AU2023263495A1 - Cross-linkers and their uses - Google Patents

Abstract

CROSS-LINKERS AND THEIR USES Abstract Charged or pro-charged cross-linking moieties and conjugates of cell binding agents and drugs comprising the charged or pro-charged cross-linking moieties and method of making the same.

Description

CROSS-LINKERS AND THEIR USES

This application claims priority to United States Provisional

Application No. 61/049,291, filed April 30, 2008 and to United States

Provisional Application No. 61/147,966, filed January 28, 2009.

FIELD OF THE INVENTION

[01] The present invention relates to the synthesis of novel charged

cross linkers and cross linkers which can be processed by a target cell to give

charged moieties. The present invention also relates to methods of making

cell-binding agent-drug conjugates comprising modification of cell-binding

agents with these cross-linkers, followed by reaction with drugs, or

modification of the drugs with these crosslinkers, followed by reaction with

cell-binding agents. The improved method of making conjugates provides the

ability to link a higher number of drug molecules per cell-binding agent

resulting in greater potency and providing greater aqueous solubility to the

conjugates.

BACKGROUND OF THE INVENTION

[021 The bifunctional modification reagent N-succinimidyl 3-(2

pyridyldithio) propionate (SPDP) has been used to link two proteins together

through a disulfide bond. The reagent is reacted with the first protein to

introduce an active disulfide-containing group in the modification step. A

second protein, which contains a free thiol group, is then added to form a disulfide bond between the two proteins in the conjugation step. Many derivatives of SPDP and imide versions of SPDP have been described (U.S.

Patent 4,563,304; J. Carlsson et al. 173 Biochem. J. 723-737 (1978); Goff D.

A., Carroll, S. F. 1 BioConjugate Chem. 381-386 (1990); L. Delprino et al. 82

J. Pharm. Sci. 506-512 (1993); S. Arpicco et al., 8 BioConjugate Chem

327-337 (1997)).

[03] Conjugates of cell-binding agents with highly cytotoxic drugs

have been described (U.S. Patent Nos. 5,208,020, 5,416,064; 5,475,092,

5,585,499, 6,436,931, 6,372,738 and 6,340,701; R.V.J. Chari et al., 52 Cancer

Res. 127-131 (1992)). In these conjugates, the cell-binding agents are first

modified with a bifunctional agent such as SPDP, SPP or SMCC to introduce

an active disulfide or a maleimido moiety. Reaction with a thiol-containing

cytotoxic drug provides a conjugate in which the cell-binding agent, such as a

monoclonal antibody, and drug are linked via disulfide bonds or thioether

bonds.

[041 Heterobifunctional cross-linkers comprising a

nitropyridyldithio, dinitropyridyldithio, N.N-dialkylcarboxamidopyridyldithio

or di-(N.N-dialkylcarboxamido) pyridyldithio group and a reactive carboxylic

ester group such as aN-succinimidyl ester group or a N-sulfosuccinimidyl

ester group have been described (U.S. Patent No. 6,913,748). The presence of

a N-sulfosuccinimidyl group was claimed to provide higher aqueous solubility

to these cross-linkers. However, once the cell-binding agent has been reacted with these cross-linkers, the N-sulfosuccinimidyl group is displaced and the solubility advantage is lost, both for the modified cell-binding agent and its drug conjugate. Since cytotoxic drugs used in cell-binding agent-drug conjugates are often only sparingly soluble in aqueous solutions, it is often difficult to link a sufficient number of drug molecules to the cell-binding agent and still maintain aqueous solubility. In addition, reactions have to be conducted in dilute solutions, which are cumbersome to scale up because of the need to use large volumes of solution.

SUMMARY OF THE INVENTION

1051 The present invention provides charged linkers, wherein the

charges are retained both after modification of the cell-binding agent and in

the resulting drug conjugate. More specifically, the present invention relates

to the use of charged linkers to link drugs to a cell-binding agent (e.g., an

antibody). In one aspect of the invention, the charged linkers are used to

modify cell-binding agents and link them to drugs. In another aspect of the

invention, the charged linkers are used to modify drugs and link them to cell

binding agents. In yet another aspect of the invention, the charged linkers are

used to simultaneously link drugs and the cell-binding agents. In all instances,

the preferred end result is a drug-charged linker-cell-binding agent conjugate,

which can be represented by the formula, CB-(-L°-D)q, wherein CB is a cell

binding agent, Lcis a charged linker, D is a drug molecule, and q is an integer

from 1 to 20. The presence of a charged group(s) in the linker in the cell binding agent-drug conjugate provides several advantages, such as i) greater water solubility of the final product, ii) ability to operate at a higher concentration in aqueous solutions, iii) ability to link a greater number of drug molecules per molecule of cell-binding agent, resulting in higher potency, iv) potential for the charged conjugate species to be retained inside the target cell, resulting in higher potency, and v) improved sensitivity of multidrug resistant cells, which would be unable to export the charged drug species from the cell.

The invention also describes linkers, which can be coupled to a drug and a cell

binding agent to give a conjugate which can be metabolized in a cell to

produce a drug metabolite containing one or more charged moieties. These

linkers will be referred to as pro-charged linkers. Moieties of the linker which

will become charged after cell processing will be referred to as pro-charged

moieties.

[06] In one aspect of the present invention, the charged or pro

charged cross linker is represented by formula (I) wherein Y' can react with a

cell-binding agent and Q can react with a cytotoxic drug:

R7 8 R3 R4

Y' Zs Q

R9 RIO R5R 6 R, R2

(I)

wherein:

Y' represents a functional group that enables reaction with a

cell-binding agent;

Q represents a functional group that enables linkage of a

cytotoxic drug via a disulfide, thioether, thioester, peptide, hydrazone, ether,

ester, carbamate or amide bond;

R 1, R 2, R 3, R 4, R 5, R 6, R 7, R8, R9, and Rio are the same or

different and are H, linear alkyl having from 1-6 carbon atoms, branched or

cyclic alkyl having from 3 to 6 carbon atoms, linear, branched or cyclic

alkenyl or alkynyl having from 2 to 6 carbon atoms, anions, such as but not 2 2 2 2 limited to, S0 3~, X-S3, OP0 3 -, X-OP0 3 , P0 3 , X-P0 3 , CO2, and cations,

such as but not limited to, a nitrogen containing heterocycle, N*R 1 R 12R1 3 or

X-N*R 1 R 12R 31, or a phenyl, wherein:

Ra, R 12 and R13 are the same or different and are H, linear

alkyl having from 1 to 6 carbon atoms, or branched or cyclic alkyl having

from 3 to 6 carbon atoms and X represents phenyl or a linear alkyl having

from 1 to 6 carbon atoms, or a branched or cyclic alkyl having from 3 to 6

carbon atoms;

1, m and n are 0 or an integer from 1 to 4; and

A is a phenyl or a substituted phenyl, wherein the substituent is

a linear alkyl having from 1 to 6 carbon atoms, or a branched or cyclic alkyl

having from 3 to 6 carbon atoms, or a charged substituent selected from 2 2 anions, such as but not limited to, S0 3~, X-SO3~, OPO2-, X-OPO 3 -, P0 3 , X

P0 32-, C0 2~, and cations, such as but not limited to, a nitrogen containing

heterocycle, NR11R 12 R 13 or X-N+R 1 1R 12R 31, wherein X has the same

definition as above, and wherein g is 0 or 1;

Z is an optional polyethyleneoxy unit of formula (OCH 2CH 2)p,

wherein p is 0 or an integer from 2 to about 1000, or F-El-P-E2-F2 unit in

which El and E2 are the same or different and are C=,0, or NR 14, wherein

R 14 is H, a linear alkyl having from 1-6 carbon atoms, a branched or cyclic

alkyl having from 3 to 6 carbon atoms, a linear, branched or cyclic alkenyl or

alkynyl having from 2 to 6 carbon atoms; P is a peptide unit between 2 and 20

amino acids in length, wherein El or E2 can be linked to the peptide through

the terminal nitrogen, terminal carbon or through a side chain of one of the

amino acids of the peptide; and F Iand F2 are the same or different and are an

optional polyethyleneoxy unit of formula (OCH 2CH2)p, wherein p is 0 or an

integer from 2 to about 1000, provided that when Z is not F1-El-P-E2-F2, at

least one of R 1, R 2, R 3, R4, R5 , R 6, R7, R8 , R9 , and RIO is a charged substituent

5 , R 6, R 7, R8 , R9, and Rio is a 1 , R 2, R3, R 4, R or when g is 1, at least one of A, R

charged substituent.

[07] In another aspect, the present invention provides a cell-binding

agent-drug conjugate of formula (II), in which the cell-binding agent, CB, and

the drug, D, have reacted at the two ends of the charged or pro-charged cross

linker:

R 7 R8 R3 R 1 Y

R_99 RIOR i RP mW Z

R, D

R2 j q 5&

(II)

wherein:

CB represents a cell-binding agent;

D represents the drug linked to the cell-binding agent by a disulfide,

thioether, thioester, peptide, hydrazone, ether, ester, carbamate, or amide

bond;

R 1, R2, R3, R 4 , R 5, R 6 , R7, R 8, R9, and Rio are the same or different and

are H, linear alkyl having from 1-6 carbon atoms, branched or cyclic alkyl

having from 3 to 6 carbon atoms, linear, branched or cyclic alkenyl or alkynyl

having from 2 to 6 carbon atoms, anions, such as but not limited to, S0 3 -. X

2 S03 . OP0 32 , X-OP0 32 -, P0 32 , X-P0 3 , C0 2 , cations, such as but limited to,

a nitrogen containing heterocycle, N+RR 12R 13 or X-N+RnR12R13, or a

phenyl, wherein:

Ro, R 12 and RI3 are the same or different and are H, linear

alkyl having from 1 to 6 carbon atoms, branched or cyclic alkyl having from 3

to 6 carbon atoms and X represents phenyl or a linear alkyl having from 1 to 6

carbon atoms, or a branched or cyclic alkyl having from 3 to 6 carbon atoms;

1, m and n are 0 or an integer from 1 to 4; and

A is a phenyl or substituted phenyl, wherein the substituent is a

linear alkyl having from 1 to 6 carbon atoms, or a branched or cyclic alkyl

having from 3 to 6 carbon atoms, or a charged substituent selected from

anions, such as but not limited to, S03~. X-S3-. OP032-, X-OP0 32-, P0 32-, X

P0 3 2 , C0 2-, cations, such as but not limited to, a nitrogen containing

heterocycle, N+R 1 R 12R 31 or X-N*RIR1 2RI3, wherein X has the same

definition as above, and wherein g is 0 or 1;

Z is an optional polyethyleneoxy unit of formula (OCH 2CH2)p,

wherein p is 0 or an integer from 2 to about 1000, or F1-El-P-E2-F2 unit in

which El and E2 are the same or different and are C=O,0, or NR14, wherein

R 14 is H, a linear alkyl having from 1-6 carbon atoms, a branched or cyclic

alkyl having from 3 to 6 carbon atoms, a linear, branched or cyclic alkenyl or

alkynyl having from 2 to 6 carbon atoms; P is a peptide unit between 2 and 20

amino acids in length, wherein El or E2 can be linked to the peptide through

the terminal nitrogen, terminal carbon or through a side chain of one of the

amino acids of the peptide; and Fl and F2 are the same or different and are an

optional polyethyleneoxy unit of formula (OCH 2CH2)p, wherein p is 0 or an

integer from 2 to about 1000, provided that when Z is not F1-El-P-E2-F2, at

least one of R1 , R2, R3, R4, R5 , R6, R7, Rs, R9, and Rio is a charged substituent

or when g is 1, at least one of A, 1R , R2, R3, 4, R5, R6, R7, R8, R 9, and RiO is a

charged substituent;

Y represents a carbonyl, thioether, amide, disulfide, or

hydrazone group; and q represents an integer from 1 to 20.

[081 In a further aspect, the present invention provides a modified

cell-binding agent of formula (III), in which the cell-binding agent, CB, has

reacted with the cross linker, which still has Q, a group capable of reacting

with a cytotoxic drug:

. R7 R3 1 ~

Zn zY Q

. R9 RIO R5R"6R R2 _q

(III)

wherein the substituents are as defined above.

[09] In an even further aspect, the present invention provides a

modified drug of formula (IV), in which the drug, D, has reacted with the

cross linker, which still has Y', a group capable of reacting with the cell

binding agent:

R 7 R8 R3 R4 Y' Z' D 1m AAg R 9 R 10 R 5 R 6 R1 R2

(IV)

wherein the substituents are as defined above.

[10] The present invention further relates to a method of making a

cell-binding agent drug conjugate of formula (II), wherein the drug is linked to

a cell-binding agent via a charged or pro-charged linker.

[11] The present invention also relates to a method of making a

modified cell-binding agent of formula (II), wherein the cell-binding agent is

reacted with the charged or pro-charged linker.

[12] The present invention also relates to a method of making a

modified drug of formula (IV), wherein the drug is reacted with the charged or

pro-charged linker.

[13] The present invention includes a composition (e.g., a

pharmaceutical composition) comprising conjugates or derivatives thereof

(and/or solvates, hydrates and/or salts thereof) and a carrier (a

pharmaceutically acceptable carrier). The present invention also includes a

composition (e.g., a pharmaceutical composition) comprising conjugates or

derivatives thereof, (and/or solvates, hydrates and/or salts thereof) and a

carrier (a pharmaceutically acceptable carrier), further comprising a second therapeutic agent. The present compositions are useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (e.g., human).

[14] The present invention includes a method of inhibiting abnormal

cell growth or treating a proliferative disorder in a mammal (e.g., human)

comprising administering to said mammal a therapeutically effective amount

of the conjugates or derivatives thereof, (and/or solvates and salts thereof) or a

composition thereof, alone or in combination with a second therapeutic agent.

[15] The compounds of this invention, derivatives thereof, or

conjugates thereof, and compositions comprising them, are useful for treating

or lessening the severity of disorders, such as, characterized by abnormal

growth of cells (e.g., cancer). Other applications for compounds or conjugates

of this invention include, but are not limited to, treating osteoporosis,

depression, anxiety, stress, phobias, panic, dysphoria, psychiatric disorders,

and pain or as antiepileptics, antibacterials, diuretics and hypotensives,

hypolipidemics, and anti-depressants.

BRIEF DESCRIPTION OF THE DRAWINGS

[16] Figure 1 shows the synthesis of sulfonic acid-containing cross

linking reagents that contain a nitropyridyldisulfide group and a reactive

carboxylic acid ester. Hydroxyalkanoate esters are first converted into dibromoalkanoate esters as shown, followed by conversion of the a-bromo substituent into a sulfonic acid.

[17] Figure 2 shows the synthesis of sulfonic acid-containing cross

linking reagents that contain a pyridyldisulfide group and a reactive carboxylic

acid ester.

[181 Figures 3, 4 and 5 show various routes for the synthesis of

charged cross-linking agents bearing a reactive carboxylic acid ester and

maleimido substituent, enabling linkage via thioether bonds.

[19] Figures 6 and 7 show the synthesis of phosphate-containing

cross-linking reagents that contain a pyridyldisulfide group and a reactive

carboxylic acid ester.

[201 Figure 8 shows the synthesis of phosphate-containing cross

linking reagents that contain a nitropyridyldisulfide group and a reactive

carboxylic acid ester

[21] Figures 9 and 10 show different routes for the synthesis of

phosphate-containing charged cross-linking agents bearing a reactive

carboxylic acid ester and a maleimido substituent, enabling linkage via

thioether bonds.

[22] Figure I Ishows the synthesis of sulfonic acid-containing

cross-linking reagents, where the sulfonate substituent is attached to a

branched alkyl group. These reagents also bear a pyridyldisulfide group and a

reactive carboxylic acid ester.

[231 Figure 12 shows the synthesis of sulfonic acid-containing

cross-linking reagents, where the sulfonate substituent is attached to a

branched alkyl group. These reagents also bear a reactive carboxylic acid

ester and a maleimido group that allows for linkage via thioether bonds.

[24] Figure 13 shows the synthesis of quartenary amine-containing

cross-linking reagents that contain a pyridyldisulfide group and a reactive

carboxylic acid ester.

[25] Figure 14 shows the synthesis of quartenary amine cross

linking agents bearing a reactive carboxylic acid ester and maleimido

substituent, enabling linkage via thioether bonds.

[26] Figure 15 shows the synthesis of sulfonic acid-containing

cross-linking reagents that contain a pyridyldisulfide group and a reactive

carboxylic acid ester. In these compounds, the sulfonate substituent is on the

carbon atom on the position P to the carboxyl ester.

[27] Figure 16 shows the synthesis of phosphate-containing cross

linking reagents that contain a pyridyldisulfide group and a reactive carboxylic

acid ester. In these compounds, the phosphate substituent is on the p-position

relative to the carboxyl ester.

[28] Figures 17, 18 and 19 show the synthesis of various sulfonic

acid-containing cross-linking reagents that contain a polyethyleneglycol

(PEG) chain, along with a nitropyridyldisulfide group and a reactive

carboxylic acid ester.

[29] Figures 20 and 21 show the synthesis of various sulfonic acid

containing cross-linking reagents that contain a polyethyleneglycol (PEG)

chain, along with a maleimido group and a reactive carboxylic acid ester.

[30] Figure 22 shows the synthesis of phosphate-containing cross

linking reagents, where the phosphate substituent is attached to an aromatic

group. These reagents also bear a reactive carboxylic acid ester and a

nitropyridyldithio group that allows for linkage via disulfide bonds.

[31] Figure 23 shows the synthesis of phosphate-containing cross

linking reagents, where the phosphate substituent is attached to a branched

alkyl group. These reagents also bear a reactive carboxylic acid ester and a

nitropyridyldithio group that allows for linkage via disulfide bonds.

[32] Figures 24 - 31 show the synthesis of sulfonate-containing

cross-linking reagents that also incorporate a hydrazide moiety allowing for

linkage via acid-labile bonds.

[33] Figures 32 - 36 show the synthesis of phosphate-containing

cross-linking reagents that also incorporate a hydrazide moiety allowing for

linkage via acid-labile bonds.

[34] Figures 37 - 38 show the synthesis of quartenary amine

containing cross-linking reagents that also incorporate a hydrazide moiety

allowing for linkage via acid-labile bonds.

[351 Figures 39 - 42 show the synthesis of charged cross-linking

reagents that also incorporate a polyethyleneglycol (PEG) moiety.

[361 Figures 43-44 show the synthesis of phosphate-containing

cross-linking reagents, where the phosphate substituent is attached to an

aromatic residue or to an alkyl group. These reagents also bear a reactive

carboxylic acid ester and a nitropyridyldithio group that allows for linkage via

disulfide bonds.

[37] Figures 45-49 show the synthesis of charged cross-linking

agents bearing reactive carboxylic acid ester and a haloacetyl substituent,

enabling linkage via thioether bonds.

[38] Figure 50 shows the synthesis of a procharged linker that would

generate a negatively charged carboxylate metabolite.

[39] Figure 51 shows a conjugate of linker 158 to a drug and a

monoclonal antibody and how the conjugate would be processed in the

lysosome of a target cell to give a metabolite containing the drug bearing a

negatively charged carboxylate.

[40] Figure 52 shows the synthesis of a procharged linker that would

generate a positively charged amine-containing metabolite.

[41] Figure 53 shows a conjugate of a procharged linker to a drug

and a monoclonal antibody and how the conjugate would be processed in the

lysosome of a target cell to give a metabolite of the drug bearing a positively

charged amine.

[421 Figure 54 shows the synthesis of a procharged linker that would

generate a charged carboxylate metabolite.

[43] Figure 55 shows a conjugate of linker 172 to a drug and a

moloclonal antibody and how the conjugate would be processed in the

lysosome of a target cell to give a metabolite containing the drug bearing a

carboxylic acid and a lysine residue.

[441 Figure 56 shows the use of charged linker in modifying a cell

binding agent and producing a cell-binding agent-drug conjugate bearing a

charged linker.

[45] Figures 57(A), (B) and (C) show the in vitro potency of cell

binding agent-drug conjugates in which a charged crosslinker is incorporated.

[46] Figure 58 shows the in vitro potency and target selectivity of

cell-binding agent-drug conjugates bearing a charged crosslinker.

[47] Figure 59 shows the mass spectrum of cell-binding agent-drug

conjugates bearing a charged crosslinker.

[48] Figure 60 shows the cytotoxicity of Anti-CanAg (huC242)

sulfonate linker-maytansinoid conjugates with increasing maytansinoids load

(E:A) toward COL0205 cells.

[49] Figure 61 shows the cytotoxicity of Anti-CanAg (huC242)

sulfonate linker-maytansinoid conjugates with increasing maytansinoids load

(E:A) toward multi-drug resistant COL0205-MDR cells.

[50] Figure 62 compares cytotoxicity of Anti-CanAg (huC242)

maytansinoid conjugates with or without sulfonate group in the linker toward

multi-drug resistant COL0205-MDR cells.

[51] Figure 63 compares the cytotoxicity of Anti-EpCAM (B38.1)

maytansinoid conjugates with or without sulfonate group in linker toward

multi-drug resistant COL0205-MDR cells.

[52] Figure 64 compares the cytotoxicity of Anti-EpCAM (B38.1)

maytansinoid conjugates with or without sulfonate group in linker toward

multi-drug resistant HCT15 cells.

[53] Figure 65 compares the cytotoxicity of Anti-EpCAM (B38.1)

maytansinoid conjugates with or without sulfonate group in linker toward

multi-drug resistant COL0205-MDR cells.

[541 Figure 66 shows the in vivo anti-tumor activity of anti-EpCAM

antibody-maytansinoid conjugates on COL0205 mdr xenografts (individual

tumors).

[551 Figure 67 shows the in vivo anti-tumor activity of anti-EpCAM

antibody-maytansinoid conjugates on COL0205 xenografts (individual

tumors).

[56] Figures 68 - 70 show the methods of synthesis of sulfonic acid

containing cross-linking reagents. These reagents bear a reactive carboxylic

acid ester and a maleimido group that allows for linkage via thioether bonds.

157] Figure 71 shows the methods of synthesis of quartenary amine

containing cross-linking reagents. These reagents also bear a reactive

carboxylic acid ester and a pyridyldithio group that allows for linkage via

disulfide bonds.

[58] Figures 72(A) and (B) show Plasma pharmacokinetics of

huC242 Antibody-Sulfo-Mal-[ 3H-labeled]-DM4 conjugates with 3.5 DM4/Ab

or 6.4 DM4/Ab dosed at 12.9 mg/kg and 7.9 mg/kg (i.v.) respectively in CD-1

mice. A. Ab concentrations (measured by ELISA or by 3H counts) versus

time after administration. B. Maytansinoid (DM4)/Antibody (Ab) ratio

versus time after administration.

[59] In Figures 1-71, where applicable, n reperesents 0 or an integer

from 1 to 10, and m represents 0 or an integer from 1 to 2000.

DETAILED DESCRIPTION OF THE INVENTION

[60] The novel conjugates disclosed herein use charged or pro

charged cross-linkers. Examples of some suitable cross-linkers and their

synthesis are shown in Figures 1 to 10. Preferably, the charged or pro-charged

cross-linkers are those containing sulfonate, phosphate, carboxyl or quaternary

amine substituents that significantly increase the solubility of the modified

cell-binding agent and the cell-binding agent-drug conjugates, especially for

monoclonal antibody-drug conjugates with 2 to 20 drugs/antibody linked.

Conjugates prepared from linkers containing a pro-charged moiety would produce one or more charged moieties after the conjugate is metabolized in a cell.

Cross-linkers

[61] The synthetic routes to produce charged crosslinkers of the

present invention are shown in Figures 1-49. Synthetic routes to produce

linkers with pro-charged moieties are shown in figures 50, 52, and 54. Figures

51, 53 and 55 show a conjugate of each of the respective pro-charged linkers

with a drug and a monoclonal antibody and how these conjugates would be

metabolized in a target cell to give charged metabolites. The crosslinkers

possess three elements: a) a substituent that is either charged or will become

charged when conjugates employing these linkers are metabolized in cells.

The charge will be either anionic, such as but not limited to, carboxylate,

sulfonate or phosphate, or cationic, such as but not limited to, a tertiary,

quaternary, or primary amine or a nitrogen-containing heterocycle, b) a group,

such as a N-hydroxysuccimimide ester, maleimido group, haloacetyl group,

and hydrazide, capable of reaction with a cell-binding agent, and c) a group,

such as but not limited to, a disulfide, maleimide, haloacetyl, and hydrazide,

capable of reaction with a drug. The charged or pro-charged substituent can

be introduced by methods described herein. For example, a sulfonate charge

can be introduced by first treating a commercially available haloester

compound with thioacetate to produce a thioacetyl compound, followed by

oxidation of the thioacetyl group, using hydrogen peroxide, to a sulfonate group. Phosphate containing crosslinkers can be synthesized by methods described herein. First the desired reactive group, such as but not limited to, thiol, maleimide, haloacetyl and hydrazide, is introduced by the reactions shown in Figures 6-10, followed by hydrolysis of the phosphate ester to give the charged crosslinker bearing a phosphate. A positively charged quaternary amine substituent can be introduced in the crosslinker by reaction of an amine with an a,p-unsaturated ketone (see, for example, Figures 13 and 37).

Alternatively a charged amine substituent can be introduced by displacement

of a halogen with the amine or nitrogen containing heterocycle of choice.

[621 Preferably, the cross-linkers are compounds of the formula (I)

below:

R 7 R8 R3 R4 Y' Z Q 1 A m R 9 RIO R 5 R6 R, R2

(I)

wherein Y' represents a functional group that enables reaction

with a cell-binding agent;

Q represents a functional group that enables linkage of a drug

via a disulfide, thioether , thioester, peptide, hydrazone, ester, ether, carbamate

or amide bond;

R 1, R 2, R 3, R4, R 5, R 6, R7, R 8, R9, and RIO are the same or

different and are H, linear alkyl having from 1-6 carbon atoms, branched or

cyclic alkyl having from 3 to 6 carbon atoms, linear, branched or cyclic

alkenyl or alkynyl having from 2 to 6 carbon atoms, anions, such as but not

limited to, So3 -, X-SO3~, OPO32-, X-OPO 2 ,P 2 , X-P0 2 , CO 2-, cations, 3 3 3

such as but not limited to, a nitrogen containing heterocycle, N*RR 1 2R1 3 or

X-N*R 11R12R 13 or a phenyl, wherein:

R 11, R 12 and R 13 are the same or different and are H, linear

alkyl having from 1 to 6 carbon atoms, or branched or cyclic alkyl having

from 3 to 6 carbon atoms and X represents phenyl or a linear alkyl having

from 1 to 6 carbon atoms, or a branched or cyclic alkyl having from 3 to 6

carbon atoms;

1, m and n are 0 or an integer from 1 to 4;

A is a phenyl or substituted phenyl, wherein the substituent is a

linear alkyl having from 1 to 6 carbon atoms, or a branched or cyclic alkyl

having from 3 to 6 carbon atoms, or a charged substituent selected from 2 2 2 anions, such as but not limited to, SO. X-S0 3 . OP0 3 , X-OP0 3 , P0 3 ,X

P0 32 , C0 2-, and cations, such as but not limited to, a nitrogen containing

heterocycle, N*R 11R 12R 31 or X-N*R11R12R 3, wherein X has the same

definition as above, and wherein g is 0 or 1;

Z is an optional polyethyleneoxy unit of formula (OCH 2 CH 2 )p,

wherein p is 0 or an integer from 2 to about 1000, or F1-El-P-E2-F2 unit in which El and E2 are the same or different and are C=O,0, or NR14, wherein

R 14 is H, a linear alkyl having from 1-6 carbon atoms, a branched or cyclic

alkyl having from 3 to 6 carbon atoms, a linear, branched or cyclic alkenyl or

alkynyl having from 2 to 6 carbon atoms; P is a peptide unit between 2 and 20

amino acids in length, wherein El or E2 can be linked to the peptide through

the terminal nitrogen, terminal carbon or through a side chain of one of the

amino acids of the peptide; and Fl and F2 are the same or different and are an

optional polyethyleneoxy unit of formula (OCH2CH2)p, wherein p is 0 or an

integer from 2 to about 1000, provided that when Z is not F-El-P-E2-F2, at

least one of R1 , R2, R3, R4, R5 , R 6 , R 7, R8 , R9 , and Rio is a charged substituent

or when g is 1, at least one of A, R 5 , R6, R 7 , R8, R9, and Rio is a 1 , R 2 , R 3 , R4 , R

charged substituent.

[631 Examples of the functional group, Y', that enables reaction

with a cell-binding agent include amine reacting agents such as but not limited

to N-hydroxysuccinmide esters, p-nitrophenyl esters, dinitrophenyl esters,

pentafluorophenyl esters; thiol reactive agents such as but not limited to

pyridyldisulfides, nitropyridyldisulfides, maleimides, haloacetates and

carboxylic acid chlorides.

[64] Examples of the functional group, Q, which enables linkage of

a cytotoxic drug, include groups that enable linkage via a disulfide, thioether,

thioester, peptide, hydrazone, ester, carbamate, or amide bond. Such functional groups include, but are not limited to, thiol, disufide, amino, carboxy, aldehydes, maleimido, haloacetyl, hydrazines, and hydroxy.

[65] Examples of linear alkyls include methyl, ethyl, propyl, butyl,

pentyl and hexyl. Examples of branched or cyclic alkyls having 3 to 6 carbon

atoms include isopropyl, sec-butyl, isobutyl, tert-butyl, pentyl, hexyl,

cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[66] Examples of linear alkenyls having 2 to 6 carbon atoms

includeethenyl, propenyl, butenyl, pentenyl, hexenyl. Examples of branched

or cyclic alkenyls having 2 to 6 carbon atoms include isobutenyl, isopentenyl,

2-methyl-I-pentenyl, 2-methyl-2-pentenyl.

[67] Examples of linear alkynyls having 2 to 6 carbon atoms include

ethynyl, propynyl, butynyl, pentynyl, hexynyl. Examples of branched or

cyclic alkynyls having up to 6 carbon atoms include 3-methyl--butynyl, 3

methyl-i-penynyl, 4-methyl-2-hexynyl.

[681 In preferred embodiments, one of R 1, R2 , R3, R4, R9, RIO is a

charged substituent selected from sulfonate, phosphate or trialkylammonium,

and the rest are H, 1, g and m are each 0, n = 1, Q and Y' are each

independently, a disulfide substituent, a maleimido, a haloacetyl group, or a

N-hydroxy succinimide ester. In another more preferred embodiment, one of

R 1, R 2, R3, R 4, R 9, Rio is a sulfonate, and the rest are H, 1, g and m are each 0,

n = 1, Q is a disulfide, maleimido or haloacetyl moiety, and Y' is a maleimido

moiety or a N-hydroxy succinimide ester. In a further more preferred

1 R9 , Rio is a sulfonate, and the rest are H, 1, embodiment, one of R 1, R2, R3, 4,

g and m are each 0, n = 1, Q is a pyridyldithio or nitropyridyldithio group,

maleimido or haloacetyl moiety, and Y' is a N-hydroxy succinimide ester.

[69] The synthesis of 2-dithionitropyridyl and 2-dithio

dinitropyridyl containing cross-linkers of formulae (I) is shown, for example,

in Figures 1, 2 and the synthesis of the corresponding 4-dithionitropyridyl and

4-dithio-dinitropyridyl containing cross-linkers of the formula (I) is shown, for

example, in Figure 6. The synthesis of maleimido-containing charged cross

linkers of the formula (I) with a sulfonate group is shown, for example, in

Figures 3, 4 and 5. The synthesis of maleimido-containing charged cross

linkers of the formula (I) with a phosphate group is shown, for example, in

Figures 9 and 10. The synthesis of quaternary amine-containing charged

crosslinkers of formula (I) is shown, for example, in Figures 13 and 14. The

synthesis of polyethylene glycol-containing charged cross linkers of formula

(I) are shown, for example, in Figures 17 -21. The synthesis of charged cross

linkers of formula (I) bearing a hydrazide moiety enabling linkage via acid

labile bonds is shown, for example, in Figures 24-36.

Cell-binding agent drug -conjugates

[70] Using the charged or pro-charged crosslinkers a high number

(>6) of drug molecules can be introduced. In non limiting examples, Figure

57 exemplifies that cell-binding agent-drug conjugates prepared using a

charged crosslinker of the present invention display high potency. In addition, the potency is target selective (see, for example, Figure 58), since, even after linkage of a high number of drug molecules, the conjugate is highly potent towards target cells, but much less potent towards non-target cells. As exemplified in Figure 59, mass spectral analysis demonstrates that the drugs are linked covalently to the cell-binding agent via the charged crosslinker.

[71] The conjugates of the present invention can be represented by

the following formula, CB-(-L-D), wherein CB is a cell-binding agent, L' is

a charged or pro-charged linker, D is a drug molecule, and q is an integer from

i to 20.

[72] Preferably, the conjugates have the following formula (H):

R7 R 8 R3 D 1 Y

R9 RIO R5R 6 m D ], R2 _ q

(II)

wherein CB represents a cell-binding agent,

D represents a drug linked to the cell-binding agent by a

disulfide, thioether, thioester, peptide, hydrazone, ester, carbamate or amide

bond;

R 1, R2, R3, R4, R 5, R,, R 7, R 8, R9 , and RIO are the same or

different and are H, linear alkyl having from 1-6 carbon atoms, branched or

cyclic alkyl having from 3 to 6 carbon atoms, linear, branched or cyclic

alkenyl or alkynyl having from 2 to 6 carbon atoms, anions, such as but not

limited to, So 3 . X-S0 3 . OP0 3 , X-OP0 3 , P0 32 -, X-P0 32 , C0 2 , cations,

such as but not limited to, a nitrogen containing heterocycle, N RiIR12R13 or

X-N'R 1 R 12R 3 , or a phenyl, wherein:

RI, R1 2 and R 13 are same or different and are H, linear alkyl

having from 1 to 6 carbon atoms, branched or cyclic alkyl having from 3 to 6

carbon atoms and X represents phenyl or a linear alkyl having from 1 to 6

carbon atoms, or a branched or cyclic alkyl having from 3 to 6 carbon atoms;

1, m and n are 0 or an integer from I to 4;

A is a phenyl or substituted phenyl, wherein the substituent is a

linear alkyl having from 1 to 6 carbon atoms, or a branched or cyclic alkyl

having from 3 to 6 carbon atoms, or a charged substituent selected from 2 anions, such as but not limited to, S03~. X-S0 3 ~. OP0 3 , X-OP0 3 2-, P0 32-, X

P0 32 , C0 2 ~, cations, such as but not limited to, a nitrogen containing

heterocycle, N+RR 12R 13 or X-N*R 1 R 12R 3, wherein X has the same

definition as above, and wherein g is 0 or 1;

Z is an optional polyethyleneoxy unit of formula (OCH 2 CH2 )p,

wherein p is 0 or an integer from 2 to about 1000, or F-El-P-E2-F2 unit in

which El and E2 are the same or different and are C=O,0, or NR14, wherein

R 14 is H, a linear alkyl having from 1-6 carbon atoms, a branched or cyclic

alkyl having from 3 to 6 carbon atoms, a linear, branched or cyclic alkenyl or

alkynyl having from 2 to 6 carbon atoms; P is a peptide unit between 2 and 20

amino acids in length, wherein E1 or E2 can be linked to the peptide through

the terminal nitrogen, terminal carbon or through a side chain of one of the

amino acids of the peptide; and F1 and F2 are the same or different and are an

optional polyethyleneoxy unit of formula (OCH 2 CH2 )p, wherein p is 0 or an

integer from 2 to about 1000, provided that when Z is not F1-E1-P-E2-F2, at

least one of R1 , R 2, R3 , R 4, R5 , R6, R7, R8 , R9 , and RIO is a charged substituent

1 , R 2, R 3, R4, R 5, R 6, R 7, R8, R 9, and Rio is or when g is 1, at least one of A, R a

charged substituent;

Y represents a carbonyl, thioether, amide, disulfide, or

hydrazone group; and q is an integer from 1 to 20.

[731 As described in more detail below, the drug can be any of many

small molecule drugs, including, but not limited to, maytansinoids, CC-1065

analogs, morpholinos, doxorubicins, taxanes, cryptophycins, epothilones,

calicheamicins, auristatins, and pyrrolobenzodiazepine dimers.

[74] In preferred embodiments, one of R1 , R2 , R 3, R4 , R9, RIO is a

charged substituent selected from sulfonate, phosphate, carboxylate or

trialkylammonium, and the rest are H, 1, g and m are each 0, n = 1, D is a

maytansinoid, a CC-1065 analog or a pyrrolobenzodiazepine dimer. In

another more preferred embodiment, one of R1 , R2, R 3, R4 , R 9, Rio is a sulfonate, and the rest are H, 1, g and m are each 0, n = 1, D is a maytansinoid,

CC-1065 analog or a pyrrolobenzodiazepine dimer linked via a disulfide,

thioester, or thioether bond. In a further more preferred embodiment, one of

R 1, R2, R 3, R 4, R 9, Rio is a sulfonate, and the rest are H, 1, g and m are each 0,

n = 1, and Q is a maytansinoid, a CC-1065 analog, or a taxane.

[75] In a preferred embodiment, when Z is an F1-E1-P-E2-F2 unit,

El and E2 are the same or different and are C=O or NR14, wherein R 14 is H,

a linear alkyl having from 1-6 carbon atoms, a branched or cyclic alkyl having

from 3 to 6 carbon atoms, P is a peptide unit between 2 and 8 amino acids in

length, wherein El or E2 can be linked to the peptide through the terminal

nitrogen, terminal carbon or through a side chain of one of the amino acids of

the peptide, preferred amino acid residues are glycine (gly), alanine (ala),

leucine (leu), glutamic acid (glu), or lysine (lys), which can be used in any

combination or any order (e.g., gly-gly-gly or ala-leu-ala-leu, etc.); and Fl and

F2 are the same or different and are an optional polyethyleneoxy unit of

formula (OCH 2CH 2)p, wherein p is 0 or an integer from 2 to about 1000.

[76] In a more preferred embodiment, when Z is an F1-El-P-E2-F2

unit, El and E2 are the same or different and are C=O or NR14, wherein R14

is H or a linear alkyl having from 1-6 carbon atoms, P is a peptide unit

between 2 and 5 amino acids in length, wherein El or E2 can be linked to the

peptide through the terminal nitrogen, terminal carbon or through a side chain

of one of the amino acids of the peptide; and F1 and F2 are the same or different and are an optional polyethyleneoxy unit of formula (OCH 2CH2)p, wherein p is 0 or an integer from 2 to 24.

[77] Preferably, q, the number of drugs bound to each cell-binding

agent is 1-20, more preferably 2-18, and even more preferably 2-16, and most

preferably 2-10.

[781 To synthesize the conjugate, the cell-binding agent can be

modified with the crosslinkers of the present invention to introduce reactive

disulfide groups, maleimido, haloacetyl or hydrazide groups. Synthesis of the

cell-binding agent-drug conjugates linked via disulfide bonds is achieved by a

disulfide exchange between the disulfide bond in the modified cell-binding

agent and a drug containing a free thiol group. Synthesis of the cell-binding

agent-drug conjugates linked via thioether is achieved by reaction of the

maleimido or haloacetyl modified cell-binding agent and a drug containing a

free thiol group. Synthesis of conjugates bearing an acid labile hydrazone link

can be achieved by reaction of a carbonyl group with the hydrazide moiety in

the linker, by methods known in the art (see, for example, P. Hamann et al.,

BioConjugateChem., 13; 40-46, 2002; B. Laguzza et al., J.Med Chem., 32;

548-555, 1959; P. Trail et al., Cancer Res., 57; 100-105, 1997).

[79] Alternatively, the drug can be modified with the crosslinkers of

the present invention to give a modified drug of formula (IV) bearing a

functionality capable of reacting with a cell binding agent. For example a

thiol-containing drug can be reacted with the charged or pro-charged crosslinker of formula (I) bearing a maleimdo substituent at neutral pH in aqueous buffer to give a drug connected to the charged linker via a thioether link. A thiol-containg drug can undergo disulfide exchange with a charged linker bearing a pyrdiyldithio moiety to give a modified drug attached via a disulfide bond to the charged crosslinker. A drug bearing a hydroxyl group can be reacted with a charged or pro-charged crosslinker bearing a halogen, in the presence of a mild base, to give a modified drug bearing an ether link. A hydroxyl group containing drug can be condensed with a charged crosslinker of formula (I) bearing a carboxyl group, in the presence of a dehydrating agent, such as dicyclohexylcarbodimide, to give an ester link. An amino group containing drug can similarly undergo condensation with a carboxyl group on the charged or pro-charged crosslinker of formula (I) to give an amide bond.

[801 The conjugate may be purified by standard biochemical means,

such as gel filtration on a Sephadex G25 or Sephacryl S300 column,

adsorption chromatography, and ion exchange or by dialysis as previously

described. In some cases (e.g. folic acid, melanocyte stimulating hormone,

EGF etc) the cell-binding agent-drug conjugates can be purified by

chromatography such as by HPLC, medium pressure column chromatography

or ion exchange.

Modified cell-binding agents

[811 The cell-binding agent modified by reaction with crosslinkers

of the present invention are preferably represented by the formula (III)

YW - R3 Q R 2

. R RIO R5p6 R2 q

(III)

wherein the substituents are as described above for the charged

or pro-charged linker and the cell-binding agent drug conjugate.

[82] In preferred embodiments, one of R1 , R2 , R3 , R4, R9, RIO is a

charged substituent selected from sulfonate, phosphate, carboxyl or

trialkylammonium, and the rest are H, 1, g and m are each 0, n = 1, Q is a

disulfide substituent, a maleimido, haloacetyl group, or a N-hydroxy

succinimide ester, and Y is thioether, amide, or disulfide. In another more

preferred embodiment, one of R1 , R2 , R3, R4, R 9, RIO is a sulfonate, and the

rest are H, 1, g and m are each 0, n = 1, Q is a disulfide, maleimido or

haloacetyl moiety, and Y is thioether, amide, or disulfide. In a further more

preferred embodiment, one of R1, R2 , R3, R 4, R 9, RIO is a sulfonate, and the

rest are H, 1, g and m are each 0, n = 1, Q is a pyridyldithio or

nitropyridyldithio group, and Y is thioether, amide, or disulfide.

[83] In a preferred embodiment, when Z is an F1-El-P-E2-F2 unit,

El and E2 are the same or different and are C=O or NR14, wherein R1 4 is H,

a linear alkyl having from 1-6 carbon atoms, a branched or cyclic alkyl having

from 3 to 6 carbon atoms, P is a peptide unit between 2 and 8 amino acids in

length, wherein El or E2 can be linked to the peptide through the terminal

nitrogen, terminal carbon or through a side chain of one of the amino acids of

the peptide, preferred amino acid residues are glycine (gly), alanine (ala),

leucine (leu), glutamic acid (glu), or lysine (lys), which can be used in any

combination or any order (e.g., gly-gly-gly or ala-leu-ala-leu, etc.); and F1 and

F2 are the same or different and are an optional polyethyleneoxy unit of

formula (OCH 2CH2)p, wherein p is 0 or an integer from 2 to about 1000.

[84] In a more preferred embodiment, when Z is an F1-El-P-E2-F2

unit, El and E2 are the same or different and are C=O or NR14, wherein R 14

is H or a linear alkyl having from 1-6 carbon atoms, P is a peptide unit

between 2 and 5 amino acids in length, wherein El or E2 can be linked to the

peptide through the terminal nitrogen, terminal carbon or through a side chain

of one of the amino acids of the peptide; and F1 and F2 are the same or

different and are an optional polyethyleneoxy unit of formula (OCH 2 CH2 )p,

wherein p is 0 or an integer from 2 to 24.

[85] The modified cell-binding agent can be prepared by reacting

the cell-binding agent with the charged crosslinkers by methods known in the

art for other crosslinkers (U.S. Patent Nos. 6,340,701 B1, 5,846,545,

5,585,499, 5,475,092, 5,414,064, 5,208,020, and 4,563,304; R.V.J. Chari et al.

CancerResearch 52, 127-131, 1992; R.V.J. Chari et al. Cancer Research 55,

4079-4084, 1995; J. Carlsson et al. 173 Biochem. J (1978) 723-737(1978);

Goff, D. A., Carroll, S. F. 1 BioConjugate Chem. 381-386 (1990); L. Delprino

et al. 82 J. Pharm. Sci. 506-512 (1993); S. Arpicco et al., 8 BioConjugate

Chem 327-337 (1997)). Advantageously, because the cross-linker groups are

soluble in water or require only a small percentage of organic solvent to

maintain solubility in aqueous solution, the reaction between the cell-binding

agent and the cross-linker can be conducted in aqueous solution. The cross

linking reagent is dissolved in aqueous buffer, optionally containing a small

amount (typically <10% by volume) of a polar organic solvent that is miscible

with water, for example different alcohols, such as methanol, ethanol, and

propanol, dimethyl formamide, dimethyl acetamide, or dimethylsulfoxide at a

high concentration, for example 1-100 mM, and then an appropriate aliquot is

added to the buffered aqueous solution of the cell-binding agent. An

appropriate aliquot is an amount of solution that introduces 1-10 cross-linking

groups per cell-binding agent, preferably 1-5 groups, and the volume to be

added should not exceed 10 %, preferably 5 %, and most preferably 0-3 % of

the volume of the cell-binding agent solution. The aqueous solutions for the

cell-binding agents are buffered between pH 6 and 9, preferably between 6.5

and 7.5 and can contain any non-nucleophilic buffer salts useful for these pH

ranges. Typical buffers include phosphate, triethanolamine.HCl, HEPES, and

MOPS buffers, which can contain additional components, such as sucrose and

salts, for example, NaCl. After the addition the reaction is incubated at a

temperature of from 4 C to 40 C, preferably at ambient temperature. The

progress of the reaction can be monitored by measuring the increase in the

absorption at 495 nm or another appropriate wavelength. After the reaction is

complete, isolation of the modified cell-binding agent can be performed in a

routine way, using for example gel filtration chromatography, or adsorptive

chromatography.

[86] The extent of modification can be assessed by measuring the

absorbance of the nitropyridine thione, dinitropyridine dithione,

carboxamidopyridine dithione or dicarboxamidopyridine dithione group

released. In a non limiting example, Figure 56 shows the results from the

modification of the cell-binding agent, the C242 antibody, with a sulfonate

crosslinker of the present invention. The time course of linker/antibody (L/A)

incorporation is shown, for example, along with the drugs/antibody (D/A)

linked. The charged or pro-charged crosslinkers described herein have

diverse functional groups that can react with any cell-binding agent that

possesses a suitable substituent. For example cell-binding agents bearing an

amino or hydroxyl substituent can react with crosslinkers bearing an

N-hydroxysuccinimide ester, cell-binding agents bearing a thiol substituent

can react with crosslinkers bearing a maleimido or haloacetyl group.

Additionally, cell-binding agents bearing a carbonyl substituent can react with crosslinkers bearing a hydrazide. One skilled in the art can readily determine which crosslinker to use based on the known reactivity of the available functional group on the cell-binding agent.

[87] Crosslinkers bearing a positive charge (for example, compound

214, Figure 71) can be directly reacted with a cell binding agent in aqueous

buffer at a pH between 5 and 9, optionally containing an organic cosolvent

(such as 1 to 20% dimethylaceatmide or ethanol) to provide a modified cell

binding agent bearing a positive charge and a thiol group. The thiol group of

the cell binding agent can be reacted with a cytotoxic drug bearing either a

maleimido, haloacetamido or an active disulfide (example pyridyldithio,

nitropyridyldithio group) to provide a conjugate. The conjugate can be

purified by the methods described above.

[881 Alternatively, crosslinkers bearing a positive charge and a

reactive ester (for example, compound 216, Figure 71) can be directly reacted

with a cell binding agent (for example, through its lysine amino group) to

introduce a positive charge and an active disulfide. Reaction with a thiol

containing cytotoxic drug as described above can provide a conjugate bearing

a positive charge.

Modified Cytotoxic Drugs

[89] The cytotoxic drugs modified by reaction with crosslinkers of

the present invention are preferably represented by the formula (IV):

R R8 R R4

1 Ag n m R9 R1 0 R5 R 6 R1 R2

(IV)

wherein the substituents are as described above for the charged

or pro-charged linker and the cell-binding agent drug conjugate.

[90] In preferred embodiments, one of R1 , R 2, R 3, R4 , R9 , RIO is a

charged substituent selected from sulfonate, phosphate, carboxyl or

trialkylammonium, and the rest are H, 1, g and m are each 0, n = 1, and Y' is a

disulfide substituent, a maleimido, haloacetyl group, or a N-hydroxy

succinimide ester. In another more preferred embodiment, one of R1 , R2, R 3 ,

R4, R9, RIO is a sulfonate, and the rest are H, 1, g and m are each 0, n = 1, and

Y' is a maleimido moiety or a N-hydroxy succinimide ester. In a further more

preferred embodiment, one of R1 , R2 , R3, R4, R 9, RIO is a sulfonate, and the

rest are H, 1, g and m are each 0, n = 1, and Y' is a N-hydroxy succinimide

ester.

[911 In a preferred embodiment, when Z is an F-El-P-E2-F2 unit,

El and E2 are the same or different and are C=O or NR14, wherein R1 4 is H,

a linear alkyl having from 1-6 carbon atoms, a branched or cyclic alkyl having

from 3 to 6 carbon atoms, P is a peptide unit between 2 and 8 amino acids in

length, wherein El or E2 can be linked to the peptide through the terminal

nitrogen, terminal carbon or through a side chain of one of the amino acids of

the peptide, preferred amino acid residues are glycine (gly), alanine (ala),

leucine (leu), glutamic acid (glu), or lysine (lys), which can be used in any

combination or any order (e.g., gly-gly-gly or ala-leu-ala-leu, etc.); and F1 and

F2 are the same or different and are an optional polyethyleneoxy unit of

formula (OCH 2CH2)p, wherein p is 0 or an integer from 2 to about 1000.

[92] In a more preferred embodiment, when Z is an F-El-P-E2-F2

unit, El and E2 are the same or different and are C=O or NR14, wherein R1 4

is H or a linear alkyl having from 1-6 carbon atoms, P is a peptide unit

between 2 and 5 amino acids in length, wherein El or E2 can be linked to the

peptide through the terminal nitrogen, terminal carbon or through a side chain

of one of the amino acids of the peptide; and F1 and F2 are the same or

different and are an optional polyethyleneoxy unit of formula (OCH 2CH2)p,

wherein p is 0 or an integer from 2 to 24.

[93] The modified drugs can be prepared by reacting the drug with

the crosslinkers of the present invention to give a modified drug of formula

(IV) bearing a functionality capable of reacting with a cell binding agent. For example a thiol-containing drug can be reacted with the charged or pro charged crosslinker of formula (I) bearing a maleimdo substituent at neutral pH in aqueous buffer to give a drug connected to the charged or pro-charged linker via a thioether link. A thiol-containg drug can undergo disulfide exchange with a charged or pro-charged linker bearing a pyrdiyldithio moiety to give a modified drug attached via a disulfide bond to the charged or pro charged crosslinker. A drug bearing a hydroxyl group can be reacted with a charged crosslinker bearing a halogen, in the presence of a mild base, to give a modified drug bearing an ether link. A hydroxyl group containing drug can be condensed with a charged crosslinker of formula (I) bearing a carboxyl group, in the presence of a dehydrating agent, such as dicyclohexylcarbodimide, to give an ester link. An amino group containing drug can similarly undergo condensation with a carboxyl group on the charged or pro-charged crosslinker of formula (I) to give an amide bond. The modified drug can be purified by standard methods such as column chromatography over silica gel or alumina, crystallization, preparatory thin layer chromatography, ion exchange chromatography or HPLC.

Cell-binding Agents

[94] The cell-binding agent that comprises the conjugates and the

modified cell-binding agents of the present invention may be of any kind

presently known, or that become known, and includes peptides and non-peptides.

The cell-binding agent may be any compound that can bind a cell, either in a

specific or non-specific manner. Generally, these can be antibodies (especially

monoclonal antibodies and antibody fragments), adnectins (US Publication No.:

20070082365), interferons, lymphokines, hormones, growth factors, vitamins,

nutrient-transport molecules (such as transferrin), or any other cell-binding

molecule or substance.

[951 Where the cell-binding agent is an antibody (for example, a

murine, human humanized, resurfaced or a chimeric or any other antibody

known to one of skill in the art), it binds to an antigen that is a polypeptide and

may be a transmembrane molecule (e.g. receptor) or a ligand such as a growth

factor. Exemplary antigens include molecules such as renin; a growth

hormone, including human growth hormone and bovine growth hormone;

growth hormone releasing factor; parathyroid hormone; thyroid stimulating

hormone; lipoproteins; alpha-1-antitrypsin; insulin A-chain; insulin B-chain;

proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone;

glucagon; clotting factors such as factor vme, factor IX, tissue factor (TF), and

von Willebrands factor; anti-clotting factors such as Protein C; atrial

natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase

or human urine or tissue-type plasminogen activator (t-PA); bombesin;

thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta;

enkephalinase; RANTES (regulated on activation normally T-cell expressed

and secreted); human macrophage inflammatory protein (MIP-1-alpha); a serum albumin, such as human serum albumin; Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin associated peptide; a microbial protein, such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6

(NT-3, NT4, NT-5, or NT-6), or a nerve growth factor such as NGF-p;

platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF

and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF)

such as TGF-alpha and TGF-beta, including TGF-1, TGF-p2, TGF- P3,

TGF-p4, or TGF- P5; insulin-like growth factor-I and -II (IGF-I and IGF-II);

des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins,

EpCAM, GD3, FLT3, PSMA, PSCA, MUC1, MUC16, STEAP, CEA,

TENB2, EphA receptors, EphB receptors, folate receptor, mesothelin, cripto,

alphavbeta, integrins, VEGF, VEGFR, tarnsferrin receptor, IRTA1, IRTA2,

IRTA3, IRTA4, IRTA5; CD proteins such as CD2, CD3, CD4, CD5, CD6,

CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD23, CD25, CD26, CD28,

CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59,

CD70, CD79, CD80, CD81, CD103, CD105, CD134, CD137, CD138, CD152

or an antibody which binds to one or more tumor-associated antigens or cell

surface receptors disclosed in US Publication No. 20080171040 or US

Publication No. 20080305044 and are incorporated in their entirety by

reference; erythropoietin; osteoinductive factors; immunotoxins; a bone

morphogenetic protein (BMP); an interferon, such as interferon-alpha, -beta,

and -gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and

G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase; T-cell

receptors; surface membrane proteins; decay accelerating factor; viral antigen

such as, for example, a portion of the HIV envelope; transport proteins;

homing receptors; addressins; regulatory proteins; integrins, such as CD11a,

CD11b, CD11c, CD18, an ICAM, VLA-4, EpCAM and VCAM; a tumor

associated antigen such as HER2, HER3 or HER4 receptor; and fragments of

any of the above-listed polypeptides.

[96] Preferred antigens for antibodies encompassed by the present

invention also include CD proteins, such as CD3, CD4, CD8, CD19, CD20,

CD34, and CD46; members of the ErbB receptor family, such as the EGF

receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules, such as

LFA-1, Mac1, p150. 9 5 , VLA-4, ICAM-1, VCAM, EpCAM, alpha4/beta 7

integrin, and alpha v/beta3 integrin including either alpha or beta subunits

thereof (e.g. anti-CD11a, anti-CD18 or anti-CDllb antibodies); growth

factors, such as VEGF; tissue factor (TF); TGF-p.; alpha interferon (alpha

IFN); an interleukin, such as IL-8; IgE; blood group antigens Apo2, death

receptor; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4;

protein C etc. Preferred antibodies that can be used are antibodies to CD2,

CD3, CD4, CD5, CD6, CDL1, CD19, CD20, CD22, CD26, CD30, CD33,

CD37, CD38, CD40, CD44, CD56, CD79, CD105, CD138, EphA receptors

(e.g., EphA2 receptor), EphB receptors, EGFr, EGFRvIII, HER2, HER3,

trastuzumab, pertuzumab mesothelin, cripto, alphavbeta6, integrins, VEGF,

VEGFR, folate receptor (for example, FOLR1), transferrin receptor, GD3,

EpCAM or an antibody which binds to one or more tumor-associated antigens

or cell-surface receptors disclosed in US Publication No. 20080171040 or US

Publication No. 20080305044 and are incorporated in their entirety by

reference.

[97] Additional examples of cell-binding agents that can be used

include:

-resurfaced antibodies (U.S. patent no. 5,639,641);

-humanized or fully human antibodies, selected from but not

limited to, huMy9-6, huB4, huC242, huN901, DS6, CD38, IGF-IR, CNTO 95,

B-B4, trastuzumab, pertuzumab, bivatuzumab, sibrotuzumab, and rituximab

(see, e.g., U.S. Patent Nos. 5,639,641, 5,665,357; and 7,342,110, U.S.

Provisional Patent Application No. 60/424,332, International Patent

Application WO 02/16,401, U.S. Patent Publication Number 20060045877,

U.S. Patent Publication Number 20060127407, U.S. Patent Publication

Number 20050118183, Pedersen et al., (1994) J. Mol. Biol. 235, 959-973,

Roguska et al., (1994) Proceedings of the NationalAcademy ofSciences, Vol

91, 969-973, supra, Colomer et al., Cancer Invest., 19: 49-56 (2001), Heider et al., Eur. J Cancer, 31A: 2385-2391 (1995), Welt et al., J Clin. Oncol., 12:

1193-1203 (1994), and Maloney et al., Blood, 90: 2188-2195 (1997)); and

-epitope-binding fragments of antibodies such as sFv, Fab,

Fab', and F(ab') 2 (Parham, I Immunol. 131:2895-2902 (1983); Spring et al, J

Immunol. 113:470-478 (1974); Nisonoff et al, Arch. Biochem. Biophys.

89:230-244 (1960)).

Additional cell-binding agents include other cell-binding proteins and

polypeptides exemplified by, but not limited to:

- Ankyrin repeat proteins (DARPins; Zahnd et al., J.Biol.

Chem., 281, 46, 35167-35175, (2006); Binz, H.K., Amstutz, P. & Pluckthun,

A. (2005) Nature Biotechnology, 23, 1257-1268) or ankyrin-like repeats

proteins or synthetic peptides described, for example, in U.S. Patent

Publication Number 20070238667; U.S. Patent No. 7,101,675; and

WO/2007/147213; WO/2007/062466);

-interferons (e.g. a, P, y); -lymphokines such as IL-2, IL-3, IL-4, IL-6;

-hormones such as insulin, TRH (thyrotropin releasing

hormones), MSH (melanocyte-stimulating hormone), steroid hormones, such

as androgens and estrogens;

-vitamins such as folic acid;

-growth factors and colony-stimulating factors such as EGF,

TGF-a, G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5:155

158 (1984)); and

-transferrin (O'Keefe et al, J Biol. Chem. 260:932-937 (1985)).

[98] Monoclonal antibody techniques allow for the production of

specific cell-binding agents in the form of monoclonal antibodies. Particularly

well known in the art are techniques for creating monoclonal antibodies

produced by immunizing mice, rats, hamsters or any other mammal with the

antigen of interest such as the intact target cell, antigens isolated from the

target cell, whole virus, attenuated whole virus, and viral proteins such as viral

coat proteins. Sensitized human cells can also be used. Another method of

creating monoclonal antibodies is the use of phage libraries of sFv (single

chain variable region), specifically human sFv (see, e.g., Griffiths et al, U.S.

Patent No. 5,885,793; McCafferty et al, WO 92/01047; and Liming et al,

WO 99/06587.)

[991 Selection of the appropriate cell-binding agent is a matter of

choice that depends upon the particular cell population that is to be targeted,

but in general monoclonal antibodies and epitope binding fragments thereof

are preferred, if an appropriate one is available.

[1001 For example, the monoclonal antibody My9 is a murine IgG 2a

antibody that is specific for the CD33 antigen found on Acute Myeloid

Leukemia (AML) cells (Roy et al. Blood 77:2404-2412 (1991)) and can be used to treat AML patients. Similarly, the monoclonal antibody anti-B4 is a murine IgG 1, which binds to the CD19 antigen on B cells (Nadler et al, J.

Immunol. 131:244-250 (1983)) and can be used if the target cells are B cells or

diseased cells that express this antigen such as in non-Hodgkin's lymphoma or

chronic lymphoblastic leukemia. Similarly, the antibody N901 is a murine

monoclonal IgG1 antibody that binds to CD56 found on small cell lung

carcinoma cells and on cells of other tumors of the neuroendocrine origin (Roy

et al.J.Nat. Cancer Inst. 88:1136-1145 (1996)), C242 antibody that binds to

the CanAg antigen, pertuzumab, trastuzumab that binds to HER2/neu, and

anti-EGF receptor antibody.

[101] Additionally, GM-CSF, which binds to myeloid cells, can be

used as a cell-binding agent to diseased cells from acute myelogenous

leukemia. IL-2, which binds to activated T-cells, can be used for prevention of

transplant graft rejection, for therapy and prevention of graft-versus-host

disease, and for treatment of acute T-cell leukemia. MSH, which binds to

melanocytes, can be used for the treatment of melanoma. Folic acid, which

targets the folate receptor expressed on ovarian and other cancers is also a

suitable cell-binding agent.

[102] Cancers of the breast and testes can be successfully targeted

with estrogen (or estrogen analogues) or androgen (or androgen analogues),

respectively, as cell-binding agents.

Drugs

[103] Drugs that can be used in the present invention include

chemotherapeutic agents. "Chemotherapeutic agent" is a chemical compound

useful in the treatment of cancer. Examples of chemotherapeutic agents

include alkylating agents, such as thiotepa and cyclophosphamide

(CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and

piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and

uredopa; ethylenimines and methylamelamines including altretamine,

triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide

and trimethylolomelamine; acetogenins (especially bullatacin and

bullatacinone); a camptothecin (including the synthetic analogue topotecan);

bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and

bizelesin synthetic analogues); cryptophycins (particularly cryptophycin I and

cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues,

KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;

spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,

cholophosphamide, estramustine, ifosfamide, mechlorethamine,

mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine,

prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine,

chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics,

such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin

.gammal and calicheamicin theta I, see, e.g., Angew Chem Intl. Ed. Engl.

33:183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin; chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, nitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimnidine analogs such as, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals, such as aminoglutethimide, mitotane, trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK*; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,

2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A,

roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine;

mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");

cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL*, Bristol-Myers

Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE*, Rhone

Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine;

mercaptopurine; methotrexate; platinum analogs such as cisplatin and

carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin

C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide;

daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase

inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid;

capecitabine; and pharmaceutically acceptable salts, acids or derivatives of

any of the above. Also included in this definition are anti-hormonal agents

that act to regulate or inhibit hormone action on tumors, such as anti-estrogens

including for example tamoxifen, raloxifene, aromatase inhibiting

4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,

onapristone, and toremifene (Fareston); and anti-androgens, such as flutamide,

nilutamide, bicalutamide, leuprolide, and goserelin; siRNA and

pharmaceutically acceptable salts, acids or derivatives of any of the above.

Other chemotherapeutic agents that can be used with the present invention are

disclosed in US Publication No. 20080171040 or US Publication No.

20080305044 and are incorporated in their entirety by reference.

[1041 In a preferred embodiment, chemotherapeutic drugs are

essentially small molecule drugs. A "small molecule drug" is broadly used

herein to refer to an organic, inorganic, or organometallic compound that may

have a molecular weight of for example 100 to 1500, more suitably from 120

to 1200, favorably from 200 to 1000, and typically having a molecular weight

of less than about 1000. Small molecule drugs of the invention encompass

oligopeptides and other biomolecules having a molecular weight of less than

about 1000. Small molecule drugs are well characterized in the art, such as in

W005058367A2, European Patent Application Nos. 85901495 and 8590319,

and in U.S. Patent No. 4,956,303, among others and are incorporated in their

entirety by reference.

[1051 Preferable small molecule drugs are those that allow for linkage

to the cell-binding agent. The invention includes known drugs as well as those

that may become known. Especially preferred small molecule drugs include

cytotoxic agents.

[1061 The cytotoxic agent may be any compound that results in the

death of a cell, or induces cell death, or in some manner decreases cell

viability, wherein each cytotoxic agent comprises a thiol moiety.

[1071 Preferred cytotoxic agents are maytansinoid compounds, taxane

compounds, CC-1065 compounds, daunorubicin compounds and doxorubicin

compounds, pyrrolobenzodiazepine dimers, calicheamicins. Auristatins and

analogues and derivatives thereof, some of which are described below.

[108] Other cytotoxic agents, which are not necessarily small

molecules, such as siRNA, are also encompassed within the scope of the

instant invention. For example, siRNAs can be linked to the crosslinkers of

the present invention by methods commonly used for the modification of

oligonucleotides (see, for example, US Patent Publications 20050107325 and

20070213292). Thus the siRNA in its 3' or 5'-phosphoromidite form is

reacted with one end of the crosslinker bearing a hydroxyl functionality to

give an ester bond between the siRNA and the crosslinker. Similarly reaction

of the siRNA phosphoramidite with a crosslinker bearing a terminal amino

group results in linkage of the crosslinker to the siRNA through an amine.

siRNA are described in detail in U.S. Patent Publication Numbers:

20070275465,20070213292,20070185050,20070161595,20070054279,

20060287260,20060035254,20060008822,20050288244,20050176667,

which are incorporated herein in their entirety by reference.

Maytansinoids 1109] Maytansinoids that can be used in the present invention are

well known in the art and can be isolated from natural sources according to

known methods or prepared synthetically according to known methods.

[1101 Examples of suitable maytansinoids include maytansinol and

maytansinol analogues. Examples of suitable maytansinol analogues include

those having a modified aromatic ring and those having modifications at other

positions.

[1111 Specific examples of suitable analogues of maytansinol having

a modified aromatic ring include:

(1) C-19-dechloro (U.S. Patent No. 4,256,746) (prepared by

LAH reduction of ansamitocin P2);

(2) C-20-hydroxy (or C-20-demethyl)+/-C-19-dechloro (U.S.

Patent Nos. 4,361,650 and 4,307,016) (prepared by demethylation using

Streptomyces orActinomyces or dechlorination using LAH); and

(3) C-20-demethoxy, C-20-acyloxy (-OCOR), +/-dechloro

(U.S. Patent No. 4,294,757) (prepared by acylation using acyl chlorides).

[1121 Specific examples of suitable analogues of maytansinol having

modifications of other positions include:

(1) C-9-SH (U.S. Patent No. 4,424,219) (prepared by the

reaction of maytansinol with H2S or P2S5);

(2) C-14-alkoxymethyl (demethoxy/CH2OR) (U.S. Patent No.

4,331,598);

(3) C-14-hydroxymethyl or acyloxymethyl (CH2OH or

CH20Ac) (U.S. Patent No. 4,450,254) (prepared from Nocardia);

(4) C-15-hydroxy/acyloxy (U.S. Patent No. 4,364,866)

(prepared by the conversion of maytansinol by Streptomyces);

(5) C-15-methoxy (U.S. Patent Nos. 4,313,946 and 4,315,929)

(isolated from Trewia nudiflora);

(6) C-18-N-demethyl (U.S. Patent Nos. 4,362,663 and

4,322,348) (prepared by the demethylation of maytansinol by Streptomyces);

and

(7) 4,5-deoxy (U.S. Patent No. 4,371,533) (prepared by the

titanium trichloride/LAH reduction of maytansinol).

[1131 The synthesis of thiol-containing maytansinoids useful in the

present invention is fully disclosed in U.S. Patent Nos. 5,208,020, 5,416,064,

and U. S. Patent Application No. 20040235840.

[1141 Maytansinoids with a thiol moiety at the C-3 position, the C-14

position, the C-15 position or the C-20 position are all expected to be useful.

The C-3 position is preferred and the C-3 position of maytansinol is especially

preferred. Also preferred are an N-methyl-alanine-containing C-3 thiol moiety

maytansinoid, and an N-methyl-cysteine-containing C-3 thiol moiety

maytansinoid, and analogues of each.

[1151 Specific examples of N-methyl-alanine-containing C-3 thiol

moiety maytansinoid derivatives useful in the present invention are

represented by the formulae M1, M2, M3, M6 and M7.

CH 3 0

N (CH 2)SH 0) HI 0 CH 3

May

M1

wherein:

I is an integer of from 1 to 10; and

may is a maytansinoid.

CH 3 0 R, R2 0lj II HN CH-CH-(CH 2)mSH ) CH 3

May M2

wherein:

R 1 and R 2 are H, CH3 or CH 2CH3, and may be the same or different;

m is 0, 1, 2 or 3; and

may is a maytansinoid.

0

N (CH 2), SH 0 Fhay

M3 wherein: n is an integer of from 3 to 8; and may is a maytansinoid.

0 0, oN (CH2)/SH YO \ 0 o X3o N O

NH o MeO M6

wherein:

lis1,2or3;

Yo is Cl or H; and

X3 is H or CH3 .

CH 3 0 0 I N CH-H-(CR 3R 4)mSH 0 CH 3 May M7

wherein:

R 1, R 2, R 3, R4 are H, CH3 or CH2CH 3, and may be the same or

different; mis0, 1,2 or3; and may is a maytansinoid.

[1161 Specific examples of N-methyl-cysteine-containing C-3 thiol

moiety maytansinoid derivatives useful in the present invention are

represented by the formulae M4 and M5.

SH (OH 3) 0 0

N (CH 2)pCH 3 0 -nay M4

wherein:

ois1,2or3;

p is an integer of 0 to 10; and

may is a maytansinoid.

SH

N0(CH2)qCH3 YO \ 0 0 N 00O X3o

MeO M5

wherein:

ois1,2or3;

q is an integer of from 0 to 10;

Yo is Cl or H; and

X3 is H or CH 3 .

Preferred maytansinoids are those described in U.S. Patent Nos.

5,208,020; 5,416,064; 6,333.410; 6,441,163; 6,716,821; RE39,151 and

7,276,497.

Taxanes

[117] The cytotoxic agent according to the present invention may

also be a taxane.

[1181 Taxanes that can be used in the present invention have been

modified to contain a thiol moiety. Some taxanes useful in the present

invention have the formula T Ishown below:

R2 0 0 OR5 O 40 R "K NH 10 9 876 R4 NH O

5 O\R3 R3 \ OH UAc T 00 OR6 IRI"

R{ R' TI

[119] Four embodiments of these novel taxanes are described below.

[120] In embodiments (1), (2), (3), and (4), R 1,Ri', and R1 " are the

same or different and are H, an electron withdrawing group, such as F, NO 2

, CN, Cl, CHF 2, or CF 3 or an electron donating group, such as -OCH 3

, -OCH2CH3 , -NR7RS, -OR 9, wherein R7 and Rs are the same or different and

are linear, branched, or cyclic alkyl groups having 1 to 10 carbon atoms or

simple or substituted aryl having 1 to 10 carbon atoms. Preferably the number

of carbon atoms for R 7 and R8 is 1 to 4. Also, preferably R 7 and R8 are the

same. Examples of preferred -NR7R8 groups include dimethyl amino, diethyl

amino, dipropyl amino, and dibutyl amino, where the butyl moiety is any of

primary, secondary, tertiary or isobutyl. R 9 is linear, branched or cyclic alkyl

having 1 to 10 carbon atoms.

[1211 R 1 preferably is OCH3, F, NO 2 , or CF3 .

[122] Also preferably, R 1is in the meta position and R 1' and R1 " are

H or OCH3 .

[1231 R 2 in embodiments (1), (2) and (4), is H, heterocyclic, a linear,

branched, or cyclic ester having from 1 to 10 carbon atoms or heterocyclic, a

linear, branched, or cyclic ether having from 1 to 10 carbon atoms or a

carbamate of the formula -CONRioR, ,wherein Rio and R 1 are the same or

different and are H, linear alkyl having from 1-6 carbon atoms, branched, or

cyclic alkyl having 3 to 10 atoms or simple or substituted aryl having 6 to 10

carbon atoms. For esters, preferred examples include -COCH 2CH3 and

-COCH 2CH 2CH3. For ethers, preferred examples include -CH 2CH3 and

-CH 2CH2CH 3. For carbamates, preferred examples include -CONHCH 2CH3,

CONHCH 2CH2 CH3, -CO-morpholino, -CO-piperazino, -CO-piperidino, or

CO-N-methylpiperazino.

[1241 R2 in embodiment (3), is a thiol-containing moiety.

[1251 R 3 in embodiments (1), (3) and (4), is aryl, or is linear,

branched or cyclic alkyl having I to 10 carbon atoms, preferably

-CH 2CH(CH 3) 2 .

[1261 R 3 in embodiment (2), is -CH=C(CH 3)2 .

[127] R4 in all four embodiments, is -OC(CH 3) 3 or -C 6 H.

[1281 R 5 in embodiments (1) and (2), is a thiol-containing moiety and

R6 has the same definition as above for R2 for embodiments (1), (2) and (4).

[129] R 5 and R6 in embodiment (3), are the same or different, and

have the same definition as above for R2 for embodiments (1), (2) and (4).

[1301 R5 in embodiment (4), has the same definition as above for R2

for embodiments (1), (2) and (4) and R6 is a thiol moiety.

[1311 The preferred positions for introduction of the thiol-containing

moiety are R 2 and R 5, with R 2 being the most preferred.

[1321 The side chain carrying the thiol moiety can be linear or

branched, aromatic or heterocyclic. One of ordinary skill in the art can readily

identify suitable side chains. Specific examples of thiol moieties include

-(CH 2)nSH, -CO(CH 2 )nSH, -(CH 2)nCH(CH 3)SH, -CO(CH 2)nCH(CH 3)SH,

-(CH 2 )nC(CH 3 ) 2 SH, -CO(CH 2)nC(CH 3) 2SH, -CONR 2 (CH2),SH,

-CONR 12(CH 2),CH(CH 3)SH, or -CONR 2(CH 2)nC(CH 3) 2SH, -CO

morpholino-XSH, -CO-piperazino-XSH, -CO-piperidino-XSH, and -CO-N

methylpiperazino-XSH wherein

X is a linear alkyl or branched alkyl having 1-10 carbon atoms.

R 12 is a linear alkyl, branched alkyl or cyclic alkyl having 1 to

10 carbon atoms, or simple or substituted aryl having from 1 to 10 carbon

atoms or heterocyclic, and can be H, and

n is an integer of 0 to 10.

[1331 Examples of linear alkyls include methyl, ethyl, propyl, butyl,

pentyl and hexyl.

[134] Examples of branched alkyls include isopropyl, isobutyl,

sec.-butyl, tert.-butyl, isopentyl and 1-ethyl-propyl.

[1351 Examples of cyclic alkyls include cyclopropyl, cyclobutyl,

cyclopentyl and cyclohexyl.

[1361 Examples of simple aryls include phenyl and naphthyl.

[1371 Examples of substituted aryls include aryls such as those

described above substituted with alkyl groups, with halogens, such as Cl, Br,

F, nitro groups, amino groups, sulfonic acid groups, carboxylic acid groups,

hydroxy groups or alkoxy groups.

[138] Examples of heterocyclics are compounds wherein the

heteroatoms are selected from 0, N, and S, and include morpholino,

piperidino, piperazino, N-methylpiperazino, pyrrollyl, pyridyl, furyl and

thiophene.

[1391 The taxanes having a thiol moiety can be synthesized according

to known methods. The starting material for the synthesis is the commercially

available 10-deacetylbaccatin III. The chemistry to introduce various

substituents is described in several publications (Ojima et al, J Med Chem.

39:3889-3896 (1996); Ojima et al., J Med Chem. 40:267-278 (1997); Ojima

et al., Proc. Nat. Acad. Sci., 96:4256-4261 (1999); U.S. Patent No. 5,475,011

and U.S. Patent No. 5,811,452).

[140] The substituent R 1 on the phenyl ring and the position of the

substituent R, can be varied until a compound of the desired toxicity is

obtained. Furthermore, the degree of substitution on the phenyl ring can be

varied to achieve a desired toxicity. That is, the phenyl ring can have one or more substituents (e.g., mono-, di-, or tri-substitution of the phenyl ring) which provide another means for achieving a desired toxicity. One of ordinary skill in the art can determine the appropriate chemical moiety for R1 and the appropriate position for R1 using only routine experimentation.

[1411 For example, electron withdrawing groups at the meta position

increase the cytotoxic potency, while substitution at the para position is not

expected to increase the potency as compared to the parent taxane. Typically,

a few representative taxanes with substituents at the different positions (ortho,

meta and para) will be initially prepared and evaluated for in vitro

cytotoxicity.

[142] The thiol moiety can be introduced at one of the positions

where a hydroxyl group already exists. The chemistry to protect the various

hydroxyl groups, while reacting the desired one, has been described previously

(see, for example, the references cited supra). The substituent is introduced

by simply converting the free hydroxyl group to a disulfide-containing ether, a

disulfide-containing ester, or a disulfide-containing carbamate. This

transformation is achieved as follows. The desired hydroxyl group is

deprotonated by treatment with the commercially-available reagent lithium

hexamethyldisilazane (1.2 equivalents) in tetrahydrofuran at -40°C as

described in Ojima et al. (1999), supra. The resulting alkoxide anion is then

reacted with an excess of a dihalo compound, such as dibromoethane, to give a

halo ether. Displacement of the halogen with a thiol (by reaction with potassium thioacetate and treatment with mild base or hydroxylamine) will provide the desired thiol-containing taxane.

[143] Alternatively, the desired hydroxyl group can be esterified

directly by reaction with an acyl halide, such as 3-bromopropionyl chloride, to

give a bromo ester. Displacement of the bromo group by treatment with

potassium thioacetate and further processing as described above will provide

the thiol-containing taxane ester. Preferred taxoids are those described in U.S.

Patent Nos. 6,340,701; 6,372,738; 6.436,931; 6,596,757; 6,706,708;

7,008,942; 7,217,819 and 7,276,499.

CC-1065 analogues

[144] The cytotoxic agent according to the present invention may

also be a CC-1065 analogue.

[1451 According to the present invention, the CC-1065 analogues

contain an A subunit and a B or a B-C subunit. The A subunits are CPI

(cyclopropapyrroloindole unit) in its natural closed cyclopropyl form or in its

open chloromethyl form, or the closely related CBI unit

(cyclopropylbenzindole unit) in the closed cyclopropyl form or the open

chloromethyl form. The B and C subunits of CC-1065 analogues are very

similar and are 2-carboxy-indole and 2-carboxy-benzofuran derivatives. For

activity, the analogues of CC-1065 need at least one such 2-carboxy-indole

subunit or 2-carboxy-benzofuran subunit, although two subunits (i.e., B-C)

render the analogue more potent. As is obvious from the natural CC-1065 and from the analogues published (e.g., Warpehoski et al, J Med. Chem.

31:590-603 (1988), D. Boger et al., J. Org. Chem; 66; 6654-6661, 2001; U. S.

Patent Nos 5,739,350; 6,060,608; 6.310.209), the B and C subunits can also

carry different substituents at different positions on the indole or benzofuran

rings.

[1461 CC-1065 analogues containing a thiol moiety can be any of the

following A subunits of the formulae A-1 {CPI (Cyclopropyl form)}, A-2

{CPI (Chloromethyl form)), A-3 {CBI (Cyclopropyl form)}, and A-4 {CBI

(Chloromethyl form)} covalently linked via an amide bond from the

secondary amino group of the pyrrole moiety of the A subunit to the C-2

carboxy group of either a B subunit of the formula F-1 or a B-C subunit of the

formulae F-3 or F-7.

A subunits

CH 3 CH 3 CI

NH NH

NH NH O A-1 OH A-2

-CI

NH NH 0 A-3 OH A-4

B and covalently bound B and C subunits

R7 R6

R4

RR R5

HOOC HOOC R4

W1 R2 W1 R2

R1 F-1 F-3 0

N Rs

/\ R4 N W2

HOOC O R3

WS W1#11R2 R1 F-7

wherein each W, and W2 may be the same or different and may

be 0 or NH; and

wherein, in Formula F-1 R 4 is a thiol moiety, in Formula F-3

one of R or R4 is a thiol moiety, in Formula F-7 one of R' or R 4 is a thiol

containing moiety; when R or R' is a thiol moiety, then R1 to R 6, which may

be the same or different, are hydrogen, C1 -C 3 linear alkyl, methoxy, hydroxyl,

primary amino, secondary amino, tertiary amino, or amido; and when R4 is a

thiol moiety, R, R 1, R 2, R 3, R 4, R 5 and R 6, which may be the same or different,

are hydrogen, C 1 -C 3 linear alkyl, methoxy, hydroxyl, primary amino,

secondary amino, tertiary amino, or amido, and R' is NH 2 , alkyl, O-alkyl,

primary amino, secondary amino, tertiary amino, or amido. In addition, the chlorine atom in A-2 and A-4 subunits can be replaced with another suitable halogen.

[1471 In a preferred embodiment, R and R' are thiol moieties and Ri

and R2 are each hydrogen. In another preferred embodiment, R and R' are

thiol moieties and R, to R6 are each hydrogen.

[1481 In an especially preferred embodiment, R or R4 is

-NHCO(CH2)SH, -NHCOC 6 H4 (CH 2 )ISH, or -O(CH 2);SH, and R' is

-(CH 2)SH, -NH(CH 2);SH or -O(CH2),SH wherein I is an integer of 1 to 10.

[1491 Examples of primary amines include methyl amine, ethyl

amine and isopropyl amine.

[1501 Examples of secondary amines include dimethyl amine,

diethylamine and ethylpropyl amine.

[1511 Examples of tertiary amines include trimethyl amine, triethyl

amine, and ethyl-isopropyl-methyl amine.

[1521 Examples of amido groups include N-methylacetamido,

N-methyl-propionamido, N-acetamido, and N-propionamido.

[153] Examples of alkyl represented by R', when R' is not a linking

group, include CI-C 5 linear or branched alkyl.

[1541 Examples of O-alkyl represented by R' when R' is not a linking

group, include compounds where the alkyl moiety is a Ci-C 5 linear or

branched alkyl.

[1551 The above-described CC-1065 analogues may be isolated from

natural sources and methods for their preparation, involving subsequent

modification, synthetic preparation, or a combination of both, are well

described (see, e.g., U.S. patent nos. 5,475,092, 5,585,499 and 5,846,545).

Preferred CC-I065 analogs are those described in U.S. Patent Nos. 5,475,092;

5,595,499; 5,846,545; 6,534,660; 6,586,618; 6,756,397 and 7,049,316

Daunorubicin/DoxorubicinAnalogues

[156] The cytotoxic agent according to the present invention may

also be a daunorubicin analogue or a doxorubicin analogue.

[1571 The daunorubicin and doxorubicin analogues of the present

invention can be modified to comprise a thiol moiety.

[158] The modified doxorubicin/daunorubicin analogues useful in the

present invention have the formula D1 shown below:

O OH O

CH2X OH

OMe O OH O

CH3 O -N

R Y R' D1

wherein,

X is H or OH;

Y is 0 or NR 2, wherein R2 is linear or branched alkyl having 1

to 5 carbon atoms;

R is a thiol moiety, H, or liner or branched alkyl having 1 to 5

carbon atoms; and

R' is a thiol moiety, H, or -OR1, wherein R1 is linear or

branched alkyl having 1 to 5 carbon atoms;

provided that R and R' are not thiol moieties at the same time.

[1591 In a preferred embodiment, NR2 is NCH 3. In another preferred

embodiment, R' is -0.

[1601 In an especially preferred embodiment, the thiol moiety is

-(CH 2)nSH, -O(CH 2)nSH, -(CH 2)nCH(CH 3)SH, -O(CH 2)nCH(CH 3)SH,

-(CH 2)nC(CH 3)2 SH, or -O(CH 2)nC(CH 3)2 SH, wherein n is an integer of 0 to

10.

[1611 Examples of the linear or branched alkyl having 1 to 5 carbon

atoms, represented by R, R1 , and R2, include methyl, ethyl, n-propyl,

isopropyl, n-butyl, isobutyl, sec.-butyl, tert.-butyl, and pentyl, in any of its

eight isomeric arrangements.

11621 R 1 and R2 preferably are methyl.

[163] Examples of linear alkyls include methyl, ethyl, propyl, butyl,

pentyl and hexyl.

[1641 Examples of branched alkyls include isopropyl, isobutyl,

sec.-butyl, tert.-butyl, isopentyl and I-ethyl-propyl.

[1651 When either R or R' is not a linking group, the substituent in

that position can be varied until a compound of the desired toxicity is

obtained. High toxicity is defined as having an IC 50 towards cultured cancer

cells in the range of 1 x 1012 to 1 x 10~ M, upon a 72 hour exposure time.

Representative examples of substituents are H, alkyl, and O-alkyl, as

described above. One of ordinary skill in the art can determine the appropriate

chemical moiety for R and R' using only routine experimentation.

[166] For example, methyl and methoxy substituents are expected to

increase the cytotoxic potency, while a hydrogen is not expected to increase

the potency as compared to the parent daunorubicin analogues with

substituents at the different positions will be initially prepared and evaluated

for in vitro cytotoxicity.

[1671 The modified doxorubicin/daunorubicin analogues of the

present invention, which have a thiol moiety, are described in WO 01/38318.

The modified doxorubicin/daunorubicin analogues can be synthesized

according to known methods (see, e.g., U.S. Patent No. 5,146,064).

[1681 Auristatin include auristatin E, auristatin EB (AEB), auristatin

EFP (AEFP), monomethyl auristatin E (MMAE) and are described in U.S.

Patent No. 5,635,483, Int. J. Oncol. 15:367-72 (1999); Molecular Cancer

Therapeutics, vol. 3, No. 8, pp. 921-932 (2004); U.S. Application Number

11/134826. U.S. Patent Publication Nos. 20060074008, 2006022925.

[1691 The cytotoxic agents according to the present invention include

pyrrolobenzodiazepine dimers that are known in the art (US Patent Nos

7,049,311; 7.067.511; 6,951,853; 7,189,710; 6,884,799; 6,660,856.

Analogues and derivatives

[1701 One skilled in the art of cytotoxic agents will readily

understand that each of the cytotoxic agents described herein can be modified

in such a manner that the resulting compound still retains the specificity and/or

activity of the starting compound. The skilled artisan will also understand that

many of these compounds can be used in place of the cytotoxic agents

described herein. Thus, the cytotoxic agents of the present invention include

analogues and derivatives of the compounds described herein.

Therapeutic Use

[1711 The cell-binding agent drug conjugates (e.g.,

immunoconjugates) of this invention can also be used in combination with

other chemotherapeutic agents. Such chemotherapeutic agents are listed

above or are described in U.S. Patent No. 7,303,749.

[1721 The cell-binding agent drug conjugates (e.g.,

immunoconjugates) of the present invention can be administered in vitro, in

vivo and/or ex vivo to treat patients and/or to modulate the growth of selected

cell populations including, for example, cancer of the lung, blood, plasma,

breast, colon, prostate, kidney, pancreas, brain, bones, ovary, testes, and

lymphatic organs; autoimmune diseases, such as systemic lupus, rheumatoid

arthritis, and multiple sclerosis; graft rejections, such as renal transplant

rejection, liver transplant rejection, lung transplant rejection, cardiac transplant

rejection, and bone marrow transplant rejection; graft versus host disease; viral

infections, such as CMV infection, HIV infection, and AIDS; and parasite

infections, such as giardiasis, amoebiasis, schistosomiasis, and the like.

Preferably, the immunoconjugates and chemotherapeutic agents of the

invention are administered in vitro, in vivo and/or ex vivo to treat cancer in a

patient and/or to modulate the growth of cancer cells, including, for example,

cancer of the blood, plasma, lung, breast, colon, prostate, kidney, pancreas,

brain, bones, ovary, testes, and lymphatic organs; more preferably lung, colon

prostrate, plasma, blood or colon cancer.

[173] "Modulating the growth of selected cell populations" includes

inhibiting the proliferation of selected cell populations (e.g., multiple myeloma

cell populations, such as MOLP-8 cells, OPM2 cells, H929 cells, and the like)

from dividing to produce more cells; reducing the rate of increase in cell

division as compared, for example, to untreated cells; killing selected cell

populations; and/or preventing selected cell populations (such as cancer cells)

from metastasizing. The growth of selected cell populations can be modulated

in vitro, in vivo or ex vivo.

[174] In the methods of the present invention, the cell-binding agent

drug conjugates (e.g., immunoconjugates) can be administered in vitro, in

vivo, or ex vivo. The cell-binding agent drug conjugates (e.g.,

immunoconjugates) can be used with suitable pharmaceutically acceptable

carriers, diluents, and/or excipients, which are well known, and can be

determined, by one of skill in the art as the clinical situation warrants.

Examples of suitable carriers, diluents and/or excipients include: (1)

Dulbecco's phosphate buffered saline, pH about 6.5, which would contain

about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9% saline (0.9% w/v

NaCl), and (3) 5% (w/v) dextrose.

[1751 The compounds and compositions described herein may be

administered in appropriate form, preferably parenterally, more preferably

intravenously. For parenteral administration, the compounds or compositions

can be aqueous or nonaqueous sterile solutions, suspensions or emulsions.

Propylene glycol, vegetable oils and injectable organic esters, such as ethyl

oleate, can be used as the solvent or vehicle. The compositions can also

contain adjuvants, emulsifiers or dispersants.

[1761 The compositions can also be in the form of sterile solid

compositions that can be dissolved or dispersed in sterile water or any other

injectable sterile medium.

[177] The "therapeutically effective amount" of the cell-binding agent

drug conjugate (e.g., immunoconjugates) described herein refers to the dosage

regimen for modulating the growth of selected cell populations and/or treating

a patient's disease, and is selected in accordance with a variety of factors,

including the age, weight, sex, diet and medical condition of the patient, the

severity of the disease, the route of administration, and pharmacological

considerations, such as the activity, efficacy, pharmacokinetic and toxicology

profiles of the particular compound used. The "therapeutically effective

amount" can also be determined by reference to standard medical texts, such

as the Physicians Desk Reference 2004. The patient is preferably an animal,

more preferably a mammal, most preferably a human. The patient can be male

or female, and can be an infant, child or adult.

[1781 Examples of suitable protocols of cell-binding agent drug

conjugates (e.g., immunoconjugate) administration are as follows. The

conjugates can be given daily for about 5 days either as an i.v., bolus each day

for about 5 days, or as a continuous infusion for about 5 days.

[1791 Alternatively, the conjugates can be administered once a week

for six weeks or longer. As another alternative, the conjugates can be

administered once every two or three weeks. Bolus doses are given in about 50

to about 400 ml of normal saline to which about 5 to about 10 ml of human

serum albumin can be added. Continuous infusions are given in about 250 to

about 500 ml of normal saline, to which about 25 to about 50 ml of human

serum albumin can be added, per 24 hour period. Dosages will be about 10 pg

to about 1000 mg/kg per person, i.v. (range of about 100 ng to about 100

mg/kg).

[180] About one to about four weeks after treatment, the patient can

receive a second course of treatment. Specific clinical protocols with regard to

route of administration, excipients, diluents, dosages, and times can be

determined by the skilled artisan as the clinical situation warrants.

[181] The present invention also provides pharmaceutical kits

comprising one or more containers filled with one or more of the ingredients

of the pharmaceutical compounds and/or compositions of the present

invention, including, one or more immunoconjugates and one or more

chemotherapeutic agents. Such kits can also include, for example, other

compounds and/or compositions, a device(s) for administering the compounds

and/or compositions, and written instructions in a form prescribed by a

governmental agency regulating the manufacture, use or sale of

pharmaceuticals or biological products.

[182] The compounds and conjugates (e.g., immunoconjugates could

also be used for the manufacture of a medicament useful for treating or

lessening the severity of disorders, such as, characterized by abnormal growth

of cells (e.g., cancer).

[1831 Cancer therapies and their dosages, routes of administration

and recommended usage are known in the art and have been described in such

literature as the Physician's Desk Reference (PDR). The PDR discloses

dosages of the agents that have been used in treatment of various cancers. The

dosing regimen and dosages of these aforementioned chemotherapeutic agents

and conjugates that are therapeutically effective will depend on the particular

cancer being treated, the extent of the disease and other factors familiar to the

physician of skill in the art and can be determined by the physician. For

example, the 2006 edition of the Physician's Desk Reference discloses that

Taxotere (see p. 2947) is an inhibitor of tubulin depolymerization;

Doxorubicin (see p 786), Doxil (see p 3302) and oxaliplatin (see p 2908) are

DNA interacting agents, Irinotecal (see p. 2602) is a Topoisomerase I

inhibitor, Erbitux (see p 937) and Tarceva (see p 2470) interact with the

epidermal growth factor receptor. The contents of the PDR are expressly

incorporated herein in their entirety by reference. One of skill in the art can

review the PDR, using one or more of the following parameters, to determine

dosing regimens and dosages of the chemotherapeutic agents and conjugates, which can be used in accordance with the teachings of this invention. These parameters include:

1. Comprehensive index

a) by Manufacturer

b) Products (by company's or trademarked drug name)

c) Category index (for example, "antihistamines", "DNA

alkylating agents" taxanes etc.)

d) Generic/chemical index (non-trademark common drug names)

2. Color images of medications

3. Product information, consistent with FDA labeling

a) Chemical information

b) Function/action

c) Indications & Contraindications

d) Trial research, side effects, warnings

[1841 All references cited herein and in the examples that follow are

expressly incorporated by reference in their entireties.

EXAMPLES

[1851 The invention will now be described by reference to non

limiting examples. Unless otherwise specified, all percents and ratios are by

volume.

Example 1: Materials and Methods

Methyl 2-(acetylthio)-4-bromobutanoate

Br SAc

Br HSAc/DIPEA0 Br O 0 THF, -20°C-0°C >95% 0

11861 10.0 g (38.4 mmol) of methyl 2,4-dibromobutanoate in 100 ml

of dry THF at 20°C was added drop wise the mixture of 2.75 ml (38.5 mmol)

of thiolacetic acid in 8.5 ml (48.9 mmol) of DIPEA and 50 ml of dry THF in

1.5 hour. After stirring overnight at -20 0C then 0C for 2 hours under Ar, the

mixture was concentrated, diluted with EtAc/Hexane, washed with 1.0 M

NaH 2PO 4, dried over MgSO 4 , filtered, evaporated, and SiO 2 chromatographic

purification (1:12 to 1:10 EtAc/Hexane) to afford 9.5 g (96%) of the title

compound. 1HNMR(CDC3)4.38(1H,t,J= 7.1Hz),3.74(s,3H),3.40(m,

2H), 2.57 ~ 2.47 (in, 1H), 2.37 (s, 3H), 2.36 ~ 2.21 (in, 1H); 13C NMR

193.24, 171.36, 53.15, 44.45, 34.67, 30.46, 29.46; MS m/z+ 276.9 (M+Na),

278.9 (M+2+Na)

4-Bromo-1-methoxy-1-oxobutane-2-sulfonic acid

SAc SO 3 H

0O-I H 20 2/HOAc Br O Br 0 ~ >90%0

[1871 9.2 g (36.3 mmol) of methyl 2-(acetylthio)-4-bromobutanoate

in 80 ml of acetic acid was added 40 ml of hydrogen peroxide (35% in water).

The mixture was stirred overnight, then evaporated, diluted with water, neutralized with NaHC03, washed with 1:1 Hexane/EtAc. The aqueous solution was evaporated, dissolved in methanol, concentrated, and crystallized with methanol/toluene to afford 8.6 g (90% yield) of the title compound. m.p.

= 288~ 293 (decomp); 1H NMR (D20) 4.12 (dd, 1H, J = 4.8, 9.3 Hz), 3.83 (s,

3H), 3.64 (in, 1H), 3.53 (in, IH), 2.54 (m, 2H); 13C NMR 172.16, 66.73,

55.66, 33.39, 32.70; MS m/z- 260.8 (M-1).

4-(Acetylthio)-1-methoxy-1-oxobutane-2-sulfonicacid

SO3 H S3 Br HSAc/DIPEA 0 S0 3 H

Br DMA, 700 C ] >90%

[1881 5.0 g (19.2 mmol) of 4-bromo-l-methoxy-1-oxobutane-2

sulfonic acid in 100 ml of THF was added 3.0 ml of thioacetic acid and 9.0 ml

of DIPEA in 100 ml of THF. The mixture was stirred overnight then refluxed

at 70°C for 1 hr, evaporated and co-evaporated with 3 x 100 ml of water after

being neutralized to pH 7 with NaHCO 3 . The mixture was redissolved in

methanol, filtered through celite, concentrated and purified with SiO 2

chromatography eluted with CH 30H/CH 2Cl 2/HCOOH 37.5:250:1 to 50:250:1)

to afford 4.4 g (90% yield) of the title compound. 1H NMR(D20) 3.95 (dd,

1H, J = 4.1, 10.3 Hz), 3.83 (s, 3H), 3.74 (in, 2H), 3.22 (dd, 2H, J = 7.4, 14.9

Hz), 2.39 (s, 3H); 13C NMR 203.88, 172.91, 67.32, 56.17, 29.04, 20.61; MS

m/z- 254.8 (M-H)

4-((5-nitropyridin-2-yl)disulfanyl)-2-sulfobutanoicacid

SO 3 H 1)NaOH 02 N IN 0

2) (SPyNO 2) 2, OH' OH 0 pH 7. S SO 3 H

11891 3.0 g (11.7 mmol) of 4-(Acetylthio)-1-methoxy-1-oxobutane-2

sulfonic acid in 100 ml of water was added 50 ml of 3 M NaOH. After being

stirred under Ar for 3 h, the mixture was neutralized with 1 M H 2PO4 to pH

7.2 under Ar. The mixture was added dropwise to the solution of 10.0 g (32.2

mmol) of 1,2-bis(5-nitropyridin-2-yl)disulfane in 200 ml of DMA. After

being stirred for 4 h under Ar, the mixture was concentrated, diluted with

water, filtered, evaporated and purified with C-18 4.0 x 20 cm column eluted

with water/methanol (95:5) to afford 3.1 g (75% yield) of the title compound.

m.p. = 288 ~ 291°C (decomp.) 1H NMR (DMF-d7) 9.29 (d, 1H, J = 2.2 Hz),

8.63 (dd, 1H, J = 2.7, 8.9 Hz), 8.17 (d, 1H, J = 8.9 Hz), 3.73 (t, 1H, J = 7.2

Hz), 3.22 - 3.17 (m, 1H), 3.15 - 3.10 (m, 1H), 2.41 - 2.33 (m, 2H); 13C NMR

170.92, 169.10, 146.04, 143.67, 133.65, 120.72, 64.22, 37.82, 29.26; MS m/z

352.8 (M-H).

1-(2,5-dioxopyrrolidin-1-yloxy)-4-((5-nitropyridin-2-yl)disulfanyl)-l

oxobutane-2-sulfonic acid

02N 0 0 0 2N S 0 S1- -- OH --- N '

, SO 3H EDC/DMA SO 3 H 0

[190] 220 mg (0.62 mmol) of 4-((5-nitropyridin-2-yl)disulfanyl)-2

sulfobutanoic acid in 15 DMA was added 130 mg (1.13 mmol) of NHS and

480 mg (2.50 mmol) of EDC. The mixture was stirred under Ar overnight,

evaporated and purified on SiO 2 chromatography eluted with

CH2 CH2/CH 30H/HCOOH (10000:1000:1 to 10000:1500:1) to afford 227 mg

(82% yield) of the title compound. 1H NMR (DMSO-d6) 9.25 (d, 1H, J= 5.2

Hz), 8.57 (dd, 111, J = 2.5, 8.9 Hz), 8.04 (t, 1H, J = 8.0 + 8.9 Hz), 3.86 (dd,

1H, J= 4.9, 9.7 Hz), 3.13 ~ 3.12 (m, 2H), 2.76 (s, 4H), 2.36 -2.30 (m, 1H),

2.25'~ 2.21 (m, 1H); 13C NMR 166.96, 165.01, 144.93, 142.26, 132.63,

119.61, 61.00, 35.03, 29.30, 25.39; MS m/z- 449.8 (M-H).

Methyl 2-(acetylthio)-4-bromobutanoate

Br SAc

Br O HSAc/DIPEA 0 THF, -20°C-0°C >95% 0

[191] 10.0 g (38.4 mmol) of methyl 2,4-dibromobutanoate in 100 ml

of dry THF at -20°C was added dropwise the mixture of 2.75 ml (38.5 mmol)

of thiolacetic acid in 8.5 ml (48.9 mmol) of DIPEA and 50 ml of dry THF in

1.5 hour. After stirring overnight at -20°C then 0C for 2 hours under Ar, the

mixture was concentrated, diluted with EtAc/Hexane, washed with 1.0 M

NaH 2PO 4, dried over MgSO 4 , filtered, evaporated, and Si02 chromatographic

purification (1:12 to 1:10 EtAc/Hexane) to afford 9.5 g (96%) of the title

compound. 1I NMR (CDC3) 4.38 (111, t, J = 7.1Hz), 3.74 (s, 3H), 3.40 (m,

2H), 2.57 ~ 2.47 (m, 1H), 2.37 (s, 3H), 2.36 ~ 2.21 (m, 1H); 13C NMR

193.24, 171.36, 53.15, 44.45, 34.67, 30.46, 29.46; MS m/z+ 276.9 (M+Na),

278.9 (M+2+Na)

4-Bromo-1-methoxy-1-oxobutane-2-sulfonic acid

SAc SO 3 H

Br 0 H 20 2 /HOAc Br O >90% O

[192] 9.2 g (36.3 mmol) of methyl 2-(acetylthio)-4-bromobutanoate

in 80 ml of acetic acid was added 40 ml of hydrogen peroxide (35% in water).

The mixture was stirred overnight, then evaporated, diluted with water,

neutralized with NaHCO3 , washed with 1:1 Hexane/EtAc. The aqueous

solution was evaporated, dissolved in methanol, concentrated, and crystallized

with methanol/toluene to afford 8.6 g (90% yield) of the title compound. m.p.

= 288~ 293 (decomp); 1H NMR (D20) 4.12 (dd, IH, J = 4.8, 9.3 Hz), 3.83 (s,

3H), 3.64 (m, 1H), 3.53 (m, 111), 2.54 (m, 2H); 13C NMR 172.16, 66.73,

55.66, 33.39, 32.70; MS m/z- 260.8 (M-1).

4-(Acetylthio)-1-methoxy-1-oxobutane-2-sulfonic acid

SO3 H Sa Bo HSAc/DIPEA 0 S03H 0 Br DMA, 70 C b >90%

[1931 5.0 g (19.2 mmol) of 4-bromo--methoxy-1-oxobutane-2

sulfonic acid in 100 ml of THF was added 3.0 ml of thioacetic acid and 9.0 ml

of DIPEA in 100 ml of THF. The mixture was stirred overnight then refluxed

at 70C for 1 hr, evaporated and co-evaporated with 3 x 100 ml of water after

neutralized to pH 7 with NaHCO 3 . The mixture was redissolved in methanol,

filtered through celite, concentrated and purified with SiO 2chromatography

eluted with CH30H/CH 2C 2/HCOOH 37.5:250:1 to 50:250:1) to afford 4.4 g

(90% yield) of the title compound. 1H NMR(D20) 3.95 (dd, IH, J= 4.1, 10.3

Hz), 3.83 (s, 3H), 3.74 (in, 2H), 3.22 (dd, 2H, J = 7.4, 14.9 Hz), 2.39 (s, 3H);

13C NMR 203.88, 172.91, 67.32, 56.17, 29.04, 20.61; MS m/z- 254.8 (M-H)

4-((5-nitropyridin-2-yl)disulfanyl)-2-sulfobutanoic acid

0 SO 3H0N0 03 -1) NaOH --I-0 N I S ON S 2) (SPYNO 2) 2, S OH 0 pH 7.0 SO 3 SO3H

11941 3.0 g (11.7 mmol) of 4-(Acetylthio)-1-methoxy-1-oxobutane-2

sulfonic acid in 100 ml of water was added 50 ml of 3 M NaOH. After stirring

under Ar for 3 h, the mixture was neutralized with 1 M H 2PO4 to pH 7.2 under

Ar. The mixture was added dropwise to the solution of 10.0 g (32.2 mmol) of

1,2-bis(5-nitropyridin-2-yl)disulfane in 200 ml of DMA. After stirring for 4 h under Ar, the mixture was concentrated, diluted with water, filtered, evaporated and purified with C-18 4.0 x 20 cm column eluted with water/methanol (95:5) to afford 3.1 g (75% yield) of the title compound. m.p.

= 288 - 291 0C (decomp.) 1H NMR (DMF-d7) 9.29 (d, 1H, J = 2.2 Hz), 8.63

(dd, 1H, J = 2.7, 8.9 Hz), 8.17 (d, 1H, J = 8.9 Hz), 3.73 (t, 1H, J = 7.2 Hz),

3.22 - 3.17 (m, 1H), 3.15 ~ 3.10 (m, 1H), 2.41 ~ 2.33 (m, 2H); 13C NMR

170.92, 169.10, 146.04, 143.67, 133.65, 120.72, 64.22, 37.82, 29.26; MS m/z

352.8 (M-H).

1-(2,5-dioxopyrrolidin-1-yloxy)-4-((5-nitropyridin-2-yl)disulfanyl)-l

oxobutane-2-sulfonicacid

ON N 0 Oo 0 N 0 0 2N S SOH .--.-- S-N0N SOH EDC/DMA SO3 H

[1951 220 mg (0.62 mmol) of 4-((5-nitropyridin-2-yl)disulfanyl)-2

sulfobutanoic acid in 15 DMA was added 130 mg (1.13 mmol) of NHS and

480 mg (2.50 mmol) of EDC. The mixture was stirred under Ar overnight,

evaporated and purified on SiO 2 chromatography eluted with

CH 2CH2/CH 30H/HCOOH (10000:1000:1 to 10000:1500:1) to afford 227 mg

(82% yield) of the title compound. 1H NMR (DMSO-d6) 9.25 (d, 1H, J = 5.2

Hz), 8.57 (dd, 1H, J = 2.5, 8.9 Hz), 8.04 (t, 1H, J = 8.0 + 8.9 Hz), 3.86 (dd,

1H, J = 4.9, 9.7 Hz), 3.13 - 3.12 (m, 2H), 2.76 (s, 4H), 2.36 -2.30 (m, 1H),

2.25 ~ 2.21 (in, 1H); 13C NMR 166.96, 165.01, 144.93, 142.26, 132.63,

119.61, 61.00,35.03,29.30,25.39; MS m/z- 449.8 (M-H).

4-(pyridin-2-yldisulfanyl)-2-sulfobutanoic acid

0 SO 3H 1)NaOH 0

2) PySSPy, S OH SO 3H

11961 1.5 g (5.85 mmol) of 4-(Acetylthio)--methoxy-1-oxobutane-2

sulfonic acid was added to 100 ml of 0.5 M NaOH solution. After stirring

under Ar for 3 h, the mixture was concentrated to ~ 50 ml and neutralized with

I M H 2 PO4 to pH 7.2 under Ar. The mixture was added dropwise to the

solution of 4.0 g (18.1 mmol) of 2,2'-dithiodipyridine in 60 ml of DMA. After

stirring for 4 h under Ar, the mixture was concentrated, diluted with water,

filtered, evaporated and purified with C-18 4.0 x 20 cm column eluted with

water/methanol (99:1 to 90:10) to afford 1.32 g (73% yield) of the title

compound. 1H NMR (DMF-d7) 8.39 (dd, 1H, J = 3.5, 4.8 Hz), 7.86 (in, 2H),

7.25 (in, 1H), 3.59 (dd, 1H, J= 5.2, 9.4 Hz), 2.90 (in, 2H), 2.28 (in, 2H); 13C

NMR 172.60, 159.16, 148.93, 138.09, 121.03, 119.38, 67.49, 36.39, 28.666;

MS m/z- 307.8 (M-H).

2 1-(2,5-dioxopyrrolidin-1-yloxy)-1-oxo-4-(pyridin-2-yldisulfanyl)butane-

sulfonic acid

OHN HO-N~ 0 0 SS OH - .. O SO3 H EDC/DMA SO3 H 0

[1971 680 mg (2.20 mmol) of 4-(pyridin-2-yldisulfanyl)-2

sulfobutanoic acid in 50 DMA was added 300 mg (2.60 mmol) of NHS and

800 mg (4.16 mmol) of EDC. The mixture was stirred under Ar overnight,

evaporated and purified on SiO 2 chromatography eluted with

CH2CH2/CH 30H/HCOOH (10000:1000:1 to 10000:1500:1) to afford 720 mg

(80% yield) of the title compound. 1H NMR (DMSO-d6) 8.40 (dd, 1H, J =

3.5, 4.7 Hz), 7.85 (in, 2H), 7.24 (in, 1H), 3.58 (dd, 1H, J= 5.1, 9.4 Hz), 2.94 ~

2.90 (m, 2H), 2.74 (s, 4H), 2.31 -2.27 (in, 2H); 13C NMR 168.16, 161.11,

147.91, 139.22, 121.63, 119.31, 66.80, 36.30, 28.36, 25.42; MS m/z- 404.9

(M-H).

3, 6-endoxo-A-tetrahydrophthalhide

P 0 0 20

NH OO 1NH 00

[1981 Maleimide (5.0 g, 51.5 mmol) in ethylether (200 ml) was added

furan (5.5 ml, 75.6 mmol). The mixture was heated inside a 1 L of autoclave

bomb at 100C for 8 h. The bomb was cooled down to room temperature, and

the inside solid was rinsed with methanol, concentrated and crystallized in

ethyl acetate/hexane to afford 8.4 g (99%) of the title compound. 1H NMR

(DMF-d7):11.08 (s, 1H) (NH), 6.60 (m, 2H), 5.16 (m, 2H), 2.95 (m, 2H). 13C

NMR 178.84, 137.69, 82.00, 49.92. MS m/z+ 188.4 (MW + Na).

Methyl 4-N-(3, 6-endoxo-A-tetrahydrophthalido)-2-sulfo-butyrate

o o O0 0 o ~J~$NH KC03 /KI/DM F I~ 0 INH B0 Br-,^ 1 0 S03H1 SOH

[199] 3, 6-Endoxo-A-tetrahydrophthalhide (0.80 g, 4.85 mmol) in

DMA (20 ml) was added K2 C03 (1.4 g, 10.13 mmol) and KI (0.19 g, 1.14

mmol). After stirring under Ar for 1 hr, methyl 4-bromo-2-sulfo-butyrate (0.98

g, 3.77 mmol) in DMA (10 ml) was added. The mixture was stirred under Ar

overnight, evaporated, re-dissolved in 1% HAc in methanol, filtered,

evaporated and purified by SiO 2 chromatography and eluted with 1:5:0.01 to

1:4:0.01 CH30H/CH 2Cl 2/HA to afford 0.98 (75%) g of the title compound.

1H NMR (DMF-d7): 6.59 (m, 2H), 5.16 (dd, 2H, J =0.8, 7.8 Hz), 3.65-3.63

(m, 3H), 3.47 (m, 2H), 3.01 (s, 3H), 2.83 (m, 2H). 13C NMR 172.94, 162.86,

137.68, 81.98, 52.39, 49.91, 48.58, 36.01, 21.97. MS m/z- 343.9 (MW - H).

Methyl 4-N-maleimido-2-sulfo-butyrate 09 0 0 '0 0 Reflux

0 SO 3H 0 SO3 H

[200] In an opened round bottom flask, methyl 4-N-(3, 6-endoxo-A

tetrahydrophthalido)-2-sulfo-butyrate (0.30 g, 0.87 mmol) in 20 ml of 1:1

DMA/ 100 mM NaH 2 PO4 , pH 7.0 was heated at 120 - 140°C for 4 h. During

the reaction time, 5 x 10 ml of water was gradually added to keep the reaction

volume around 15 ml. The mixture was concentrated to dryness and purified

by SiO 2 chromatography eluted with 1:5:0.01 to 1:4:0.01 1 CH30H/CH 2C 2/HAc to afford 0.230 g (95%) of the title compound. H NMR

(DMF-d7): 6.60 (s, 2H), 4.06 (d, 1H), 3.60 (in, 3H), 3.47 (in, 2H), 2.43 (in,

2H); C NMR 171.59, 164.96, 136.10, 66.20, 51.71, 34.82, 22.10. MS m/z 13

276.6 (MW - H).

Methyl 4-azido-2-sulfo-butyrate

0 0 Br NN 3, N3 /

SO 3H DMA, >90% SO 3H

[2011 Methyl 4-bromo-2-sulfo-butyrate (1.07 g, 4.11 mmol) and

sodium azide (0.70 g (10.7 mmol) in DMF (50 ml) was stirred overnight. The

mixture was evaporated and purified by SiO 2 chromatography and eluted with

1:5:0.01 CH30H/CH 2C2/HAc and crystallized with CH 30H/Toluene/Hexane

to afford 1.00 g (95%) of the title compound. m.p = 267 -272 C (decomp). 1H

NMR (DMF-d7): 12.06 (br, 1H), 3.65 (s, 3H), 3.59 (dd, 1H, J = 5.4, 8.9 Hz),

3.47 (in, 2H), 2.24 (in,2H). "C NMR 171.10, 64.29, 52.24, 50.64, 21.35. ESI

MS m/z+ 267.9 (M + 2Na-H), m/z- 222.0 (M-H). HRMS m/z- (C 5H9 N 30 5S

H) calcd 222.0185, found 222.0179.

4-azido-2-sulfo-butyric acid

0 0 N3 O cH 0 N3 OH HAc,lIOOC SO3H >95% SO 3H

[202] Methyl 4-azido-2-sulfo-butyrate (1.00 g, 4.08 mmol) in the

mixture of HC (50 ml, 1.0 M) and HAC (5 ml) was heated at 100°C for 8 hrs.

The mixture was evaporated and co-evaporated 3x 50 ml of water, and

crystallized with water/acetone to afford 1.0 g (99%) of the title compound. 'H

NMR (DMF-d 7): 3.60 (m, 2H), 3.52 (m, 1H), 2.24 (m, 2H). "C NMR 170.96,

63.04, 50.66, 29.12. ESI MS m/z- 207.7 (MW -H); HRMS m/z- (C 4 H7N 3 0 5 S

H) called 208.0028, found 208.0021.

4-Amino-2-sulfo-butyric acid

0 0 N3 OH H2/Pd/C : H 2N OH SO 3H H 20, >95% SO3H

[2031 4-Azido-2-sulfo-butyric acid (500 mg, 2.40 mmol), water (20

ml) and Pd/C (110 mg, 10% Pd, 50% water based) were placed into a 250 ml

hydrogenation shaking bottle. After the air in the bottle was sucked out by a

vacuum, 20 psi of hydrogen was let into the bottle. The mixture was shaken

for 8 h, then filtered through celite, washed with DMF, evaporated and co

evaporated with dry DMF to afford 476 mg (91% HCI salt) of the title

product. ESI MS m/z- 181.8 (MW -H). This product was used directly without

furtherpurification.

(Z)-4-(3-carboxy-3-sulfopropylamino)-4-oxobut-2-enoicacid

H2 N OH H OH SO3H DMF, >90% o SO3H

[204] The above 4-Amino-2-sulfo-butyric acid, HCI salt (476 mg,

2.16 mmol) in dry DMF (20 ml) was added maleic anhydride (232 mg, 2.36

mmol). The mixture was stirred under Ar overnight, evaporated and purified

on self packed c-18, $1.0 x 25 cm column, eluted with water. The fractions contained product were pooled, evaporated and crystallized with H 20/acetone to afford 552 mg (91%) of the title product. 'H NMR (DMF-d7): 9.70 (br,

1H), 6.73 (d, 1H, J = 12.8 Hz), 6.32 (d, 1H, J = 12.8 Hz), 3.69 (m, 1H), 3.47

(m, 2H), 2.27 (m, 2H). 13 C NMR 171.47, 167.32, 165.87, 135.44, 133.07,

63.82,39.13,27.62. ESI MS m/z- 279.8 (MW -H); HRMS m/z- (C 8HIIN0 8S

H) calcd 280.0127, found 280.0121.

4-N-Maleimido-2-sulfo-butanoic acid

o0 0

uH OH SO 3H OH 1). HMDS/ZnC/DMA 2). CH3 OH/HCI (cat) N SO3H OH

0 > 85%

[2051 (Z)-4-(3-carboxy-3-sulfopropylamino)-4-oxobut-2-enoic acid

(310 mg, 1.10 mmol) in mixture dry DMA (5 ml) and dry toluene (20 ml) was

heated. After the temperature reached at 80°C, HMDS (hexamethyldisilazane)

(1.40 ml, 6.71 mmol) and ZnCl2 (1.85 ml, 1.0 M in diethyl ether, 1.85 mmol)

was added. The mixture was continued heated to 115 ~ 125 0C and toluene was

collected through Dean-Stark trap. The reaction mixture was fluxed at 120 C

for 6 h. During this period, 2 x 20 ml of dry toluene was added to keep the

mixture volume around 8 - 10 ml. Then the mixture was cooled, 1 ml of 1:10

HCI (cone)/CH 30H was added, evaporated, purified on Si02 chromatography

eluted with CH 30H/CH2 Cl 2/HAc (1:5:0.01 to 1:4:0.01) to afford 260mg

(92%) of the title product. H NMR (DMF-d 7): 10.83(br, 1H), 6.95 (s, 2H) ,

1H, J= 12.8 Hz), 3.65 (in, 1H), 3.54 (m, 2H), 2.27 (in,2H). 13 C NMR 173.61,

172.04,135.47,64.18, 37.1, 27.89. ESI MS m/z- 261.8 (MW -H). HRMS m/z

(C 8H 9NO 7 S -H) calcd 262.0021, found 262.0027.

Succinimidyl4-N-maleimido-2-sulfo-butyrate 00

04~ 0 HO-N 0 OH HO N ON

O SO 3 H EDC/DMA o SO 3H o

[206] 4-N-maleimido-2-sulfo-butanoic acid (260 mg, 0.99 mmol) in

DMA (10 ml) was added to NHS (220 mg, 1.91 mmol) and EDC (500 mg,

2.60 mmol). The mixture was stirred under Ar overnight, evaporated and

purified on SiO2 chromatography eluted with CH2CH 2/CH 30H/HAc

(10000:1000:1 to 10000:2000:1), then crystallized with DMA/EtAc/Hexane to

afford 285 mg (81% yield) of the title compound. 'H NMR (DMF-d7) 6.99 (s,

1H), 3.83 (in, 1H), 3.64 (in, 2H), 2.75 (s, 4H), 2.34 (in,2H); "C NMR 171.97,

171.82, 166.64, 135.58, 62.00, 36.66, 26.62; ESI MS m/z- 358.9 (M-H);

HRMS m/z- (C1 2 H 12 N 2 09 S -H) called 359.0185, found 359.0178

(E)-Methyl 4-azidobut-2-enoate

0 0 Br-- N3 0

[2071 To the solution of NaN 3 (2.80 g, 43.01 mmol) in 100 ml of

DMF at -20°C was added methyl 4-bromocrotonate (5.00 ml, 85%, 36.10 mmol). After stirred at -20°C for 30 min, the mixture was stirred at 0C for 4 h, evaporated, suspended with EtAc/Hexane (1:1), filtered, evaporated and chromatographic purification on SiO 2 column eluted with EtAc/Hexane (1:25 to 1: 10 ) to afford HRMS for 4.08 g (80%) of the title product. 'H NMR

(CDC 3) 6.88 (m, 1H), 6.06 (ddd, 1H, J -= 1.7, 3.4, 15.6 Hz), 3.97 (dd, 2H, J = 3 1.2, 4.96 Hz), 3.73 (s, 3H); C NMR 166.23, 140.86, 123.49, 51.95, 51.36;

ESI MS m/z+ 182.5 (M+ Na + H 20);HRMS m/z+ (C 5H7N 30 2 + H 2 0 + Na)

caled 182.0542, found 182.0548.

Methyl 3-(acetylthio)-4-azidobutanoate

0 SAc O N, O - N3 0

[2081 To the solution of (E)-Methyl 4-azidobut-2-enoate (4.00g,

28.37 mmol) in 60 ml of THF at0C was added the mixture of thiolacetic acid

(3.0 ml, 42.09 mmol) and DIPEA (8.0 ml, 45.92 mmol) in 60 ml of THF in 20

min. After stirred at 0C for 1 hr, the mixture was stirred at RT overnight,

evaporated, redissolved in CH2 C12 , washed with NaHCO3 (sat.) and 1 M

NaH 2PO4/NaCI (sat.), pH 4 respectively, dried over MgSO4, filtered,

evaporated and chromatographic purification on SiO2 column eluted with

EtAc/Hexane (1:8 to 1: 4) to afford HRMS for 4.98 g (81%) of the title

product. 1H NMR (CDCl 3) 3.66 (m, 1H), 3.62 (s, 3H), 3.40 (dd, 1H, J = 7.5,

12.7 Hz), 3.31 (m, 1H), 2.78 (m, 1H), 2.60 (m, 1H), 2.32 (s, 3H); "C NMR

(DMF-d7) 192.20, 172.48, 56.56, 53.60, 51.31, 34.58, 30.56; ESI MS m/z+

240.0 (M+ Na), 255.9 (M+ K); HRMSm/z+ (C 7H1 1 N 3 0 3 S+ Na) calcd

240.0419, found 240.0415.

Azido-4-methoxy-4-oxobutane-2-sulfonic acid

SAc 0 SO 3H O N3 O/ 0 N3 0

[2091 Methyl 3-(acetylthio)-4-azidobutanoate (4.00 g, 18.43 mmol) in

75 ml of acetic acid was added 25 ml of H20 2 (30%). The mixture was stirred

overnight, evaporated and co-evaporated with EtOH/toluene and purified on

SiO 2 chromatography eluted with CH 30H/CH 2Cl 2/HAc (100:800:1 to

100:500:1) to afford 3.85 (93%) g the title compound. IH NMR (CD 30D) 3.78

(dd, 1H, J = 5.0, 12.7 Hz), 3.62 (s, 3H), 3.44 (dd, 1H, J = 7.5, 12.7 Hz), 3.33 13 (m, 1H), 2.84 (dd, 1H, J = 5.6, 16.5 Hz), 2.57 (dd, 1H, J = 7.5, 16.5 Hz); C

NMR (DMF-d7) 173.37, 57.31, 52.54, 52.49, 34.51; ESI MS m/z- 221.7 (M+

H),

4-Azido-3-sulfobutanoic acid

SO3H 0 SO 3H 0 N3 Of 3 N3 OH

[210] Azido-4-methoxy-4-oxobutane-2-sulfonic acid (3.80 g, 17.04

mmol) in 150 ml of 1.0 M HC was added 8.0 ml of HAc. The mixture was

refluxed at 120°C overnight, evaporated and co-evaporated with water, EtOH,

EtOH/toluene respectively and purified on SiO 2 chromatography eluted with

CH 30H/CH 2C 2/HAc (100:500:1 to 100:400:1) to afford 3.02 (85%) g the title

compound. 'H NMR (CD 30D) 3.77 (dd, 1H, J 5.1, 12.8 Hz), 3.45 (dd, 1H, J

= 7.0,12.8 Hz), 3.31 (m, 1H), 2.86 (dd, 1H, J 4.7,16.7 Hz), 2.51 (dd, lH, J

= 8.4, 16.7 Hz); 13 C NMR (DMF-d7) 173.98, 67.50, 59.78, 27.82; ESI MS

m/z- 207.7 (M -H).

4-amino-3-sulfobutanoic acid

SO3H O SO3 HO N3 1 OH H2N 2 OH

[2111 In a 500 ml of hydrogenation bottle was added 4-azido-3

sulfobutanoic acid (3.00 g, 14.35 mmol), 150 ml of methanol and 0.32 g of

Pd/C (10% Pd, 50% wet). After sucked out air, 30 psi of H2 was conducted,

and the mixture was shaken overnight, filtered through celite, evaporated, and

coevaporated with dry EtOH to afford about 2.50 g (95%) of 4-amino-3

sulfobutanoic acid. 'H NMR (CD 30D) 3.24 (m, 1H), 3.17 (m, 1H), 2.90 (dd,

1H, J = 2.6, 16.5 Hz), 2.33 (dd, 1H, J = 10.1, 16.5 Hz), ESI MS m/z- 181.60

(M-H). The resulted compound was unstable and was used directly without

further purification.

2 -enoic acid (Z)-4-(3-carboxy-2-sulfopropylamino)-4-oxobut-

SO3H0 'OH H2N N K)OH O O

H03S OH

[212] To the solution of 4-amino-3-sulfobutanoic acid (- 2.50 g,

13.66 mmol) in 100 ml of DMA was added maleic anhydride (1.48 g, 15.10

mmol) and the mixture was stirred over night, evaporated, purified on C-18

column (2 x 30 cm) eluted with 1% HAc in water and crystallized with

MeOH/Acetone/toluene to afford 3.34 g (83%) of (Z)-4-(3-carboxy-2

sulfopropylamino)-4-oxobut-2-enoic acid. 'HNMR (CD 30D) 6.33 (d, 1H, J =

12.6 Hz), 6.10 (d, 1H, J = 12.6 Hz), 3.64 (dd, IH, J = 5.8, 14.0 Hz), 3.54 (in,

1H), 3.30 (in, 1H), 2.78 (dd, 1H, J = 4.9, 16.8 Hz), 2.39 (in, 1H); "C NMR

173.52, 168.68, 167.98, 135.59, 127.79, 57.31, 40.56, 34.52; ESI MS m/z

279.7 (M -H).

4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-sulfobutanoicacid

0 0 (OH

HO 3 S OH HO 3S OH

[2131 (Z)-4-(3-carboxy-2-sulfopropylanino)-4-oxobut-2-enoic acid

(450 mg, 1.60 mmol) in mixture of 10 ml of dry DMA and 50 ml of dry

toluene was heated. After the temperature reached at 80°C, HMDS

(hexamethyldisilazane, 1.80 ml, 8.63 mmol, ) and ZnCl2 (3.2 ml, 1.0 M in

diethyl ether) were added. The mixture was continued heated to 115 ~ 125 °C and toluene was collected through Dean-Stark trap. The reaction mixture was fluxed at 120 C for 6 h. During this period, 2 x 20 ml of dry toluene was added to keep the mixture volume around 8 - 10 ml. Then the mixture was cooled, 1 ml of 1:10 HCI (con)/CH 30H was added, evaporated, purified on

SiO2 chromatography eluted with 1:5:0.01 CH30H/CH 2C12/HAc to afford 315

mg (75%) of the title product. 'H NMR (DMF-d7) 6.96 (s, 2H), 4.04 (dd, 1H,

J = 4.3, 13.8 Hz), 3.47 (m, 1H), 3.23 (dd, IH, J = 7.4, 14.7Hz), 2.99 (dd, 1H, J

= 3.3 , 16.8 Hz), 2.35 (dd, 1H, J = 8.1, 16.9 Hz); "C NMR 173.58, 172.18,

135.54,54.61,40.24,32.43,ESI MS m/z- 261.70 (M -H).

1-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-(2,5-dioxopyrrolidin-1-yloxy)

4-oxobutane-2-sulfonicacid

0 0 HO 3S OH HO 3S ONHS

[214] 4-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-sulfobutanoic acid

(110 mg, 0.418 mmol), EDC (240 mg, 1.25 mmol) and N-hydroxysuccinimide

(58 mg, 0.504 mmol) was stirred in 10 ml of DMA for overnight, evaporated

and purified on SiO2 chromatography eluted with CH30H/CH 2C 2/HAc 1 (100:900:1 to 100: 600:1) to afford 112 mg (75%) of the title product. H

NMR (DMF-d7) 6.93 (s, 2H), 4.06 (dd, 1H, J = 4.8, 13.1 Hz), 3.80 (dd, 1H, J

10.7, 13.9 Hz), 3.35 (dd, 1H J= 3.3, 17.8 Hz), 3.25 (m, 1H), 3.10 (dd, 1H, J

= 2.2, 16.4 Hz), 2.87 (in, 4H); C NMR 172.27, 170.88, 169.29, 135.55,

55.28, 40.22, 32.69, 26.66; ESI MS m/z- 261.70 (M -H).

Ethyl 3-(acetylthio)-3-cyanopropanoate

CN 0 HSAc/Et3N CN 0

THF, O*C, 65%

[2151 (Z)-ethyl 3-cyanoacrylate (5.01 g, 40.00 mmol) in 80 ml of

THF at -20°C was added the solution of thiol acetic acid (5.0 ml, 70.15 mmol)

and DIPEA (16.0 ml, 92.03 mmol) in 20 ml of THF in 30 min. The reaction

was kept at -20°C for 4 hr then room temperature overnight. The mixture was

concentrated, diluted with CH2 Cl2 , washed with saturated NaHCO 3, dried over

MgSO4 , filtered, evaporated and purified by SiO 2 chromatography (1:4

EtAC/Hexane) to afford 5.22 g (65%) of the title compound. Rf =0.25 (1:4

EtAC/Hexane); H NMR (CDC 3), 4.44 (in, 1H), 4.11 (dd, 2H, J = 7.1, 14.3

Hz), 3.38 (in, 1H), 3.15 (in, 1H), 2.17 (s, 3H), 1.19 (t, 3H, J = 7.2 Hz); 1 3 C

NMR 194.12, 173.21, 119.82, 61.35, 33.52, 30.08, 14.62; MS m/z+ 225.9

(MW + Na), m/z- 201.7 (MW-H).

Cyano-3-ethoxy-3-oxopropane-1-sulfonic acid

CN 0 CN 0

AcSKA O HOS'> O-

[216] Ethyl 3-(acetylthio)-3-cyanopropanoate (2.00g, 9.95 mmol) in

acetic acid (40 ml) was added H 2 02 (12 ml, 30%). The mixture was stirred overnight, evaporated and purified on silica gel chromatography eluted with methanol/dichloromethane/acetic acid (1:8:0.01 to 1:5:0.01) to afford 1.72 g

(84%) of the title compound. 1H NMR (DMSO), 4.63 (m, 1H), 4.12 (dd, 2H, J

= 7.1, 14.3 Hz), 3.27 (m, 111), 3.05 (in, 1H), 1.28 (t, 3H, J = 7.2 Hz); 13C NMR

173.15,113.85, 61.38, 48.32,26.33, 14.15; MS m/z- 205.7 (MW-H).

1-(tert-Butoxycarbonylamino)-4-ethoxy-4-oxobutane-2-sulfonicacid

ON 0 BOC-HN

H03S1 OHOsS OC2H,

12171 In a hydrogenation bottle was added Cyano-3-ethoxy-3

oxopropane-1-sulfonic acid (2.50 g, 12.06 mmol), ethanol (80 ml), fresh

filtered Raney Nickel (0.40 g) and BOC anhydride (3.30 g, 15.12 mmol).

After the air inside the bottle was sucked out by vacuum, 20 psi of hydrogen

was conducted to the bottle. The bottle was shaken over night, filtered through

celite, evaporated, and purified on silica gel chromatography eluted with

methanol/dichloromethane/acetic acid (1:6:0.01) to afford 3.18 g (85%) of the

title compound. 1H NMR (DMS), 6.82 (s, 1H), 4.26 (m, 1H), 4.11 (dd, 2H, J

= 7.1, 14.3 Hz), 3.53 (dd, 1H, J = 4.2, 13.4 Hz), 3.36 (in, 1H), 2.86 (in, 1H),

2.51 (in, 1H), 1.38 (s, 9H), 1.22 (t, 3H, J= 7.2 Hz); "C NMR 173.35, 155.72,

80.44, 62.05, 52.55, 41.61, 34.50, 28.85, 14.52; MS m/z- 309.8 (MW-H).

4-(tert-butoxycarbonylamino)-3-sulfobutanoic acid

BOC-HN BOC-HN

HO 3 S OC2H 5 HO 3S OH

2

[218] 1-(tert-Butoxycarbonylamino)-4-ethoxy-4-oxobutane-

sulfonic acid (402 mg, 1.29 mmol) in the mixture of THF/H20(1:2, 60 ml)

was added lithium hydroxide monohydrate (2.0 g, 47.6 mmol). The mixture

was stirred under Ar overnight, concentrated, purified on C-18 column (2 x 30

cm) eluted with from 100% water to 10% methanol in water to afford 328 mg

(90%) of the title compound. 'H NMR (DMSO), 6.78 (s, 1H), 4.03 (m, 1H),

3.57 (dd, 1H, J= 4.2, 13.4 Hz), 3.41 (m, 1H), 2.89 (m, 1H), 2.61 (m, 1H), 1.39 13 C NMR 174.21, 155.82, 79.85, 59.95, 42.06, 32.52, 28.88, 14.55; (s, 9H);

ESI MS 281.8 (M-H);

(Z)-4-(3-carboxy-2-sulfopropylamino)-4-oxobut-2-enoic acid

0 SO3 H O BOC-HN OHOH ' O OH HO 3S OH 28 0

[219] 4-(Tert-butoxycarbonylamino)-3-sulfobutanoic acid (321 mg,

1.13 mmol) was stirred in the mixture of HCl (conc)/Dioxane (1:4, 15 ml) for

30 min, evaporated and coevaporated with EtOH/Toluene (1:1, 4 x 20 ml) to

dryness. To the dryness material was added maleic anhydride (121 mg, 1.23

mmol) and DMA (20 ml) and the mixture was stirred overnight, evaporated

and run through C-18 column eluted with water and crystallized with

EtOH/Hexane to afford 263 mg (83%) of the title compound. ESI MS 279.8

(M- H). The NMR data are the same through the route with 4-azido-3

sulfobutanoic acid.

N,N,N-trimethyl-2-oxotetrahydrothiophen-3-aminium

0 HCI*H 2N s CH 31

[2201 , 3-aminodihydrothiophen-2(3H)-one hydrochloride (6.00 g,

39.1 mmol), sodium bicarbonate (3.28 g, 39.1 mmol) and iodomethane (13

mL, 209 mmol) were stirred in dry methanol (100 ml) overnight, filtered

through celite, evaporated, purified on SiO2 column eluted with

MeOH/CH 2C 2/HAc(1:5:0.01), and crystallized with EtOH/Hexane to afford

5.25 g (84%) of the title product. mp 228- 231°C. 'H NMR (CD 30D) 4.27 (m, 3 1H), 3.25 (s, 9H), 2.56 - 2.47 (m, 2H), 2.34 (m, 111), 2.26 (m, 1H); C NMR

168.97, 75.06, 53.25, 30.85, 16.46; ESI MS m/z+ 160.0 (M+).

1-carboxy-N,N,N-trimethyl-3-(pyridin-2-yldisulfanyl)propan--aminium HC H 3C O H3 CH 3 H 3CN 1).NaOI H 3 C' H3C", H S P- S " COOH 2). PySSPy, pH 7 N

[2211 N,N,N-trimethyl-2-oxotetrahydrothiophen-3-aminium acetate

(2 g, 9.13 mmol) was stirred in 75 ml of 1 M NaOH (3 g NaOH in 75 ml H 2 0)

for 45 min. neutralized with 4 M H 3 PO4 to pH 7.4, concentrated, added to 1,2 di(pyridin-2-yl)disulfane (11 g, 49.9 mmol) in 200 ml of MeOH. The mixture was stirred over night, extracted with EtAc. The aqueous solution was evaporated, suspended with MeOH, filtered salt, evaporated and purified on

C-18 column (2 cm x 30 cm) eluted with water/methanol (100 water to 20%

methanol/water) to afford 2.6 g (75%) of the title product. ESI MS m/z+ 309.1

(M +Na-H).

1. Modification of antibody with sulfo linker

[2221 The huC242 is modified with sulfo linker at 8 mg/mL antibody,

a 15 fold molar excess of sulfo linker (~30mM stock solution in DMA). The

reaction is carried out in 100 mM NaPi, pH8.0 buffer with DMA (5% v/v) for

15, 30, 120, and 200 minutes at 25 °C. The modified huC242 was purified by

G25 column with 50 mM NaPi, 50 mM NaCl, and 2 mM EDTA, pH6.5 to

remove the excess sulfo linker.

2. Measurement of releasable Spy-NO2 and antibody concentration of

modified huC242

[223] The assay and spectral measurement were carried in 100 mM

NaPi, pH7.5 at room temperature. The molar ratio of Spy-NO2 released per

mole of huC242 antibody was calculated by measuring the A 2 80 of the sample

and then the increase in the A394 of the sample after adding DTT (50 pL of 1

M DTT/mL of sample). The concentration of DTT-released 2

1 The mercaptopyridine is calculated using a E394 nm of 14,205 M-1cm .

concentration of antibody can then be calculated using a 6280 nmof 217,560 M~

Icm- 1 after subtracting the contribution of Spy-NO 2 absorbance at 280 nm

(A 394 nmpost DTT x 3344/14205) from the total A 2 80 nm measured before DTT

addition. The molar ratio of Spy-N0 2 :Ab can then be calculated. The mg/mL

(g/L) concentration of huC242 is calculated using a molecular weight of

147,000 g/mole.

3. Conjugation reaction

[2241 The modified huC242 was reacted with a 1.7-fold molar excess

of DM4 (based on DM4 stock SH concentration) over Spy-NO 2. The reaction

is carried out at 2.5 mg/mL antibody in 50 mM NaPi, 50 mM NaCl, 2 mM

EDTA, pH6.5 and DMA (5% v/v). After addition of DM4, the reaction was

incubated 25°C for ~20 hours. The final conjugate was purified by G25

column with 10 mM Histidine, 130 mM Glycine, 5% sucrose, pH5.5 to

remove the excess DM4 drug.

4. Calculation of huC242 and DM4 concentration

[2251 The huC242 and DM4 both absorb at the two wavelengths used

to measure each component separately, i.e., 280 and 252 nm. The extinction 1 coefficient at 280 nm for huC242 is 217,560 and for DM4 is 5180 M- . The

252 nm/280 nm absorbance ratios of huC242 and DM4 are 0.368 and 5.05

respectively. The concentrations were calculated with following equation

CD= A? 2- 0.368A 8 o CAb= A28 - 5180C_

24692.4 217,560

Results

Modification L/A D/A Monomer ratio Free drug

% time 15 min 5.0 4.1 96.7% N/D* 30 min 6.1 5.4 96.2% <1% 120 min 6.6 6.8 95.7% <1% 200 min 6.6 6.3 95.9% <1%

C242-Sulfo-DM4 linker titration

Linker DM4 mg/mL jig/mL Free Excess L:A xs D:A Ab DM4 %Monomer Drug 5 2.4 1.7 1.9 0.83 8.2 95 0 10 4.1 1.7 3.3 0.83 14.4 94 0 15 5.6 1.7 4.6 0.82 20.0 93 0 20 7.3 1.7 6.0 0.82 25.8 91 0 25 9.1 1.3 6.6 0.79 27.7 92 0.6 30 10.4 1.3 7.6 0.68 27.5 94 1.1 35 12.2 1.3 8.2 0.67 26.7 95 1.6

Conjugationprotocol:

[2261 Modification was done at pH 8.0, buffer A and 5% DMA for 90

min at room temperature, the antibody concentration is 7 mg/ml. The

modificed antibody was purified by NAP column using Buffer A pH6.5. The

conjugation was down at Buffer A, pH6.5 with 5-10% DMA at room

temperature overnight. The drug to linker ratio ranged from 1.3 to 1.7

deepening on the total drug added.

Example 2: Conjugate Synthesis.

[2271 SPP or SSNPP linker was dissolved in ethanol at a

concentration of approximately 10 mM. Antibody was dialyzed into buffer A

(50 mM KPi, 50 mM NaCl, 2 mM EDTA, pH 6.5). For the linker reaction, the

antibody was at 8 mg/ml, and 7 equivalents of linker were added while stirring

in the presence of 5% (v/v) ethanol. The reaction was allowed to proceed at

ambient temperature for 90 minutes. Unreacted linker was removed from the

antibody by Sephadex G25 gel filtration using a Sephadex G25 column

equilibrated with Buffer A at pH 6.5 or 150 mM potassium phosphate buffer

containing 100 mM NaCl, pH 7.4 as indicated. For the SPP linker, the extent

of modification was assessed by release of pyridine-2-thione using 50 mM

DTT and measuring the absorbance at 343 nm as described below (6343 =

8080 M 1 cm 1 for free pyridine-2-thione). For SSNPP, modification was

assessed directly by measuring the absorbance at 325 nm (E325 = 10,964 M

cm1 for the 4-nitropyridyl-2-dithio group linked to antibody). For the

conjugation reaction, thiol-containing drug (either DM1 or DC4) was

dissolved in DMA (N, N-dimethylacetamide) at a concentration of

approximately 10 mM. The drug (0.8 - 1.7-fold molar excess relative to the

number of linker molecules per antibody as indicated) was slowly added with

stirring to the antibody which was at a concentration of 2.5 mg/ml in buffer A

(pH 6.5 or pH 7.4) in a final concentration of 3% (v/v) DMA. The reaction was allowed to proceed at ambient temperature for the indicated times. Drug conjugated antibody was purified using a Sephadex G25 column equilibrated with buffer B (PBS, pH 6.5). For DML, the extent of drug conjugation to antibody was assessed by measuring A 252 and A 28 0 of the conjugate as described below. A similar approach was used for DC4 (see below).

Measurement of Releasable Pyridine-2-thione and Ab Concentration of SPP

Modified Ab.

[2281 The molar ratio of pyridine-2-thione released per mole of

antibody is calculated by measuring the A 2 8 0 of the sample and then the

increase in the A3 43 of the sample after adding DTT (50 pL of 1 M DTT/mL of

sample). The concentration of DTT-released pyridine-2-thione is calculated

using an 6343 of 8080 Mcm. The concentration of antibody can then be

calculated using an 6280 of 194,712 M 1cm 1 after subtracting the contribution

of pyridine-2-thione absorbance at 280 nm (A3 4 3 nm post DTT x 5100/8080)

from the total A 2 80nm measured before DTT addition. The molar ratio of

pyridine-2-thione:Ab can then be calculated. The mg/mL (g/L) concentration

of Ab is calculated using a molecular weight of 147,000 g/mole.

Measurement of antibody-linked 5-Nitropyridyl-2-dithio Groups and Ab

Concentration of SSNPP-Modified Ab.

[229] The molar ratio of the 4-nitropyridyl-2-dithio groups linked per

mole of antibody is calculated by measuring the A 280 and A 325 of the sample

without DTT treatment. The number of antibody-bound 4-nitropyridyl-2

dithio groups is calculated using an E325m of 10,964 M- 1cm-1. The

concentration of antibody can then be calculated using an 6280 nm of 194,712

M~1cm~1 after subtracting the contribution of the 5-nitropyridyl-2-dithio group

absorbance at 280 nm (A 325s a x 3344/10964) from the total A 280 nm measured.

The molar ratio of 4-nitropyridyl-2-dithio groups :Ab can then be calculated.

The mg/mL (g/L) concentration of Ab is calculated using a molecular weight

of 147,000 g/mole.

Calculating Ab and DM1 component concentrations of Ab-DMl.

[2301 The Ab and DM1 both absorb at the two wavelengths used to

measure each component separately, i.e., 280 and 252 nm. The components

are quantified using the following algebraic expressions which account for the

contribution of each component at each wavelength (CAb is the molar

concentration of Ab and CD is the molar concentration of DM1):

1) Total A 2 80 =1 9 4 ,7 1 2 CAb + 5,700CD

2) Total A 2 52=(194,712 x 0. 3 7 )CAb+ (4.7 x 5,700) CD

Each equation is solved for CAb:

I a) CAb= A280 - 5,700CD 194,712

2a) Ca= A_52- 26,790Cn 72,043

and an equality is set up (equation 1a= equation 2a) and solved for CD:

CD= A252_- 0.37A28o 24,681

[2311 Once the CD is calculated, the value is used to solve for CAb in

equation 1a (or 2a) above. The ratio of DM:Ab can then be calculated. The

mg/mL (g/L) concentration of antibody is calculated using a molecular weight

of 147,000 g/mole and the concentration of DM1 is calculated using a

molecular weight of 736.5 g/mole (linked DM1)

Efficiency of disulfide exchange is increased with SSNPP.

[232] As shown in Table 1, the efficiency of conjugation is enhanced

in reactions where SSNPP is used as the cross-linker compared to reactions

using SPP. The percent efficiency was calculated by dividing the value for

DM1 per antibody by the linker per antibody ratio times 100. Conjugations of

the N901 antibody using SSNPP resulted in cross-linking efficiencies of 93%

at both pH 6.5 and 7.4. The efficiency of conjugation of N901 with SPP in

these experiments was 70% at pH 6.5 and 77% at pH 7.4. The increased

efficiency with SSNPP demonstrates that a target DM Ito antibody ratio can

be achieved using antibody that is modified with a reduced number of linker

molecules. In fact, a similar drug to antibody ratio (4.3) was achieved in the

final conjugate with an antibody preparation having 4.2 (5-nitropyridyl-2 dithio)-groups per antibody introduced with SSNPP compared to an antibody having 5.6 pyridyl-2-dithio groups introduced with SPP (Table 2). The amount of drug required to obtain comparable conjugation results was therefore 25% lower for the SSNPP-modified antibody than the SPP-modified antibody under these conditions. An additional potential benefit of the increased efficiency with SSNPP is that a reduced molar excess of DM1 may be used in the conjugation reaction. A comparison of the DM1 per antibody ratios following conjugation with a range of drug equivalents in the reaction

(0.8 - 1.7 fold excess) shows that a 1.1-fold molar excess is sufficient to

achieve 100% conjugation efficiency using the SSNPP cross-linker (Figure 7).

A comparison of the time course of the reaction of DM1 with antibody that

had been modified with SSNPP or SPP is shown, for example, in Figure 8. In

each case the modified antibody was treated with a 1.1-fold molar excess of

DM1 per mole of linker incorporated. The reaction with the SSNPP-modified

antibody is considerably faster than with the SPP-modified antibody (Figure

8). Even, a molar excess of 1.7-fold is not sufficient to achieve a similar

efficiency using SPP. The ability to use 1) a lower molar excess of DM1 and

2) fewer linkers per antibody allows a reduction in the amount of drug needed

to achieve a target DM Ito antibody ratio by as much as 50% when using

SSNPP as the cross-linker instead of SPP.

[233] The increased efficiency of conjugation using the SSNPP linker

is accomplished without compromise in the monomeric character of the conjugate and in the amount of unconjugated (free) drug associated with the antibody conjugate. SEC analysis is used to determine the amount of monomer, dimer, trimer, or higher molecular weight aggregates. Typical results of greater than 90% monomer were obtained with either linker as shown in Table 1. The level of unconjugated drug was measured by reverse phase HPLC analysis of the conjugate sample. The percent free drug for either reaction was less than 2%. In addition, shorter conjugation reaction times are possible with SSNPP compared with SPP (U.S. Patent

No.6,913,748), which may decrease loss of some antibodies that are sensitive

to prolonged exposure to organic solvent required in the conjugation reaction.

Shorter reaction times should also decrease drug loss due to DM1

dimerization, which is a competing side reaction during conjugation. The

resulting increases in yield and reduced side reactions should further

contribute to reduced DM1 requirements.

[234] The enhanced rate and efficiency of conjugation when using

SSNPP was also observed when conjugating a different drug to the antibody

demonstrating the broad applicability of this new linker reagent. A

comparison of conjugation efficiencies using SSNPP and SPP when

conjugating the N901 antibody with the DNA-alkylating drug, DC4, a

CC-1065 analogue, is shown, for example, in Table 3. By2hoursthe

reaction using the SSNPP cross-linking reagent was complete whereas the

reaction using the SPP reagent showed only 73% completeness by 2 hours and significant incorporation of drug beyond 2 hours (91% after 18 hours).

Only much prolonged reaction times may lead to 100% completeness.

Example 3. In vitro Cytotoxicity Evaluation of Maytansinoid Conjugates

of Antibodies with Thioether (Non-Cleavable) and Disulfide Linkers

Containing sulfonate group:

[235] The cytotoxic effects of the antibody-maytansinoid conjugates

with thioether and disulfide linkers containing a sulfonate groupwere typically

evaluated using a WST-8 cell-viability assay after a 4-5 day continuous

incubation of the cancer cells with the conjugates. The antigen-expressing

cancer cells (~1000-5000 cells per well) were incubated in 96-well plates in

regular growth medium containing fetal bovine serum with various

concentrations of the antibody-maytansinoid conjugates for about 5 days. The

WST-8 reagent was then added and the plate absorbance was measured at 450

nm after ~2-5 h. The survival fraction was plotted versus conjugate

concentration to determine the IC50 value (50% cell killing concentration) of

the conjugate.

[236] Figures 60 and 61 show the enhancement in cytotoxicities of

Anti-CanAg (huC242) - maytansinoid conjugates with the sulfonate

containing disulfide-bonded linker (huC242-Sulfo-SPDB-DM4) bearing 6.0 to

7.6 maytansinoid/Ab compared to the conjugate with 3.3 maytansinoid/Ab

toward CanAg-positive COL0205 and COL0205-MDR cells. The potency of the conjugates with high maytansinoids loads indicate that the decoration of the antibody with up to 8 maytansinoid molecules did not affect the conjugate binding to the target COL0205 cells.

12371 Figure 64 shows the cytotoxic activities of anti-CanAg Ab

maytansinoid conjugates with similar maytansinoid load against CanAg

antigen-positive COL0205-MDR cells. The presence of sulfonate group in

disulfide linker significantly enhanced conjugate potency toward these

multiple drug resistant cells. The enhanced potency of the sulfonate-linked

conjugate is a novel finding and potentially very promising for therapeutic

applications.

[238] Figure 63 shows the cytotoxic activities of anti-EpCAM Ab

maytansinoid conjugates with similar maytansinoid load against EpCAM

antigen-positive COL0205-MDR cells. The presence of a sulfonate group in

disulfide linker significantly enhanced conjugate potency toward these

multiple drug resistant cells. The enhanced potency of the sulfonate-linked

conjugate is a novel finding and potentially very promising for therapeutic

applications.

[239] Figure 64 shows the cytotoxic activities of anti-EpCAM Ab

maytansinoid conjugates with similar maytansinoid load against EpCAM

antigen-positive HCT cells. The presence of a sulfonate group in the disulfide

linker significantly enhanced conjugate potency toward these multiple drug resistant cells. The enhanced potency of the sulfonate-linked conjugate is a novel finding and potentially very promising for therapeutic applications.

[240] Figure 65 shows the cytotoxic activities of anti-EpCAM Ab

maytansinoid conjugates with similar maytansinoid load against EpCAM

antigen-positive COL0205-MDR cells. The presence of a sulfonate group in

the thioether linker significantly enhanced conjugate potency toward these

multiple drug resistant cells. The enhanced potency of the sulfonate-linked

conjugate is a novel finding and potentially very promising for therapeutic

applications.

Example 4. Comparison of in vivo anti-tumor activity of the anti

EpCAM-maytansinoid conjugates, B38.1-SPDB-DM4 and B38.1-sulfo

SPDB-DM4, on colon cancer, COL0205 and COL0205-MDR,

xenografts:

[241] The anti-tumor effect of B38.1-SPDB-DM4 and B38.1-sulfo

SPDB-DM4 conjugates was evaluated in a xenograft model of human colon

carcinoma, COL0205 and COL0205-MDR, which was engineered to

overexpress P-glycoprotein. The cells were injected subcutaneously in the

area under the right shoulder of SCID mice. When the tumor's volume

reached approximately 200 mm 3 in size, the mice were randomized by tumor

volume and divided into three groups. Each group was treated with a single

i.v. bolus of either B38.1-SPDB-DM4 (10 mg conjugate protein/kg), B38.1 sulfo-SPDB-DM4 (10 mg conjugate protein/kg) or phosphate-buffered saline

(vehicle control). Tumor growth was monitored by measuring tumor size

twice per week. Tumor size was calculated with the formula: length x width x

height x 2.

[2421 The changes in volumes of individual COL0205-MDR tumors

are shown in Figure 66. Treatment with either conjugate resulted in

significant tumor growth delay. B38.1-sulfo-SPDB-DM4 was more

efficacious than B38.1-sulfo-SPDB-DM4 in this human colon cancer

xenograft model.

12431 The changes in volumes of individual COL0205 tumors are

shown in Figure 67. Treatment with either conjugated resulted in significant

tumor growth delay. Two of six animals treated with B38.1-sulfo-SPDB-DM4

had complete tumor regressions. Thus, B38.1-sulfo-SPDB-DM4 was

significantly more efficacious than B38.1-sulfo-SPDB-DM4 in this model.

Example 5. Synthesis of procharged linkers (CX1-1):

Z-Gly-Gly-Gly-P -Ala-OtBu 0 0 0 H 0 H H ON N N 'N O -rOH H2 N O EDC , I N H 0 H 0 0 H 0 H 0

[244] 1.3 g (4.0 mmol) of Z-Gly-Gly-Gly-OH, 0.583 g (4.0 mmol) of

tert-butyl-3-aminopropionate 0.651 g (4.25 mmol) of hydroxybenzotriazole

and 0.81 g (4.23 mmol) of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride were weighed into a 50 mL flask then dissolved in 20 mL of dimethylformamide with magnetic stirring under a nitrogen atmosphere. After

3 hours the reaction mixture was purified in 5 mL portions by reverse phase

HPLC using a 5.0 cm x 25 cm C18 column. The column was run at 100

mL/min with deionized water containing 0.3 % formic acid 5% acetonitrile for

10 min followed by a 15 min linear gradient from 5% acetonitrile to 90%

acetonitrile. Product fractions (retention time of 19 min) were combined and

solvent was removed by rotary evaporation under vacuum to give 1.35 g

(75%) of the title compound. 'HNMR(d 6 -DMSO)8.16(t,J= 5.2Hz,1H),

8.10(t,J=5.2Hz,lH),7.82(t,J=5.2Hz,1H),7.25-7.4(m,5H),5.04(s,

2H), 3.74 (d, J= 5.6 Hz, 2H), 3.67 (t, J= 6.4 Hz, 4H), 3.25 (q, J= 6.1 Hz,

2H), 2.35 (t, J= 6.8 Hz, 2H), 1.39 (s, 9H). "C NMR (d6 -DMSO) 170.45,

169.61, 169.00, 168.63, 156.49, 136.94, 128.30, 127.76, 127.69, 79.89, 65.51,

43.56, 42.10, 41.90, 34.89, 34.78, 27.70. HRMS ( M +Na+) Calc. 473.2012

found 473.1995.

H-Gly-Gly-Gly-p-Ala-OtBu

0 H 0 H 0 H H o Pd-c , H2 N NjN yO / ON NN 10% 0 H H 0 0 H 0 0 0

[245] 1.3 g (2.89 mmol) of Z-Gly-Gly-Gly-p-Ala-OtBu was disolved

in 80 mL of 95:5 methanol:deionized water in a 250 mL parr shaker flask to which was added 0.12 g of 10% palladium on carbon. The flask was shaken under a hydrogen atmosphere (42 PSI) for 7 hours. The mixture was vacuum filtered through celite filter aid and the filtrate was concentrated by rotary evaporation under vacuum to give 0.88 g (96%) of the title compound. 1H

NMR (d 6-DMSO) 8.12 (t, J= 1.6Hz 2H), 8.08 (t, J=1.6 Hz, 1H), 3.75 (s,2H),

3.64 (d, J= 5.9 2H), 3.28 (bs, 2H), 3.24 (q, J= 6.0 Hz, 2H), 3.13 (s, 2H), 2.35

(t, J = 6.8 Hz, 2H), 1.39 (s, 9H). 1C NMR (d-DMSO) 173.38, 170.46,

169.18, 168.70, 79.89, 44.65, 41.95, 34.88, 34.78, 27.71. HRMS (M + H)

Cale. 317.1825, found 317.1801

Mal-Gaba-Gly-Gly-Gly-p -Ala-OtBu

0 0 H H EEIC 0 0 H EDC N N N O 'N OHH+ H 2 N 0 H010 H 0 H 0

[246] 513 mg (2.8 mmol) of 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1

yl)butanoic acid, 800 mg (0.2.8 mmol) tert-butyl 3-(2-(2-(2

aminoacetamido)acetamido)acetamido)propanoate and 583 mg (3.0 mmol) N

(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride were disolved

in 12 mL of dimethyl formamide and stirred for 3 hours. The reaction mixture

was purified in four equal portions by reverse phase HPLC using a 5.0 cm x

25 cm C18 column. The column was eluted at 100 mL/min with deionized water containing 0.3 % formic acid and 5% acetonitrile for 10 min followed by a 13 min linear gradient from 5% acetonitrile to 33 % acetonitrile. Product fractions (retention time of 21 min) were combined and solvent was removed by rotary evaporation under vacuum to give 832 mg (62 %) of the title compound. 'H NMR (d6 -DMSO) 8.10-8.16 (m, 2H), 8.07 (t, J= 4.8 Hz, 1H),

7.0 - 7.15(m, 1H), 3.747 (t, J= 6.0 Hz, 3H), 3.64 (d, J= 5.6 Hz, 2H), 3.41 (t,

J= 6.8, 2H), 3.1-3.33 (m, 1H), 3.19-3.26 (m, 2H), 2.348 (t, J= 6.8, 2H), 2.132 13 (t, J= 7.2 Hz, 2H), 1.67 - 1.76 (m, 2H), 1.39 (s, 9H). C NMR (d6 -DMSO)

171.80, 170.98, 170.39, 169.48, 168.96, 168.56, 134.37, 79.83, 42.05, 41.83,

37.38, 34.82, 34.71, 32.26, 27.83, 23.95. HRMS (M + Na) Calc. 504.2070

found 504.2046

Mal-Gaba-Gly-Gly-Gly-P-Ala-OH

0 0

VN0 H0 H TFA / 0 N TA -N OH 0H 0 H 0 0 0 H 0 H 0 06

[2471 820 mg (1.7 mmol) of Mal-Gaba-Gly-Gly-Gly-p-Ala-OtBu was

disolved in 9.0 mL of 95:5 trifluoroacetic acid: deionized water and

magnetically stirred for 3 hours. Solvent was removed by rotary evaporation

under vacuum to give 730 mg (100%) of the title compound. 'H NMR (d6

DMSO) 12.1 (bs, 1H), 8.05-8.20 (m, 3H), 7.82 (t, J= 6.0 Hz, 1H), 7.00 (s,

2H), 3.71 (t,J=6.0Hz, 4H), 3.65 (d,J=6.0Hz, 2H), 3.41 (t,J=7.2Hz, 2H),

3.26 (q, J= 5.6 Hz, 2H), 2.38 (t, J= 7.2 Hz, 2H,), 2.14 (q, J= 8.0 Hz, 2H),

1.67-1.77 (m, 2H). "C NMR (d6-DMSO) 172.70, 171.83, 171.01, 169.50,

168.99, 168.51, 134.38, 42.07, 41.84, 36.75, 34.70, 33.69, 32.28, 23.97

HRMS (M + Na) Calc. 448.1444 found 448.1465

Mal-Gaba-Gly-Gly-Gly-p -Ala-ONHS (CX1-1) 0 0 0 0 0 H0

H 0 N OH EDC, NHS N N N 00N N JN0 H 0 H 0 0 0 H 0 H 0 0

[2481 76 mg (0.18 mmol) of Mal-Gaba-Gly-Gly-Gly-p-Ala-OH, 72

mg, (0.376 mmol) of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide

hydrochloride and 66 mg (0.575 mmol) of N-hydroxysuccinimide were

disolved in 1.0 mL of dimethyformamide with magnetic stirring. After 2

hours the reaction mixture was purified in two equal portions by reverse phase

HPLC using a 1.9 cm x 10 cm C8 column. The column was eluted at 18

mL/min with deionized water containing 0.3 % formic acid and 5% 1,4

dioxane for 3 min followed by a 15 min linear gradient from 5% 1,4-dioxane

to 30 % 1,4-dioxane. Product fractions (retention time 6.5 min) were collected

in a flask and immediately frozen in a dry ice acetone bath. Solvent was

removed by lyophilization at ambient temperature to give 40 mg (42%) of the

title compound. 1 H NMR (d 6-DMSO) 8.08-8.11 (m, 3H), 7.99 (t, J = 6.4

Hz,1H), 7.00 (s, 2H), 3.6-3.75 (m, 6H), 3.0-3.2 (m, 4H), 2.84 ( s, 4H), 2.13 (t,

J= 7.6 Hz), 1.83-1.93 (m, 2H), 1.69-1.72 (m, 2H). HRMS (M + Na) calc.

545.1608 found 545.1638

Z-Glu(OtBu)-Gly.Gly-NH2

O HCI N OIL O- N- - N=C=N- O 0 H 2 N~ 0 0\N OH + N OOH2 ON HN\/N 2

H NH20 0 H

[249] 40 mL of Dimethyl formamide was added to 2.52 g (7.47

mmol) of Z-Glu(OtBu)-OH, 1.3 g (8.49 mmol) of hydroxybenzotriazole, 1.3 g

(7.76 mmol) of H-Gly-GlyNH2, and 1.52 g ( 7.93 mmol) of N-(3

dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride. 2.5 mL (14.3

mmol) of diisopropyl ethyl amine was added and the reaction was stirred over

night. The reaction mixture was purified in three equal portions by direct

injection on a preparative 5 cm x 25 cm C18 HPLC column. The column was

run at 100 mL/min with deionized water containing 0.3 % formic acid with

5% acetonitrile for 10 min followed by a 15 min linear gradient from 5%

acetonitrile to 90% acetonitrile. Product fractions (retention time 18 - 20 min)

were combined and solvent was removed by rotary evaporation under vacuum

to give 2.9 g (83%) of the title compound. 'H NMR (400 MHz, CDCl 3) 6 7.79

- 7.68 (m, 1H), 7.64 (s, 1H), 7.27 (q, J= 4.9, 5H), 6.90 (s, 1H), 6.42 (s, 1H),

6.35 (d, J= 6.8, 1H), 5.08 (d, J= 12.0, 1H), 4.98 (d, J= 12.2, 1H), 4.20 (dd, J

= 12.9,7.6, 1H), 3.84-3.95 (m, 2H), 3.83 (d, J= 5.0,2H), 2.42 - 2.19 (m, 2H),

2.07 (d, J= 6.9, 1H), 1.96 - 1.83 (m, 1H), 1.39 (s, 9H). 1C NMR (101 MHz,

DMSO) 6 171.79, 171.65, 170.82, 168.87, 163.04, 156.08, 136.86, 128.31,

127.74, 79.64, 65.58, 53.96, 42.17, 41.81, 31.25, 27.73, 27.01.

H-Glu(OtBu)-Gly-Gly-NH 2

0 Y

0 O 10% Pd-C 0

NH2 H2N HNAN H2 H OHN 0 H 0 0 H 0

[250] 940 mg (2.09 mmol) of Z-Glu(OtBu)-Gly-GlyNH2 was

dissolved in 40 mL of 95:5 methanol:de-ionized water in a 250 mL glass

PARR hydrogenation shaker flak. 222 mg of 10% palladium on carbon was

added to the flask and the contents were hydrogenated with shaking under

hydrogen (40 PSI) for 4 hours. The mixture was vacuum filtered though celite

filter aid and solvent was removed from the filtrate by rotary evaporation to

give 640 mg (94%) of the title compound. 'H NMR (400 MHz, DMSO) 6

4.03 (s, 1H), 3.75 (d, J= 3.3, 2H), 3.63 (s, 2H), 3.30 - 3.22 (in, J= 3.6, 1H),

3.14 - 3.10 (in, 1H), 2.27 (t, J= 7.9,2H), 1.84 (td, J= 13.6,7.4, 1H), 1.63 (td, 13 J = 15.0, 7.5, 1H), 1.39 (s, 9H). C NMR (101 MHz, MeOD) 8 176.53,

174.24, 172.00, 170.32, 81.82, 55.21, 43.64, 43.16, 40.44, 32.31, 30.45, 28.41.

.HRMS (M + H) Calc. 317.1825 found 317.1800.

E001008-28 Mal-Gaba-Gu(OtBu)-Gy-Gly-NH2

0 0 O O 10% Pd-C 0

0 'HN NH 2 H2N O HN, NH 2 H N HN N*, H50 0 0 H 0

[2511 603 mg (1.9 mmol) of H-Glu(OtBu)-Gly-Gly-NH2, 372 mg

(2.03 mmol) of Mal-Gaba-OH and 430 mg (2.24 mmol) of N-(3

dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride were dissolved in

4.5 mL of dimethyl formamide and 800 pL of dichloromethane. The reaction

was stirred for 3 hours at ambient temperature. The reaction mixture was

purified in two equal portions by direct injection on a preparative 5 cm x 25

cm C18 HPLC column. The column was run at 100 mL/min with deionized

water containing 0.3 % formic acid 5% acetonitrile for 10 min followed by a

15 min linear gradient from 5% acetonitrile to 90% acetonitrile. Product

fractions (retention time 17.4 - 19.2 min) were combined and solvent was

removed by rotary evaporation under vacuum to give 2.9 g (83%) of the title

compound. 1H NMR (400 MHz, CDC 3) 8 8.16 (t, J= 5.7, 1H), 8.06 (d, J=

7.4, 1H), 7.99 (t, J= 5.8, 1H), 7.19 (s, 1H), 7.06 (s, 2H), 4.18 (dd, J= 13.4,

7.9, 1H), 3.70 (d, J= 5.7, 2H), 3.62 (d, J= 5.8, 2H), 3.42 - 3.37 (in, 2H), 2.23

(t, J= 8.0, 2H), 2.12 (dd, J= 8.1, 6.4, 2H), 1.87 (dt, J= 14.2, 7.9, 1H), 1.70

(dt, J= 13.7, 6.8, 2H), 1.38 (s, 9H). "C NMR (101 MHz, DMSO) 8 173.12,

171.77, 171.65, 171.03, 170.79, 168.89, 134.43, 79.62, 52.02, 42.14, 41.81,

36.80, 32.29, 31.22, 27.73, 26.95, 24.02. HRMS (M + Na) Calc. 504.2070

found 504.2053.

Mal-Gaba-Glu(OH)-Gy-Gy-NH 2 0

0 TFA 0 OH o N N HN N NH 2

N N HN NH 2 O H O H 0 H N H 0 H 0

[2521 105 mg (0.218 mmol) of Mal-Gaba-Glu(OtBu)-Gly-Gly-NH2

was dissolved in 5 mL of 95:5 trifluoroacetic acid:de-ionized water and

magnetically stirred for 2 hours. Solvent was removed by rotary evaporation

and residue was taken up in 6 mL acetonitrile + 1.5 mL toluene to give a

suspension. Solvent was evaporated from the suspension by rotary

evaporation under vacuum to give 92 mg (100%) of the title compound. 'H

NMR (400 MHz, DMSO) 8 6.99 (s, 2H), 4.18 (dd, J= 8.2, 5.7, 1H), 3.70 (s,

2H), 3.61 (s, 2H), 3.40 (t, J= 6.8, 2H), 2.26 (t, J= 7.8, 2H), 2.19 - 2.05 (m,

2H), 1.90 (dt, J= 13.7, 7.4, 1H), 1.73 (dt, J= 14.2, 7.5, 3H). "C NMR (101

MHz, DMSO) 5 173.76, 171.72, 170.99, 170.70, 168.81, 134.37, 52.00, 41.97,

41.63, 36.75, 32.19, 29.95, 26.79, 23.93.

Mal-Gaba-Glu(ONHS)-Gy-Gy-NH2 0 0 N-OH O OH HCl O N

N N HN )k/N>NH 2 0 O O O H 0 H N H N 0 O H 0

[2531 94 mg (0.22 mmol) of Mal-Gaba-Glu(OH)-Gly-Gly-NH 2 , 75

mg (0.65 mmol) N-hydroxysuccinimide and 110 mg (0.57 mmol) of N-(3

dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride were magnetically

stirred in 1 mL of dimethyl formamide for 3 hours. The crude reaction

mixture was purified in three equal portions by direct injection on a 1.9 cm x

10 cm C8 column. The column was run at 18 m/min with deionized water

containing 0.3% formic acid and 5% 1,4-dioxane for 3 min followed by an 18

min linear gradient from 5% 1,4-dioxane to 30% 1,4-dioxane. Product

fractions (retention time 7.3 min) were collected in a flask and immediately

frozen in a dry ice/acetone bath. The combined frozen material was

lyophilized to give 80 mg (70 %) of the title compound. 1H NMR (400 MHz,

DMSO) 6 8.20 (t, J= 5.4, 1H), 8.13 (d, J= 7.3, 1H), 8.03 (t, J= 5.6, 1H), 7.21

(s, 1H), 7.06 (s, 1H), 7.01 (s, 2H), 4.29 (dd, J= 13.7, 6.5, 1H), 3.84 - 3.69 (m,

2H), 3.63 (d, J= 5.7, 2H), 3.57 (s, 2H), 3.41 (t, J= 6.8, 2H), 2.81 (s, 3H), 2.78

- 2.69 (m, 2H), 2.15 (dd, J= 9.1, 6.2, 1H), 2.10 - 1.95 (m, 1H), 1.88 (dt, J=

17.0, 7.5, 1H), 1.73 (dd, J= 14.0, 6.9, 2H). HRMS (M + Na) Calc. 545.1608

found 545.1627.

Example 6. Synthesis of positively charged linker

H 3C, O HCl*H 2N 37% HCHO N 1). NaOH s bo %up H / S NaB(CN)H 3 H 3C 2). PySSPy, pH 7 213 217

H 3 C, ,CH 3 H 3 C, ,CH 0 N NH/CN 3

SS COOH DMA S -N 218 -"N 219 0 0

3-(Dimethylamino)dihydrothiophen-2(3H)-one(217).

[254] 3-aminodihydrothiophen-2(3H)-one hydrochloride (213) (1.0 g,

6.51 mmol) and formaldehyde (3 ml, 40.3 mmol) in methanol was added

sodium cynoboronhydride (0.409 g, 6.51 mmol) in five portions in 1 h. After

being stirred for 2 h, the mixture was evaporated, redissolved in EtAc, washed

with I M NaH 2PO 4, dried over MgSO 4 , filtered, concentrated and purified by

SiO2 column eluted with MeOH/DCM (1:30) to afford 0.812 g (86%) of the

title compound. 1H NMR (CDCl 3) 3.49 (dd, 1H, J = 6.3, 12.1 Hz), 3.24 (m,

2H), 2.42 (s, 6H), 2.38 (m, 1H), 2.21 (m, 1H); 13C NMR 206.58, 73.24,

41.62, 27.47, 25.51; ESI MS m/z+146.0 (M +H), 168.0 (M +Na).

2-(dimethylamino)-4-(pyridin-2-yldisulfanyl)butanoic acid(218).

[2551 3-(dimethylamino)dihydrothiophen-2(3H)-one (217) (0.95 g,

6.54 mmol) was stirred in 15 ml of 0.5 M NaOH and 10 ml of methanol

solution for 30 min, nutralized with H3 PO4 to pH 7.2, and 1,2-di(pyridin-2

yl)disulfane (5.76 g, 26.2 mmol) in 50 ml of methanol was added. The

mixture was stirred overnight, concentrated, washed with EtAc and the

aquoues solution was loaded on C-18 column, eluted from 5% methanol in

0.01% formic acid to 30% methanol in 0.01% formic acid to afford the title

product (368 mg, 20.65 % yield). 1 H NMR (CD1 30D) 8.31 (dd, 1H, J = 0.7,

4.7 Hz), 7.77 (m, 2H), 7.15 (dd, 1H, J = 0.8, 5.8 Hz), 3.22 (m, 1H), 2.85 (m,

2H), 2.51 (s, 611), 2.05 (m, 2H); "C NMR 175.00, 161.28, 150.46, 139.40,

122.60, 121.49, 71.20, 42.46, 36.29, 29.88; ESI MS m/z+ 272.9 (M + H),

295.0 (M+Na).

2,5-dioxopyrrolidin-1-yl 2-(dimethylamino)-4-(pyridin-2

yldisulfanyl)butanoate (219)

[256] 2-(dimethylamino)-4-(pyridin-2-yldisulfanyl)butanoic acid

(218) (92 mg, 0.338 mmol), 1-hydroxypyrrolidine-2,5-dione (65 mg, 0.565

mmol) and EDC (185 mg, 0.965 mmol) was stirred in 3 ml of DMA at 50°C

overnight. The mixture was evaporated and purified on a SiO 2 column eluted

with from1:10 to 1:4 of methanol/CH 2Cl2 to afford 43 mg (35%) of the title

product. 'H NMR (CD130D) 8.40 (m, 111), 7.83 (m, 2H), 7.22 (m, 1H), 3.34

(in, 1H), 2.82 (in, 211), 2.75 (s, 4H), 2.66 (s, 6H), 1.98 (in, 2H); "C NMR

177.21, 161.78, 161.12, 150.68, 139.37, 122.70, 121.66, 70.80, 44.16, 43.15,

36.06, 27.38; ESI MS m/z+ 369.2 (M + H).

Example 7. Preparation of huMy9-6-CX1-1-DM1 procharged linker

conjugates:

[2571 The following stock solutions were used: 39.6 mM DM1 in

DMA; (2) 17.8 mM solution of CX1-1 linker in DMA; (3) 200 mM succinate

buffer pH 5.0 with 2 mM EDTA. The reaction mixture containing between 8,

12 or 16 equivalents of linker to antibody were added to a solution of the

antibody at 4 mg/ml in 90% phosphate buffer pH 6.5)/ 10% DMA and allowed

to react for 2h at 25°C. pH 5.0, followed by reaction with DM1.

[258] The Ab conjugate was separated from excess small molecule

reactants using a G25 column equilibrated in PBS pH 7.4. The purified

conjugate was allowed to hold for 2d at 25'C to allow any labile drug linkages

to hydrolyze and then the conjugate was further purified from free drug by

dialysis in PBS overnight, and then 10 mM histidine/130 mM glycine buffer

pH 5.5 (1x o/n). The dialyzed conjugate was filtered using a 0.2 um filter and

assayed by UV/Vis to calculate number of maytansinoids per Ab using known

extinction coefficients for maytansinoid and antibody at 252 and 280 nm. The

recovery was ~70% and number of maytansinoids/antibody measured for each

conjugate ranged from 3.7 to 6.8 depending on the linker excess used.

Example 8. In vivo Pharmacokinetics:

[2591 The plasma pharmacokinetics of charged Sulfo-Mal linker

conjugates of a humanized antibody C242 with 3H-labeled-DM4 (3.5 and 6.4

DM4/Ab) in CD-i mice were analyzed by antibody ELISA and by 3H

counting (Figure 72). The Ab-Sulfo-Mal-[ 3H]-DM4 conjugates bearing 3.5

and 6.4 D/A were dosed i.v. at 12.9 and 7.9 mg/kg (antibody dose)

respectively. The antibody values of plasma samples were measured by

ELISA (based on capture using goat-anti-huIgG antibody and detection using

donkey-anti-huIgG antibody-horseradish peroxidase conjugate) and by 3H

counting (scintillation counting). Figure 72 A shows that these two 3 H-counting measurements of conjugate concentrations by ELISA and by

showed similar values for each conjugate. Both the 3.5 and 6.4 D/A

Antibody-Sulfo-Mal-DM4 conjugates showed good plasma stability over 4

weeks with half-life of approximately 14.9 days and 9.7 days respectively,

which are similar to the half-life of approximately 11.8 days for the

unconjugated antibody. The DM4/Ab ratio of the two Ab-Sulfo-Mal-DM4

conjugates (initially 3.5 and 6.4 D/A) were also stable over 4 weeks in plasma

circulation, importantly even at the relatively high 6.4 D/A load (Figure 72 B).

The half life of Sulfo-Mal-linked huC242 Ab-Sulfo-Mal-DM4 conjugate with

3.5 D/A load dosed at 12.9 mg/kg was 14.9 days (AUC = 38449 hr.tg/mL),

compared to a half life of 12.6 days (AUC = 25910 hr.Lg/mL) for SMCC

linked huC242 Ab-SMCC-DM1 conjugate with a similar 4.2 D/A load dosed at 12 mg/kg, and thus was much improved over that of the SMCC conjugate

(Figure 38 B).

Table 1. Comparison of SSNPP and SPP linker in the conjugation of N901 antibody with DM1. Conjugation was conducted for 2 hours at the indicated pH using a 1.7-fold molar excess of DM1 per linker. % SEC Analysis Linker pH Linker/Ab DMl/Ab % free Efficiency drug Monomer Dimer Trimer HMW

SSNPP 7.4 4.1 3,8 93 0.8 91.9 6.3 0.6 0.1

SPP 7.4 5.6 4.3 77 1.8 93.6 4.9 0.4 0.2 SSNPP 6.5 4.0 3.7 93 0.9 - - -

SPP 6.5 6.6 4.6 70 1.9 - - -

Table 2. Reduced linker to antibody ratio required to reach target DM1 to antibody ratio with SSNPP as linker. Conjugation was conducted for 2 hours at pH 7.4 using a 1.1-fold molar excess of DM1 per linker.

Linker Linker/Ab DM1/Ab SSNPP 4.2 4.3 SPP 5.6 4.3

Table 3. Comparison of SSNPP and SPP linker in the conjugation of N901 antibody with DC4. Conjugation was conducted for the indicated time at pH 7.4 using a 1.4-fold molar excess of DC4 per linker.

Linker Time, h Linker/Ab DC4/Ab efficiency SSNPP 2 4.2 4.3 102 SSNPP 18 4.2 4.1 98 SPP 2 5.6 4.1 73 SPP 18 5.6 5.1 91

Claims (16)

WHAT IS CLAIMED IS:

1. A cell-binding agent-drug conjugate of formula (II)

R7 R8 R3 R4

CBZ 1 Ag nl LR-9 % Ri R R6M" RioR 5 R, R22 J q

(II)

wherein:

CB represents a cell-binding agent;

D represents the drug linked to the cell-binding agent by a disulfide,

thioether, thioester, peptide, hydrazone, ester, ether, carbamate, or amide

bond;

R 1, R 2, R3, R4 ,R 5, R6, R 7, R 8, R 9, and RIO are the same or different and

are H, linear alkyl having from 1-6 carbon atoms, branched or cyclic alkyl

having from 3 to 6 carbon atoms, linear, branched or cyclic alkenyl or alkynyl

having from 2 to 6 carbon atoms, a charged substituent selected from anions

selected from SO3~. X-SO3 , OP03 2, X-OP 3 2 , P03 2, X-Po32, C0 2-, and

cations selected from a nitrogen containing heterocycle, NR1 1 R 12R1 3 and X

N*RuR12R1 3 , or a phenyl; wherein:

Rn, R1 2and R 3are same or different and are H, linear alkyl having

from 1 to 6 carbon atoms, branched or cyclic alkyl having from 3 to 6 carbon

atoms and X represents phenyl or a linear alkyl from 1 to 6 carbon atoms, or

branched or cyclic alkyl having from 3 to 6 carbon atoms;

1, m and n are 0 or an integer from 1 to 4;

A is a phenyl or substituted phenyl, wherein the substituent is a linear

alkyl having from 1 to 6 carbon atoms, or a branched or cyclic alkyl having

from 3 to 6 carbon atoms, or a charged substituent selected from anions

selected from S03 , X-S0 3 , OP0 32 , X-OP0 3 2 ,P03 , X-P0 32 , C0 2-, and

cations selected from a nitrogen containing heterocycle, N*RnR1 2R 13 and X

N*R 1IR 12R , 3wherein X has the same definition as above, and wherein g is 0

or 1;

Z is an optional polyethyleneoxy unit of formula (OCH 2CH 2)p, wherein

p is 0 or an integer from 2 to about 1000, or F1-E1-P-E2-F2 unit in which El

and E2 are the same or different and are C=O,0, or NRI4, wherein R 14 is H,

a linear alkyl having from 1-6 carbon atoms, a branched or cyclic alkyl having

from 3 to 6 carbon atoms, a linear, branched or cyclic alkenyl or alkynyl

having from 2 to 6 carbon atoms; P is a peptide unit between 2 and 20 amino

acids in length, wherein El or E2 can be linked to the peptide through the

terminal nitrogen, terminal carbon or through a side chain of one of the amino

acids of the peptide; and Fl and F2 are the same or different and are an

optional polyethyleneoxy unit of formula (OCH 2CH2)p, wherein p is 0 or an integer from 2 to about 1000, provided that when Z is not FI-El-P-E2-F2, at least one of R1 , R2 , R3 , R4, R5 , R 6, R 7, R8 , R 9, and RIO is a charged substituent

5 , R6, R 7, R8 , R9 , and Rio is a 1 , R2 , R3, R4, R or when g is 1, at least one of A, R

charged substituent;

Y represents a carbonyl, thioether, amide, disulfide, or hydrazone

group; and q represents an integer from 1 to 20.

2. The conjugate of claim 1, wherein D is selected from thiotepa;

cyclophosphamide (CYTOXANC TM)); alkyl sulfonates selected from busulfan,

improsulfan and piposulfan; aziridines selected from benzodopa, carboquone,

meturedopa, and uredopa; ethylenimines; and methylamelamines; acetogenins

selected from bullatacin and bullatacinone; a camptothecin; bryostatin;

callystatin; CC-1065; adozelesin, carzelesin or bizelesin synthetic analogues

of CC-1065; cryptophycins; dolastatin; duocarmycin; KW-2189 or CBI-TMI

synthetic analogues of duocarmycin,; eleutherobin; pancratistatin; a

sarcodictyin; spongistatin; nitrogen mustards selected from chlorambucil,

chlornaphazine, cholophosphamide, estramustine, ifosfamide,

mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,

novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;

nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine,

nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g.

calicheamicin, especially calicheamicin gammal and calicheamicin theta I; dynemicin, including dynemicin A; an esperamicin; neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin; chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L norleucine, doxorubicin epirubicin, esorubicin, idarubicin, marcellomycin, nitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites selected from methotrexate and 5-fluorouracil (5-FU); folic acid analogues selected from denopterin, methotrexate, pteropterin, trimetrexate; purine analogs selected from fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimnidine analogs selected from ancitabine, azacitidine,

6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine,

floxuridine, 5-FU; androgens selected from calusterone, dromostanolone

propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as

aminoglutethimide, mitotane, trilostane; folic acid replenisher selected from

frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid;

amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;

diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium

nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids selected from

maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid;

2-ethylhydrazide; procarbazine; PSK*; razoxane; rhizoxin; sizofiran;

spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"-trichlorotriethylamine;

trichothecenes selected from T-2 toxin, verracurin A, roridin A and

anguidine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol;

mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");

cyclophosphamide; thiotepa; taxanes; chlorambucil; gemcitabine;

6-thioguanine; mercaptopurine; methotrexate; platinum analogs selected from

cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16);

ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine;

novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate;

CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO);

retinoic acid; capecitabine; anti-hormonal agents selected from anti-estrogens

including tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,

4-hydroxytamoxifen, trioxifene, keoxifene, LYl17018, onapristone, and

toremifene (Fareston); and anti-androgens selected from flutamide,

nilutamide, bicalutamide, leuprolide, and goserelin, siRNA or a combination

thereof; and pharmaceutically acceptable salts, acids or derivatives of any of

the above.

3. The conjugate of claim Ior 2, wherein D is selected from

maytansinoids, CC-1065 analogs, morpholino doxorubicin, taxanes,

calicheamicins, auristatins, pyrrolobenzodiazepine dimmer, siRNA or a combination thereof, and pharmaceutically acceptable salts, acids or derivatives of any of the above.

4. The conjugate of claim 1, wherein the cell-binding agent binds

to target cells selected from tumor cells, virus infected cells, microorganism

infected cells, parasite infected cells, autoimmune cells, activated cells,

myeloid cells, activated T-cells, B cells, or melanocytes, cells expressing one

or more of IGF-IR, CanAg, EGFR, EphA2 receptor, MUCI, MUC16, VEGF,

TF, MY9, anti-B4, EpCAM, CD2, CD3, CD4, CD5, CD6, CDll, CD lla,

CD18, CD19, CD20, CD22, CD26, CD30, CD33, CD37, CD38, CD40, CD44,

CD56, CD79, CD105, CD138, EphA receptors, EphB receptors, EGFr,

EGFRvIII, HER2/neu, HER3, mesothelin, cripto, alphavbeta 3, integrin,

alphavbetas integrin, alphavbeta integrin, Apo2, and C242 antigens; and cells

expressing insulin growth factor receptor, epidermal growth factor receptor, or

folate receptor.

5. The conjugate of claim 1, wherein the cell-binding agent is an

antibody, a single chain antibody, an antibody fragment that binds to the target

cell, a monoclonal antibody, a single chain monoclonal antibody, or a

monoclonal antibody fragment that binds the target cell, a chimeric antibody, a

chimeric antibody fragment that binds to the target cell, a domain antibody, a

domain antibody fragment that binds to the target cell, adnectins that mimic

antibodies, DARPins, a lymphokine, a hormone, a vitamin, a growth factor, a

colony stimulating factor, or a nutrient-transport molecule.

6. The conjugate of claim 5, wherein the antibody is a resurfaced

antibody, a resurfaced single chain antibody, or a resurfaced antibody

fragment thereof.

7. The conjugate of claim 5, wherein the antibody is a monoclonal

antibody, a single chain monoclonal antibody, or a monoclonal antibody

fragment thereof.

8. The conjugate of claim 5, wherein the antibody is a human

antibody, a humanized antibody or a resurfaced antibody, a humanized single

chain antibody, or a humanized antibody fragment thereof.

9. The conjugate of claim 5, wherein the antibody is a chimeric

antibody, a chimeric antibody fragment, a domain antibody, or a domain

antibody fragment thereof.

10. The conjugate of claim 8, wherein the antibody is My9-6, B4,

C242, N901, DS6, EpCAM, EphA2 receptor, CD38, IGF-IR, CNTO 95, B

B4, trastuzumab, pertuzumab, bivatuzumab, sibrotuzumab, pertuzumab, or

rituximab.

11. The conjugate of claim 4, wherein the tumor cells are selected

from breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal

cancer cells, gastric cancer cells, squamous cancer cells, small-cell lung cancer

cells, and testicular cancer cells.

12. A method of treating a tumor comprising administering to a

subject in need of treatment a therapeutically effective amount of a conjugate

of claim 1.

13. A cross linker represented by formula (I)

R 7 R8 R3 R4 Y Z Q

R9 Rio0 R5R6R1 2

(I)

wherein:

Y' represents a functional group that enables reaction with a cell

binding agent;

Q represents a functional group that enables linkage of a cytotoxic drug via a disulfide, thioether, thioester, peptide, hydrazone, ester, ether,

carbamate or amide bond;

R1, R2, R3, R4, R5, R6, R7, R8, R9, and RIO are the same or different and

are H, linear alkyl having from 1-6 carbon atoms, branched or cyclic alkyl

having from 3 to 6 carbon atoms, linear, branched or cyclic alkenyl or alkynyl

having from 2 to 6 carbon atoms, a charged substituent selected from

anionsselected from SO3, X-S0 3 , OP0 32 , X-OPO 32-, P032-, X-pO3 2 -, CO 2 -,

and cations selected from a nitrogen containing heterocycle, N+RIR1 2R 3,and

X-N+Ri R12R13, or a phenyl, wherein:

Rn, R 12 and R 13 are same or different and are H, linear alkyl having

from 1 to 6 carbon atoms, or branched or cyclic alkyl having from 3 to 6

carbon atoms and X represents phenyl or a linear alkyl having from 1 to 6

carbon atoms, or branched or cyclic alkyl having from 3 to 6 carbon atoms;

1, m and n are 0 or an integer from 1 to 4;

A is a phenyl or substituted phenyl, wherein the substituent is a linear

alkyl having from 1 to 6 carbon atoms, or a branched or cyclic alkyl having

from 3 to 6 carbon atoms, or a charged substituent selected from anions

selected from SO3, X-S0 3 , OP0 32 , X-OP0 3 , P0 32 , X-P0 3 , C0 2-, and

cations selected from a nitrogen containing heterocycle, N*RnR1 2R 3 , andX

N+R 1 R 12R , 3wherein X has the same definition as above, and wherein g is 0

or 1; and

Z is an optional polyethyleneoxy unit of formula (OCH 2CH2 )p, wherein

p is 0 or an integer from 2 to about 1000, or F1-E1-P-E2-F2 unit in which El

and E2 are the same or different and are C=O,0, or NR14, wherein R 14 is H,

a linear alkyl having from 1-6 carbon atoms, a branched or cyclic alkyl having

from 3 to 6 carbon atoms, a linear, branched or cyclic alkenyl or alkynyl

having from 2 to 6 carbon atoms; P is a peptide unit between 2 and 20 amino

acids in length, wherein El or E2 can be linked to the peptide through the

terminal nitrogen, terminal carbon or through a side chain of one of the amino

acids of the peptide; and F1 and F2 are the same or different and are an

optional polyethyleneoxy unit of formula (OCH 2 CH2)p, wherein p is 0 or an integer from 2 to about 1000, provided that when Z is not F-El-P-E2-F2, at least one of R 1, R 2, R3, R 4, R5 ,R6 , R 7, R8 , R9 , and RIO is a charged substituent or when g is 1, at least one of A, R 1, R 2, R 3, R 4, RS, 16, R7, R8, R9, and Rio is a charged substituent.

14. A compound of formula (III):

R7 R R3 D4

Y Z NN I Q . R9 RIO R5 "R R2 _ q

(III) wherein:

CB represents a cell-binding agent;

Q represents a functional group that enables linkage of a cytotoxic drug via a disulfide, thioether, thioester, peptide, hydrazone, ester, ether,

carbamate or amide bond;

R1, R 2, R3, R4, R5, 16, R7, R 8, R 9, and Rio are the same or different and

are H, linear alkyl having from 1-6 carbon atoms, branched or cyclic alkyl

having from 3 to 6 carbon atoms, linear, branched or cyclic alkenyl or alkynyl

having from 2 to 6 carbon atoms, a charged substituent selected from anions 2 2 selected from S03 , X-S0 3 ~, OP0 32-, X-OP 3 - P 3 2-, X-P0 3 , C02 -, and

cations selected from a nitrogen containing heterocycle, N+RI R1 2Ri3 and X

N*RIIR2R13, or a phenyl, wherein:

Rn, R1 2and R 1 3are same or different and are H, linear alkyl having

from 1 to 6 carbon atoms, or branched or cyclic alkyl having from 3 to 6

carbon atoms and X represents phenyl or a linear alkyl having from 1 to 6

carbon atoms, or branched or cyclic alkyl having from 3 to 6 carbon atoms;

1, m and n are 0 or an integer from 1 to 4;

A is a phenyl or substituted phenyl, wherein the substituent is a linear

alkyl having from 1 to 6 carbon atoms, or a branched or cyclic alkyl having

from 3 to 6 carbon atoms, or a charged substituent selected from anions

selected from S0 3 , X-S0 3 , OP0 32-, X-OP 2 3 -, P0 32 , X-P0 32 , C0 2-, and

cations selected from a nitrogen containing heterocycle, N*RIR1 2R 3 and X

N*RIR 12R 3, wherein X has the same definition as above, and wherein g is 0

or 1;

Z is an optional polyethyleneoxy unit of formula (OCH 2CH2)p, wherein

p is 0 or an integer from 2 to about 1000, or F-El-P-E2-F2 unit in which El

and E2 are the same or different and are C=,0, or NR14, wherein R14 is H,

a linear alkyl having from 1-6 carbon atoms, a branched or cyclic alkyl having

from 3 to 6 carbon atoms, a linear, branched or cyclic alkenyl or alkynyl

having from 2 to 6 carbon atoms; P is a peptide unit between 2 and 20 amino

acids in length, wherein El or E2 can be linked to the peptide through the

terminal nitrogen, terminal carbon or through a side chain of one of the amino

acids of the peptide; and F1 and F2 are the same or different and are an

optional polyethyleneoxy unit of formula (OCH 2 CH2 )p, wherein p is 0 or an integer from 2 to about 1000, provided that when Z is not F1-El-P-E2-F2, at least one of R 1, R 2, R3, R 4, R5 , R6 , R7 , R8 , R9, and Rio is a charged substituent or when g is 1, at least one of A, R1 , R 2, R3, R 4, R5, R6, R 7, R8, R9, and RIO is a charged substituent; and

Y represents a carbonyl, thioether, amide, disulfide, or hydrazone

group; and q represents an integer from 1 to 20.

15. A compound of formula (IV):

R7 R 8 R R4 Y1 D Ag n m R 9 R10 R 5 R 6 R1 R2 (IV)

wherein:

Y' represents a functional group that enables reaction with a cell

binding agent;

D represents the drug linked to the cell-binding agent by a disulfide,

thioether, thioester, peptide, hydrazone, ester, ether, carbamate, or amide

bond;

R1, R2, R3, R4, R5, R 6, R7, R8, R9, and RIO are the same or different and

are H, linear alkyl having from 1-6 carbon atoms, branched or cyclic alkyl

having from 3 to 6 carbon atoms, linear, branched or cyclic alkenyl or alkynyl

having from 2 to 6 carbon atoms, a charged substituent selected from anions and selected from S03-, X-S0 3 , OP0 3 , X-OPO 3 -, PO 3 , X-PO 3 CO, 2 -, cations selected from a nitrogen containing heterocycle, N+RuR1 2 RI and X

N-RjR12R13, or a phenyl, wherein:

R,, R12 and R 13 are same or different and are H, linear alkyl having

from 1 to 6 carbon atoms, or branched or cyclic alkyl having from 3 to 6

carbon atoms and X represents phenyl or a linear alkyl having from 1 to 6

carbon atoms, or branched or cyclic alkyl having from 3 to 6 carbon atoms;

1, m and n are 0 or an integer from I to 4;

A is a phenyl or substituted phenyl, wherein the substituent is a linear

alkyl having from 1 to 6 carbon atoms, or a branched or cyclic alkyl having

from 3 to 6 carbon atoms, or a charged substituent selected from SO3, X-S0 3~,

OP0 3 , X-OP0 3 , P0 32 -, X-P0 3 2, C0 -,2 a nitrogen containing heterocycle,

N*RI Ri2R13 or X-N*R 1 R 12R13, wherein X has the same definition as above,

and wherein g is 0 or 1; and

Z is an optional polyethyleneoxy unit of formula (OCH 2 CH2 )p, wherein

p is 0 or an integer from 2 to about 1000, or F1-E1-P-E2-F2 unit in which El

and E2 are the same or different and are C=O,0, or NR14, wherein R 14 is H,

a linear alkyl having from 1-6 carbon atoms, a branched or cyclic alkyl having

from 3 to 6 carbon atoms, a linear, branched or cyclic alkenyl or alkynyl

having from 2 to 6 carbon atoms; P is a peptide unit between 2 and 20 amino

acids in length, wherein El or E2 can be linked to the peptide through the

terminal nitrogen, terminal carbon or through a side chain of one of the amino acids of the peptide; and F1 and F2 are the same or different and are an optional polyethyleneoxy unit of formula (OCH 2 CH2 )p, wherein p is 0 or an integer from 2 to about 1000, provided that when Z is not F1-El-P-E2-F2, at least one of R1 , R2 , R3 , R4 , R5 , R6 , R 7, R8 , R9 , and RIO is a charged substituent or when g is 1, at least one of A, R 5 ,R 6 , R7 ,R 8 , R 9 , and RIO is a 1 , R 2, R 3, R4 , R charged substituent.

16. A pharmaceutical composition comprising an effective amount

of the conjugate of claim 1, a pharmaceutically acceptable salt or solvate

thereof, and a pharmaceutically acceptable carrier, diluent or excipient.