ZA200609483B

ZA200609483B - Chemical linkers and conjugates thereof

Info

Publication number: ZA200609483B
Application number: ZA200609483A
Authority: ZA
Inventors: Boyd Sharon; Chen Liang; Gangwar Sanjeev; Guerlavais Vincent; Horgan Killian; Li Zhi-Hong; Sufi Bilal
Original assignee: Medarex Inc
Priority date: 2004-05-19
Filing date: 2006-11-15
Publication date: 2008-01-30
Also published as: CN1997402A; CN1997402B; UA85716C2

Description

CHEMICAL LINKERS AND CONJUGATES THEREOF

This application claims the benefit of U.S. Provisional Patent Applications Serial Nos. 60/572,667, 60/661,174, and 60/669,871, which are herein incorporated by reference.

FIELD OF THE INVENTION

The present invention provides linkers that attach to a drug and a ligand and are . cleaved in vivo. The linkers are of use in forming prodrugs and conjugates of the cytotoxins of ® 10 the invention as well as other diagnostic and therapeutic moieties.

BACKGROUND OF THE INVENTION

Many therapeutic agents, particularly those that are especially effective in cancer chemotherapy, often exhibit acute toxicity in vivo, especially bone marrow and mucosal toxicity, as well as chronic cardiac and neurological toxicity. Such high toxicity can limit their applications. Development of more and safer specific therapeutic agents, particularly antitumor agents, is desirable for greater effectiveness against tumor cells and a decrease in the number and severity of the side effects of these products (toxicity, destruction of non-tumor cells, etc.).

Another difficulty with some existing therapeutic agents is their less than optimal stability in plasma. Addition of functional groups to stabilize these compounds resulted in a significant lowering of the activity. Accordingly, it is desirable to identify ways to stabilize compounds @ while maintaining acceptable therapeutic activity levels. :

The search for more selective cytotoxic agents has been extremely active for many decades, the dose limiting toxicity (i.e. the undesirable activity of the cytotoxins on normal tissues) being one of the major causes of failures in cancer therapy. For example, CC-1065 and the duocarmycins are known to be. extremely potent cytotoxins.

CC-1065 was first isolated from Streptomyces zelensis in 1981 by the Upjohn

Company (Hanka et al., J. Antibiot. 31: 1211 (1978); Martin ef al, J. Antibiot. 33: 902 (1980);

Martin et al., J. Antibiot. 34: 1119 (1981)) and was found to have potent antitumor and antimicrobial activity both in vitro and in experimental animals (Li et al., Cancer Res. 42: 999 1

J

(1982)). CC-1065 binds to double-stranded B-DNA within the minor groove (Swenson ef al.,

Cancer Res. 42: 2821 (1982)) with the sequence preference of 5'-d(A/GNTTA)-3' and 5'- d(AAAAA)-3' and alkylates the N3 position of the 3'-adenine by its CPI left-hand unit present in the molecule (Hurley et al., Science 226: 843 (1984)). Despite its potent and broad antitumor activity, CC-1065 cannot be used in humans because it causes delayed death in experimental animals.

Many analogues and derivatives of CC-1065 and the duocarmycins are known in the art. The research into the structure, synthesis and properties of many of the compounds has been reviewed. See, for example, Boger et al., Angew. Chem. Int. Ed. Engl. 35: 1438 (1996); ® 10 and Boger et al., Chem. Rev. 97: 787 (1997).

A group at Kyowa Hakko Kogya Co., Ltd. has prepared a number of CC-1065 derivatives. See, for example, U.S. Pat. No. 5,101, 038; 5,641,780; 5,187,186; 5,070,092; 5,703,080; 5,070,092; 5,641,780; 5,101,038; and 5,084,468; and published PCT application, WO 96/10405 and published European application 0 537 575 Al.

The Upjohn Company (Pharmacia Upjohn) has also been active in preparing derivatives of CC-1065. See, for example, U.S. Patent No. 5,739,350; 4,978,757, 5,332, 837 and 4,912,227.

Research has also focused on the development of new therapeutic agents which are in the form of prodrugs, compounds that are capable of being converted to drugs (active therapeutic compounds) in vivo by certain chemical or enzymatic modifications of their structure. For purposes of reducing toxicity, this conversion is preferably confined to the site of ® action or target tissue rather than the circulatory system or non-target tissue. However, even prodrugs are problematic as many are characterized by a low stability in blood and serum, due to the presence of enzymes that degrade or activate the prodrugs before the prodrugs reach the desired sites within the patient’s body.

Bristol-Myers Squibb has described particular lysosomal enzyme-cleavable antitumor drug conjugates. See, for example, U.S. Patent No. 6,214,345. This patent provides an aminobenzyl oxycarbonyl.

Seattle Genetics has published applications U.S. Pat. Appl. 2003/0096743 and

U.S. Pat. Appl. 2003/0130189, which describe p-aminobenzylethers in drug delivery agents. The linkers described in these applications are limited to aminobenzyl ether compositions.

Other groups have also described linkers. See for example de Groot et al, J.

Med. Chem. 42, 5277 (1999); de Groot et al. J. Org. Chem. 43, 3093 (2000); de Groot et al, J

Med. Chem. 66, 8815, (2001); WO 02/083180; Carl et al., J. Med. Chem. Lett. 24, 479, (1981);

Dubowchik et al., Bioorg & Med. Chem. Lett. 8,3347 (1998). These linkers include aminobenzyl ether spacer, elongated electronic cascade and cyclization spacer systems, cyclisation eliminations spacers, such as w-amino aminocarbonyls, and a p aminobenzy oxycarbonyl linker.

Stability of cytotoxin drugs, including in vivo stability, is still an important issue that needs to be addressed. In addition, the toxicity of many compounds makes them less useful, ® 10 so compositions that will reduce drug toxicity, such as the formation of a cleaveable prodrug, are needed. Therefore, in spite of the advances in the art, there continues to be a need for the development of improved therapeutic agents for the treatment of mammals, and humans in particular, more specifically cytotoxins that exhibit high specificity of action, reduced toxicity, and improved stability in blood relative to known compounds of similar structure. The instant invention addresses those needs.

SUMMARY OF THE INVENTION

The present invention relates to drug-ligand conjugates where the drug and ligand are linked through a peptidyl, hydrazine, or disulfide linker. These conjugates are potent cytotoxins that can be selectively delivered to a site of action of interest in an active form and then cleaved to release the active drug. The new linker arms of the invention can be cleaved from the cytotoxic drugs by, for example, enzymatic or reductive means in vivo, releasing an active drug moiety from the prodrug derivative. Furthermore, the invention includes conjugates between the linker arms and the cytotoxins of the invention, and conjugates between the linker arms, the cytotoxin and a targeting agent, such as an antibody or a peptide.

The invention also relates to groups useful for stabilizing therapeutic agents and markers. The stabilizing groups are selected, for example, to limit clearance and metabolism of the therapeutic agent or marker by enzymes that may be present in blood or non-target tissue.

The stabilizing groups can serve to block degradation of the agent or marker and may also act in providing other physical characteristics of the agent or marker, for example to increase the solubility of the compound or to decrease the aggregation properties of the compound. The stabilizing group may also improve the agent or marker's stability during storage in either a formulated or non-formulated form.

In a first aspect, the invention provides a cytotoxic drug-ligand compound having a structure according to any of Formulas 1-3:

XH —6Uigto ey

XH n—buho 2 ® xo 3 wherein the symbol D is a drug moiety having pendant to the backbone thereof a chemically reactive functional group, said functional group selected from the group consisting of a primary or secondary amine, hydroxyl, sulfhydryl, carboxyl, aldehyde, and a ketone.

The symbol L! represents a self-immolative spacer where m is an integer of 0, 1, 2,3,4,5,0r6.

The symbol X* represents a member selected from the group consisting of protected reactive functional groups, unprotected reactive functional groups, detectable labels, and targeting agents.

The symbol L* represents a linker member, and p is 0 or 1. L* is a moiety that ® imparts increased solubility or decreased aggregation properties to the conjugates. Examples of

L* moieties include substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroalkyl, or unsubstituted heteroalkyl, any of which may be straight, branched, or cyclic, a positively or negatively charged amino acid polymer, such as polylysine or polyargenine, or other polymers such as polyethylene glycol.

The symbols F, H, and J represent linkers, as described further herein.

In one embodiment, the invention pertains to peptide linker conjugate of the structure:

Xi (F =D wherein

D is a drug moiety having pendant to the backbone thereof a chemically reactive functional group, said functional group selected from the group consisting of a primary or secondary amine, hydroxyl, thiol, carboxyl, aldehyde, and a ketone;

L! is a self-immolative linker; m is an integer 0, 1, 2, 3, 4, 5, or 6; ® F is a linker comprising the structure: 10]

HoH e 7 or uf)

Cc H [o] wherein

AA! is one or more members independently selected from the group consisting of natural amino acids and unnatural a-amino acids; c is an integer from 1 to 20;

C L? is a self-immolative linker;

L? is a spacer group comprising a primary or secondary aminé or a carboxyl functional group; wherein if L? is present, m is 0 and either the amine of L3 forms an amide bond with a pendant carboxyl functional group of D or the carboxyl of 1.3 forms an amide bond with a pendant amine functional group of D; oisOorl;

L* is a linker member, wherein L* does not comprise a carboxylic acyl group directly attached to the N-terminus of (AAD; pisOorl; and

X* is a member selected from the group consisting of protected reactive: functional groups, unprotected reactive functional groups, detectable labels, and targeting agents.

In one embodiment, the peptide linker conjugate comprises the following structure: i : xi—ufa Hoc)

P c 0 m RN

In another embodiment, the peptide linker conjugate comprises the following structure: 4 4 1 3 ® X — Jaa =n _ } o

P Cc H [4] .

In a preferred embodiment, L? comprises an aromatic group. For example, L? can comprise a benzoic acid group, an aniline group, or an indole group. Non-limiting examples of -L3_NH- include structures selected from the following group:

HN HN

. i; ® H ® Pt } La 0

Hy it \_# 3 § 5 $-NH o] wherein Z is a member selected from O, S and NR?, and wherein R? is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl.

In preferred embodiments of the peptide linker, (AA'). is a peptide sequence cleavable by a protease expressed in tumor tissue. A preferred protease is a lysosomal protease. In preferred embodiments, ¢ is an integer from 2 to 6, or ¢ is 2, 3 or 4. In certain embodiments, the amino acid in (AA). located closest to the drug moiety is selected from the group consisting of: Ala,

Asn, Asp, Cit, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. In preferred embodiments, (AA). is a peptide sequence selected from the group consisting of Val-

Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N°- tosyl-Arg, Phe-N’-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, lle-Ala- ® Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 1), B-Ala-Leu-Ala-Leu (SEQ ID NO: 2) and Gly-Phe-Leu-Gly (SEQ ID NO: 3). In particularly preferred embodiments, (AAD, is Val-Cit or

Val-Lys. .

In some preferred embodiments, the peptide linker, F, comprises the structure:

NS

Hf © R24 : wherein o R* is selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, and unsubstituted heteroalkyl;

Each K is a member independently selected from the group consisting of substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO,, NR¥R%, NR¥CORZ,

OCONR*R*, OCOR?, and OR?! wherein

R* and R? are independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl,

substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl; and ais an integer of 0,1, 2, 3, or 4.

In other preferred embodiments, F-(LNa- comprises the structure:

R24 R24 R% fi Lol % ® { NH oe R24 Ny : and x R 0) ¢ Ley wherein each R* is a member independently selected from the group consisting of

H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, and unsubstituted heteroalkyl.

In another aspect, the invention pertains to hydrazine linker conjugates of the structure:

X4 ——(LH—H—(L")yy—D wherein

D is a drug moiety having pendant to the backbone thereof a chemically ® reactive functional group, said function group selected from the group consisting of a primary or secondary amine, hydroxyl, thiol, carboxyl, aldehyde, and a ketone;

L! is a self-immolative linker; m is an integer selected from 0, 1, 2, 3, 4, 5, or 6;

X* is a member selected from the group consisting of protected reactive functional groups, unprotected reactive functional groups, detectable labels, and targeting agents;

L! is a linker member; pisOorl;

H is a linker comprising the structure:

C(R*%)q

R¥ R24 p24 rR?

PN hk ~ ~~ n; N 0, hl wherein n, is an integer from 1 — 10; ® 5 nis 0, 1, or 2; each R?* is a member independently selected from the group consisting of

H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, and unsubstituted heteroalkyl; and 1 is either a bond or: :

R24 R%

Ax 24 N24 ” 5 R#* R wherein nz is 0 or 1 with the proviso that when n; is 0, n; is not 0; and ng is 1, 2, or 3, ® : wherein when 1 is a bond, n; is 3 and n is 1, D can not be

HaC. CO,CHs — ~C § OR

HN 2

HO” lo) 0 where R is Me or CHz- CH;-NMe;.

In some preferred embodiments, the substitution on the phenyl ring is a para substitution. In some preferred embodiments, n; is 2, 3, or4 orny is 3 ornz is 1.

In certain embodiments, 1 is a bond. In other embodiments, nj is 0 and ny is 2,

In various aspects, the invention provides hydrazine linkers, H, that can form a 6- membered self immolative linker upon cleavage, or two 5-membered self immolative linkers upon cleavage, or a single 5-membered self immolative linker upon cleavage, or a single 7- membered self immolative linker upon cleavage, or a 5S-membered self immolative linker and a 6-membered self immolative linker upon cleavage.

In a preferred embodiment, H comprises a geminal dimethyl substitution.

In a preferred embodiment, H comprises the structure:

C(R™) ® $ . R% RZ R2¢ R24 R¥ R%# O 2 EN x A A ny N rR? OR J & RMR® Ra

Preferably, n, is 2, 3, or 4, more preferably n, is 3. Preferably, each Ry is independently selected from CH; and H. In certain preferred embodiments, each Raq is H.

In another preferred embodiment, H comprises the structure: : CRMs 24 524 0

Io x R* R

N

24 24 24 ® R® R 6 R

Preferably, n; is 3. Preferably, each Ry is independently selected from CHz and H.

In yet other preferred embodiments, H comprises the structure:

Me le) | Me

Ney n N 24 © R

Preferably, each R** independently an H or a substituted or unsubstituted alkyl.

Xt —(L*)y=H—L")y—D wherein

D is a drug moiety having pendant to the backbone thereof a chemically reactive functional group, said function group selected from the group consisting of a primary or ® secondary amine, hydroxyl, thiol, carboxyl, aldehyde, and a ketone;

X*is a member selected from the group consisting of protected reactive functional groups, unprotected reactive functional groups, detectable labels, and targeting agents;

L*is a linker member; pisOorl; and

H comprises the structure:

R24 0 i I,

O .

N N

Ny ® ©

R24 where q is 0, 1,2, 3, 4, 5, or 6; and each R* is a member independently selected from the group consisting of

In yet another aspect, the invention pertains to disulfide linker conjugates of the structure:

X= ho wherein

D is a drug moiety having pendant to the backbone thereof a chemically reactive functional group, said function group selected from the group consisting of a primary or secondary amine, hydroxyl, thiol, carboxyl, aldehyde, and a ketone;

L* is a linker member;

PisOorl; ® 10 J is a linker comprising the structure:

R24 R* EN

Lea

Ka wherein each R?* is a member independently selected from the group consisting of

H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, and unsubstituted heteroalkyl; each K is a member independently selected from the group consisting of

H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted ® heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO, NRZ'RZ, NR*COR?%,

OCONR*R?*, OCOR?, and ORY wherein .

R? and R? are independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl and unsubstituted heterocycloalkyl; ais an integer of 0,1, 2, 3, or 4; and d is an integer of 0, 1, 2, 3,4, 5, or 6.

In various embodiments, J can comprise one of the following structures:

0)

NY

R24 R24 . “

WY AS aS

AN

Ka 0)

R24 R24 A : S ® “Phe 0) wherein d is 1 or 2; } 24

R O

R24 R24 7 0)

Mes re

Ee 4S AN

Ka or

R24 ® R?* R?¢ 24 oP

JN

S 0] 7 “Ps

In all of the foregoing linker conjugates, D preferably is a cytotoxic drug. In preferred embodiments, D has a chemically reactive function group selected from the group consisting of a primary or secondary amine, hydroxyl, sulfhydryl and carboxyl. Non-limiting examples of preferred drugs, D, include duocarmycins and duocarmycin analogs and derivatives, CC-1065,

CBl-based duocarmycin analogues, MCBI-based duocarmycin analogues, CCBI-based duocarmycin analogues, doxorubicin, doxorubicin conjugates, morpholino-doxorubicin, cyanomorpholino-doxorubicin, dolastatins, dolestatin-10, combretastatin, calicheamicin, maytansine, maytansine analogues, DM-1, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), 5-benzoylvaleric acid-AE ester (AEVB), tubulysins, disorazole, epothilones, Paclitaxel, docetaxel, SN-38, Topotecan, rhizoxin, echinomycin, colchicine, vinblastin, vindesine, estramustine, cemadotin, eleutherobin, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, daunorubicin conjugates, ® 10 mitomycin C, mitomycin A, carminomycin, aminopterin, tallysomycin, podophyllotoxin, podophyllotoxin derivatives, etoposide, etoposide phosphate, vincristine, taxol, taxotere retinoic acid, butyric acid, N® -acetyl spermidine and camptothecin.

In a preferred embodiment, D is a duocarmycin analog or derivative that comprises a structure: i” rR

RrR*

N

R® ® wherein the ring system A is a member selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl groups;

E and G are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, a heteroatom, a single bond, or E and G are joined to form aring system selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl;

X is a member selected from O, S and NRZ ;

R® is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl;

R’ is a member selected from the group consisting of (=O), SR, NHR and OR", wherein

R'is a member selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, diphosphates, triphosphates, acyl, C(O)R'?R'?, C(O)OR'2, C(O)NR'?R'?, P(O)(OR'2),, , C(O)CHR'’R", SR"? and SiR'?R"RY, in which

RZ, R! and R™ are members independently selected from H, substituted ® 10 or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted aryl, wherein RZ and R'? together with the nitrogen or carbon atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 members, optionally containing two or more heteroatoms;

R*,R*, R® and R* are members independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO,, NR¥R!®, NC(O)R', OC(O)NR'*R*¢, OC(O)OR"’, C(O)R”,

SRY, OR'’, CR"=NR'¢, and O(CH,),N(CHs) wherein n is an integer from 1 to 20;

RY and R® are independently selected from H, substituted or ® unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted peptidyl, wherein RS and RS together with the nitrogen atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 members, optionally containing two or more heteroatoms;

RS is a single bond which is either present or absent and when present R® and R’ are joined to form a cyclopropyl ring; and

R’ is CHp-X! or —CH,- joined in said cyclopropyl ring with RS, wherein

X! is a leaving group,

wherein at least one of R'!, RZ, RY, RY or R! links said drug to Lif present, or to F, H, or J.

In a preferred embodiment, D has the structure:

Rr? . ~~

HN 8 .

R® R% 4 . Pe

X Z RS

R® wherein

Z is a member selected from O, S and NR? wherein

R? is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl;

R!is H, substituted or unsubstituted lower alkyl, C(O)R}, or CO4RS, wherein R® is a member selected from group consisting of substituted alkyl, unsubstituted alkyl,

NR’R'®, NR°NHR'’, and OR’ : in which ® 15 R’® and R'? are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; and

R? is H, substituted alkyl or unsubstituted lower alkyl; wherein at least one of Rr! RY, RE, RY or R*® links said drug to LY if present, or to F, H, or J.

In a preferred embodiment of the above, R? is an unsubstituted lower alkyl.

In another preferred embodiment, D has the structure:

Ry fe

Ry

R® ©, R*

X z RE" : R® wherein

Z is a member selected from O, S and NR? ® wherein

R! is H, substituted or unsubstituted lower alkyl, C(O)R®, or COR’, wherein R® is a member selected from NR°R*® and OR?, in which

R’ and R!? are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

Ris H, substituted or unsubstituted lower alkyl, or C(O)R®, wherein R® is a member selected from NR’R!? and OR’, in which -

R’ and R'? are members independently selected from H, substituted or ® unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

R?is H, or substituted or unsubstituted lower alkyl or unsubstituted heteroalkyl or cyano or alkoxy; and

RY? is H, or substituted or unsubstituted lower alkyl or unsubstituted heteroalkyl, - wherein at least one of R} R12, RE, RY or R! links said drug to LLif present, or to F, H, or J.

In all of the foregoing linker conjugate structures, L* preferably comprises a non- cyclic moiety. L* preferably increases solubility of the compound as compared to the compound lacking L* and/or L* decreases aggregation of the compound as compared to the compound lacking L*. In a preferred embodiment, L* comprises a polyethylene glycol moiety. The polyethylene glycol moiety can contain, for example, 3-12 repeat units, or 2-6 repeat units or, more preferably, 4 repeat units.

In yet another aspect, the invention provides a cytotoxic drug-ligand compound having a structure according to the following formula: x S-a—L go ® wherein the symbol L' represents a self-immolative spacer where m is an integer of0,1,2,3,4,5,0r6.

The symbol L* represents a linker member, and pis OQ or 1. L* is a moiety that imparts increased solubility or decreased aggregation properties to the conjugates. Examples of

L* moieties include substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroalkyl, or unsubstituted heteroalkyl, any of which may be straight, branched, or cyclic, a positively or negatively charged amino acid polymer, such as polylysine or polyargenine, or other polymers such as polyethylene glycol. [ 20 The symbol Q represent any cleavable linker including, but not limited to, any of the peptidyl, hydrozone, and disulfide linkers described herein. Cleavable linkers include those that can be selectively cleaved by a chemical or biological process and upon cleavage separate the drug, D!, from X*.

The symbol D' represents a drug having the following formula:

Ry re

R,

R® @, RY 4 2504

X z RE

RS wherein X and Z are members independently selected from O, S and

NR; o R2 is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl;

R!is H, substituted or unsubstituted lower alkyl, C(O)R®, or CO,R®,

R" is H, substituted or unsubstituted lower alkyl, or C(O)R?, wherein R® is a member selected from NR’R'® and OR’ and R® and R'? are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

Ris H, or substituted or unsubstituted lower alkyl or unsubstituted heteroalkyl or cyano or alkoxy;

R? is H, or substituted or unsubstituted lower alkyl or unsubstituted heteroalkyl,

R® is a member selected from the group consisting of SRY, NHR! and o OR!!, wherein R!! is a member selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, diphosphates, triphosphates, acyl, C(O)RR™, C(O)OR'2, C(O)NR'*R”, P(0)(OR'),, , C(O)CHR’R", SR® and SiRR®RY, in which RZ RY, and R!* are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted aryl, wherein R!? and R" together with the nitrogen or carbon atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 members, optionally containing two or more heteroatoms; wherein at least one of R"?, RZ, and R? links said drug to LL if present, or t0Q,

R® is a single bond which is either present or absent and when present R® and R’ are joined to form a cyclopropyl ring; and

Ris CH,-X" or -CHa- joined in said cyclopropyl ring with RS, wherein

X! is a leaving group,

R*,R*, R® and R*’ are members independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO,, NRIR'¢, NC(O)R'*, OC(O)NR'*R®, OC(O)OR"?, C(O)R”, ® SR!®, OR", CR*=NRS, and O(CH,).NR**R* wherein n is an integer from 1 to 20;

RY and R!® are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted peptidyl, wherein RS and R!® together with the nitrogen atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 members, optionally containing two or more heteroatoms; and R?* and R?’ are independently selected from unsubstituted alkyl, and wherein at least one of R* , R*, R® and R” is O(CH,).NR*R”.

Yet another embodiment is a compound having a structure according to Formula 1: ° Ly

R1 rR? @ R* ‘

X z RS

R® (1) wherein X and Z are independently selected from O, S and NR? wherein R” is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl;

R! is H, substituted or unsubstituted lower alkyl, C(O)R®, or COR’,

RY is H, substituted or unsubstituted lower alkyl, or C(O)R?, each R? is a member independently sélected from NR’R' and OR’ and R® and R'° are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

S R? is H, substituted or unsubstituted lower alkyl, unsubstituted heteroalkyl, cyano, or alkoxy;

R? is H, substituted or unsubstituted lower alkyl, or unsubstituted heteroalkyl,

R® is a member selected from the group consisting of SRY, NHR!! and OR, wherein RM is a member selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, ® 10 substituted heteroalkyl, unsubstituted heteroalkyl, diphosphates, triphosphates, acyl, C(O)RR",

C(O)OR'?, C(O)NR’RY, P(O)(OR?),, , C(O)CHR'?R'?, SR"? and SiR"’R"R", in which R”,

RY, and R* are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted aryl, or RZ and RP together with the nitrogen or carbon atom to which they are attached are joined to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 members, optionally containing two or more heteroatoms;

R® is a single bond which is either present or absent and when present R® and R7 are joined to form a cyclopropyl ring; and

Ris CH,-X! or —-CH;- joined in said cyclopropyl ring with RS, wherein X! is a leaving group,

R*, R*, R® and R® are members independently selected from the group consisting of H, ® substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen,

NO, NRR'S, NC(O)RY, OC(O)NR RS, OC(O)ORY, C(O)R*, SR'*, OR”, CR¥=NR'’, and O(CH).NR*R? wherein n is an integer from 1 to 20;

R' and R!® are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted peptidyl, wherein R!® and R'® together with the nitrogen atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 members, optionally containing two or more heteroatoms;

and R* and R? are independently selected from unsubstituted alkyl, and wherein at least one of R*, R*, R® and R®” is O(CHz).NR*R”,

In yet another aspect, the invention pertains to pharmaceutical formulations. Such formulations typically comprise a conjugate compound of the invention and a pharmaceutically acceptable carrier.

In still a further aspect, the invention pertains to methods of using the conjugate compounds of the invention. For example, the invention provides a method of killing a cell, wherein a conjugate compound of the invention is administered to the cell an amount sufficient to kill the cell. In a preferred embodiment, the cell is a tumor cell. In another embodiment, the ® 10 invention provides a method of retarding or stopping the growth of a tumor in a mammalian subject, wherein a conjugate compound of the invention is administered to the subject an amount sufficient to retard or stop growth of the tumor.

Other aspects, advantages and objects of the invention will be apparent from review of the detailed description below.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations

As used herein, “Ala,” refers to alanine. “Boc,” refers to -butyloxycarbonyl. “CPL? refers to cyclopropapyrroloindole. ® “Cbz,” is carbobenzoxy.

As used herein, “DCM,” refers to dichloromethane. “DDQ,” refers to 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.

DIPEA is diisopropylethalamine “DMDA” is N,N’-dimethylethylene diamine “RBF” is around bottom flask “DMF” is N,B-dimethylformamide “HATU” is N-[[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1- yl]methylene]-N-methylmethanaminium hexafluorophosphate N-oxide

As used herein, the symbol “E,” represents an enzymatically cleaveable group.

“EDCI” is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

As used herein, “FMOC,” refers to 9-fluorenylmethyloxycarbonyl. “FMOC? irefers to 9-fluorenylmethoxycarbonyl. “HOA” is 7-Aza-1-hydroxybenzotriazole. “Leu” is leucine. “PABA” refers to para-aminobenzoic acid.

PEG refers to polyethylene glycol “PMB,” refers to para-methoxybenzyl. “TBAF,” refers to tetrabutylammonium fluoride. ® 10 The abbreviation “TBSO,” refers to t-butyldimethylsilyl ether.

As used herein, “TEA,” refers to triethylamine. “TFA,” refers to trifluororoacetic acid.

The symbol “Q” refers to a therapeutic agent, diagnostic agent or detectable label.

Definitions

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described below are those well known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, enzymatic reactions and purification steps are ® performed according to the manufacturer's specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (1989)

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y, which is incorporated herein by reference), which are provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry, and organic synthetic described below are those well known and commonly employed in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses.

The term "therapeutic agent" is intended to mean a compound that, when present in a therapeutically effective amount, produces a desired therapeutic effect on a mammal. For treating carcinomas, it is desirable that the therapeutic agent also be capable of entering the target cell.

The term "cytotoxin" is intended to mean a therapeutic agent having the desired effect of being cytotoxic to cancer cells. Cytotoxic means that the agent arrests the growth of, or kills the cells. Exemplary cytotoxins include, by way of example and not limitation, combretastatins, duocarmycins, the CC-1065 anti-tumor antibiotics, anthracyclines, and related compounds. Other cytotoxins include mycotoxins, ricin and its analogues, calicheamycins, doxirubicin and maytansinoids.

The term "prodrug" and the term "drug conjugate" are used herein ® 10 interchangeably. Both refer to a compound that is relatively innocuous to cells while still in the conjugated form but which is selectively degraded to a pharmacologically active form by conditions, e.g., enzymes, located within or in the proximity of target cells.

The term "marker" is intended to mean a compound useful in the characterization of tumors or other medical condition, for example, diagnosis, progression of a tumor, and assay of the factors secreted by tumor cells. Markers are considered a subset of “diagnostic agents.”

The term "selective" as used in connection with enzymatic cleavage means that the rate of rate of cleavage of the linker moiety is greater than the rate of cleavage of a peptide having a random sequence of amino acids.

The terms "targeting group" and “targeting agent” are intended to mean a moiety that is (1) able to direct the entity to which it is attached (e.g., therapeutic agent or marker) toa target cell, for example to a specific type of tumor cell or (2) is preferentially activated at a target ® tissue, for example a tumor. The targeting group or targeting agent can be a small molecule, which is intended to include both non-peptides and peptides. The targeting group can also bea macromolecule, which includes saccharides, lectins, receptors, ligand for receptors, proteins such as BSA, antibodies, and so forth. In a preferred embodiment of the current invention, the targeting group is an antibody or an antibody fragment, more preferably a monoclonal antibody or monoclonal antibody fragment

The term "self-immolative spacer” refers to a bifunctional chemical moiety that is capable of covalently linking two chemical moieties into a normally stable tripartate molecule.

The self-immolative spacer is capable of spontaneously separating from the second moiety if the bond to the first moiety is cleaved.

The term “detectable label” is intended to mean a moiety having a detectable physical or chemical property.

The term "cleaveable group" is intended to mean a moiety that is unstable in vivo.

Preferably the “cleaveable group” allows for activation of the marker or therapeutic agent by cleaving the marker or agent from the rest of the conjugate. Operatively defined, the linker is preferably cleaved in vivo by the biological environment. The cleavage may come from any process without limitation, e.g., enzymatic, reductive, pH, etc. Preferably, the cleaveable group is selected so that activation occurs at the desired site of action, which can be a site in or near the target cells (e.g., carcinoma cells) or tissues such as at the site of therapeutic action or marker ® 10 activity. Such cleavage may be enzymatic and exemplary enzymatically cleaveable groups include natural amino acids or peptide sequences that end with a natural amino acid, and are attached at their carboxyl terminus to the linker. While the degree of cleavage rate enhancement is not critical to the invention, preferred examples of cleaveable linkers are those in which at least about 10% of the cleaveable groups are cleaved in the blood stream within 24 hours of administration, most preferably at least about 35%.

The term "ligand" means any molecule that specifically binds or reactively associates or complexes with a receptor, substrate, antigenic determinant, or other binding site on a target cell or tissue. Examples of ligands include antibodies and fragments thereof (e.g., a monoclonal antibody or fragment thereof), enzymes (e.g. fibrinolytic enzymes), biologic response modifiers (e.g., interleukins, interferons, erythropeoitin, or colony stimulating factors), ® peptide hormones, and antigen-binding fragments thereof.

The terms “hydrazine linker” and “self-cyclizing hydrazine linker” are used interchangeably herein. These terms refer to a linker moiety that, upon a change in condition, : such as a shift in pH, will undergo a cyclization reaction and form one or more rings. The hydrazine moiety is converted to a hydrazone when attached. This attachment can occur, for example, through a reaction with a ketone group on the L* moiety. Therefore, the term hydrazone linker can also be used to describe the linker of the current invention because of this conversion to a hydrazone upon attachment.

The term “five-membered hydrazine linker” or “5-membered hydrazine linker” refers to hydrazine-containing molecular moieties that, upon a change in condition, such as a shift in pH, will undergo a cyclization reaction and form one or more S-membered rings. 25 i

Alternatively, this five membered linker may similarly be described as a five-membered hydrazone linker or a 5-membered hydrazone linker.

The term “six-membered hydrazine linker” or “6-membered hydrazine linker” refers to hydrazine-containing molecular moieties that, upon a change in condition such as a shift in pH, will undergo a cyclization reaction and form one or more 6-membered rings. This six membered linker may similarly be described as a six-membered hydrazone linker or a 6- membered hydrazone linker.

The term “cyclization reaction,” when referring to the cyclization of a peptide, hydrazine, or disulfide linker, indicates the cyclization of that linker into a ring and initiates the ® 10 separation of the drug-ligand complex. This rate can be measured ex situ, and is completed when at least 90%, 95%, or 100% of the product is formed.

The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. These terms also encompass the term “antibody.” :

The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, v- carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the ® same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. One amino acid that may be used in particular is citrulline, which is a precursor to arginine and is involved in the formation of urea in the liver. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid. The term "unnatural amino acid" is intended to represent the "D" stereochemical form of the twenty naturally occurring amino acids described above. It is further understood that 26

Cd the term unnatural amino acid includes homologues of the natural amino acids, and synthetically modified forms of the natural amino acids. The synthetically modified forms include, but are not limited to, amino acids having alkylene chains shortened or lengthened by up to two carbon atoms, amino acids comprising optionally substituted aryl groups, and amino acids comprised halogenated groups, preferably halogenated alkyl and aryl groups. When attached to a linker or conjugate of the invention, the amino acid is in the form of an "amino acid side chain", where the carboxylic acid group of the amino acid has been replaced with a keto (C(O) group. Thus, for example, an alanine side chain is -C(0)-CH(NH,)-CH, and so forth.

Amino acids and peptides may be protected by blocking groups. A blocking ® 10 group is an atom or a chemical moiety that protects the N-terminus of an amino acid or a peptide from undesired reactions and can be used during the synthesis of a drug-ligand conjugate. It should remain attached to the N-terminus throughout the synthesis, and may be removed after completion of synthesis of the drug conjugate by chemical or other conditions that selectively achieve its removal. The blocking groups suitable for N-terminus protection are well known in the art of peptide chemistry. Exemplary blocking groups include, but are not limited to, hydrogen, D-amino acid, and carbobenzoxy (Cbz) chloride. “Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the ® reference nucleotides, Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer ef al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 27

Ir

(1985); Rossolini ef al., Mol. Cell. Probes 8: 91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

The symbol "VV, whether utilized as a bond or displayed perpendicular to a bond indicates the point at which the displayed moiety is attached to the remainder of the molecule, solid support, etc.

The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e. C,-Cjo means one to ten ® 10 carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n- hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term “alkyl,” unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below, such as “heteroalkyl.” Alkyl groups, which are limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means a divalent ® radical derived from an alkane, as exemplified, but not limited, by -CH,CH;CH,CH,-, and further includes those groups described below as “heteroalkylene.” Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.

The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom selected from the group consisting of O, N, Si and S, and wherein the nitrogen, carbon and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH,-CH,-0-CH3, -CH,-CH,-NH-CHj, -

CH;-CH,-N(CH3)-CHj, -CH,-S-CH>-CH;, -CHz-CHz,-8(0)-CHs, -CH,-CH,-S(0),-CHs, -

CH=CH-O-CHj, -Si(CH3)s, -CH;-CH=N-OCHj, and ~-CH=CH-N(CH3)-CHj;. Up to two

S heteroatoms may be consecutive, such as, for example, -CH,-NH-OCH3 and —CH,-0-Si(CHa)s.

Similarly, the term “heteroalkylene” by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH;-CH,-S-CH,-CHy- and _CH,-S-CH,-CH,-NH-CH;-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and ® 10 the like). The terms “heteroalkyl” and “heteroalkylene” encompass poly(ethylene glycol) and its derivatives (see, for example, Shearwater Polymers Catalog, 2001). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula —C(O)R’- represents both —C(O),R’- and —R’C(O);,-.

The term “lower” in combination with the terms “alkyl” or “heteroalkyl” refers to a moiety having from 1 to 6 carbon atoms.

The terms “alkoxy,” “alkylamino,” “alkylsulfonyl,” and “alkylthio” (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, an SO, group or a sulfur atom, respectively. The term “arylsulfonyl” refers to an aryl group attached to the remainder ofhte molecule via an SO, group, and the term “sulfhydryl” refers to an SH group. ® In general, an “acyl substituent” is also selected from the group set forth above.

As used herein, the term “acyl substituent” refers to groups attached to, and fulfilling the valence . of a carbonyl carbon that is either directly or indirectly attached to the polycyclic nucleus of the compounds of the present invention.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of substituted or unsubstituted “alkyl” and substituted or unsubstituted “heteroalkyl”, respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of 29

Br heterocycloalkyl include, but are not limited to, 1 —(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2- piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran- 3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1 —piperazinyl, 2-piperazinyl, and the like. The heteroatoms and carbon atoms of the cyclic structures are optionally oxidized.

The terms “halo” or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(C;-Cy4)alkyl” is mean to include, but not be limited to, trifluoromethyl, 2,2,2- trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. ® 10 The term “aryl” means, unless otherwise stated, a substituted or unsubstituted polyunsaturated, aromatic, hydrocarbon substituent which can be a single ring or multiple rings (preferably from 1 to 3 rings) which are fused to gether or linked covalently. The term “heteroaryl” refers to aryl groups (or rings) that contain from one to four heteroatoms selected from N, O, and S, wherein the nitrogen, carbon and sulfur atoms are optionally oxidized, and the npitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3- pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5- oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3- furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5- benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2- ® quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. “Aryl” and “heteroaryl” also encompass ring systems in which one or more non-aromatic ring systems are fused, or otherwise bound, to an aryl or heteroaryl system.

For brevity, the term “aryl” when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term “arylalkyl” is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethy}, 3-( 1-naphthyloxy)propyl, and the like). 30

Ne

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and “heteroaryl” include both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

Substituents for the alkyl, and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are generally referred to as “alkyl substituents” and “heteroalkyl substituents,” respectively, and they can be one or more ofa variety of groups selected from, but not limited to: -OR’, =O, =NR’, =N-OR’, -NR’R”, -SR’, - halogen, -SiR’R”R”™, -OC(O)R’, -C(O)R’, -CO;R’, -CONR’R”, -OC(O)NR’R?, -NR”C(O)R’, ® 10 -NR’-C(O)NR’R’’, -NR”C(O);R’, -NR-C(NR'R”R’”)=NR””, -NR-C(NR’R”)=NR"", -S(O)R’, -

S(0)R’, -S(0):NR’R”, -NRSO,R’, -CN and -NO, in a number ranging from zero to (2m’+1), where m’ is the total number of carbon atoms in such radical. R’, R”, R”” and R”” each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each

R’, R”, R’” and R”” groups when more than one of these groups is present. ‘When R’ and R” are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6- , or 7-membered ring. For example, -NR’R” is meant to include, but not be limited to, 1- pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term “alkyl” is meant to include groups including carbon atoms bound to ® groups other than hydrogen groups, such as haloalkyl (e.g., -CF; and —CH,CF3) and acyl (e.g., -

C(O)CHs, -C(O)CFs, -C(0)CH,OCHj, and the like). -

Similar to the substituents described for the alkyl radical, the aryl substituents and heteroaryl substituents are generally referred to as “aryl substituents” and “heteroaryl substituents,” respectively and are varied and selected from, for example: halogen, -OR’, =O, =NR’, =N-OR’, -NR’R”, -SR’, -halogen, -SiR’R”R*”’, -OC(O)R’, -C(O)R’, -CO2R’, -CONR’R”, -OC(O)NR’R”, -NR”’C(O)R’, -NR’-C(O)NR"R™, -NR”C(O).R’, -NR-C(NR’R”)=NR’”, -

S(O)R’, -S(0)2R’, -S(0)NR’R”, -NRSO;R’, -CN and -NO;, -R’, -N3, -CH(Ph),, fluoro(C;- Cy)alkoxy, and fluoro(C,-Cs)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R’, R”, R™’ and R”” are preferably independently selected from hydrogen, (C;-Cg)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C;-Cas)alkyl, and (unsubstituted aryl)oxy-(C;-Cs)alkyl. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R’, R”, R’” and R”” groups when more than one of these groups is present.

Two of the aryl substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula —T-C(0)-(CRR”)¢-U-, wherein T and U are independently -NR-, -O-, -CRR’- or a single bond, and q is an integer of from 0 to 3.

Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may ® 10 optionally be replaced with a substituent of the formula ~A-(CH,),-B-, wherein A and B are independently —-CRR’-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O).NR’- or a single bond, and ris an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CRR’)s-X- (CR”R’”)4-, where s and d are independently integers of from 0 to 3, and X is -O-, -NR’-, -S-, -

S(0)-, -S(0),-, or —S(0);NR’-. The substituents R, R’, R” and R’” are preferably independently selected from hydrogen or substituted or unsubstituted (Cy-Ce) alkyl.

As used herein, the term “diphosphate” includes but is not limited to an ester of phosphoric acid containing two phosphate groups. The term “triphosphate” includes but is not limited to an ester of phosphoric acid containing three phosphate groups. For example, particular ) drugs having a diphosphate or a triphosphate include:

PANE CO,Me ot

R20-p=0 N

OL &

R'2g © N

Rr i»

X Zz

Diphosphate 5

R

CO,Me ® 0 OR"? )= xX’ \ [

R'20-P—-0-P=0 N

OR" O, &

R20 ho) N 1 X R*

R z

R%

Triphosphate

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).

The symbol “R” is a general abbreviation that represents a substituent group that is selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocyclyl groups. ® The term "pharmaceutically acceptable carrier”, as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent. Pharmaceutically acceptable carriers include pharmaceutically acceptable salts, where the term “pharmaceutically acceptable salts” includes salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium,

potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, ® 10 benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge ef al., “Pharmaceutical Salts”, Journal of

Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compounds, which are in ® . a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed ina transdermal patch reservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are encompassed within the scope of the present invention.

The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium CH, jodine-125 (**°I) or carbon-14 (**C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

The term "attaching moiety" or "moiety for attaching a targeting group" refers to a moiety which allows for attachment of a targeting group to the linker. Typical attaching groups include, by way of illustration and not limitation, alkyl, aminoalkyl, aminocarbonylalkyl, carboxyalkyl, hydroxyalkyl, alkyl-maleimide, alkyl-N-hydroxylsuccinimide, poly(ethylene glycol)-maleimide and poly(ethylene glycol)-N-hydroxylsuccinimide, all of which may be further substituted. The linker can also have the attaching moiety be actually appended to the targeting group.

As used herein, the term “leaving group” refers to a portion of a substrate that is cleaved from the substrate in a reaction.

The term “antibody” as referred to herein includes whole antibodies and any _ antigen binding fragment (i.e., “antigen-binding portion”) or single chains thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two Light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (Vy) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, Cui, Cra and Cys, and may be of the mu, delta, gamma, alpha or epsilon isotype. Each light chain is comprised of a light chain variable region (Vy) and a light chain constant region. The light chain constant region is comprised of one domain, Cr, which may be of the kappa or lambda isotype. The Vy and Vi, regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each Vy and Vy is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDRI, FR2, CDR2,

FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

The terms “antibody fragment” or "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antibody fragment” or "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the Vy,

Vy, C1 and Cy; domains; (ii) a F(ab"), fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the

Vy and Cy; domains; (iv) a Fv fragment consisting of the Vi, and Vy domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a

Vy domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, V and Vg, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the Vy, and Vy regions pair to form monovalent molecules ® (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston ez al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

The terms "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

For preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., 36 g

Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in MONOCLONAL ANTIBODIES AND

CANCER THERAPY, Alan R. Liss, Inc. (1985)).

Methods of production of polyclonal antibodies are known to those of skill in the art. An inbred strain of mice (e.g., BALB/C mice) or rabbits is immunized with the protein using astandard adjuvant, such as Freund’s adjuvant, and a standard immunization protocol. The animal’s immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the beta subunits. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are ® prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired.

Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see Kohler & Milstein, Eur. J.

Immunol. 6: 511-519 (1976)). Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art.

In a preferred embodiment, the antibody is a chimeric or humanized antibody.

Chimeric or humanized antibodies of the present invention can be prepared based on the sequence of a murine monoclonal antibody. DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and engineered to contain non-murine (e.g.,. human) immunoglobulin sequences using standard molecular biology [ techniques. For example, to create a chimeric antibody, the murine variable regions can be linked to human constant regions using methods known in the art (see e.g, U.S. Patent No. 4,816,567 to Cabilly et al). To create a humanized antibody, the murine CDR regions can be inserted into a human framework using methods known in the art (see e.g., U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to

Queen et al.).

In another preferred embodiment, the antibody is a human antibody. Such human antibodies can be generated by immunizing transgenic or transchromosomic mice in which the endogenous mouse immunoglobulin genes have been inactivated and exogenous human immunoglobulin genes have been introduced. Such mice are known in the art (see e.g., U.S.

Patent Nos: 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016;

5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay; U.S. Patent Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al.; and PCT Publication WO 02/43478 to Ishida ef al.) Human antibodies can also be prepared using phage display methods for screening libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies also are know in the art (see e.g., U.S. Patent Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner et al.; U.S. Patent Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Patent Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Patent Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths ef al). “Solid support,” as used herein refers to a material that is substantially insoluble ® 10 in a selected solvent system, or which can be readily separated (e.g., by precipitation) from a selected solvent system in which it is soluble. Solid supports useful in practicing the present invention can include groups that are activated or capable of activation to allow selected species to be bound to the solid support. A solid support can also be a substrate, for example, a chip, wafer or well, onto which an individual, or more than one compound, of the invention is bound. “Reactive functional group,” as used herein refers to groups including, but not limited to, olefins, acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic acids isonitriles, amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamic acids thiohydroxamic acids, allenes, ortho esters, sulfites, enamines, ynamines, ® ureas, pseudoureas, semicarbazides, carbodiimides, carbamates, imines, azides, azo compounds, azoxy compounds, and nitroso compounds. Reactive functional groups also include those used to prepare bioconjugates, e.g., N-hydroxysuccinimide esters, maleimides and the like (see, for example, Hermanson, BIOCONJUGATE TECHNIQUES, Academic press, San Diego, 1996).

Methods to prepare each of these functional groups are well known in the art and their application to or modification for a particular purpose is within the ability of one of skill in the art (see, for example, Sandler and Karo, eds. ORGANIC FUNCTIONAL GROUP PREPARATIONS,

Academic Press, San Diego, 1989). The reactive functional groups may be protected or unprotected.

The compounds of the invention are prepared as a single isomer (e.g., enantiomer, cis-trans, positional, diastereomer) or as a mixture of isomers. In a preferred embodiment, the compounds are prepared as substantially a single isomer. Methods of preparing substantially isomerically pure compounds are known in the art. For example, enantiomerically enriched mixtures and pure enantiomeric compounds can be prepared by using synthetic intermediates that are enantiomerically pure in combination with reactions that either leave the stereochemistry at a chiral center unchanged or result in its complete inversion. Alternatively, the final product or intermediates along the synthetic route can be resolved into a single stereoisomer. Techniques ) for inverting or leaving unchanged a particular stereocenter, and those for resolving mixtures of stereoisomers are well known in the art and it is well within the ability of one of skill in the art to choose and appropriate method for a particular situation. See, generally, Furniss ef al. (eds.),VOGEL’S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY 5™ED., Longman Scientific and Technical Ltd., Essex, 1991, pp. 809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

LINKERS

The present invention provides for drug-ligand conjugates where the drug is linked to the ligand through a chemical linker. This linker is either a peptidyl, hydrazine, or disulfide linker, and is depicted herein as (L)y—F— (La , (L)y—H— La, or LH—I— (LY , respectively. In addition to the linkers as being attached to the drug, the present invention also provides cleaveable linker arms that are appropriate for attachment to essentially any ® molecular species. The linker arm aspect of the invention is exemplified herein by reference to their attachment to a therapeutic moiety. It will, however, be readily apparent to those of skill in the art that the linkers can be attached to diverse species including, but not limited to, diagnostic agents, analytical agents, biomolecules, targeting agents, detectable labels and the like.

In one aspect, the present invention relates to linkers that are useful to attach targeting groups to therapeutic agents and markers. In another aspect, the invention provides linkers that impart stability to compounds, reduce their in vivo toxicity, or otherwise favorably affect their pharmacokinetics, bioavailability and/or pharmacodynamics. It is generally preferred that in such embodiments, the linker is cleaved, releasing the active drug, once the drug is delivered to its site of action. Thus, in one embodiment of the invention, the linkers of the invention are traceless, such that once removed from the therapeutic agent or marker (such as during activation), no trace of the linker's presence remains.

In another embodiment of the invention, the linkers are characterized by their ability to be cleaved at a site in or near the target cell such as at the site of therapeutic action or 5S marker activity. Such cleavage can be enzymatic in nature. This feature aids in reducing systemic activation of the therapeutic agent or marker, reducing toxicity and systemic side effects. Preferred cleaveable groups for enzymatic cleavage include peptide bonds, ester linkages, and disulfide linkages. In other embodiments, the linkers are sensitive to pH and are cleaved through changes in pH. ® 10 An important aspect of the current invention is the ability to control the speed with which the linkers cleave. For example, the hydrazine linkers described herein are particularly useful because, depending on which particular structure is used, one can vary the speed at which the linker cyclizes and thereby cleaves the drug from the ligand. WO 02/096910 provides several specific ligand-drug complexes having a hydrazine linker. However, there is no way to “tune” the linker composition dependent upon the rate of cyclization required, and the particular compounds described cleave the ligand from the drug at a slower rate than is preferred for many drug-linker conjugates. In contrast, the hydrazine linkers of the current invention provide for a range of cyclization rates, from very fast to very slow, thereby allowing for the selection of a particular hydrazine linker based on the desired rate of cyclization. For example, very fast cyclization can be achieved with hydrazine linkers that produce a single 5S-membered ring upon cleavage. Preferred cyclization rates for targeted delivery of a cytotoxic agent to cells ® are achieved using hydrazine linkers that produce, upon cleavage, either two 5-membered rings or a single 6-membered ring resulting from a linker having two methyls at the geminal position.

The gem-dimethyl effect has been shown to accelerate the rate of the cyclization reaction as compared to a single 6-membered ring without the two methyls at the geminal position. This results from the strain being relieved in the ring. Sometimes, however, substitutents may slow down the reaction instead of making it faster. Often the reasons for the retardation can be traced to steric hindrance. As shown in Example 2.4, the gem dimethyl substitution allows for 2 much faster cyclization reaction to occur compared to when the geminal carbon is a CH.

It is important to note, however, that in some embodiments, a linker that cleaves more slowly may be preferred. For example, in a sustained release formulation or in a formulation with both a quick release and a slow release component, it may be useful to provide a linker which cleaves more slowly. In certain embodiments, a slow rate of cyclization is achieved using a hydrazine linker that produces, upon cleavage, either a single 6-membered ring, without the gem-dimethyl substitution, or a single 7-membered ring.

The linkers also serve to stabilize the therapeutic agent or marker against degradation while in circulation. This feature provides a significant benefit since such stabilization results in prolonging the circulation half-life of the attached agent or marker. The linker also serves to attenuate the activity of the attached agent or marker so that the conjugate is relatively benign while in circulation and has the desired effect, for example is toxic, after ® 10 activation at the desired site of action. For therapeutic agent conjugates, this feature of the linker serves to improve the therapeutic index of the agent.

The stabilizing groups are preferably selected to limit clearance and metabolism of the therapeutic agent or marker by enzymes that may be present in blood or non-target tissue and are further selected to limit transport of the agent or marker into the cells. The stabilizing groups serve to block degradation of the agent or marker and may also act in providing other physical characteristics of the agent or marker. The stabilizing group may also improve the agent or marker's stability during storage in either a formulated or non-formulated form.

Ideally, the stabilizing group is useful to stabilize a therapeutic agent or marker if it serves to protect the agent or marker from degradation when tested by storage of the agent or marker in human blood at 37°C for 2 hours and results in less than 20%, preferably less than 10%, more preferably less than 5% and even more preferably less than 2%, cleavage of the agent ® or marker by the enzymes present in the human blood under the given assay conditions.

The present invention also relates to conjugates containing these linkers. More particularly, the invention relates to prodrugs that may be used for the treatment of disease, especially for cancer chemotherapy. Specifically, use of the linkers described herein provide for prodrugs that display a high specificity of action, a reduced toxicity, and an improved stability in blood relative to prodrugs of similar structure.

The linkers of the present invention as described herein may be present at any position within the cytotoxic conjugate.

Thus, there is provided a linker that may contain any of a variety of groups as part of its chain that will cleave in vivo, e.g., in the blood stream at a rate which is enhanced relative to that of constructs that lack such groups. Also provided are conjugates of the linker arms with therapeutic and diagnostic agents. The linkers are useful to form prodrug analogs of therapeutic agents and to reversibly link a therapeutic or diagnostic agent to a targeting agent, a detectable label, or a solid support. The linkers may be incorporated into complexes that include the cytotoxins of the invention.

In addition to the cleaveable peptide, hydrazine, or disulfide group, one or more self-immolative linker groups L’ are optionally introduced between the cytotoxin and the targeting agent. These linker groups may also be described as spacer groups and contain at least two reactive functional groups. Typically, one chemical functionality of the spacer group bonds ® 10 to a chemical functionality of the therapeutic agent, e.g., cytotoxin, while the other chemical functionality of the spacer group is used to bond to a chemical functionality of the targeting agent or the cleaveable linker. Examples of chemical functionalities of spacer groups include hydroxy, mercapto, carbonyl, carboxy, amino, ketone, and mercapto groups.

The self-immolative linkers, represented by L', are generally substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl or a substituted or unsubstituted heteroalkyl group. In one embodiment, the alkyl or aryl groups may comprise between 1 and 20 carbon atoms. They may also comprise a polyethylene glycol moiety.

Exemplary spacer groups include, for example, 6-aminohexanol, 6- mercaptohexanol, 10-hydroxydecanoic acid, glycine and other amino acids, 1,6-hexanediol, B- alanine, 2-aminoethanol, cysteamine (2-aminoethanethiol), 5-aminopentanoic acid, 6- ® aminohexanoic acid, 3-maleimidobenzoic acid, phthalide, a-substituted phthalides, the carbonyl group, aminal esters, nucleic acids, peptides and the like.

The spacer can serve to introduce additional molecular mass and chemical functionality into the cytotoxin-targeting agent complex. Generally, the additional mass and functionality will affect the serum half-life and other properties of the complex. Thus, through careful selection of spacer groups, cytotoxin complexes with a range of serum half-lives can be produced. :

The space(s) located directly adjacent to the drug moiety is also denoted as OH, wherein m is an integer selected from 0, 1, 2, 3, 4, 5, or 6. When multiple L' spacers are present, either identical or different spacers may be used. L' may be any self-immolative group.

In one embodiment, L' is preferably is a substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, and unsubstituted heteroalkyl, unsubstituted heterocycloalkyl, and substituted heterocycloalkyl. When the drug-ligand conjugate comprises a hydrazine linker, L! does not comprise a disulfide bond.

L* is a linker moiety that imparts increased solubility or decreased aggregation properties to conjugates utilizing a linker that contains the moiety. The L* linker does not have to be self immolative. In one embodiment, the L* moiety is substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroalkyl, or unsubstituted heteroalkyl, any of which may be straight, branched, or cyclic. The substitutions may be, for example, a ® 10 lower (C'-C%) alkyl, alkoxy, aklylthio, alkylamino, or dialkylamino. In certain embodiments, Lf comprises a non-cyclic moiety . In another embodiment, L* comprises any positively or negatively charged amino acid polymer, such as polylysine or polyargenine. L* can comprise a polymer such as a polyethylene glycol moiety. Additionally the L* linker comprises, for example, both a polymer component and a small chemical moiety.

In a preferred embodiment, L* comprises a polyethylene glycol (PEG) moiety.

The PEG portion of L* may be between 1 and 50 units long. Preferably, the PEG will have 1-12 repeat units, more preferably 3-12 repeat units, more preferably 2-6 repeat units, or even more preferably 3-5 repeat units and most preferably 4 repeat units. L* may consist solely of the PEG moiety, or it may also contain an additional substituted or unsubstituted alkyl or heteroalkyl. It is useful to combine PEG as part of the L* moiety to enhance the water solubility of the complex.

Additionally, the PEG moiety reduces the degree of aggregation that may occur during the ® conjugation of the drug to the antibody. (1) Peptide Linkers (I)

As discussed above, the peptidyl linkers of the invention can be represented by the general formula: (L*);—F— (LY , wherein F represents the linker portion comprising the peptidyl moiety. In one embodiment, the F portion comprises an optional additional self- immolative linker(s), L%, and a carbonyl group. In another embodiment, the F portion comprises an amino group and an optional spacer group(s), L’.

Accordingly, in one embodiment, the conjugate comprising the peptidyl linker comprises a structure of the Formula 4: i xian Hof)

Pp c [4 m . @)

In this embodiment, L'is a self-immolative linker, as described above, and Lisa moiety that imparts increased solubility, or decreased aggregation properties, as described above.

I? represents a self-immolative linker(s). mis 0, 1, 2, 3, 4, 5, or 6; o and p are independently 0 ( or 1. In one embodiment, mis 3, 4, 5 or 6. AA! represents one or more natural amino acids, and/or unnatural a-amino acids; ¢ is an integer between 1 and 20.

In the peptide linkers of the invention of the above Formula 4, AA! is linked, at its amino terminus, either directly to L* or, when L* is absent, directly to the x4 group (i.e., the targeting agent, detectable label, protected reactive functional group or unprotected reactive functional group). In some embodiments, when Lis present, L* does not comprise a carboxylic acyl group directly attached to the N-terminus of (AA").. Thus, it is not necessary in these embodiments for there to be a carboxylic acyl unit directly between either L* or X* and AA!, as is necessary in the peptidic linkers of U.S. Patent No. 6,214,345.

In another embodiment, the conjugate comprising the peptidyl linker comprises a structure of the Formula 5: ® =» mm p c H ° (5)

In this embodiment, L* is a moiety that imparts increased solubility, or decreased aggregation properties, as described above; L* is a spacer group comprising a primary or secondary amine or a carboxyl functional group, and either the amine of L* forms an amide bond with a pendant carboxyl functional group of D or the carboxyl of 13 forms an amide bond with a 44

A pendant amine functional group of D; and o and p are independently 0 or 1. AA! represents one or more natural amino acids, and/or unnatural a-amino acids; ¢ is an integer between 1 and 20.

In this embodiment, L! is absent (i.e., m is 0 is the general formula).

In the peptide linkers of the invention of the above Formula 5, AA! is linked, at its amino terminus, either directly to L* or, when L* is absent, directly to the X* group (i.e., the targeting agent, detectable label, protected reactive functional group or unprotected reactive functional group). In some embodiments, when L* is present, L* does not comprise a carboxylic acyl group directly attached to the N-terminus of (AA). Thus, it is not necessary in these embodiments for there to be a carboxylic acyl unit directly between either L* or X* and AA, as ® 10 is necessary in the peptidic linkers of U.S. Patent No. 6,214,345.

The self-immolative linker I?

The self-immolative linker L? is a bifunctional chemical moiety which is capable of covalently linking together two spaced chemical moieties into a normally stable tripartate molecule, releasing one of said spaced chemical moieties from the tripartate molecule by means of enzymatic cleavage; and following said enzymatic cleavage, spontaneously cleaving from the remainder of the molecule to release the other of said spaced chemical moieties. In accordance with the present invention, the self-immolative spacer is covalently linked at one of its ends to the peptide moiety and covalently linked at its other end to the chemical reactive site of the drug moiety whose derivatization inhibits pharmacological activity, so as to space and covalently link together the peptide moiety and the drug moiety into a tripartate molecule which is stable and ® pharmacologically inactive in the absence of the target enzyme, but which is enzymatically cleavable by such target enzyme at the bond covalently linking the spacer moiety and the peptide moiety to thereby effect release of the peptide moiety from the tripartate molecule. Such enzymatic cleavage, in turn, will activate the self-immolating character of the spacer moiety and initiate spontaneous cleavage of the bond covalently linking the spacer moiety to the drug moiety, to thereby effect release of the drug in pharmacologically active form.

The self-immolative linker L? may be any self-immolative group. Preferably L? is a substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, unsubstituted heterocycloalkyl, substituted heterocycloalkyl, substituted and unsubstituted aryl, and substituted and unsubstituted heteroaryl.

One particularly preferred self-immolative spacer L? may be represented by the formula 6:

N=

R24 6)

The aromatic ring of the aminobenzyl group may be substituted with one or more “K” groups. A “K” group is a substituent on the aromatic ring that replaces a hydrogen otherwise ® attached to one of the four non-substituted carbons that are part of the ring structure. The “K” group may be a single atom, such as a halogen, or may be a multi-atom group, such as alkyl, heteroalkyl, amino, nitro, hydroxy, alkoxy, haloalkyl, and cyano. Each K is independently selected from the group consisting of substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen,

NO,, NRZRZ, NRZCORZ, OCONR*'R%, OCOR?, and OR*, wherein R*' and R™ are independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl and unsubstituted heterocycloalkyl. Exemplary K substituents include, but are not limited to, F, Cl, Br, I, NO,

OH, OCHj;, NHCOCHj3, N(CH3),, NHCOCF3 and methyl. For “Ka”, ais an integer of 0, 1,2, 3, ® 20 or4. In one preferred embodiment, a is 0.

The ether oxygen atom of the structure shown above is connected to a carbonyl group. The line from the NR? functionality into the aromatic ring indicates that the amine functionality may be bonded to any of the five carbons that both form the ring and are not substituted by the -CH,-O- group. Preferably, the NR? functionality of X is covalently bound to the aromatic ring at the para position relative to the -CH-O- group. R* is a member selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, and unsubstituted heteroalkyl. In a specific embodiment, R* is hydrogen.

In a preferred embodiment, the invention provides a peptide linker of formula (4) above, wherein F comprises the structure:

ot (y od Se © boa wherein ® Ris selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, and unsubstituted heteroalkyl;

Each K is a member independently selected from the group consisting of substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO, NR2?'R%, NR2!CORZ,

OCONR?'R*?, OCOR?!, and OR?! wherein

R?! and R® are independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl; and ® a is an integer of 0,1, 2, 3, or 4. - In another embodiment, the peptide linker of formula (4) above comprises a F-(L n- that comprises the structure:

R24 R24 R# _

Ka O 0 A %

Ad oC \ R24 oT

Hmpoe= TTS © ey

Claims

WHAT IS CLAIMED IS:

1. A compound of the formula Xi —hmF— nfo wherein ® D is a drug moiety having pendant to the backbone thereof a chemically reactive functional group, said functional group selected from the group consisting of a primary or secondary amine, hydroxyl, thiol, carboxyl, aldehyde, and a ketone; L! is a self-immolative linker; m is an integer 0, 1,2,3, 4,5, or 6; F is a linker comprising the structure: i ur c ° or wr Cc H 0 @ wherein AA! is one or more members independently selected from the group consisting of natural amino acids and unnatural o-amino acids; ¢ is an integer from 1 to 20; L? is a self-immolative linker; L? is a spacer group comprising a primary or secondary amine or a carboxyl functional group; wherein if 13 is present, m is 0 and either the amine of 1? forms an amide bond with a pendant carboxy! functional group of D or the carboxyl of L* forms an amide bond with a pendant amine functional group of D; oisOorl;

L% is a linker member, wherein L* does not comprise a carboxylic acyl group directly attached to the N-terminus of (AA); pisOorl; and X4 is a member selected from the group consisting of protected reactive functional groups, unprotected reactive functional groups, detectable labels, and targeting agents.

2. The compound of claim 1, wherein the compound comprises the formula: i xian {uo )o o P [ [J m .

3. The compound of claim 1, wherein the compound comprises the formula: me P Cc H [o] .

4. The compound of claim 3, wherein L? comprises an aromatic group.

5. The compound of claim 4, wherein L? comprises a benzoic acid group, an aniline group, or an indole group.

®

6. The compound of claim 4, wherein -L-NH- comprises a group having a structure selected from the group consisting of:

HN HN LL ( N \ = \_J w z } \ 0 HE nA 3 3 . : z } -NH lo} wherein Z is a member selected from O, S and NRZ, and wherein R? is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl.

7. The compound according to any one of claims 1 to 6, wherein L* comprises a non-cyclic moiety.

8. The compound according to any one of claims 1 to 7, wherein L* increases ® solubility of the compound as compared to the compound lacking L*,

9. The compound according to any one of claims 1 to 8, wherein L* decreases aggregation of the compound as compared to the compound lacking L*

10. The compound according to any one of claims 1 to 9, wherein L* comprises a polyethylene glycol moiety.

11. The compound of claim 10, wherein the polyethylene glycol moiety contains 3-12 repeat units.

12. The compound of claim 11, wherein the polyethylene glycol moiety contains 2-6 repeat units.

13. The compound of claim 12, wherein the polyethylene glycol moiety contains 4 repeat units.

14. The compound according to any one of claims 1 to 13, wherein (AA".is a ® peptide sequence cleavable by a protease expressed in tumor tissue.

15. The compound of claim 14, wherein the protease is a lysosomal protease.

16. The compound according to any one of claims 1 to 15, wherein ¢ is an integer from 2 to 6.

17. The compound of claim 16, wherein ¢ is 2, 3 or 4.

18. The compound of according to any one of claims claim 1 to 17, wherein the amino acid in (AA), located closest to the drug moiety is selected from the group consisting of: Ala, Asn, Asp, Cit, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

19. The compound of according to any one of claims claim 1 to 18, wherein (AA. is a peptide sequence selected from the group consisting of Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N°-tosyl-Arg, Phe-N’-nitro-Arg, Phe- Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala- Leu (SEQ ID NO: 1), B-Ala-Leu-Ala-Leu (SEQ ID NO: 2) and Gly-Phe-Leu-Gly (SEQ ID NO:

3).

20. The compound according to any one of claims 1 to 19, wherein (AA). is Val-Cit or Val-Lys. :

21. The compound according to any one of claims 1 to 20, wherein D is a cytotoxic drug.

22. The compound of claim 21, wherein D comprises a chemically reactive functional group selected from the group consisting of a primary or secondary amine, hydroxyl, sulfhydryl and carboxyl.

23. The compound of claim 21, wherein D is selected from the group consisting of: duocarmycins, CC-1065, CBI-based duocarmycin analogues, MCBI-based duocarmycin ® analogues, CCBI-based duocarmycin analogues, doxorubicin, doxorubicin conjugates, morpholino-doxorubicin, cyanomorpholino-doxorubicin, dolastatins, dolestatin-10, combretastatin, calicheamicin, maytansine, maytansine analogues, DM-1, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), 5-benzoylvaleric acid-AE ester (AEVB), tubulysins, disorazole, epothilones, Paclitaxel, docetaxel, SN-38, Topotecan, rhizoxin, echinomycin, colchicine, vinblastin, vindesine, estramustine, cemadotin, eleutherobin, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, daunorubicin conjugates, mitomycin C, mitomycin A, carminomycin, aminopterin, tallysomycin, podophyllotoxin, podophyllotoxin derivatives, etoposide, etoposide phosphate, vincristine, taxol, taxotere retinoic acid, butyric acid, N® -acetyl spermidine and camptothecin. ®

24. The compound according to any one of claims 1 to 23, wherein D comprises a structure: A RS or RY 3 R Rt VV / RS wherein the ring system A is a member selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl groups; E and G are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, a heteroatom, a single bond, or E and G are joined to form a ring system selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl; X is a member selected from O, S and NRZ, R? is a member selected from H, substituted or unsubstituted alkyl, ® substituted or unsubstituted heteroalkyl, and acyl; R3 is a member selected from the group consisting of (=O), SR!!, NHR! ~ and OR", wherein R'is a member selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, diphosphates, triphosphates, acyl, C(O)RR", C(O)OR?, C(O)NR!2R", P(O)(OR'2),, , C(O)CHR?R", SR" and SiRZR"R", in which

R'? R" and R!* are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted aryl, wherein R'* and R'? together with the nitrogen or carbon atom to which they are attached

® are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 members, optionally containing two or more heteroatoms;

R*,R*, R® and R” are members independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO, NR'*R', NC(O)R", OC(O)NR¥R', 0C(0)ORY, C(O)R", SR'S, ORY, CR!*=NR', and O(CH).N(CHs)

wherein n is an integer from 1 to 20;

R!S and R'® are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted peptidyl, wherein R'S and R'® together with the nitrogen atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 members, optionally containing two or more heteroatoms; RS is a single bond which is either present or absent and when present R® and R’ are joined to form a cyclopropyl ring; and ® R is CH,-X' or —CHa- joined in said cyclopropyl ring with R®, wherein X'isa leaving group, wherein at least one of R'!, R'?, RE, RY or R! links said drug to LY if present, or to F.

25. The compound of claim 24, wherein D has the structure: R? " — HN oe . R® RY R* ox ( X Z RS RS wherein Z is a member selected from O, S and NR* wherein RZ is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl; R! is H, substituted or unsubstituted lower alkyl, C(O)RS, or COR, wherein R® is a member selected from group consisting of substituted alkyl, unsubstituted alkyl, NR°R', NR’NHR'"®, and OR’

in which R’ and R!® are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; and R?is H, substituted alkyl or unsubstituted lower alkyl; wherein at least one of R'’, R'2, R", R"® or R'® links said drug to L', if present, or to F.

26. ' The compound of claim 25, wherein R? is an unsubstituted lower alkyl. ® 27. The compound of claim 3, wherein NH;-(L*)-D has a structure selected from the group consisting of: . NH, NH, CO,Me oO CO,Me = ci =e ci HN HN H N Ho ae ol HO ! § 2 og ~z CO,Me HN N 7% ro 5 ° ® +L

NH, NH, SU WERE ® : ® : HO { o) HO N ! g z 0] Z NH el tT $ ’ as avi and [o] 4 wherein Z is a member selected from O, S and NRZ, wherein R? is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl; and wherein the NH; group on each structure reacts with (AAY). to form —(AA"-NH-.

28. The compound of claim 24, wherein D has the structure: Ra R2 Le Ry R® 7 Del N rR? 4 xX A RS" ® S wherein Z is a member selected from O, S and NR? wherein R?® is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl; R!is H, substituted or unsubstituted lower alkyl, C(O)R®, or CO,R?, wherein R® is a member selected from NR’R' and OR’, in which

R® and R'? are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; RY is H, substituted or unsubstituted lower alkyl, or C(O)R®, wherein R®is a member selected from NR°R' and OR’, in which ‘ R® and R'® are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; R? is H, or substituted or unsubstituted lower alkyl or unsubstituted heteroalkyl or cyano or alkoxy; and ® R? is H, or substituted or unsubstituted lower alkyl or unsubstituted heteroalkyl, wherein at least one of R'"'R'%, R"}, R¥ or R! links said drug to Lif present, or to F.

29. The compound of claim 2, wherein F comprises the structure: NN Hy Hu 5 : © boa ® wherein R**is selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, and unsubstituted heteroalkyl; each K is a member independently selected from the group consisting of substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO, NRZR%Z, NR®COR%, OCONR*'R%, OCOR?, and OR? wherein

R*!' and R™ are independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl; and a is an integer of 0,1, 2, 3, or 4.

30. The compound of claim 29, wherein -F (LY) comprises the structure: R24 R24 R* ® Q a | N . 24 R24 R24

_-. Yo R o) © hea wherein each R** is a member independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, and unsubstituted heteroalkyl. 3L The compound of claim 29, having the structure: ® HaC., COCH; Ra! ‘ HN R Ka R24 0] 7 Noh J xi—fLe Har) —N he ~"N"o NG Rs ple _ IL ro 2 0] R24 RS X or ay © WO 2005/112919 PCT/US2005/017804 x! SUSY 4 4 S N 5 x— Has] H— YT ~ 0 2, X wherein X' is a halogen; X is a member selected from O, S and NR%; R? is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl; and R*, R*, R’ and R*’ are members independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO,, NRR'®, NC(O)R", OC(O)NRR'S, OC(O)OR", C(O)R", OR'®, and O(CH,),N(CH3); wherein n is an integer from 1 to 20; and RY and R'® are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl, wherein R'” and R'® together with the nitrogen atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 members, optionally containing two or more heteroatoms.

32. The compound of claim 29, which is selected from the group consisting of: 180 AMENDED SHEET

NH, 2 CcQ,Me ° ° - —X JS SPONSORS WP § Ci ) 2" 0 Pa Q Oy o N , © [o} @. 5" Id CO.Me y i i i nN I NS WE NN ) "Cl. | 1 IN Y ~ 0 HY NH, ® [o] — oe PE NP ~~ "1, | 1 : Pans [e] Y ~ Q' YY [¢) o | , ON N CO,Me x1 it i A L.

NF Ab on Hm Omg On pA AN +N i | 9 AN © oxy i © [0] NH, : COsMe x Q o ‘aU Abs A ANA “1 : 9 3 0) ] ® N CO,Me 0] [®] — Xx! N = SS 0S SEH CURB: =~ 0 Oy Ne~ N gone HLT ¢} ON Y N 1 0] 0 <X SNE NS oY: Ca SO ag and NH, 0 0] J =X! (0) To 4 (a _ FCT wherein X! is Cl or Br, and Ab is an antibody, or fragment thereof. ®

33, The compound of claim 3, comprising the structure: H3CO.G CH3 xX — xfs )—{ as) Zz NH Po CJ \ N HF RS z ~( X or Xi Ty > Po CT ® NNT z ~ X wherein X! is a leaving group; 7 and X are members independently selected from O, S and NRZ, wherein R? is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl; and R? is selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted

- ub © WO 2005/112919 PCT/US2005/017804 heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO, NRR'®, NC(O)R", OC(O)NR'’R', OC(0)OR", C(O)R'?, OR", and O(CH3).N(CH3), wherein n is an integer from 1 to 20; R'> and R'® are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl, wherein R'® and R'® together with the nitrogen atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 members, optionally containing two or more heteroatoms.

34. The compound of claim 33, having the structure: 0 Cl — S cis oat Oo | b A x N Oo ~~ NS 0 HC CO,CH3 ” HN HN— Cit—Val— (PEG. A ns JID LE on 07 “A N rd nN 0 H O Ee En hi I II no a 07 “A N 183 AMENDED SHEET ; p

HaC CO,CHj 0) To —C PGA § HN—Cit—Val— pec) Ohm Ab 1 2 ~ ee ye N Oo ~ (0) A Ir or 0 ~ hn § —Cit—Val— b Jo > oP N (@) ~ 0] ~~ NA rr wherein each b is independently an integer from 0 to 20, and wherein Ab is an antibody, or fragment thereof.

35. The compound of claim 3, which is selected from the group consisting of: Li HN, pai { Y oS ) ® ie hy TU (WoT © D; {J oF i Ab Oo H nA NTS __COMs_ 1 EE HN a: HN H Yo ) 1 " Ng / AN g lo) Psy oe) IS

Lad N 1 N i: 0 i» 0 X \_NH ort" [Ses al I fal o} 0. San Ys VRS —o and QO H ® PANS 1 H 0 HN lo] OO) X y Yo wa ) RC ’ TY OF aw Ab wherein X' is Cl or Br, and Ab is an antibody, or fragment thereof.

36. The compound of claim 3, selected from the group consisting of: NH, o —~X Abe yr Og Oe, al oc ® | CATT RAA ot Re N Mo 0 = —X! Abe Ons gO SUSSUET Joy i Hx A [:} No NN mr $8 SCT NH, 0,Me 0 ~X ES ee cece ae OY He A WINS 0 | OLY and

ON N CO,Me . — x! ooo | Hs AL 0 Tl, Ne_~ x T NTO ~r oO bo wherein X' is Cl or Br, and Ab is an antibody, or fragment thereof.

37. The compound of claim 3, having the structure: ® " {2 x OCH 9 o chy NY : NOTIN SUNS SaaS OR wensty 0 N N 7 N NO NI wherein X! is Cl or Br, and Ab is an antibody, or fragment thereof.

38. A compound having the structure x8 —— (LH —H—(L")n—0D wherein D is a drug moiety having pendant to the backbone thereof a chemically reactive functional group, said function group selected from the group consisting of a primary or o secondary amine, hydroxyl, thiol, carboxyl, aldehyde, and a ketone; L! is a self-immolative linker; m is an integer selected from 0, 1, 2, 3, 4, 5, or 6; X* is a member selected from the group consisting of protected reactive functional groups, unprotected reactive functional groups, detectable labels, and targeting agents; L* is a linker member; pisOor 1; H is a linker comprising the structure:

C(R*), R24 R24 R24 R24 o | ° n, N ny | hk wherein n, is an integer from 1 — 10; nis 0, 1, or 2; ® each R* is a member independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, and unsubstituted heteroalkyl; and I is either a bond or: R24 R24 ho a3 ng 24 24 0 R R wherein nz is 0 or 1 with the proviso that when n; is 0, nz is not 0; and ny is 1,2, or3, wherein when 1 is a bond, n; is 3 and n; is 1, D can not be o HsC CO,CH3 _— —C N OR HN, 7 HO NC 0) 0 or

- _~Cl § OR Ho” NN = Io 8] where R is Me or CHz- CHz-NMes.

39. The compound of claim 38, wherein the substitution on the phenyl ring is a para ® substitution.

40. The compound according to any one of claims 38 and 39, wherein n, is 2, 3, or 4,

41. The compound of claim 40, wherein n; is 3.

42, The compound according to any one of claims 38 to 41, wherein n, is 1.

43. The compound of claim 42, wherein I is a bond.

44. The compound according to any one of claims 38 to 43, wherein H forms a 6- ® membered self immolative linker upon cleavage

45. The compound of claim 42, wherein nj is 0 and ng is 2.

46. The compound according to any one of claims 38 to 43, wherein H forms two 5- membered self immolative linkers upon cleavage.

47. The compound according to any one of claims 38 to 43, wherein H forms a 5- membered self immolative linker, H forms a 7-membered self immolative linker, or H forms a 5- membered self immolative linker and a 6-membered self immolative linker, upon cleavage.

48. The compound according to any one of claims 38 to 43, wherein H comprises the structure CR) $ o Re R24 R24 RE R% R% O 3 \ x AN N 0) N R24 R24 fo) Fo) R24 R% rR ®

49, The compound of claim 48, wherein n, is 2, 3, or 4. .

50. The compound of claim 48, wherein ny is 3.

51. The compound according to any one of claims 48 to 50, wherein each Ras is independently selected from CH; and H.

52. The compound according to any one of claims 48 to 51, wherein each Raq is H.

53. The compound according to any one of claims 38 to 43, wherein H has the structure: 2 o R R24 R24 O N Sh NT TN R* R* O boa

54. The compound of claim 53, wherein n; is 3.

55. The compound of claim 53, wherein each R* is independently selected from CH, and H.

56. The compound of claim 53, wherein H comprises the structure: Me le) | Me O \ x -N S nN" , 0 R24

57. The compound of claim 53, wherein H comprises a geminal dimethyl substitution.

58. The compound of claim 56, wherein each R?* independently an H or a substituted or unsubstituted alkyl.

59. The compound according to any one of claims 38 to 58, wherein D is a cytotoxic drug.

60. The compound according to any one of claims 38 to 59, wherein D has a chemically reactive function group selected from the group consisting of a primary or secondary amine, hydroxyl, sulfhydryl and carboxyl.

61. The compound according to any one of claims 38 to 60, wherein D is selected o from the group consisting of: duocarmycins, CC-1065, CBI-based duocarmycin analogues, MCBI-based duocarmycin analogues, CCBI-based duocarmycin analogues, doxorubicin, doxorubicin conjugates, morpholino-doxorubicin, cyanomorpholino-doxorubicin, dolastatins, dolestatin-10, combretastatin, calicheamicin, maytansine, maytansine analogues, DM-1, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), 5- benzoylvaleric acid-AE ester (AEVB), tubulysins, disorazole, epothilones, Paclitaxel, docetaxel, SN-38, Topotecan, rhizoxin, echinomycin, colchicine, vinblastin, vindesine, estramustine, cemadotin, eleutherobin, methotrexate, methopterin, dichloromethotrexate, S-fluorouracil, 6- mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, daunorubicin conjugates, mitomycin C, mitomycin A, carminomycin, aminopterin,

tallysomycin, podophyllotoxin, podophyllotoxin derivatives, etoposide, etoposide phosphate, vincristine, taxol, taxotere retinoic acid, butyric acid, N& -acetyl spermidine and camptothecin.

62. The compound according to any one of claims 38 to 61, wherein D comprises a structure: A RC i” rR? RY R* ® \ / RS wherein the ring system A is a member selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl groups; E and G are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, a heteroatom, a single bond, or E and G are joined to form a ring system selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl; X is a member selected from O, S and NR?%; R? is a member selected from H, substituted or unsubstituted alkyl, ® substituted or unsubstituted heteroalkyl, and acyl; R> is a member selected from the group consisting of (=O), SR'!, NHR! and OR", wherein R! is 2a member selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, diphosphates, triphosphates, acyl, C(O)R*R'3, C(0)OR™?, C(O)NR'?R", P(O)(OR"?),, , C(O)CHR’R**, SR" and SiR2RIRM in which

R"? RY and R'* are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted aryl, wherein R'? and R"? together with the nitrogen or carbon atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 members, optionally containing two or more heteroatoms; R*, R* Rand R>’ are members independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO, NRUR'6, NC(O)R, OC(O)NRR', OC(O)OR", C(O)R”, SRY, OR, CR!*=NR'¢, and O(CH,),N(CHs), wherein n is an integer from 1 to 20; RY and R¢ are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted peptidyl, wherein R'® and R!® together with the nitrogen atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 members, optionally containing two or more heteroatoms; Réisa single bond which is either present or absent and when present R® and R7 are joined to form a cyclopropyl ring; and ® R is CH,-X! or —CH,- joined in said cyclopropyl ring with R®, wherein X! is a leaving group, wherein at least one of R', R'2, R™, R'S or R® links said drug to L', if present, or to H.

63. The compound of claim 62, wherein D has the structure:

R? " ~ HN <5 ; R3 RY N R¢ 1259: X Zz RS RS wherein ® Z is a member selected from O, S and NR? wherein RZ is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl; R'is H, substituted or unsubstituted lower alkyl, C(O)R®, or CO,RS, wherein R® is a member selected from group consisting of substituted alkyl, unsubstituted alkyl, NR°RY, NR°NHR'?, and OR’ in which R’ and R!? are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; and R? is H, substituted alkyl or unsubstituted lower alkyl; wherein at least one of R'!, R'2, RE RP or R16 links said drug to Lif ® present, or to H.

64. The compound of claim 63, wherein R? is an unsubstituted lower alkyl.

65. The compound of claim 62, wherein D has the structure:

Ry’ A Ry R® © R® 4 2508 X z RS ks wherein Z is a member selected from O, S and NR? ® wherein R? is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl; R! is H, substituted or unsubstituted lower alkyl, C(O)R?, or CO:R%, wherein R® is a member selected from NR’R'? and OR”, in which R® and R'° are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; RY is H, substituted or unsubstituted lower alkyl, or C(O)R?, wherein R® is a member selected from NR’R!® and OR®, in which R® and R'° are members independently selected from H, substituted or ® unsubstituted alkyl and substituted or unsubstituted heteroalkyl; R? is H, or substituted or unsubstituted lower alkyl or unsubstituted heteroalkyl or cyano or alkoxy; and R? is H, or substituted or unsubstituted lower alkyl or unsubstituted heteroalkyl, wherein at least one of R!!'R'?, R"3, RY or R' links said drug to LY if present, or to H.

66. The compound according to any one of claims 38 to 65, wherein L* comprises a non-cyclic moiety.

67. The compound according to any one of claims 38 to 66, wherein L* increases solubility of the compound as compared to the compound lacking LY

68. The compound according to any one of claims 38 to 67, wherein L* decreases aggregation of the compound as compared to the compound lacking LY,

69. The compound according to any one of claims 38 to 68, wherein L* comprises a ® polyethylene glycol moiety.

70. The compound of claim 69, wherein the polyethylene glycol moiety contains 3-12 repeat units.

71. The compound of claim 70, wherein the polyethylene glycol moiety contains 2-6 repeat units.

72. The compound of claim 71, wherein the polyethylene glycol moiety contains 4 repeat units.

73. The compound of claim 63, having the structure: ® H3C CO,CH3 = X' ]? 3) ly IX RN SP AY N R2R24 O Oo R24R24 24 Z 6 X or x1 (R¥)5C RS XS ° 3 RHR2¢ R24 R?R2s R24 O p X NY oN N J x N I ng ! N N O ~~ RURH \_— 0 © RRR | ZR X

74. The compound of claim 63, having the structure: ® HaC, CO,CHj, — x! Ra 4 R* CHy R24 gas pas 0 HN Z# LS | 5° ) L P 3 x N NS o AN N | Rg’ R#* Rat N= R24 Fo) Le Iz RS or AN x? Ra) a 7 R xt, ° CHs R24 R24 R24 5 ~< TL ) 3 KX N N SN N | Rs' 24 N No© Yo R R24 = R24 0 R24 u Zz RS

75. The compound of claim 63, having the structure: H,C CO,CH, x1 J - = N CH HN z O Xfeg) o 1 [Hi fren x 2° ns SRLS fo) RS N, 1 R¥* Ru \== Oo © CH, Ir o or CO,CH / iol Px! IVAN s [0] x4 0, M HHiG, CHy PEG, fS ABE, x LD <3 RMR Ps 5 ! Hy Io wherein PEG is a polyethylene glycol moiety and X!isClor Br.

76. The compound of claim 63, having the structure: —COM_xt Ab OAS OS SO oH X ON" 0 0) | N NO Ne { 0 0 o ! / ag To wherein X! is Cl or Br, and Ab is an antibody, or fragment thereof.

77. The compound of claim 63, having a structure selected from the group consisting of: CO,Me — —X1 1 oN N ; PENI : Abr OO ° oI TN COMe x1 NH H Abr Op Ong O | bil in. ONe ° Pete o | { ® SU and COMe x1 or LX sp-N or ’ o} NTN TO N o Abs OO ° FCT ~~ wherein X! is Cl or Br, and Ab is an antibody, or fragment thereof.

78. A compound of the formula a ae wherein D is a drug moiety having pendant to the backbone thereof a chemically reactive functional group, said function group selected from the group consisting of a primary or secondary amine, hydroxyl, thiol, carboxyl, aldehyde, and a ketone; L' is a self-immolative linker; m is an integer selected from 0, 1,2, 3,4, 5, or 6; ® X* is a member selected from the group consisting of protected reactive functional groups, unprotected reactive functional groups, detectable labels, and targeting agents; L* is a linker member; PisOorl; J is a linker comprising the structure: R24 R24 Py EE Ka wherein each R?* is a member independently selected from the group consisting of ® H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, and unsubstituted heteroalkyl; cach K is a member independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO, NR?'RZ, NR?'COR%, OCONR*R%, OCOR?!, and OR! wherein R?!' and R® are independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl,

substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl and unsubstituted heterocycloalkyl; a is an integer of 0,1, 2, 3, or 4; and d is an integer of 0, 1, 2, 3, 4, 5, or 6.

79. The compound of claim 78, wherein J comprises the structure: O re J N dd R24 R24 aS EN Ky i

80. The compound of claim 78, wherein J comprises the structure: 0] ) S

81. The compound of claim 80, wherein d is 1 or 2.

82. The compound of claim 78, wherein J comprises the structure: 24 R 0) R24 R24 R24 le) Ms 7 Ka }

83. The compound of claim 82, wherein J comprises the structure: 24 R 0) rR? R% R24 S O ®

84. The compound according to any one of claims 78 to 83, wherein D is a cytotoxic : drug. :

85. The compound according to any one of claims 78 to 84, wherein D has a chemically reactive function group selected from the group consisting of a primary or secondary amine, hydroxyl, sulfhydryl and carboxyl.

86. The compound according to any one of claims 78 to 85, wherein D is selected from the group consisting of: duocarmycins, CC-1065, CBI-based duocarmycin analogues, MCBI-based duocarmycin analogues, CCBI-based duocarmycin analogues, doxorubicin, doxorubicin conjugates, morpholino-doxorubicin, cyanomorpholino-doxorubicin, dolastatins, ® dolestatin-10, combretastatin, calicheamicin, maytansine, maytansine analogues, DM-1, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethy] auristatin E (MMABE), 5- benzoylvaleric acid-AE ester (AEVB), tubulysins, disorazole, epothilones, Paclitaxel, docetaxel, SN-38, Topotecan, rhizoxin, echinomycin, colchicine, vinblastin, vindesine, estramustine, cemadotin, eleutherobin, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6- mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, daunorubicin conjugates, mitomycin C, mitomycin A, carminomycin, aminopterin, tallysomycin, podophyllotoxin, podophyllotoxin derivatives, etoposide, etoposide phosphate, vincristine, taxol, taxotere retinoic acid, butyric acid, N® -acetyl spermidine and camptothecin.

87. The compound according to any one of claims 78 to 86, wherein D comprises a structure: A) ge i. R% N i RS ® wherein the ring system A is a member selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl groups; E and G are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, a heteroatom, a single bond, or E and G are joined to form a ring system selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl; X is a member selected from O, S and NR”; R2 is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl; R® is a member selected from the group consisting of (=O), SR'!, NHR! and OR", C wherein Ris a member selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, diphosphates, triphosphates, acyl, C(O)R12R®, C(0)OR'2, C(O)NR'?R¥, P(O)(OR'*),, , C(O)CHR'?R", SR" and SiRZRPRM, : in which RY RE, and R'* are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted aryl, wherein R!? and R!? together with the nitrogen or carbon atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 members, optionally containing two or more heteroatoms; R*,R*, R® and R® are members independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO, NRPR'S, NC(O)R", OC(O)NR'’R", OC(0)ORY, C(O)R", SRY, OR!®, CR'*=NR®, and O(CH,).N(CH3), wherein n is an integer from 1 to 20; ® R'® and R'® are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted peptidyl, wherein R'S and R® together with the nitrogen atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 members, optionally containing two or more heteroatoms; RS is a single bond which is either present or absent and when present RS and R’ are joined to form a cyclopropyl ring; and R7 is CH,-X! or —CH,- joined in said cyclopropyl ring with RS, wherein X! is a leaving group, wherein at least one of R*, R', RE, RY or R® links said drug to Lif ® present, or to J.

88. The compound of claim 87, wherein D has the structure:

R? " ~~ HN a R3 R* N R FIX X Zz RS" ks wherein C Z is a member selected from O, S and NR? wherein R? is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl; R'is H, substituted or unsubstituted lower alkyl, C(O)R®, or COR, wherein R® is a member selected from group consisting of substituted alkyl, unsubstituted alkyl, NR’RY, NR*NHR', and OR’ in which R? and R™ are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; and R2is H, substituted alkyl or unsubstituted lower alkyl; wherein at least one of R'!, RZ RY, RY or R'¢ links said drug to Lif ® present, or to J.

89. The compound of claim 88, wherein R? is an unsubstituted lower alkyl.

90. The compound of claim 87, wherein D has the structure:

Ry’ re R, R® ©, R* ‘ X z RE ie wherein 7 is a member selected from O, S and NR? ® wherein R? is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl; R!is H, substituted or unsubstituted lower alkyl, C(O)R®, or CO.R?, wherein R® is a member selected from NR’R'° and OR’, in which R? and R'° are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; RY is H, substituted or unsubstituted lower alkyl, or C(O)R®, wherein R® is a member selected from NR’R! and OR’, in which R® and R'? are members independently selected from H, substituted or ® unsubstituted alkyl and substituted or unsubstituted heteroalkyl; R? is H, or substituted or unsubstituted lower alkyl or unsubstituted heteroalkyl or cyano or alkoxy; and RZ is H, or substituted or unsubstituted lower alkyl or unsubstituted heteroalkyl, wherein at least one of R' "R'2, R® RY or R!6 links said drug to Lif present, or to J.

91. The compound according to any one of claims 78 to 90, wherein L* comprises a non-cyclic moiety.

92. The compound according to any one of claims 78 to 91, wherein L* increases solubility of the compound as compared to the compound lacking LY

93, The compound according to any one of claims 78 to 92, wherein L* decreases aggregation of the compound as compared to the compound lacking L*

94, The compound according to any one of claims 78 to 93, wherein L* comprises a ® polyethylene glycol moiety.

95. The compound of claim 94, wherein the polyethylene glycol moiety contains 3-12 repeat units.

96. The compound of claim 95, wherein the polyethylene glycol moiety contains 2-6 repeat units.

97. The compound of claim 96, wherein the polyethylene glycol moiety contains 4 repeat units.

98. The compound of claim 88, having the structure: ® HsC CO,CH3 — x’ “rR 4 HN R x rl R 1% R24 R# SN 9) ~ N / R z 5 3 R . wy

Ke . or

X! aR ” ] Rq2 PY A R% R24 R24 N Oo N I ~S ZZ Tr Z RS X13 ; & | X NX Ka ®

99. A compound of claim 88, having a structure selected from the group consisting of:

H.C CO,CHs 3 __ —X oHN 2 F OMe XD NC } A O HH N° 0 Yo Pp ¢ 4H 0 bh HC, FO:LHs y1 = — E OM ~ Pig x N Ab o 1 Hey N" 0 ro TEAS ® HaC Fo x1 = OMe BEN QHsC “No NOUN AE To A o .S it ~ hn S 7) fo)

H.C CO,CH, 1 3 __ — X RN OM 2 C0 § 0 Aree s 50 HC CO,CH;, 1 3 __ — X B - OM BOR ~N JU [_ N 0 | HC H NO Yo Ab Ao FSA 0 0 ] and

H.C CO,CH, 3 _ x1 J OM oHNL 2 8 e o [HC CHa “vo he "A ~ 0} Ar No) Nas 0 50 wherein X! is Cl or Br, and Ab is an antibody, or fragment thereof. ®

100. A compound having the structure XA ——(Lp=H—(L)n—D wherein D is a drug moiety having pendant to the backbone thereof a chemically reactive functional group, said function group selected from the group consisting of a primary or secondary amine, hydroxyl, thiol, carboxyl, aldehyde, and a ketone; Lis a self-immolative linker; m is an integer selected from 0, 1,2, 3, 4, 5, or 6;

X*is a member selected from the group consisting of protected reactive functional groups, unprotected reactive functional groups, detectable labels, and targeting agents; L*is a linker member; pisOorl; H is a linker comprising the structure: RY Q I~ ~~ Al, 0) N N : ( NT q R24 R24 where qis 0, 1,2, 3,4, S, or 6; and . wherein each R* is a member independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, and unsubstituted heteroalkyl. This hydrazine structure can also form five-, six-, or seven-membered rings and additional components can be added to form multiple rings.

101. The compound of claim 100, wherein H forms a 6-membered self immolative ®o linker upon cleavage

102. The compound of claim 100, wherein H forms two 5-membered self immolative linkers upon cleavage.

103. The compound according to any one of claims 100 to 102, wherein D isa cytotoxic drug.

104. The compound according to any one of claims 100 to 103, wherein D has a chemically reactive function group selected from the group consisting of a primary or secondary amine, hydroxyl, sulfhydryl and carboxyl.

105. The compound according to any one of claims 100 to 104, wherein D is selected from the group consisting of: duocarmycins, CC-1065, CBI-based duocarmycin analogues, MCBI-based duocarmycin analogues, CCBI-based duocarmycin analogues, doxorubicin, doxorubicin conjugates, morpholino-doxorubicin, cyanomorpholino-doxorubicin, dolastatins, dolestatin-10, combretastatin, calicheamicin, maytansine, maytansine analogues, DM-1, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), 5- benzoylvaleric acid-AE ester (AEVB), tubulysins, disorazole, epothilones, Paclitaxel, docetaxel, SN-38, Topotecan, rhizoxin, echinomycin, colchicine, vinblastin, vindesine, estramustine, ® cemadotin, eleutherobin, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6- mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, daunorubicin conjugates, mitomycin C, mitomycin A, carminomycin, aminopterin, tallysomycin, podophyllotoxin, podophyllotoxin derivatives, etoposide, etoposide phosphate; vincristine, taxol, taxotere retinoic acid, butyric acid, N? -acetyl spermidine and camptothecin.

106. The compound according to any one of claims 100 to 105, wherein D comprises a structure: A R® 7 R* 3 R = ® \ J RS wherein the ring system A is a member selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl groups; E and G are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, a heteroatom, a single bond, or E and G are joined to form a ring system selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl; 209 oA Ea

X is a member selected from O, S and NR;

RZ is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl;

R® is a member selected from the group consisting of (=O), SR!! NHR!

and OR, wherein

R'is a member selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, diphosphates, triphosphates, acyl, C(O)R’R'?, C(0)OR", C(O)NR’R¥, P(O)(OR™*)z, , C(O)CHR'?R®, SR?

® and SiR1ZRVRM, in which

RY, RYE, and R'* are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted aryl, wherein R'> and R* together with the nitrogen or carbon atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 members, optionally containing two or more heteroatoms;

R*,R*, R’ and RR’ are members independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO,, NRR!, NC(O)R"®, OC(O)NR'’R", OC(O)OR', C(O)RY, SR¥, OR!S, CRI=NR'S, and O(CH,):N(CH:), ® wherein i "nis an integer from 1 to 20;

RY and R'S are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted peptidyl, wherein RY and R!® together with the nitrogen atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 members, optionally containing two or more heteroatoms;

R®is a single bond which is either present or absent and when present R® and R” are joined to form a cyclopropyl ring; and R’ is CH,-X! or —CH,- joined in said cyclopropyl ring with R®, wherein X! is a leaving group, wherein at least one of R', R™?, RB, RP or RS links said drug to LY if present, or to F.

107. The compound of claim 106, wherein D has the structure: ® ’ = HN RS . Rr? R* N R* X Z RS RS wherein Z is a member selected from 0, S and NR? wherein R? is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl; ) R! is H, substituted or unsubstituted lower alkyl, C(O)R®, or CO.R®, wherein R® is a member selected from group consisting of substituted alkyl, unsubstituted alkyl, NR’R'®, NR’NHR'?, and OR’ in which R® and R'? are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; and R? is H, substituted alkyl or unsubstituted lower alkyl; wherein at least one of R'!, R'?, RB, RP or R* links said drug to LLif present, or to H.

108. The compound of claim 107, wherein R? is an unsubstituted lower alkyl.

109. The compound of claim 106, wherein D has the structure: Ry’ fe Ry R @ RY ‘ 2504 X z RS ® 3 wherein : Z is a member selected from O, S and NR? wherein RZ is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl; R'is H, substituted or unsubstituted lower alkyl, C(O)R®, or CO,R?, wherein R® is a member selected from NR’R!? and OR, in which R® and R'° are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; RY is H, substituted or unsubstituted lower alkyl, or C(O)R®, wherein R®is C a member selected from NR°R' and OR’, in which R’ and R'® are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; R?is H, or substituted or unsubstituted lower alkyl or unsubstituted heteroalkyl or cyano or alkoxy; and R? is H, or substituted or unsubstituted lower alkyl or unsubstituted heteroalkyl, wherein at least one of RM'R'?, R", R¥ or R'® links said drug to L', if present, or to H.

110. The compound according to any one of claims 100 to 109, wherein L* comprises a non-cyclic moiety.

111. The compound according to any one of claims 100 to 110, wherein L* increases solubility of the compound as compared to the compound lacking L*

112. The compound according to any one of claims 100 to 111, wherein L* decreases ® aggregation of the compound as compared to the compound lacking LA.

113. The compound according to any one of claims 100 to 112, wherein L* comprises a polyethylene glycol moiety.

114. The compound of claim 113, wherein the polyethylene glycol moiety contains 3- 12 repeat units.

115. The compound of claim 114, wherein the polyethylene glycol moiety contains 2-6 repeat units.

116. The compound of claim 115, wherein the polyethylene glycol moiety contains 4 ® repeat units.

117. The compound of claim 109, having the structure: 0 0 /\ Ab _cl no NE in : N 0) 0] L (J) NJ N or . as O—\_ /\ Ab _Br imn— © ed o! iv : / 0 0} = N » 3 () N¢ CN © o N H he wherein Ab is an antibody, or fragment thereof.

118. A compound of the formula: xi -a—bigo wherein Lis a self-immolative linker; mis an integer 0, 1,2,3,4, 5, 0r 6; L*is a linker member, wherein L* does not comprise a carboxylic acyl group directly attached to the N-terminus of (AAY; pisOorl; X*is a member selected from the group consisting of protected reactive : C functional groups, unprotected reactive functional groups, detectable labels, and targeting agents; Q is a cleavable linker; and D!is a a drug having the following formula: Ry’ A on Ry¢' R® 7 OL N R* 4 X Z RS RS wherein X and Z are members independently selected from O, S and NR, : R23 is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl; R! is H, substituted or unsubstituted lower alkyl, C(O)R®, or CO;R", RY is H, substituted or unsubstituted lower alkyl, or C(O)R®, wherein R? is a member selected from NR’RY and OR’ and R® and R' are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; ® R? is H, or substituted or unsubstituted lower alkyl or unsubstituted heteroalkyl or cyano or alkoxy; R? is H, or substituted or unsubstituted lower alkyl or unsubstituted heteroalkyl, R3 is a member selected from the group consisting of SR'!, NHR!! and OR}, wherein RY is a member selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, diphosphates, triphosphates, acyl, C(O)R?R"*, C(O)OR"?, C(O)NR!2R", P(O)(OR'2),, , C(O)CHR’R", SR" and SiR™RR", in which R'2, R®, and R* are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted aryl, wherein R'2 and R® together with the nitrogen or carbon atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring ® system having from 4 to 6 members, optionally containing two or more heteroatoms;

wherein at least one of R!!, R'2, and R™ links said drug to L!, if present, or to Q, '

RS is a single bond which is either present or absent and when present RS and R’ are joined to form a cyclopropyl ring; and

R7 is CHp-X! or —CH,- joined in said cyclopropyl ring with R®, wherein

X! is a leaving group,

RY, R*, R® and R¥ are members independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO, NRUR'S, NC(O)R*, OC(O)NR'R'¢, OC(O)OR", C(O)R", SR'®, OR!®, CR'=NR'¢, and O(CH,),NR*‘R*’ wherein n is an integer from 1 to 20; wherein R" and R'® are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted peptidyl, wherein R' and R'® together with the nitrogen atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 members, optionally containing two or more heteroatoms; @ and R* and R? are independently selected from unsubstituted alkyl, and wherein at least one of R* , R*’, R® and R” is O(CH,)sNR**R*’.

119. The compound of claim 118, wherein n is 2.

120. The compound according to any one of claims 118 and 119, wherein R* and R® "are methyl.

121. The compound according to any one of claims 118 to 120, wherein R*,R® and R® are H and R* is O(CH),NR**R?. ®

122. The compound of claim 121, wherein R*is O(CH,);N(CHs),.

123. The compound according to any one of claims 118 to 122, wherein R, RY, RY, and R* are H.

124. The compound according to any one of claims 118 to 123, wherein the drug D! has a formula selected from:

Ry Ry’ [) ; R/ 6 1 R 7 R24 \ / of WW of 5 “R25 ® i Raz R,' Ry Ry x RA R24 \ / of W \

N . R25 n ® x1! R3 N J / ON 0) 0 x1 R3 N / SOO Re and O O ®

125. A compound of the formula Ry’ k Ry OF ‘ 2504 X Z RS RS wherein X and Z are independently selected from O, S and NR wherein R¥ is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl; . ® R! is H, substituted or unsubstituted lower alkyl, C(O)R®, or CO,R?, RY is H, substituted or unsubstituted lower alkyl, or C(O)R®, each R® is a member independently selected from NR’RY and OR’ and R® and R'® are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; R? is H, substituted or unsubstituted lower alkyl, unsubstituted heteroalkyl, cyano, or alkoxy; R?¥ is H, substituted or unsubstituted lower alkyl, or unsubstituted heteroalkyl, RS is a member selected from the group consisting of SR*!, NHR"! and OR", wherein R*! is a member selected from the group consisting of H, substituted alkyl, unsubstituted alkyl,

substituted heteroalkyl, unsubstituted heteroalkyl, diphosphates, triphosphates, acyl, C(O)RR", C(O)OR'2, C(O)NR'?R'3, P(O)(OR*),, , C(O)CHR'?R"?, SR'?and SiR'?R"R", in which R", R®, and R™ are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted aryl, or RZ and R" together with the nitrogen or carbon atom to which they are attached are joined to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 members; R® isa single bond which is either present or absent and when present Rand R are joined to form a cyclopropyl ring; and R7 is CH,-X! or ~CHj- joined in said cyclopropyl ring with RS, wherein X' is a leaving

® .. R*, R*, R® and R” are members independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO,, NRIR'S NC(O)R'*, OC(O)NR'*R'¢, OC(O)OR', C(O)R"?, SR’, OR", CR'*=NR'®, and O(CH,),NR*R* wherein n is an integer from 1 to 20; R*® and R*6 are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted peptidyl, wherein R'S and RS together with the nitrogen atom to which they are attached are optionally joined to form a substituted or unsubstituted heterocycloalkyl ring system having from 4 to 6 members, optionally containing two or more heteroatoms; and R** and R?’ are independently selected from unsubstituted alkyl, and : wherein at least one of R*, R*’, R® and R> is O(CH,).NR*R%,

126. The compound of claim 125, wherein R* is O(CHz).NR*R*.

127. The compound of claim 126, wherein R'is O(CH3),N(CH3)a.

128. The compound of claim127, wherein R*’, R® and R> are H.

129. The compound according to any one of claims 125 to 128, wherein R® is absent and R’ is CHp-X', wherein X' is F, Cl, or Br.

130. The compound according to any one of claims 125 to 129, wherein RL RY RY, and R*’ are H.

131. The compound according to any one of claims 125 to 130, wherein X isOand Z is O. ®

132. The compound according to any one of claims 125 to 131, wherein the compound has the following formula: R2 Ry Ry R¢ 6 R R7 R3 N R%* J / 0) N_ X ya R25 n :

133. The compound according to any one of claims 125 to 132, wherein the compound has the following formula: Ro Ry’ Ri Ry x1 R3 N R%4 J / ® OAM x 7 R25 n wherein X! is F, Cl, or Br.

134. The compound according to any one of claims 125 to 133, wherein the compound has the following formula: x R® ® N J / ENLN 0 Oo

135. The compound according to any one of claims 125 to 134, wherein the compound has the following formula: x R3 N / SIT ~~ ~~ AN 0) Oo

136. A pharmaceutical formulation comprising a compound according to any one of claims 1 to 135 and a pharmaceutically acceptable carrier.

137. A compound according to any one of claims 1 to 135 for use in a method of killing a cell, said method comprising administering to said cell an amount of said compound sufficient to kill said cell.

138. The compound of claim 137, wherein the cell is a tumor cell.

139. A compound according to any one of claims 1 to 135 for use in a method of retarding or stopping the growth of a tumor in a mammalian subject, comprising administering to said subject an amount of said compound , sufficient to retard or stop the growth.

140. Use of a compound according to any one of claims 1 to 135 in the manufacture of a medicament for use in killing a cell.

141. The use of claim 140, wherein the cell is a tumor cell.

142. Use of a compound according to any one of claims 1 to 135 in the manufacture of a medicament for retarding or stopping the growth of a tumor in a mammalian subject. 222 AMENDED SHEET