3H), 2.54 (m, 2H), 3.66 (d,1H), 3.78 (s, 3H), 4.21 (ABq, 2H), 4.41 (m, 1H), 4.63 (d, 1H), 4.92 (d, 1H),5.62 (d, 1H), 5.70 (m, 2H), 6.22 (s, 1H), 6.74 (d, 2H), 7.09-7.60 (m, 23H), 7.80(d, 2H), 8.09 (d, 2H). MS (FAB) 1148 (M+Na)+, 1164 (M+K)+.-77-1O15202530CA 02264610 1999-03-08WO 98/19705 7 ‘ PCTIUS97/19851Example 33 . ..MEt-IDA-(Phe-Lys(Mtr)-PABC-7-Paclitaxe|)2A solution of paclitaxel-2'-Mtr 32 (789.3 mg, 0.701 mmol) in methylenechloride (5 mL) at 0°C under argon was treated with DIEA (0.122 mL, 1equiv), pyridine (0.057 mL, 1 equiv) and diphosgene (0.043 mL, 0.5 equiv).The mixture was allowed to warm to rt for 1.5 h and then it was added, viasyringe, to a stirred suspension of MEt-IDA-(Phe-Lys(Mtr)—PABOH)2 26a(547.3 mg, 0.5 equiv) in methylene chloride (5 mL) and DIEA (0.122 mL, 1equiv) at rt. After 16 h the mixture was diluted with ethyl acetate and washedwith pH 5 buffer (0.5M biphthalate), water and brine, dried over sodiumsulfate, and concentrated in vacuo. The residue was chromatographed onsilica, eluting with 1) 5:1 and 2) 3:1 methylene chloride/(5:1 ethylacetate/methanol), to give the product as a colorless glass (859.2 mg, 63%).Also recovered was a small amount of the mono-taxol adduct (172.0 mg,18%). ‘H-NMR (CDCI3/CH3OD) 8 1.05-1.80 (4xm, 12H), 1.12 (s, 6H), 1.14(s, 6H), 1.61 (s, 6H), 1.79 (s, 6H), 1.90 (m, 2H), 2.09 (m, 4H), 2.16 (s, 6H),2.28 (s, 6H), 2.58 (m, 3H), 2.98 (brs, 4H), 3.05 (m, 4H), 3.37 (m, 2H), 3.75 (s,6H), 3.78 (s, 6H), 4.16 (d, 2H), 4.27 (d, 2H), 4.32 (m, 2H), 4.63 (d and m, 4H),4.92 (d, 2H), 5.16 (m, 4H), 5.49 (ABq, 2H), 5.62 (d, 2H), 5.71 (m, 4H), 6.33 (s,2H), 6.53 (s, 2H), 6.75 (q, 8H), 6.97 (brd, 2H), 7.05-7.65 (m, 96H), 7.81 (d,4H), 8.08 (d, 4H), 8.68 (brs, 2H),Example 34MEt-lDA-(Phe-Lys-PABC-7-Paclitaxe|)2-2ClCH2CO2HMEt-IDA-(Phe—Lys(Mtr)-PABC-7-Paclitaxe|)2 33 (333.0 mg, 0.0861 mmol) inmethylene chloride (3 mL) was treated with anisole (1.87 mL, 200 equiv) and1M chloroacetic acid in methylene chloride (1.72 mL, 20 equiv). After 4 h themixture was diluted with ether (30 mL). The resulting suspension wassonicated for several minutes and then the solid was collected by filtrationand washed with ether (256.3 mg, 97%). The product was shown todecompose slowly in solution. 1H-NMR (DMF—d7) 8 1.15 (s, 6H), 1.19 (s,6H), 1.77 (s, 6H), 1.94 (s, 6H), 1.30-2.00 (m, 12H), 2.15 (m, 4H), 2.59 (m, 4H),3.09 (m, 8H), 3.93 (d, 2H), 4.16 (s,.4H), 4.69 (m, 4H), 4.90 (d, 2H), 5.07 (d,-78-1O15202530W0 98/ 19705CA 02264610 1999-03-08PCT/U S97/ 198512H), 5.16 (m, 4H), 5.56 (ABq, 2H), 5.67 (d. 2H), 5.70 (ABq, 2H), 6.14 (-1, 2H), __6.42 (S, 2H), 6.49 (m, 2H), 7.02 (S, 2H), 7.10-7.90 (m, 44H), 8.11 (d, 4H), 9.09(br, 2H). MS (ESI) 2778.2 (MH)+_.gExample 35N-Carbobenzyloxy-N‘, N'-bis-(t-butoxycarbony|-methyl)propylenediamineThis was prepared from N—carbobenzyloxypropylenediamine hydrochloride(3.4624 g, 14.15 mmol) as described above for 22 (5.3017 g, 86%). 1H-NMR (CDCI3) 6 1.45 (s, 18H), 1.63 (m, 2H), 2.72 (t, 2H), 3.32 (t, 2H), 3.38 (s,4H), 5.11 (s, 2H), 6.26 (brt, 1H), 7.33 (m. 5H). MS (ESI) 437.5 (MH)+.Example 36N, N-Bis-(t-butoxycarbonylmethy|)propylenediaminediacetic acidA solution of N-carbobenzyloxy-N‘, N'—bis-(t-butoxycarbonylmethy|)-propylenediamine 35 ( 5.3017 g, 12.144 mmol) in ethanol (75 mL) wasfiltered through a bed of Raney nickel into a solution of acetic acid (2.1 mL, 3equiv) in ethanol (75 mL). The filtrate was degassed with nitrogen andtreated with 10% palladized charcoal (0.4 g). The mixture washydrogenated at 50 psi on a Parr apparatus for 16 h and then the catalystwas filtered off on a bed of celite. The filtrate was concentrated in vacuo andthe residue flushed with chloroform (150 mL). The crude product wascarried on without further purification.Example 37N-Maleoyl-N‘, N'-bis-(t-butoxycarbonylmethyl)-propylenediamineThis was prepared from N, N—bis—(t-butoxycarbony|—methyl)propy|enediamine diacetic acid 36 (12.14 mmol) as described abovefor 24. The crude product was chromatographed on silica. eluting with 3:1hexane/ethyl acetate, to give the product as a pale—yellow oil (2.0653 g, 44%-79-1015202530WO 98/19705CA 02264610 1999-03-08PCT/US97/19851(3 steps)). 1H-NMR (CDCI3) 8 1.42 (S, 18H), 1.74 (m, 2H), 2.72 (t,'2H), 3.40.a(3, 4H), 3.60 (t, 2H), 6.68 (s, 2H). MS (DCI) 383 (MH)"', 327 (MH-C4H9)+.Example 38N-Maleoyl-N‘, N'-bis-(carboxymethyl)propylenediamineop-toluenesulfonic acidA stirred solution of N-maleoyl-N‘, N'-bis-(t-butoxycarbonylmethyl)-propylenediamine 37 (2.06 g, 5.39 mmol) in methylene chloride (25 mL) at rtwas treated with p-toluenesulfonic acid monohydrate (3.07 g, 3 equiv). After16 h the solvent was removed in vacuo. The residue was triturated withether four times (each time the ether was decanted off). The resulting semi-sold material was flushed with chloroform (2 x 150 mL). NMR showed 1.4-1.5 moles of p-toluenesulfonic acid per mole of product (2.68 g, 97%). 1H-NMR (CD3OD) 5 1.96 (m, 2H), 2.29 (s, 4.2H), 3.31 (m, 2H), 3.51 (t, 2H), 4.13(s, 4H), 6.78 (s, 2H), 7.15 (d, 2.8H), 7.62 (d, 2.8H).Example 39MPr-IDA-(Phe-Lys(Mtr)—PABOH)2This was prepared from N-ma|eoyl—N', N'-bis-(carboxymethyl)propylenediamine—p-toluenesulionic acid 38 (2.6842 g, 5.25mmol) and Phe-Lys(Mtr)-PABOH 10a (7.0432 g, 2 equiv) as describedabove for 26a, except that the crude product was chromatographed onsilica, eluting with 1) 2:1 and 2) 1.521 methylene chloride/(5:1 ethylacetate/methanol), to give the product as a white solid (4.5253 g, 55%). 1H-NMR (CDCI3/CD3OD) 8 1.24 (m, 4H), 1.41 (m, 4H), 1.52 (m, 2H), 1.83 (m,2H), 2.06 (brt, 4H), 2.23 (bit, 2H), 2.88 (ABq, 4H), 3.09 (m, 4H), 3.27 (t, 2H),3.73 (s,"6H), 4.36 (q, 2H), 4.53 (brs, 4H), 4.71 (q, 2H), 6.61 (s, 2H), 6.75 (d,4H), 7.05-7.48 (m, 42H), 8.74 (brs, 2H). MS (ESI) 1576.4 (MH)+, 1303.6 (M-Mtr)+.-30-1015202530WO 98/19705’CA 02264610 1999-03-08PCT/US97/19851..Exam|:_>le 40 . .. __MPr—lDA-(Phe-Lys(Mtr)-PABC-PNP)2This was prepared from MPr—lDA:(Phe—Lys(Mtr)—PABOH)2 39 (1.7993 g,1.143 mmol) as described above for 27a (1.8909 g, 87°/0). 1H-NMR(CDCI3) 5W1.29 (m, 4H), 1.42 (m, 4H), 1.60 (m, 2H), 1.88 (m, 2H), 2.07 (brt,4H), 2.32 (brt, 2H), 2.92 (ABq, 4H), 3.08 (m, 4H), 3.29 (t, 2H), 3.72 (s, 6H),4.35 (q, 2H), 4.66 (q, 2H), 5.20 (s, 4H), 6.63 (s, 2H), 6.75 (d, 4H), 7.05-7.45(m, 42H), 7.52 (d, 4H), 8.21 (d, 4H), 8.71 (brs, 2H). MS (ESI) 1906.0 (MH)"',1634.4 (M-Mtr)+. MS (ESI) 1907.8 (MH)+.Example 41MPr-IDA-(Phe-Lys(Mtr)-PABC-DOX)2.. _ -This was prepared from MPr-lDA—(Phe-Lys(Mtr)—PABC-PNP)2 40 (722.7 mg,0.379 mmol) as described above for 28a. The crude product was —chromatographed on silica, eluting with 1) 20:1 and 2) 15:1 methylenechloride/methanol, to give the product as an orange solid (869.4 mg, 84%).1H-NMR (CDCI3/CD3OD) 6 1.16 (d, 10H),1.42 (m,4H), 1.49 (m, 4H), 1.82(m, 6H), 2.00, (m, 2H), 2.12 (m, 4H), 2.19 (m, 4H), 2.53 (brt, 2H), 2.89 (brs,4H), 2.99 (m, 4H), 3.25 (brs, 4H), 3.49 (m, 2H), 3.67 (s, 6H), 3.73 (m, 2H),3.99 (s, 6H), 4.12 (m, 2H), 4.31 (m, 2H), 4.55 (m, 2H), 4.71 (s, 4H), 4.92 (ABq,4H), 5.15 (brs, 2H), 5.40 (brs, 2H), 6.55 (s, 2H), 6.69 (d, 4H), 7.00-7.50 (m,44H), 7.70 (t, 2H), 7.96 (d, 2H), 8.71 (br, 2H). M8 (E81) 2716.8 (MH)+.Example 42MPr-lDA-(Phe-Lys-PABC-DOX)2 -2Cl2CHCO2HThis was prepared from MPr-lDA-(Phe-Lys(Mtr)-PABC-DOX)2 41 (714.3 mg,0.263 mmol) as described above for 29a (545.9 mg, 96%). ‘H-NMR (DMF-d7) 6 1.23 (d, 6H), 1.54 (m, 6H), 1.73 (m, 6H), 2.02 (m, 2H), 2.26 (ABq, 2H),2.48 (m, 4H), 3.09 (m, 4H), 3.12 (brs, 4H), 3.29 (m, 6H), 3.91 (m, 2H), 4.10 (s,6H), 4.32 (q, 2H), 4.56 (m, 2H), 4.80 (s and m, 6H), 5.13 (brs, 2H), 5.41 (brs,2H), 5.68 (br, 2H), 6.17 (s, 2H), 6.72 (d, 2H), 6.99 (s, 2H), 7.27 (m, 18H), 7.70-31-1015202530W0 98/19705CA 02264610 1999-03-08PCT/US97/19851(m, 4H), 7.92 (m, 2H), 8.52 (d, 2H), 8.69 (d, 2H), 10.22 (brs, 2H), MS«(ESl) ..2171.3 (MH)+.Example 43Di-t-butyliminodiacateA stirred mixture of glycine t-butyl ester hydrochloride (5.0811 g, 30.31mmol) and potassium bicarbonate (6.373O g, 2.1 equiv) in DMF (40 mL) at0°C was treated with t-butyl bromoacetate (4.9 mL, 1 equiv). The ice bathwas removed and the mixture was allowed to stir at rt for 14 h. The solventwas removed in vacuo at 30°C and the residue was partitioned betweenethyl acetate and 50% sat. sodium bicarbonate. The organic phase waswashed with water (3x) and brine, dried over sodium sulfate, andconcentrated in vacuo. The residue was chromatographed on silica, elutingwith 1) 5% and 2) 25% ehtyl acetate/hexane, to give the product as acolorless oil (4.931O g, 66%). 1H-NMR (CDCI3) 5 1.46 (s, 18H), 3.55 (s,4H), MS (DCI) 246 (MH)+, 190 (MH—C4H9)+.Example 44N-Maleoyl-N’, N'-bis-(t-butoxycarbonyl-methyl)propionamideA suspension of 2-maleimidopropionic acid (1.0007 g, 5.916 mmol) inmethylene chloride (12 mL) at rt was treated with oxalyl chloride\(1.2 mL, 2.1equiv) and DMF (1 drop). After 4 h the solvents were evaporated. Theresidue was flushed with dry methylene chloride (2 x 20 mL) and dissolvedin methylene chloride (2 mL). To this was added a solution of di-t-butyliminodiacetate 43 (1.4510 g, 1 equiv) and DIEA (1.24 mL, 1.2 equiv) inmethylene chloride (6 mL), dropwise over 15 min. After 14 h the mixture wasdiluted with ethyl acetate and washed with 15% citric acid, water and brine.The organic phase was dried over sodium sulfate and concentrated in vacuoto give a pale—ye||ow glass which was pure enough by NMR to carry to thenext step (2.3448 g, 100%). 1H—NMR (CDCI3) 8 1.43 (s, 9H), 1.46 (s, 9H),-82..1015202530W0 98/ 19705CA 02264610 1999-03-08PCT/US97/198512.84 (1, 2H), 3.88 (1, 2H), 3.97 (5, 2H), 4.04 (s, 2H), 6.69 (s, 2H). MS..(DCl) __397 (MH)+, 341 (MH-C4H9)+.Example 45N-Maleoyl-N‘, N'-bis-(carboxymethy|)- propionamideA stirred mixture of N-maleoyl-N‘, N'-bis—(t-butoxycarbonylmethy|)propionamide 44 (1.9100 g, 4.82 mmol) in methylenechloride (40 mL) at rt was treated with trifluoroacetic acid (20 mL). Themixture was sonicated at rt for 20 min and then left to stand for 3.5 h. Thereaction was concentrated in vacuo and the residue flushed with chloroform(2x5O mL) and then triturated with ether. The resulting pale-yellow solid wascollected by filtration and washed with ether (12273 g, 90%). 1H-NMR(CD3OD/CDCI3) 5 2.61 (t, 2H), 3.69 (t, 2H), 4.02 (s, 2H), 4.13 (s, 2H), 6.71(s,2H). MS (FAB') 283.1 (M-H)". HRMS Calcd: 285.0723. Found: 285.0712.Example 46MP-lDA-(Phe-Lys(Mtr)-PABOH)2This was prepared from N-ma|eoyl—N', N'—bis-(carboxymethyl)propionamide45 (142.1 mg, 0.500 mmol) and Phe-Lys(Mtr)-PABOH 10a (671.0 mg, 2equiv) as described above for 26a, except that the crude product waschromatographed on silica, eluting with 1) 2:1 and 1.521 methylenechloride/(5:1 ethyl acetate/methanol), to give the product as a white solid(349.7 mg, 44%). 1H—NMFt(CDC|3/CD3OD) 5 1.00-1.90 (m, 121-1),2.07 (brt,4H), 2.27 (bn. 2H), 3.08 (m, 4H), 3.32-3.82 (m, 6H), 3.70 (s, 6H), 4.28 (m,1H), 4.35 (m, 2H), 4.52 (d, 41-1), 4.82 (m, 1H), 8.51 (s, 2H), 8.72 (q, 4H), 7.00-7.52 (m, 42H). MS (FAB) 1812.5 (M+Na)+, 1828 (1v1+1<)+.Example 47MP-lDA-(Phe-Lys(Mtr)-PABC-PNP)2This was prepared from MP—IDA-(Phe-Lys(Mtr)-PABOH)2 46 (225.4 mg,0.142 mmol) as described above for 27a (195.5 mg, 72%). 1H-NMR(CDC|3/CD300) 5 1.05-1.95 (m, 12H), 2.05 (bl't, 4H), 2.33 (m, 2H), 3.08 (m,-83-1015202530W0 98/ 19705CA 02264610 1999-03-08PCTlUS97/ 198514H), 3.55 (t, 2H), 3.70 (s, 6H),.B.60-3.99 (m, 4H), 4.52 (m, 2H), 4.63 (m, 2H),__5.21 (d, 4H), 6.57 (s, 2H), 6.74 (q.4H), 7.00-7.65 (m, 46H), 8.20 (q. 4H). MS(ES|) 1920.8 (MH)+, 1647.8 (MH—Mtr)+.Example 48MP-IDA-(Phe-Lys(Mtr)-PABC-D0X)2This was prepared from MP-IDA—(Phe—Lys(Mtr)-PABC-PNP)2 47 (195.0 mg,0.102 mmol) as described above for 28a. The crude product waschromatographed on silica, eluting with 1) 20:1 and 2) 15:1 methylenechloride/methanol, to give the product as an orange solid (117.7 mg, 42%).1H-NMR (CDCI3/CD3OD) 61.21 (m and 0|, 10H), 1.42 (m, 4H), 1.59 (m, 2H),1.80 (m, 4H), 2.06 (m, 6H), 3.02 (m, 8H), 3.40-3.90 _(m_, 61:1), 3.69 (s, 6H), 4.00(s, 6H), 4.07 (m, 2H), 4.29 (m, 2H), 4.48 (m, 4H), 4.72 (s, 4H), 4.90 (m, 4H),5.19 (brs, 2H), 5.42 (brs, 2H), 6.51 (s, 2H), 6.71 (m, 4H), 6.90-7.55_(m, 44H),7.70 (t, 2H), 7.92 (m, 2H). MS (ESI) 1365.4 (MH2)+2, 2728.8 (MH)+.Examgle 49MP-IDA-(Phe-Lys-PABC-DOX)fi-2Cl2CHC02HThis was prepared from MP—lDA-(Phe-Lys(Mtr)-PABC-DOX)2 48 (91.0 mg,0.033 mmol) as described above for 29a (76.4 mg, 94%). 1H-NMR(CDCI3/CD3OD) 8 1.21 (d, 6H), 1.30-1.1.82 (m, 14H), 2.01 (t, 2H), 2.30 (m,4H), 2.53 (m, 2H), 3.04 (m, 12H), 3.50 (m, 2H), 3.72 (m, 2H), 3.92 (s, 6H),4.03 (m, 2H), 4.19 (m, 2H), 4.52 (m, 2H), 4.65 (s, 4H), 4.84 (m, 4H), 5.12 (brs,2H), 5.37 (brs, 2H), 5.79 (s, 2H), 6.57 (s, 2H), 6.73 (d, 2H), 6.95-7.50 (m,20H), 7.68 (1, 2H), 7.89 (m, 2H). MS (ESI) 1092.4 (MH2)+2, 2185.6 (MH)+.Example 50Z-BAlaA stirred solution of B-alanine (50.10 g, 0.562 mol) in 2M NaOH (281 mL) at0°C was treated with benzyl chloroformate (88 mL, 1.1 equiv), dropwise over1 h. Stirring was continued for 1.5 h at 0°C, followed by 1.5 h at rt. Theresulting mixture was extracted with ether (4x500 mL). The aqueous phase-34-1015202530W0 98/1 9705CA 02264610 1999-03-08PCT/US97/ 19851was acidified with 5M HCI (120 mL). The resulting white precipitate wascollected by filtration, washed with water and dissolved in methylenechloride. The solution was dried over sodium sulfate and concentrated invacuo to give a white solid (11495 g, 92%). ‘H-NMR (CDCI3) 5 2.61 (t,2H), 3.48 (q, 2H), 5.12 (s, 2H), 5.29 (br, 1H), 7.37 (s, SH). MS (DCI) 224(MH)'''.Example 51Z-(3Ala—OSuA mixture of Z-BAla 50 (37.42 g, 0.168 mol) and N-hydroxysuccinimide(19.34 g, 1 equiv) in methylene chloride (700 mL) was stirred at 0°C andtreated with DCC (34.69 g, 1 equiv). The reaction was allowed to warm to rt.After 16 h the DCU was removed by filtration and the filtrate wasconcentrated in vacuo to give a white foam (53.80 g, 100%). 1H-NMR(CDCI3) 8 2.86 (s and m, 6H), 3.60 (9, 2H), 5.12 (s, 2H), 5.41 (m, 1H), 7.37(m, SH). MS (DCI) 321 (MH)+.Example 52Z-(3Ala-IDAA stirred solution of iminodiacetic acid (16.84 g, 84.32 mmol) and lithiumhydroxide monohydrate (10.62 g, 2 equiv) in water (240 mL) at 0°C wastreated with a solution of Z-(5A|a-OSu 51 (27.01 g, 0.667 equiv) in DME (130mL). After 15 min the ice bath was removed and the reaction was stirred at rtfor 16 h. The mixture was concentrated to a volume of 200 mL on the rotaryevaporator. The resulting suspension was extracted with ethyl acetate(2x250 mL), and then the aqueous phase was acidified to pH 1-2 with 85%phosphoric acid. The mixture was extracted with 10% isopropanol/ethylacetate (3x200 mL). The combined organic phases were washed with 2%phosphoric acid and brine, dried over sodium sulfate, and evaporated togive a colorless glass which was flushed with chloroform, giving a foam(21.36 g, 75%). ‘H-NMR (CD300) 6 2.51 (t, 2H), 3.32 (t, 2H), 4.04 (s, 2H),4.15 (s, 2H), 4.98 (s, 2H), 7.25 (m, 5H). MS (FAB) 339 (MH)+, 361 (M+Na)+,-35-1015202530W0 98I19705CA 02264610 1999-03-08PCT/U S97/ 19851377 (M+K)+. Anal. Calcd for C15H13N207-H20: C—50.56, H—5.66,— N-7.86. ..Found: C-50.56, H-5.45, N-8.21.Example 53Z-[3Ala-IDA-(OSu)2This was prepared from Z-[3Ala-IDA 52 (21.36 g, 63.12 mmol) as describedabove for 51. The crude product was used without purification. 7H—NMFt(CDCI3/CD3OD) 5 2.62 (t, 2H), 2.83 (s, 8H), 3.53 (m, 2H), 4.56 (s, 4H), 5.08(s, 2H), 5.58 (brt, 1H), 7.35 (m, SH). Anal. Calcd for C23H24N4O11-0.5H2O:C-51.02, H-4.65, N—10.35. Found: C-50.83, H—5.13, N-10.75.Example 54Z-[3Ala-IDA-(BAla-O-t-Bu)2A stirred suspension of Z-BA|a—lDA-(OSu)2 53 (ca. 63.12 mmol) and B-alanine t-butyl ester hydrochloride (25.80 g, 2.25 equiv) in DME (250 mL) atrt were treated with DIEA (24.7 mL, 2.25 equiv). After 12 h the solvents wereevaporated and the residue partitioned between ethyl acetate (400 mLO and2% phosphoric acid. The organic phase was washed with water and brine,dried over sodium sulfate and evaporated. The residue waschromatographed on silica, eluting with 1) ethyl acetate, 2) 15:1, and 3) 10:1ethyl acetate/methanol. to give the product as a thick, colorless oil (26.56 g,71%). 1H-NMR (CDCI3) 8 1.41 (s, 18H), 2.46 (m, 6H), 3.49 (m, 6H), 3.88 (s,2H), 3.97 (s, 2H), 5.09 (s, 2H), 5.56 (brt, 1H), 6.89 (brt, 1H), 7.33 (m, 5H), 8.74(brt, 1H). MS (FAB) 593 (MH)+, 615 (M+Na)+, 631 (M+K)+. Anal. Calcd forC29H44N4Og-H20: C—57.04, H-7.59, N—9.17. Found: C—57.15, H—7.36, N-9.46.Example 55[3Ala-IDA-(BAla-O-t-Bu)2Z-[5Ala—lDA—(O—t-Bu)2 54 (16.93 g, 28.56 mmol) in absolute ethanol (100 mL)was degassed by bubbling nitrogen through it for 30 min. Palladizedcharcoal (10%, 1 g) was carefully added and the mixture was hydrogenated-85-1015202530WO 98/19705 A 7CA 02264610 1999-03-08PCTfUS97/ 19851on a Parr apparatus at 50 psi for 16 h. The catalyst was then filtered. off on g_bed of celite and the filtrate concentrated in vacuo. The residue was flushedwith methylene chloride, giving a colorless foam (13.11 g, 100%). 1H-NMR(CDCl3) 8 1.47 (s, 18H), 2.45 (q.6H), 3.01 (t, 2H), 3.51 (m, 4H), 3.91 (s, 2H),4.04 (s, 2H), 6.98 (brt, 1H), 8.93 (brt, 1H). MS (FAB) 459 (MH)+, 481(M+Na)+, 497 (M+K)+. Anal. Calcd for C21H38N4O7-2.5H2O: C-50.09, H-8.61, N-11.12. Found: C-50.17, H-8.61, N-10.51.Example 56Maleoyl-BAla-IDA-(BA|a-O-t-Bu)2A stirred solution of BAla-IDA-(O-t-Bu)2 55 (6.20g, 13.5 mmol) in chloroform(100 mL) at rt was treated with maleic anhydride (1.46 g, 1.1 equiv). After 1.5h the mixture was concentrated in vacuo. The residue was flushed withhexane and carried on without further purification. 1H-NMR (CDCI3) 5 1.50(s, 18H), 2.48 (m, 4H), 2.57 (m, 2H), 3.48 (m, 4H), 3.67 (q, 2H), 3.92 (s, 2H),4.06 (s, 2H), 6.38 (ABq, 2H), 7.39 (brt, 1H), 7.91 (brt, 1H), 8.22 (brt, 1H). MS(FAB) 459.1 (MH)+, 481.2 (M+Na)+, 497.1 (M+K)+.Example 57MP-IDA-(BAla-O-t-Bu)2A solution of maleoyl—[3A|a-lDA—(BAla—O-t—Bu)2 56 (7.50 g, 13.5 mmol) inacetonitrile (150 mL) under argon at 0°C was treated with trimethylsilylchloride (8.6 mL, 5.02 equiv) followed by triethylamine (9.4 mL, 5.02 equiv).The mixture was heated at reflux for 4 h, cooled to rt, and then storedovernight at -20°C. The resulting solid triethylamine hydrochloride wasfiltered off and the filtrate concentrated in vacuo. The residue waspartitioned between ethyl acetate and water. The aqueous phase wasextracted with more ethyl acetate. The combined organic phases were driedover sodium sulfate and evaporated. The residue was chromatographed onsilica, eluting with 1) 5%, 2) 7%, and 3) 10% methanol/methylene chloride, togive the product as a pale-yellow gum (4.83 g, 66%). 1H-Nl\/IR (CDC|3) 81.42 (s, 18H), 2.47 (m, 4H), 2.61 (t, 2H), 3.50 (m, 4H), 3.81 (t, 2H), 3.88 (s,-87-‘IO15202530CA 02264610 1999-03-08W0 98/ 19705 PCT/US97/198512H), 3.99 (3, 2H), 6.60 (brt, 1H), 6.69 (S, 2H), 8.82 (brt, 1H). MS (FAB) 561.2‘(M+Na)+, 577 (M+K)+. Anal. Calcd for C25H33N4O7—1.5H2O: C-53.09, H-7.30, N-9.91. Found: C-53.13, l-l_-_6.98, N-10.05.Example 58MP—lDA-(BAla)2A solution of MP-IDA-(BA|a—O—t—Bu)2 57 (3.50 g, 6.50 mmol) in methylenechloride (40 mL) at rt was treated with trifluoroacetic acid (30 mL). themixture was sonicated for 10 min and then left to stand for 1 h. The solventswere evaporated on the rotovap and the residue flushed with chloroform (3x)and ether (4x). The crude product was carried on without further purification.1H-NMR (CD3OD) 5 2.47 (m, 4H), 2.53 (m, 2H), 3.40 (m, 4H), 3.69 (t, 2H),3.89 (s, 2H), 4.04 (s, 2H), 6.71 (s, 2H). MS (FAB) 433.0 (M+Li)+. Anal. Calcdfor C17H22N4O9-3H2O: C—42.50, H-5.87, N—11.66. Found: C-42.44, H-5.92,N-11.54. 'Example 59MP-IDA-([3Ala-OSu)2A stirred solution of MP—|DA—(BA|a)2 58 (136.0 mg, 0.319 mmol)and NHS(73.5 mg, 2 equiv) in DMF (2 mL) at rt was treated with DCC (265.0 mg, 4.01equiv). The mixture was stirred at rt for 16 h and then was carried on directlyto the next step without isolation of the product.Example 60MP-IDA-(BAla-Phe-Lys(Mtr)—PABOH)2The crude reaction mixture containing MP—|DA-(BAla)2 58 (ca. 0.319 mmol)was diluted with DMF (5 mL) and then treated with Phe-Lys(Mtr)-PABOH10a (429.1 mg, 2.0 equiv). After stirring at rt for 5 h the solvents wereremoved under high vacuum and the residue was triturated with ether. Theresulting solid was collected by filtration and then chromatographed onsilica, eluting with 5% methanol/ethyl acetate to remove impurities, and 10%methanol/methylene chloride to elute the product (220.1 mg, 40%). TH-NMR-88-1015202530WO 98/19705CA 02264610 1999-03-08PCT/US97/ 19851(DMSO-d5)6 1.15-1.75 (m, 12H), 1.92 (m,4H),2.21 (m,4H),2.41.(t,2H), __2.72 (m, 2H), 3.01 (m, 2H), 3.13 (m, 4H), 3.58 (m, 2H), 3.57 (s, 6H), 3.78 (cl,2H), 3.90 (brs, 2H), 4.38 (m, 2H), 4.42 (d, 4H), 4.53 (m, 2H), 5.09 (t, 2H), 7.30(d, 4H), 5.95 (s, 2H), 7.05-7.30 (in, 32H), 7.38 (d, 6H), 7.52 (d, 4H), 8.08 (m,2H), 8.13 (d, 2H), 9.91 (brs, 2H). MS (FAB) 1754.0 (M+Na)+, 1770.0 (M+K)"'.Example 61MP-IDA-([3Ala-Phe-Lys(Mtr)-PABC-PNP)2A stirred solution of MP-IDA-(BAla-Phe—Lys(Mtr)-PABOH)2 60 (95 mg,0.0548 mmol) in 1:1 DMF/methylene chloride (6 mL) at rt was treated withbis-p-nitrophenylcarbonate (100 mg, 6 equiv) and DIEA (0.057 mL, 6.0equiv). After 16 h the solvents were removed under high vacuum and theresidue was triturated with 1:1 methylene chloride/ether until it became asolid. This suspension was stored in the freezer for 1.5 h and then the solidwas collected by filtration and washed with ether (105 mg, 95%). 1H-NMR(DMSO-d5) 5 1.20-1.75 (m, 12H), 1.93 (m, 4H), 2.21 (m, 4H), 2.41 (m, 2H),2.71 (t, 2H), 3.01 (d, 2H), 3.14 (m, 4H), 3.56 (m, 2H), 3.67 (s, 6H), 3.78 (brs,2H), 3.91 (brs, 2H), 4.37 (m, 2H), 4.56 (m, 2H), 5.23 (s, 4H), 6.80 (d, 4H), 6.94(s, 2H), 7.05-7.50 (m, 38H), 7.56 (d, 4H), 7.64 (d, 4H), 8.09 (m, 2H), 8.18 (d,2H), 8.30 (d, 4H), 10.03 (brs, 2H). MS (ESI) 2052.3 (iviH)+.Example 62MP—lDA-(BAla-Phe-Lys(Mtr)-PABC-DOX)2MP-lDA-(BA|a-Phe-Lys(Mtr)—PABC~PNP)2 61 (1.445 g, 0.701 mmol) anddoxorubicin hydrochloride (1.010 g, 2.48 equiv) in DMF (55 mL) were treatedwith DIEA (0.31 mL, 2.50 equiv). After 40 h at rt the solvent was evaporatedunder high vacuum and the residue was triturated with 1:1 methylenechloride/ether. The resulting suspension was sonicated briefly and then thesolid was collected by filtration and washed with ether. The solid wasdissolved as far as possible in 10% methanol/chloroform (100 mL). Themixture was filtered and the filtrate was concentrated on the rotovap to ca. 50mL. Chloroform (50 mL) was added and the procedure was repeated twomore times. The resulting solid was collected by filtration and washed with-39-1015202530W0 98/ 19705 ‘CA 02264610 1999-03-08PCT/US97/19851chloroform and then ether (1.22 g, 61%). 1H-NMR (DMSO-d5) 5 1213 (d, ..6H), 1.20-1.75 (m, 12H), 1.81 (m, 2H), 1.92 (m, 4H), 2.19 (m, 6H), 2.30 (m,2H), 2.72 (m, 2H), 2.98 (m, 6H), 3.12 (m, 2H), 3.45 (m, 2H), 3.56 (t, 2H), 3.69(s, 6H), 3.73 (m, 2H), 3.89 (m, 2H), 3.96 (s, 6H), 4.14 (m, 2H), 4.33 (m, 2H),4.57 (d and m, 6H), 4.70 (d, 2H), 4.84 (t, 2H), 4.91 (m, 4H), 5.22 (brs, 2H),5.46 (s, 2H), 6.79 (d, 4H), 6.93 (s, 2H), 7.05-7.30 (m, 32H), 7.33 (d, 6H), 7.52(d, 4H), 7.60 (m, 2H), 7.88 (d, 2H), 8.06 (m, 2H), 8.13 (d, 2H), 9.96 (brs, 2H),MS (ESI) 2872.6 (MH)+.Example 63MP-IDA-(BA|a-Phe-Lys-PABC-DOX)2-2Cl2CHCO2H A stirredsuspension of MP-lDA—([3A|a-Phe-Lys(Mtr)-PABC-DOX)2 62 (132 mg,0.0459 mmol) in methylene chloride (8 mL) was treated with anisole (1 mL,200 equiv) and dichloroacetic acid (0.076 mL, 20 equiv). The mixtureimmediately became homogeneous. After 5 min an orange precipitatebegan to form. After 2 h ethyl acetate (25 mL) was added and thesuspension stirred for 30 min. The solid was collected by filtration andwashed with ethyl acetate and then ether (89 mg, 80%). 1H-NMR (DMSO-d5) 8 1.12 (d, 6H), 1.22-1.90 (14H), 2.20 (m, 8H), 2.74 (m, 6H), 2.97 (m, 6H),3.15 (m, 2H), 3.43 (brs, 2H), 3.56 (m, 2H), 3.72 (m, 2H), 3.97 (s, 6H), 4.14 (m,2H), 4.38 (m, 2H), 4.56 (s and m, 6H), 4.70 (m, 2H), 4.89 (m, 4H), 5.21 (brs,2H), 5.46 (brs, 2H), 6.01 (s, 2H), 6.81 (d, 2H), 6.97 (s, 2H), 7.21 (m, 14H),7.56 (d, 4H), 7.61 (m, 2H), 7.77 (m, 2H), 7.89 (d, 2H), 8.12 (m, 2H), 10.01 (m,2H). Ms (ESI) 2325.6 (MH)+.Example 64M-Et-lDA-(Phe-Lys-PABC-DOX=B-Ala-mTEG)2MTEG-B—Ala Hydrazide (254 mg, 866 umole) was dissolved in 10 mlmethanol along with 50 ul TFA. This solution was added to a suspension ofM-Et—|DA—(Phe-Lys—PABC-DOX)2 (276 mg, 108 umole) in 40 ml anhydrousmethanol. The reaction was stirred for 3.5 hr. at room temperature. Thereaction mixture was rotary evaporated to the point of precipitation, then 1 ml-90-1O15202530W0 98/ 1 9705CA 02264610 1999-03-08PCT/US97/ 19851CH2Cl2 was added to redissolve. This was added dropwise to 250ml ethe‘r_,_precipitating a red solid. The solid was filtered, washed with ether, and driedunder high vacuum to yield M-Et-IDA-(Phe—Lys-PABC-DOX=B—Ala-mTEG)2(300 mg, 90%).1H-NMR (d7—DMF): (selected peaks) 5 1.23 (d, 6H), 2.34 (t, 4H), 3.28 (s, 6H),3.55 (s, 24H), 4.03 (s, 6H), 4.10 (t, 4H), 6.98 (s, 2H).Mass Spec.: FAB 1354.6 (ivi+2H)2+Elemental Analysis for C132H154N13O44 -4.0H20-3.0TFA: Theoretical C,53.11; H, 5.65; N, 8.08. Found C, 52.99;H, 5.96; N, 8.64.FTIR: 3412, 2938, 1702, 1681, 1616, 1520, 1412, 832, 696 cm‘1.Example 65M-Et-IDA-(Phe-Lys—PABC-D0X=[3-Ala)2B-Ala Hydrazide triflouroacetate (578 mg, 1.75 mmole) was dissolved in 10ml methanol along with 50 ul TFA. This solution was added to a suspensionof M—Et-lDA-(Phe-Lys-PABC-DOX)2 (111 mg, 43.6 umole) in 15 mlanhydrous methanol. The reaction was stirred for 1.5 hr. at roomtemperature. The reaction mixture was rotary evaporated to a volume of 3 ml,then added dropwise to 200 ml acetonitrile, precipitating a red solid. Thesolid was filtered, washed with acetonitrile, and dried under high vacuum toyield M-Et—lDA—(Phe-Lys-PABC—DOX=[3-Ala)2 (117 mg, 99%).1H-NMR (d5-DMSO): (selected peaks) 5 1.13 (d, 6H), 3.95 (s, 6H), 6.96 (s,2H).Example 66N-t—6utoxycarbonyl-3-aminopropanolA solution of 3-aminopropanol (15 mL, 0.20 mol) in methylene chloride (50mL) at rt was treated with t-butylpyrocarbonate (42.02 g, 1 equiv) inmethylene chloride (25 mL), dropwise over 2 h. After stirring overnight themixture was evaporated, flushed with heptane (3x), and dried in vacuo togive the product as a thick oil (34.31 g, 100%). ‘H NMR 61.48 (s, 9H), 3.12(br, 1H), 3.22 (q, J=7.5 Hz, 2H), 3.60 (q, J=7.8 Hz, 2H), 4.81 (br, 1H), MS-91-1015202530W0 98/19705CA 02264610 1999-03-08PCT/US97/19851(DC!) 176 (MH)+. Anal calcd for C8H17NO3: C—54.84, H-9.78, N'7.99w Found_;_C—54.80, H-9.82, N-7.97.Example 67N-t-Butoxycarbonyl-3-aminopropyl mesylateThe protected amino alcohol 64 (6.601 g, 37.67 mmol) in methylenechloride (45 mL) at 0°C under argon was treated with methanesulfonylchloride (2.92 mL, 1.0 equiv) and triethylamine (5.25 mL, 1.0 equiv),dropwise over 30 min. The mixture was stirred for 16 h and then the solventwas evaporated. The residue was partitioned between ether (200 mL) andwater (250 mL) and the organic phase was washed with 10% citric acid,water and brine, dried over sodium sulfate and evaporated. The residuewas chromatographed on silica, eluting with 2% methanol/methylenechloride. The product was found to be unstable over extended periods at rt(5.800 g, 61%). ‘H NMR 6 1.34 (s, 9H), 1.86 (q, J=7.2 Hz, 2H), 2.95 (s, 3H),3.19 (m, 2H), 4.12 (t, J=8.0 Hz, 2H), 4.79 (br, 1H),Example 68Diethyl 2-(N-t-butoxycarbonylaminopropyl)-malonateSodium hydride (60% in mineral oil, 01.84 g, 46 mmol) suspended in THF(20 mL) at 0°C under argon was carefully treated with diethyl malonate (7mL, 1.0 equiv) and the mixture was allowed to warm to rt. When it becamehomogeneous the mesylate 65 (5.84 g, 0.5 equiv) in THF (15 mL) wasadded all at once. The mixture was heated at reflux overnight, diluted with1:1 hexane/ethyl acetate and washed with water and brine, dried oversodium sulfate and evaporated. The residue was chromatographed onsilica, eluting with 25-35% EtOAo/hexane (5.751 g, 79%) ‘H NMR 6 1.18 (t,J=7.9“Hz, 6H), 1.35 (s, 9H), 1.81 (m, 2H), 3.04 (q, J=7.4 Hz, 2H), 3.24 (t,J=6.2 Hz, 1H), 4.10 (q, J=7.9 Hz, 4H), 4.62 (br, 1H), MS (FAB) 318 (MH)+,262 (M—C4H9), 218 (M-C5H9O2). Anal calcd for C15H27NO6: C—56.77, H-8.57,N-4.41. Found: C—56.65, H—8.45, N-4.42.-92-1015202530W0 98/ 19705CA 02264610 1999-03-08PCT/US97/19851._ Example 69 , .. __N-t-Butoxycarbonyl-5-amino-2-hydroxymethylpentanolThe diester 66 (4.957 g, 15.62 mmol) in ether (100 mL) under argon at 0°Cwas treated with lithium borohydride (2M in THF, 24 mL, 3 equiv) and thenwith methanol (1.9 mL, 3 equiv). The mixture was heated at reflux for 2.5 hand then carefully quenched with methanol (25 mL) followed by acetic acid(2.7 mL, 3 equiv) upon cooling to rt. The mixture was evaporated andflushed with methylene chloride (2x). The residue was dissolved inmethanol (100 mL) and the mixture heated at reflux overnight and thenevaporated. The resulting oil was dissolved as far as possible in chloroform(100 mL) with sonication and then the solid inorganics were removed byfiltration. The filtrate was evaporated and the residue chromatographed onsilica, eluting with 9% methanol/methylene chloride, to give the product as athick oil (2.770 g, 76%) ‘H NMR 51.21 and 1.41 (m, each 2H), 1.37 (s, 9H),1.60 (m, 1H), 2.98 (q, J=7.4 Hz, 2H), 3.68 (m, 4H), 3.96 (br, 1H), 4:97 (brt,1H). MS (DCI) 234 (MH)+, 178 (M—C4Hg)+, 134 (M-C5H9O2)+. Anal calcd forC11H23NO4: C-56.63, H—9.94, N—6.00. Found: C—56.16, H—9.83, N-5.94.Example 705—Amino-2-hydroxymethylpentanol-CF3C02HA solution of 5% water in 50% TFA/methylene chloride (4 mL) was added tothe diol 67 (0.145 g, 0.621 mmol) and the mixture was stirred for 1 h. Thesolvents were evaporated and the residue flushed with methylene chloride(2x) (0.153 g, 100%). ‘H NMR 81.22 and 1.50 (m, each 2H), 1.69 (m, 1H),2.82 (br, 2H), 3.51 (d, J=6.9 Hz, 4H), 7.78 (br, BH). M8 (DC!) 134 (MH)+.Example 715-Maleimido—2-hydroxymethylpentanolThe aminodiol 68 in dry acetonitrile under argon at 0°C is treated withmaleic anhydrlde (1 equiv) and DIEA (1 equiv). The mixture is allowed to stirat rt for 4 h. The reaction is re—cooled to 0°C and treated with trimethylsilylchloride (3 equiv) and DIEA (3 equiv). The mixture is heated at reflux for 4 hand then, after cooling to rt, is evaporated. The residue is partitioned-93-1015202530W0 98/ 19705CA 02264610 1999-03-08PCT/US97/ 19851between ethyl acetate and water. The organic phase is washed with ...10°/ocitric acid, water and brine, dried over sodium sulfate and evaporated. Theresidue is chromatographed on silica gel to give the product.Example 725-Maleirnido-2-hydroxymethylpentanol bis-chloroformateA solution of the diol 69 in dry methylene chloride is added dropwise to avigorously stirred solution of 1M phosgene in toluene (20 equiv) at 0°C.After addition is complete the mixture is stirred at rt for 16 h and then thesolvents are removed on the rotary evaporator. The residue is flushed withdry methylene chloride and dried in vacuo. The crude bis—chloroformate isused without further purification.Example 73MPr-BHP-(Phe-Lys(MMT)-PABOH)2To a stirred solution of the crude bis-chloroformate 70 in dry methylenechloride at 0°C is added a solution of Phe-Lys(MMT)-PABOH 10a (2 equiv)and DIEA (2 equiv) in methylene chloride all at once. The mixture is allowedto stir at rt for 16 h and then the solvent is evaporated. The residue ispartitioned between ethyl acetate and water. The organic phase is washedwith brine, dried over sodium sulfate and evaporated. The product is purifiedby column chromatography using silica gel.Example 74MPr-BHP-(Phe-Lys(MMT)-PABC-PNP);A stirred mixture of 71, bis-p-nitrophenylcarbonate (6 equiv) and freshly-activated 4Apowdered sieves in dry methylene chloride under argon at rt istreated with DIEA (6 equiv). After continued stirring for 16 h the mixture isfiltered and the filtrate evaporated. The residue is dried in vacuo for severalhours and then dissolved in a minimum volume of methylene chloride. Tothis is added two-times the volume of ether. The resulting solid is collectedby filtration, washed with 2:1 ether/methylene chloride, and dried in vacuo.-94-i1015202530WO 98/19705CA 02264610 1999-03-08PCT/US97/ 19851Example 75 , __MPr—BHP-(Phe-Lys(MMT)-PABC-DOX)2A stirred solution of the bis-p—nitrophenylcarbonate 72 and DOX Mhydrochloride (1.1 equiv) in DMF_is treated with DIEA (1.1 equiv). Afterstirring for 2 d the mixture is diluted with ethyl acetate. The solution iswashed with water (4x) and brine, dried over sodium sulfate and evaporated.The residue is chromatographed on silica gel to give the product.Example 76MPr-BHP-(Phe-Lys-PABC-D0X)2-2Cl2CHCOZHA stirred solution of 73 in methylene chloride is treated with anisole (100equiv) and then dichloroacetic acid (10 equiv). After 1.5 h the mixture isdiluted with ethyl acetate. The resulting suspension is stirred at rt for 1 h andthen the orange solid product is collected by filtration, washed with ethylacetate and dried in vacuo.Example 77N-Adpoc-3-hydroxyaminopropaneA stirred solution of 3-hydroxyaminopropane and sodium bicarbonate (1equiv) in water at rt is treated with 1-(1'-adamanty|)—1—methy|—ethoxycarbony|chloride (1 equiv). After 6 h the resulting solid product is collected byfiltration, washed with water, and lyophilized in vacuo. The crude product iscarried on without further purification.Example 78N-Adpoc-3-methanesulfonyloxyaminopropaneTo a stirred solution of the alcohol 75 in methylene chloride at 0°C is addedmethanesulfonyl chloride (1.05 equiv) and then triethylamine (1.05 equiv).The mixture is allowed to warm to rt and stirred overnight at rt. The solventsis evaporated and the residue partitioned between ethyl acetate and pH 5buffer (biphthalate). The organic phase is washed with water and brine,dried over sodium sulfate and evaporated to give the product which iscarried on without further purification.-95-1015202530WO 98/19705CA 02264610 1999-03-0sPCT/US97/19851—— Example 79t-Butyl N-adpoc-5-amino-2-t-butoxycarbonyl-pentanoateA stirred suspension of sodium hydride in dry tetrahydrofuran at 0°C underargon is carefully treated with di-t-butylmalonate (1 equiv). The mixture isgradually allowed to warm to rt. When the mixture is homogeneous it is re-cooled to 0°C and then treated all at once with a solution of the mesylate 76(0.8 equiv) in tetrahydrofuran. The mixture is allowed to warm to rt and thenis heated at reflux for 16 h. Upon cooling- to rt the mixture is diluted withether and the solution is washed with sat. ammonium chloride, water andbrine, dried over sodium sulfate and evaporated. The residue ischromatographed on silica gel to give the product.Example 80t-Butyl 5-amino-2-t-butoxycarbonyl-pentanoateThe Adpoc substrate 77 is dissolved in 5% trifluoroacetic acid/methylenechloride containing anisole (100 equiv). After 30 min the solvents areremoved under high vacuum at rt and the residue dried in vacuo for severalhours. The crude product is carried on immediately without furtherpurification.Example 81t-Butyl 5-maleimido-2-t-butoxycarbonyl-pentanoate 79The aminodiester 78 in dry acetonitrile under argon at 0°C is treated withmaleic anhydride (1 equiv) and the stirred mixture is allowed to stir at rt for 4h. The mixture is re-cooled to 0°C and treated with trimethylsilyl chloride (3equiv) and DIEA (3 equiv). The mixture is heated at reflux for 4 h and then,after cooling to rt, is evaporated. The residue is partitioned between ethylacetate and water. The organic phase is washed with 10% citric acid, waterand brine, dried over sodium sulfate and evaporated. The residue ischromatographed on silica to give the product.-95-. W0 98/ 197051015202530CA 02264610 1999-03-08I’CT/US97/ 19851Example 82 . 2-(3'-maleimidopropyl)-malonic acidThe di-t-butyl ester 79 is dissolved in 20% trifluoroacetic acid and themixture is stirred at rt for 5 h. The solvents are evaporated and the residueflushed with methylene chloride several times. The residue is triturated withether and the resulting solid product is collected by filtration, washed withether, and dried in vacuo.Example 83MPr-Mal-(Phe-Lys(MMT)-PABOH)2A stirred solution of the diacid 80 in dimethoxyethane at 0°C is treated withn-hydroxysuccinimide (2.2 equiv) and 0.5 M DCC in methylene chloride (2.2equiv). The mixture is allowed to warm to rt. After 4 h a solution of Phe-Lys(MMT)-PABOH 10a (2 equiv) in dimethoxyethane is added and themixture is stirred at rt for 16 h. The mixture is then filtered and the filtrateevaporated. The residue is partitioned between ethyl acetate and water.The organic phase is washed with water and brine, dried over sodiumsulfate and evaporated. The product_is purified by chromatography on silicagel.Example 84MPr-Mal-(Phe—Lys(MMT)-PABC-PNP)2A stirred mixture of 81, bis—p-nitrophenylcarbonate (6 equiv) and freshlyactivated 4A powdered sieves in dry methylene chloride under argon at rt istreated with DIEA (6 equiv). After continued stirring for 16 h the mixture isfiltered and the filtrate evaporated. The residue is dried in vacuo for severalhours and then dissolved in a minimum volume of methylene chloride. Tothis is added two—times the volume of ether. The resulting solid is collectedby filtration, washed with 2:1 ether/methylene chloride, and dried in vacuo.Example 85MPr-Mal-(Phe-Lys(MMT)-PABC-D0X)2A stirred solution of the bis—p—nitrophenylcarbonate 82 and DOXhydrochloride (1.1 equiv) in DMF is treated with DIEA (1.1 equiv). After-97-CA 02264610 1999-03-08WO 98119705 PCT/US97/19851stirring for 2 d the mixture is diluted with ethyl acetate. The solutionis«&1015202530washed with water (4x) and brine, dried over sodium sulfate and evaporated.The residue is chromatographed on silica gel to give the product.Example 86MPr-Mal-(Phe-Lys-PABC-DOX)2-2Cl2CHC02HA stirred solution of 83 in methylene chloride is treated with anisole (100equiv) and then dichloroacetic acid (10 equiv). After 1.5 h the mixture isdiluted with ethyl acetate. The resulting suspension is stirred at rt for 1 h andthen the orange solid product is collected by filtration, washed with ethylacetate and dried in vacuo.Example 87N-Carbobenzyloxy-N‘, N'-bis-(carboxymethyl)-ethylenediamineThe di-t-butyl ester 22 is dissolved in 20% trifluoroacetic acid and themixture is stirred at rt for 5 h. The solvents are evaporated and the residueflushed with methylene chloride several times. The residue is triturated withether and the resulting solid product is collected by filtration, washed withether, and dried in vacuo.Example 88N-Carbobenzyloxy-N‘, N'-bis-(2-hydroxyethyl)-ethylenediamine 86A solution of the bis-carboxylic acid 85 in diglyme at 0°C is treated with N-hydroxysuccinimide (2.2 equiv) and DCC (2.2 equiv). The mixture is allowedto warm to rt and then stirred for 16 h. The resulting solid is removed byfiltration. The filtrate is cooled to 0°C under an argon atmosphere and thentreated with 0.5 M sodium borohydride in diglyme. The mixture is allowed towarm to rt for 16 h and is then poured into ice water. The resulting mixture isextracted with ethyl acetate. The organic phase is washed with water andbrine, dried over sodium sulfate and evaporated to give the product which iscarried on without further purification.-98-1015202530WO 98/19705CA 022674610 1999-03-08PCTIU S97/ 19851Example 89Bis-(2-hydroxyethy|)ethylenediamine hydrochlorideA solution of diol 86 and conc. l-lCl (1 equiv) in methanol is degassedibybubbling nitrogen through it for 30 min. To this is added 10% palladizedcharcoal (5 wt. percent). The resulting mixture is shaken on a Parrapparatus under 50 psi of hydrogen gas for 16 h. The mixture is then filteredand the filtrate evaporated. The residue is flushed several times withchloroform to drive off traces of methanol. The crude product is carried onwithout further purification.Example 90Bis-(2-hydroxyethyl)-2'-maleimidoethylamineThe aminodiol 87 in dry acetonitrile under argon at 0°C is treated withmaleic anhydride (1 equiv) and DIEA (1 equiv). The mixture is allowed to stirat rt for 4 h. The mixture is re—cooled to 0°C and treated with trimethylsilylchloride (3 equiv) and DIEA (3 equiv). The reaction is heated at reflux for 4 hand then, after cooling to rt, is evaporated. The residue is partitionedbetween ethyl acetate and water. The organic phase is washed with 10%citric acid, water and brine, dried over sodium sulfate and evaporated. Theresidue is chromatographed on silica gel to give the product.Example 91Bis-(2-chlorocarbonyloxyethyl)-2'-maleimidoethylamineA solution of the diol 88 in dry methylene chloride is added dropwise to avigorously stirred solution of 1M phosgene in toluene (20 equiv) at 0°C.After addition is complete the mixture is stirred at rt for 16 h and then thesolvents are removed on the rotary evaporator. The residue is flushed withdry methylene chloride and dried in vacuo. The crude bis-chloroformate isused without further purification.-99-1015202530W0 98/19705CA 02264610 1999-03-08PCT/US97/19851Example 92 , .. ‘_MEt-lBHE-(Phe-Lys(MMT)-PABOH)2To a stirred solution of the crude bis—ch|oroformate 89 in dry methylenechloride at 0°C is added a solution of Phe-l_ys(MMT)-PABOH 10a (2 equiv)and DIEA (2 equiv) in methylene chloride all at once. The mixture is allowedto stir at rt for 16 h and then the solvent is evaporated. The residue ispartitioned between ethyl acetate and water. The organic phase is washedwith brine, dried over sodium sulfate and evaporated. The product is purifiedby column chromatography using silica gel.Example 93MEt-lBHE-(Phe-Lys(MMT)-PABC-PNP)2A stirred mixture of 90, bis—p—nitrophenylcarbonate (6 equiv) and freshlyactivated 4A powdered sieves in dry methylene chloride under argon at rt istreated with DIEA (6 equiv). After continued stirring for 16 h the mixture isfiltered and the filtrate evaporated. The residue is dried in vacuo for severalhours and then dissolved in a minimum volume of methylene chloride. Tothis is added two-times the volume of ether. The resulting solid is collectedby filtration, washed with 2:1 ether/methylene chloride, and dried in vacuo.Example 94MEt-IBHE-(Phe-Lys(MMT)-PABC-DOX);A stirred solution of the bis-p—nitrophenylcarbonate 91 and DOXhydrochloride (1.1 equiv) in DMF is treated with DIEA (1.1 equiv). Afterstirring for 2 d the mixture is diluted with ethyl acetate. The solution iswashed with water (4x) and brine, dried over sodium sulfate and evaporated.The residue is chromatographed on silica gel to give the product.Example 95MEt-lBHE-(Phe-Lys-PABC-D0X)2-2C|2CHCO2HA stirred solution of 92 in methylene chloride is treated with anisole (100equiv) and then dichloroacetic acid (10 equiv). After 1.5 h the mixture isdiluted with ethyl acetate. The resulting suspension is stirred at rt for 1 h and-100-1015202530wo 98/19705 ’ ‘CA 02264610 1999-03-08PCT/US97/19851then the orange solid product is collected by filtration, washed with.ethylcaacetate and dried in vacuo.Example 961_1_4 (257 mg, 0.25 mmole) and Doxorubicin HCI (377 mg, 0.65 mmole) arestirred in 25 ml dry methanol for 24 hour. The reaction is concentrated byrotary evaporation to 4 ml, then purified in two equal portionsflon SephadexLH-20 (1" x 18") with methanol. Fractions containing pure product arepooled, rotary evaporated, and dried under high vacuum to yield 11:5 (222mg, 50%).Example 97Conjugation of BR96 or IgG and Branched Peptide LinkerThiolationzBR96 and lgG were thiolated (reduced) by a variation of previously reportedmethod1. In a typical example, 1.27 g BR96 (75 ml at 105.7 uM, 7.93 umole)was de—oxygenated by several cycles of alternating vacuum and Aratmosphere. This was then treated with 7.4 mM DTT (8.6 ml, 63.6 umole inAr-bubbled PBS, pH 7.0) and stirred at 37°C under Ar for 3 hr. Removal oflow molecular weight compounds was accomplished by ultrafiltration againstPBS, pH 6.0 in an Amicon stirred cell at 4°C. A 400 ml Amicon cell was fittedwith an Amicon YM30 filter (molecular weight cut-off 30,000), and charged to40 psi with Ar. Cell eluant was monitored for thiol content with Ellman'sreagent-2 until a baseline reading at 412 nm was obtained. Concentration ofprotein and thiol groups were determined according to the previouslyreported method‘. In this example, 1.10 g reduced BR96 (85 ml at 80.8 uMMAb, 619.1 uM thiol) was obtained, for a yield of 87% and a thiol titer of 7.7mole thiol groups/mole BR96.Example 98Conjugation:The following procedure,1 for the conjugation of BR96 and M—Et-lDA-(Phe-Lys-PABC-DOX=B-Ala-mTEG)2, is typical of that used for all branched-101-CA 022646l0 1999-03-08W0 98l19705 PCT/U S97/ 19851peptide linkers modified with the B-Ala—mTEG hydrazone or [5-Ala , ., __hydrazone. To reduced BR96 (83 ml, 6.70 umole MAb, 51.6 umole thiol) wasadded dropwise at 0°C under Ar a solution of M—Et-|DA—(Phe-Lys-PABC—DOX=B—Ala-mTEG)2 (239 mg, 77.3 umole) in 4.0 ml DMSO. After stirring for5 30 min., the reaction was filtered through a O.22u sterile filter. Conjugate waspurified at 4°C by percolation (approximately 2 ml/min.) through a 1" x 24"Bio-Beads column (initially prepared by swelling and packing in methanol,then equilibrated in H20, and finally PBS, pH 7.4). The purified conjugatewas filtered again through a O.22u sterile filter to yield 95 ml of BFl96—S—Et—10 lDA—(Phe-Lys-PABC—DOX=B—Ala—mTEG)2 (BR96, 76.1 uM; DOX, 1.11 mM;MR, 14.6 mole DOX/mole BR96; yield, 100%). Conjugate was frozen in liquidN2 and stored at —80°C.For more guidance, see Willner, D., Trail, P. A., Hofstead, S. J., King,H. D., Lasch, Braslawsky, G. R., Greenfield, R. S., Kaneko, T., Firestone, R. A.15 (1993) (6—Maleimidocaproyl)hydrazone of Doxorubicin: A new derivative forthe preparation of immunoconjugates of Doxorubicin. Bioconjugate Chem.,4, 521 and Fliddles, P. w., Blakeley, R. L., Zerner, B., (1979) .E|lman'sreagent: 5,5'—Dithiobis(2-nitrobenzoic acid)-A reexamination. Anal.Blochem., 94, 75.20-102-

Claims (57)

What is Claimed is:

1. A branched peptide linker for linking a thiol group derived from a targeting ligand to two or more drug moieties which comprises a compound having a terminus containing a thiol acceptor for binding to a thiol group derived from a targeting ligand, at least one point of branching which is a polyvalent atom allowing for a level of branching of 2n, wherein n is a positive integer, at least two amino acid moieties providing an enzymatic cleavage site, and at least two other termini containing groups capable of forming covalent bonds with chemically reactive functional groups derived from a drug moiety.

2. The branched peptide linker of Claim 1 wherein n is 1, 2, 3, or 4.

3. The branched peptide linker of Claim 1 wherein n is 1, 2, or 3.

4. The branched peptide linker of Claim 1 wherein n ia 1 or 2.

5. The branched peptide linker of Claim 1 linked to an antibody or fragment thereof.

6. The branched peptide linker of Claim 1 wherein said polyvalent atom is nitrogen or carbon.

7. The branched peptiide linker of Claim 1 wherein said polyvalent is nitrogen.

8. The branched peptide linker of Claim 1 wherein said polyvalent is carbon.

9. The branched peptide linker of Claim 1 linked to drug moieties which are anthracyclines.

10. The branched peptide linker of Claim 1 wherein n is 1 or 2, said tatgeting ligand is an antibody or fragment thereof, said polyvalent atom is nitrogen and said drug moieties are anthracyclines.

11. A compound of the formula wherein A is a thiol acceptor;
W is a bridging moiety;
c is an integer of 0 to 1;
a is an integer of 2 to 12;
Q is O, NH, or N-lower alkyl;
p is an integer of 0 or 1;
d is an integer of 0 or 1;
E is a polyvalent atom;
each b is an integer of 1 to 10;
each X is of the formula -CO-Y-Zm-Gn wherein Y is two amino acid residues in the L form;
Z is one or two amino acid residues;
m is an integer of 0 or 1;
G is a self-immolative spacer; and n is a integer of 0 or 1; provided that when G is 0 then -Y-Z m is ala-leu-ala-leu or gly-phe-leu-gly;
or each X is of the formula wherein each X1 is of the formula -CO-Y-Z m-G n; -and wherein Y, Z, Q, E, G, m, d, p, a, b and n are as defined above;
or each X1 is of the formula wherein each X2 is of the formula -CO-Y-Z m-G n;
and wherein Y, Z, G, Q, E, m, d, p, a, b and n are as defined above;
or each X2 is of the formula wherein each X3 is of the formula -CO-Y-Z m-G n;
and wherein Y, Z, G, Q, E, m, d, p, a, b and n are as defined above;
or each X3 is of the formula wherein each X4 is of the formula -CO-Y-Z m-G n;
and wherein Y, Z, G. Q, E, m, d, p, a, b and n are as defined above.

12. The compound of Claim 11 wherein each X is of the formula -CO-Y-Z m-G n wherein Y, m, Z, and n are as defined above.

13. The compound of Claim 11 wherein each X1 is of the formula -CO-Y-Z m-G n wherein Y, m, Z, and n are as defined above.

14. The compound of Claim 11 wherein each X2 is of the formula -CO-Y-Z m-G n wherein Y, m, Z, and n are as defined above.

15. The compound of Claim 11 wherein each X3 is of the formula -CO-Y-Z m-G n wherein Y, m, Z, and n are as defined above.

16. The compound of Claim 11 wherein -Y-Z m is a dipeptide, tripeptide or tetrapeptide containing amino acids selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, lysine, lysine protected with acetyl or formyl, arginine, arginine protected with tosyl or nitro groups, histidine, ornithine, ornithine protected with acetyl or formyl, and citrulline.

17. The compound of Claim 11 wherein the -Y-Z m group is phenylalanine-lysine.

18. The compound of Claim 11 wherein the -Y-Z m group is valine-citrulline.

19. The compound of Claim 11 wherein the -Y-Z m group is valine-lysine.

20. The compound of Claim 11 wherein E is nitrogen.

21. The compound of Claim 11 wherein E is carbon.

22. A compound of the formula wherein A is a thiol acceptor;
W is a bridging moiety;
c is an integer of 0 to 1;
a is an integer of 2 to 12;
Q is O, NH, or N-lower alkyl;
p is an integer of 0 or 1;
d is an integer of 0 or 1;
E is a polyvalent atom;
each b is an integer of 1 to 10;
each X is of the formula -CO-Y-Z m-G nD
wherein Y is two amino acid residues in the L form;
Z is one or two amino acid residues;
m is an integer of 0 or 1;
G is a self-immolative spacer;
n is a integer of 0 or 1; provided that when G is 0 then -Y-Z m-is ala-leu-ala-leu or gly-phe-leu-gly; and D is a Drug moiety having a backbone and at least one chemically reactive functional group pendant thereto chemically reacted to the self-immolative spacer or terminal amino acid residue to form a covalent bond, said functional group selected from the group consisting of a primary or secondary amine, hydroxyl, sulfhydryl, carboxyl, aldehyde or ketone;
or each X is of the formula wherein each X1 is of the formula -CO-Y-Z m-G n-D;
wherein Y, Z, G, D, Q, E, m, d, p and n are as defined above;
or each X1 is of the formula wherein each X2 is of the formula -CO-Y-Z m-G n-D;
and wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as defined above;
or each X2 is of the formula wherein each X3 is of the formula -CO-Y-Z m-G n-D;
and wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as defined above;
or each X3 is of the formula and wherein each X4 is of the formula -CO-Y-Z m-G n-D;
and wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as defined above.

23. The compound of Claim 22 wherein each X is of the formula -CO-Y-Z m-G n-D
wherein Y, m, Z, n, and D are as defined above.

24. The compound of Claim 22 wherein each X1 is of the formula -CO-Y-Z m-G n-D
wherein Y, m, Z, n, and D are as defined above.

25. The compound of Claim 22 wherein each X2 is of the formula CO-Y-Z m-G n-D
wherein Y, m, Z, n, and D are as defined above.

26. The compound of Claim 22 wherein each X3 is of the formula -CO-Y-Z m-G n-D
wherein Y, m, Z, n, and D are as defined above.

27. The compound of Claim 22 wherein -Y-Z m is a dipeptide, tripeptide or tetrapeptide containing amino acids selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, lysine, lysine protected with acetyl or formyl, arginine, arginine protected with tosyl or nitro groups, histidine, ornithine, ornithine protected with acetyl or formyl, and citrulline.

28. The compound of Claim 17 wherein the -Y-Z m group is phenylalanine-lysine.

29. The compound of Claim 17 wherein the -Y m-Z m group is valine-citrulline.

30. The compound of Claim 17 wherein the -Y-Z m group is valine-lysine.

31. The compound of Claim 22 wherein E is nitrogen.

32. The compound of Claim 22 wherein E is carbon.

33. The compound of Claim 22 wherein D is an amino containing drug moiety selected from the group consisting of mitomycin-C, mitomycin-A, daunorubicin, doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, aminopterin, actinomycin, bleomycin, 9-amino camptothecin, N8-acetyl spermidine, 1-(2 --chloroethyl)-1,2-dimethanesulfonyl hydrazide, tallysomycin, and derivatives thereof.

34. The compound of Claim 22 wherein D is doxorubicin.

35. The compound of Claim 22 wherein D is mitomycin-C.

36. The compound of Claim 22 wherein D is mitomycin-A.

37. The compound of Claim 22 wherein D is tallysomycin.

38. The compound of Claim 22 wherein D is N-(5,5-diacetoxypentyl)doxorubicin.

39. The compound of Claim 22 wherein D is a hydroxyl containing drug moiety selected from the group consisting of etoposide, camptothecin, taxol, esperamicin, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4-ene-2,6-diyne-13-one, anguidine, doxorubicin, morpholine-doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, vincristine, vinblastine and derivatives thereof.

40. The compound of Claim 22 wherein D is a sulfhydryl containing drug moiety selected from the group consisting of esperamicin and 6-mercaptopurine, and derivatives thereof.

41. The compound of Claim 22 wherein D is a carboxyl containing drug moiety selected from the group consisting of methotrexate, campothecin (ring-opened form of the lacton), butyric acid, retinoic acid, and derivatives thereof.

42. The compound of Claim 22 wherein D is an anthracycline.

43. The compound of Claim 22 wherein G is

44. The compound of Claim 22 wherein G is p-aminobenzyl-carbonyl.

45. The compound of Claim 22 wherein G has the formula wherein R1 is C1-C5 alkyl, T is O, N or S.

46. The compound of Claim 22 wherein G has the formula wherein R2 is H or C1-C5 alkyl,

47. The compound of Claim 22 wherein G is

48. The compound of Claim 22 wherein A is

49. A compound of the formula wherein L is a ligand;
q is an integer of 1 to 10;
A is a thiol acceptor;
W is a bridging moiety;
c is an integer from 0 to 1;
a is an integer of 2 to 12;
Q is O, NH, or N-lower alkyl;
p is an integer of 0 or 1;
d is an integer of 1 or 2;
E is a polyvalent atom;
each b is an integer of 1 to 10;
each X is of the formula -CO-Y-Z m-G n-D
wherein Y is two amino acid residues in the L form;
Z is one or two amino acid residues;
m is an integer of 0 or 1;
G is a self-immolative spacer; and n is a integer of 0 or 1; provided that when G is 0 then -Y-Z m- is ala-leu-ala-leu or gly-phe-leu-gly;
D is a drug moiety having a backbone and at least one chemically reactive functional group pendant thereto reacted to the self-immolative spacer to form a covalent bond, said funtional group selected from the group consisting of a primary or secondary amine, hydroxyl, carboxyl, sulfhydryl, aldehyde, or ketone;
or X is of the formula wherein each X1 is of the formula -CO-Y-Z m-G n-D;
and wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as defined above;
or each X1 is of the formula wherein each X2 is of the formula -CO-Y-Z m-G n-D;
and wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as defined above;
or each X2 is of the formula wherein each X3 is of the formula -CO-Y-Z m-G n-D;
wherein Y, Z, G, D, Q, E, m, d. p, a, b and n are as defined above;
or each X3 is of the formula wherein end X4 is of the formula -CO-Y m-Z m-G n-D;
wherein Y, Z, G, D, Q, E, m, d, p, a, b and n are as defined above.

50. The compound of Claim 49 wherein L is an immunoglobulin, or a fragment thereof.

51. The compound of claim 49 wherein L is an immunoglobulin selected from the group consisting of BR96, BR64, L6, a relaxed BR96, a relaxed BR64, a chimeric L6, a relaxed chimeric BR96, a relaxed chimeric BR64, a relaxed chimeric L6; and, fragments thereof.

52. The compound of claim 49 wherein L is a ligand selected from the group consisting of bombesin, EDG, transferrin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, I1-6, TFG-.alpha., TFG-.beta., VGF, insulin, insulin-like growth factors I and II, carbohydrates, lectins, and apoproteins from low density lipoproteins.

53. A pharmaceutical composition comprising a compound of Claim 49 and a pharmaceutically acceptable carrier.

54. A method of controlling the growth of undesirable cells which comprises administering a compound of Claim 49 to a host in need of such treatment.

55. The method of claim 54 wherein the undesirable cells are cancer cells.

56. A method of treatment of a warm blooded animal in need thereof which comprises administering to said animal an effective tumor inhibiting amount of a compound of Claim 49.

57. A method of targeting a pharmaceutical agent to a desired site in a host or sample comprising administering to the host or contacting the sample with a compound of Claim 49.