AU2019202435B2 - Novel benzodiazepine derivatives - Google Patents

Abstract

5 The invention relates to novel benzodiazepine derivatives with antiproliferative activity and more specifically to novel benzodiazepines of formula (I) and (II), in which the diazepine ring (B) is fused with a heterocyclic ring (CD), wherein the heterocyclic ring is bicyclic or a compound of formula (III), in which the diazepine ring (B) is fused with a heterocyclic ring (C), wherein the heterocyclic ring is monocyclic. The invention 10 provides cytotoxic dimers of these compounds. The invention also provides conjugates of the monomers and the dimers. The invention further provides compositions and methods useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal using the compounds or conjugates of the invention. The invention further relates to methods of using the compounds or conjugates for in vitro, in situ, and in vivo is diagnosis or treatment of mammalian cells, or associated pathological conditions. 11228189_1 (GHMatters) P87639.AU.3 2/04/19

Description

NOVEL BENZODIAZEPINE DERIVATIVES FIELD OF THE INVENTION

[01] The present invention relates to novel cytotoxic compounds and cytotoxic conjugates comprising these cytotoxic compounds and cell-binding agents. More specifically, this invention relates to novel benzodiazepine compounds (e.g., indolinobenzodiazepines or oxazolidinobenzodiazepines), derivatives thereof, intermediates thereof, conjugates thereof, and pharmaceutically acceptable salts thereof, which are useful as medicaments, in particular as anti-proliferative agents.

RELATED APPLICATIONS

[Ola] The present application is a divisional of Australian Patent Application No. 2017206212, the entirety of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[02] Benzodiazepine derivatives are useful compounds for treating various disorders, and include medicaments such as, antiepileptics (imidazo [2,1 b][1,3,5]benzothiadiazepines, U.S. Pat. No. 4,444,688; U.S. Pat. No. 4,062,852), antibacterials (pyrimido[1,2-c][1,3,5]benzothiadiazepines, GB 1476684), diuretics and hypotensives (pyrrolo(1,2-b)[1,2,5]benzothiadiazepine 5,5 dioxide, U.S. Pat. No. 3,506,646), hypolipidemics (WO 03091232), anti-depressants (U.S. Pat. No. 3,453,266); osteoporosis (JP 2138272).

[03] Recently, it has been shown in animal tumor models that benzodiazepine derivatives, such as pyrrolobenzodiazepines (PBDs), act as anti-tumor agents (N-2 imidazolyl alkyl substituted 1,2,5-benzothiadiazepine-1,1-dioxide, U.S. Pat. No. 6,156,746), benzo-pyrido or dipyrido thiadiazepine (WO 2004/069843), pyrrolo [1,2-b]

[1,2,5] benzothiadiazepines and pirrole [1,2-b][1,2,5] benzodiazepine derivatives (W02007/015280),tomaymycin derivatives

1 11228189_1(GHMattes) P87639.AU.3 2/04/19

(e.g., pyrrolo[1,4]benzodiazepines), such as those described in WO 00/12508,

W02005/085260, W02007/085930, and EP 2019104. Benzodiazepines are also known to

affect cell growth and differentiation (Kamal A., et al., Bioorg Med Chem. 2008 Aug

;16(16):7804-10 (and references cited therein); Kumar R, Mini Rev Med Chem. 2003

Jun;3(4):323-39 (and references cited therein); Bednarski J J, et al., 2004; Sutter A. P, et al.,

2002; Blatt N B, et al., 2002), Kamal A. et al., Current Med. Chem., 2002; 2; 215-254, Wang

J-J., J.Med. Chem., 2206; 49:1442-1449, Alley M.C. et al., Cancer Res. 2004; 64:6700-6706,

Pepper C. J., Cancer Res 2004; 74:6750-6755, Thurston D.E. and Bose D.S., Chem Rev

1994; 94:433-465; and Tozuka, Z., et al., Journal of Antibiotics, (1983) 36; 1699

1708. General structure of PBDs is described in US Publication Number 20070072846. The

PBDs differ in the number, type and position of substituents, in both their aromatic A rings

and pyrrolo C rings, and in the degree of saturation of the C ring. Their ability to form an

adduct in the minor groove enables them to interfere with DNA processing, hence their

potential for use as antiproliferative agents.

[04] There still exists a need for novel benzodiazepine derivatives as effective and safe

therapeutics for treating a variety of proliferative disease states, such as cancer.

SUMMARY OF THE INVENTION

[05] One aspect of the invention provides novel benzodiazepines of formula (I) and (II), in

which the diazepine ring (B) is fused with a heterocyclic ring (CD), wherein the heterocyclic

ring is bicyclic,

2 17477638_1 (GHMatters) P87639.AU.3

RR 1 R2 y RC 5 Z R N3 A OeB3 C \/ R R6 W- /\ R2 BNC

R4 R3

(I) (II)

wherein:

the double line = between N and C represents a single bond or a double bond, provided that

when it is a double bond X is absent and Y is H, and when it is a single bond, X is H, or an

amine protecting moiety that converts the compound into a prodrug that can be transformed into

the free amine in vitro or in vivo;

Y is selected from -OR, an ester represented by -OCOR', a carbonate represented by -OCOOR',

a carbamate represented by -OCONR'R", an amine or a hydroxyl amine represented by NR'R",

amide represented by -NRCOR', a peptide represented by NRCOP, wherein P is an amino acid

or a polypeptide containing between 2 to 20 amino acid units, a thioether represented by SR', a

sulfoxide represented by SOR', a sulfone represented by -SO 2R', a sulfite -SO 3, a bisulfite

-OSO3, a halogen, cyano, an azido, or a thiol, wherein R, R' and R" are same or different and

are selected from H, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or

alkynyl having from 1 to 20 carbon atoms a polyethylene glycol unit (-OCH 2CH 2)., wherein n is

an integer from 1 to 2000, a 5- or 6-membered heteroaryl ring containing one or more

heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5 to 18 membered fused

ring system, wherein at least one of the rings is aromatic, containing one or more heteroatoms

independently selected from nitrogen, oxygen, and sulfur, aryl having from 6 to 18 carbon atoms, a 3 to 18-membered heterocyclic ring having I to 6 heteroatoms selected from 0, S, N and P wherein the substituent is selected from halogen, OR 7, NRsR, NO 2 , NRCOR', SRio, a sulfoxide represented by SOR', a sulfone represented by -SO2 R', a sulfite -SO3, a bisulfite -OSO3 , a sulfonamide represented by SO 2NRR', cyano, an azido, -COR, OCOR Ior OCONRR1 2

, wherein R 7, Rs, R 9, RIO, Ru and R 12 are each independently selected from H, linear, branched or

cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit(

OCH 2CH2)n, wherein n is an integer from 1 to 2000, a 5- or 6-membered heteroaryl ring

containing one or more beteroatoms independently selected from nitrogen, oxygen, and sulfur. a

to 18 membered fused ring system, wherein at least one of the rings is aromatic.containing one

or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, aryl having from

6 to 18 carbon atoms 3 to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected

from 0, S, N and P and RIO optionally is SR1 3 or COR1 3 , wherein R13 is selected from linear,

branched or cyclic alkyl, alkenyl or alkynyl having from I to 10 carbon atoms, a polyethylene

glycol unit (-OCH2CH2), wherein n is an integer from 1 to 2000, , a 5- or 6-membered

heteroaryl ring containing one or more heteroatoms independently selected from nitrogen,

oxygen, and sulfur, a 5 to 18 membered fused ring system, wherein at least one of the rings is

aromatic, containing one or more heteroatoms independently selected from nitrogen, oxygen, and

sulfur, 3 to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected from 0, S, N and

P and R1 can also be OR 14 , wherein R 14 is H or has the same definition as R, optionally, R" is

an OH;

W is C=O, C=S, CH2, BH (B=Boron), SO or SO 2;

R 1, R 2, R 3, R4 , are each independently selected from H, substituted or unsubstituted linear,

branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene

glycol unit (-OCH 2CH )., 2 wherein n is an integer from I to 2000, or a substituent selected from a

halogen, OR7 , NRsR 9, NO2, NRCOR', SR 10, a sulfoxide represented by SOR', a sulfone

represented by -SO2R', a sulfite -SO3, a bisulfite -OSO3, a sulfonamide represented by

SO2NRR', cyano, an azido, guanidinium [-NH(C=NH)NH 2], -CORI,, -OCOR or

OCONR 1 1R 12 wherein R 7, Rs, R 9, Rio, R11 and R 12 have the same definitions as given above,

optionally, any one of R 1, R2 , R 3 , R4 is a linking group that enables linkage to a cell binding

agent via a covalent bond or is selected from a polypyrrolo, poly-indolyl, poly-imidazolyl,

polypyrollo-imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolyl unit optionally bearing a

linking group that enables linkage to a cell binding agent;

R 5 is selected from OR1 5 , CRR'OH, SH, CRR'SH, NHR1 5 or CRR'NHRi5 , wherein R15 has the

same definition as R., R and R' have the same definition as given above; optionally, R 5 is a

linking group that enables linkage to a cell binding agent via a covalent bond or is selected from

a polypyrrolo, poly-indolyl, poly-imidazolyl, polypyrollo-imidazolyl, poly-pyrollo-indolyl or

polyimidazolo-indolyl unit optionally bearing a linking group that enables linkage to a cell

binding agent;;

R 6 is OR, SR, NRR', wherein R and R' have the same definition as given above, or optionally

R 6 is a linking group;

Z is selected from (CH 2), wherein n is 1, 2 or 3, CR15 R1 6 , NR17, 0 or S, wherein R1 5 , R1 6 and

R 17 are each independently selected from H, linear, branched or cyclic alkyl having from 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH2CH2)n, wherein n is an integer from 1 to

2000; or their pharmaceutically acceptable solvates, salts, hydrates or hydrated salts, their

optical isomers, racemates, diastereomers, enantiomers of these compounds.

provided that the compound has no more than one linking group that enables linkage to a cell

binding agent via a covalent bond.

[06] A second aspect of the invention provides novel benzodiazepines of formula (III), in

which the diazepine ring (B) is fused with a heterocyclic ring (C), wherein the heterocyclic

ring is monocyclic,

x Y

A B C Y' (III)

wherein:

the double line between N and C represents a single bond or a double bond, provided that

when it is a double bond X is absent and Y is H, and when it is a single bond, X is H or an

amine protecting moiety that converts the compound into a prodrug;

Y is selected from -OR, an ester represented by -OCOR', a carbonate represented by

OCOOR', a carbamate represented by -OCONR'R", an amine or a hydroxyl amine

represented by NR'R", amide represented by -NRCOR', a peptide represented by NRCOP,

wherein P is an amino acid or a polypeptide containing between 2 to 20 amino acid units, a

thioether represented by SR', a

6 17477638_1 (GHMatters) P87639.AU.3 carbon atoms, a polyethylene glycol unit (-OCH 2CH2)n, wherein n is an integer from 1 to 2000; or their pharmaceutically acceptable solvates, salts, hydrates or hydrated salts, their optical isomers, racemates, diastereomers, enantiomers of these compounds.

provided that the compound has no more than one linking group that enables linkage to a cell

binding agent via a covalent bond.

[06] A second object of the invention is to provide novel benzodiazepines of formula (III), in

which the diazepine ring (B) is fused with a heterocyclic ring (C), wherein the heterocyclic ring

is monocyclic,

x y

B C ' (III)

wherein:

the double line = between N and C represents a single bond or a double bond, provided that

when it is a double bond X is absent and Y is H, and when it is a single bond, X is H or an amine

protecting moiety that converts the compound into a prodrug;

Y is selected from -OR, an ester represented by -OCOR', a carbonate represented by -OCOOR',

a carbamate represented by -OCONR'R", an amine or a hydroxyl amine represented by NR'R",

amide represented by -NRCOR', a peptide represented by NRCOP, wherein P is an amino acid

or a polypeptide containing between 2 to 20 amino acid units, a thioether represented by SR', a sulfoxide represented by SOR', a sulfone represented by -SO 2R', a sulfite -SO 3, a bisulfite

OSO3, a halogen, cyano, an azido, or a thiol, wherein R, R' and R" are same or different and

selected from H, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl

having from 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH 2CH 2)n, wherein n is an

integer from 1 to 2000, a 5- or 6-membered heteroaryl ring containing one or more heteroatoms

independently selected from nitrogen, oxygen, and sulfur, a 5 to 18 membered fused ring system,

wherein at least one of the rings is aromatic, containing one or more heteroatoms independently

selected from nitrogen, oxygen, and sulfur, aryl having from 6 to 18 carbon atoms 3 to 18

membered heterocyclic ring having 1to 6 heteroatoms selected from 0, S, N and P, wherein the

substituent is selected from halogen, OR, NRR9 , NO 2, NRCOR', SRio, a sulfoxide represented

by SOR', a sulfone represented by -SO 2R', a sulfite -SO 3, a bisulfite -OSO3, a sulfonamide

represented by SO 2NRR', cyano, an azido, -COR1 , OCOR11 or OCONR R 12 , wherein R7 , R8

, R 9, Rio, R 1 and R 12 are each independently selected from H, linear, branched or cyclic alkyl,

alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH 2CH 2),

wherein n is an integer from 1 to 2000, a 5- or 6-membered heteroaryl ring containing one or

more heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5 to 18 membered

fused ring system, wherein at least one of the rings is aromatic, containing one or more

heteroatoms independently selected from nitrogen, oxygen, and sulfur, aryl having from 6 to 18

carbon atoms 3 to 18-membered heterocyclic ring having I to 6 heteroatoms selected from 0, S,

N and P and RIO optionally is SR 13 or COR13 , herein R 13 is selected from linear, branched or

cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit(

OCH 2CH2)n, wherein n is an integer from 1 to 2000, a 5- or 6-membered heteroaryl ring

containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, a

to 18 membered fused ring system, wherein at least one of the rings is aromatic, containing

one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, aryl having

from 6 to 18 carbon atoms, 3 to 18-membered heterocyclic ring having 1 to 6 heteroatoms

selected from 0, S, N and P and R1 can also be OR 14, wherein R 14 is H or has the same

definition as R, optionally R" is OH;

W is C=O, C=S, CH2, BH, SO or SO 2 ;

R 5 is selected from OR1 5 , CRR'OH, SH, CRR'SH, NHR15 or CRR'NHR1 5 , wherein R15 has the

same definition as R. or is a linking group that enables linkage to a cell binding agent via a

covalent bond or is selected from a polypyrrolo, poly-indolyl, poly-imidazolyl, polypyrollo

imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolyl unit optionally bearing a linking group

that enables linkage to a cell binding agent;;

R 6 is OR, SR or NRR', wherein R and R' have the same definition as given above, optionally R 6

is a linking group;

X' is CH 2, NR, CO, BH, SO or SO 2 ;

Y' is 0, CH 2, NR or S;

Z' is CH 2 or (CH 2)n, wherein n is 2, 3 or 4; or their pharmaceutically acceptable solvates, salts,

hydrates or hydrated salts, their optical isomers, racemates, diastereomers, enantiomers or the

polymorphic crystalline structures of these compounds; provided that the compound has no more than one linking group that enables linkage to a cell binding agent via a covalent bond.

[07] A third aspect of the invention provides cytotoxic dimers (IV), (V) and (VI)

x Y x Y R 1' z ---N A-D-L-D'-A' N

R2' N-W a Re R W -N R2

R 3' R 4' (IV) R4 R3

R2' R 1'y x x y R1 R2 - -N N---,. R3. A-D-L-D'-A' N , R3

R4' W R6 W (V)

Y X x Y

Zl_N. A-D-L-D'-A'

Y' R6 R6 W NX. (VI)

of the benzodiazepine monomers of formulas (I) and (II) and (III), respectively, in which the

dimer compounds optionally bear a linking group that allows for linkage to cell binding

agents,

wherein:

9 17477638_1 (GHMatters) P87639.AU.3 the double line = between N and C represents a single bond or a double bond, provided that when it is a double bond X is absent and Y is H, and when it is a single bond, X is H or an amine protecting moiety that converts the compound into a prodrug;

Y is selected from -OR, an ester represented by -OCOR', a carbonate represented by -OCOOR',

a carbamate represented by -OCONR'R", an amine or a hydroxyl amine represented by NR'R",

amide represented by -NRCOR', a peptide represented by NRCOP, wherein P is an amino acid

or a polypeptide containing between 2 to 20 amino acid units, a thioether represented by SR', a

sulfoxide represented by SOR', a sulfone represented by -SO 2R', a sulfite -SO 3, a bisulfite

OSO3, a halogen, cyano, an azido, or a thiol, wherein R, R' and R" are same or different and are

selected from H, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl

having from I to 10 carbon atoms, a polyethylene glycol unit (-OCH 2CH 2)n, wherein n is an

integer from 1 to 2000, a 5- or 6-membered heteroaryl ring containing one or more heteroatoms

independently selected from nitrogen, oxygen, and sulfur, a 5 to 18 membered fused ring system,

wherein at least one of the rings is aromatic, containing one or more heteroatoms independently

selected from nitrogen, oxygen, and sulfur, aryl having from 6 to 18 carbon atoms, 3 to 18

membered heterocyclic ring having 1to 6 heteroatoms selected from 0, S, N and P wherein the

substituent is selected from halogen, OR, NRsR9 , NO 2, NRCOR', SRio, a sulfoxide represented

by SOR', a sulfone represented by -SO 2R', a sulfite -SO 3, a bisulfite -OSO 3, a sulfonamide

represented by SO 2NRR', cyano, an azido, -COR1 , OCOR1 1 or OCONRR 12 , wherein of R7 , Rs,

R 9, Rio, R 1 and R 12 are each independently selected from H, linear, branched or cyclic alkyl,

alkenyl or alkynyl having from I to 10 carbon atoms, a polyethylene glycol unit (-OCH 2CH 2)n, wherein n is an integer from 1 to 2000, a 5- or 6-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5 to 18 membered fused ring system, wherein at least one of the rings is aromatic, containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, aryl having from 6 to 18 carbon atoms, 3 to 10-membered heterocyclic ring having 3 to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected from 0, S, N and P and RIO is optionally SR13 or COR1

, wherein R1 is selected from linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to

carbon atoms, a 5- or 6-membered heteroaryl ring containing one or more heteroatoms

independently selected from nitrogen, oxygen, and sulfur, a 5 to 18 membered fused ring system,

wherein at least one of the rings is aromatic, containing one or more heteroatoms independently

selected from nitrogen, oxygen, and sulfur, aryl having from 6 to 18 carbon atoms, 3 to 18

membered heterocyclic ring having 1 to 6 heteroatoms selected from 0, S, N and P, optionally

RI is OR 14, wherein R 14 has the same definition as R, optionally R" is OH;

W is C=O, C=S, CH2, BH, SO or SO2 ;

R 1 , R 2, R 3, R4 , Ri', R 2 ', R 3 ' and R 4 ' are each independently selected from H, substituted or

unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon

atoms, a polyethylene glycol unit (-OCH 2 CH 2)n, wherein n is an integer from 1 to 2000, or a

substituent selected from a halogen, guanidinium [-NH(C=NH)NH 2], OR, NRsR9 , NO 2 ,

NRCOR', SRio, a sulfoxide represented by SOR', a sulfone represented by -S0 2R', a sulfite

SO3, a bisulfite -OS0 3, a sulfonamide represented by SO 2NRR', cyano, an azido, -CORI1 ,

OCOR Ior OCONRIR 12 wherein R7 , Rs, R9, Rio, R 1 and R 12 are as defined aboveoptionally, any one of R 1, R 2, R 3, R4, R', R 2', R 3', or R4 ' is a linking group that enables linkage to a cell binding agent via a covalent bond or is selected from a polypyrrolo, poly-indolyl, poly imidazolyl, polypyrollo-imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolyl unit optionally bearing a linking group that enables linkage to a cell binding agent,

Z is selected from (CH 2),, wherein n is 1, 2 or 3, CR1 5 R 16, NR 17, 0 or S, wherein R1 5 , R1 6 and

R 17 are each independently selected from H, linear, branched or cyclic alkyl having from 1 to 10

carbon atoms, a polyethylene glycol unit (-OCH 2CH2)n, wherein n is an integer from 1 to 2000;

R 6 is OR, SR or NRR', wherein R and R' have the same definition as given above, optionally R 6

is a linking group;

X' is selected from CH 2, NR, CO, BH, SO or S02 wherein R has the same definition as given

above;

Y' is 0, CH 2, NR or S, wherein R has the same definition as given above;

Z' is CH 2 or (CH 2)n, wherein n is 2, 3 or 4, provided that X', Y' and Z' are not all CH2 at the

same time;

A and A' are the same or different and are selected from 0, -CRR'O, S, -CRR'S, -NR15 or

CRR'NHR 1 5, wherein R and R' have the same definition as given above and wherein R1 5 has the

same definition as R.

D and D' are same or different and independently selected from linear, branched or cyclic alkyl,

alkenyl or alkynyl having 1 to 10 carbon atoms, optionally substituted with any one of halogen,

OR 7 , NRsR 9, NO2, NRCOR', SR 1 ,0 a sulfoxide represented by SOR', a sulfone represented by

S0 2R', a sulfite -SO 3 , a bisulfite -OSO 3, a sulfonamide represented by SO 2NRR', cyano, an azido, -CORii, OCORii or OCONRiiR12, wherein the definitions of R7, R8, R9, Rio, Riu and

R12 are as defined above, or a polyethylene glycol unit (-OCH2CH2)n, wherein n is an integer

from 1 to 2000;

L is an optional phenyl group or 3 to 18-membered heterocyclic ring having 1 to 6

heteroatoms selected from 0, S, N and P that is optionally substituted, wherein the

substituent is a linking group that enables linkage to a cell binding agent via a covalent bond,

or is selected from linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10

carbon atoms, optionally substituted with any one of halogen, OR7, NRR9, N02, NRCOR',

SRio, a sulfoxide represented by SOR', a sulfone represented by -SO2R', a sulfite -S03, a

bisulfite -OS0 3 , a sulfonamide represented by SO2NRR', cyano, an azido, -CORii, OCORii

or OCONRiiR12, wherein R7, R8, R9, Rio, Ri and R12 have the same definitions as given

above, a polyethylene glycol unit (-OCH2CH2)n, wherein n is an integer from 1 to 2000;

optionally, L itself is a linking group that enables linkage to a cell binding agent via a

covalent bond; or their pharmaceutically acceptable solvates, salts, hydrates or hydrated salts,

their optical isomers, racemates, diastereomers, enantiomers or the polymorphic crystalline

structures of these compounds; provided that the compound has no more than one linking

group that enables linkage to a cell binding agent via a covalent bond.

[72] A fourth aspect of the invention provides conjugates of cell binding agents with the

novel benzodiazepine compounds or derivatives thereof of the present invention. These

conjugates are useful as therapeutic agents, which are delivered specifically to target cells and

are cytotoxic.

13 17477638_1 (GHMatters) P87639.AU.3

[73] The present invention includes a composition (e.g., a pharmaceutical composition)

comprising novel benzodiazepine compounds, derivatives thereof, or conjugates 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

novel benzodiazepine compounds, derivatives thereof, or conjugates 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). The present

compositions are also useful for treating depression, anxiety, stress, phobias, panic, dysphoria,

psychiatric disorders, pain, and inflammatory diseases in a mammal (e.g., human).

[74] 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 novel benzodiazepine compounds, derivatives thereof, or

conjugates thereof, (and/or solvates and salts thereof) or a composition thereof, alone or in

combination with a second therapeutic agent.

[75] The present invention includes a method of synthesizing and using novel benzodiazepine

compounds, derivatives thereof, and conjugates thereof for in vitro, in situ, and in vivo diagnosis

or treatment of mammalian cells, organisms, or associated pathological conditions.

[76] 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 and 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.

The present invention as claimed herein is described in the following items 1 to 13:

1. A process for the preparation of compound of formula (8):

HN1

MeO 8 comprising the steps of: a) converting a compound of formula (6): OHQ BnO NO

6 0

into a compound of formula (7):

BnuOC

MeQ N D

7 ;and b) deprotecting the benzyl protecting group of the compound of formula (7) to form a compound of formula (8).

2. The process of item 1, further comprising the steps of: a) coupling a compound of formula (2): MeO 2C

H2® C D

and a compound of formula (4): BnO NO 2 C1 MeO C (4)

15 17477638_1 (GHMatters) P87639.AU.3 to give a compound of formula (5):

MeO 2C BnQ N~j Meo 5 0 ; and b) reducing the compound of formula (5) to form an aldehyde of formula (6):

OHC BnO NO, Me ~ N/ 6 0

3. The method of item 1, wherein the compound of formula (6) is reacted with sodium dithionite to the compound of formula (7).

4. A compound represented by formula (1):

BnO HN

MeOw N/\

1

5. A process for preparing a compound represented by the following formula:

BnO HN-.

MeO N

comprising the step of reducing a compound represented by formula 7:

BnO N

MeO

7 to form the compound of formula:

BnO HN

MeO

6. The process of item 5, wherein the step of reducing is carried out by using NaBH4.

15a 17477638_1 (GHMatters) P87639.AU.3

7. The process of item 6, wherein the reduction reaction is carried out in a mixture of ethanol and dichloromethane.

8. The process of item 7, wherein the reaction is carried out at a temperature between 0 °C and room temperature.

9. The process of item 7, wherein the reaction is quenched by addition of saturated ammonium chloride.

10. A compound represented by formula 7:

BnO N

MeO

7

11. A compound represented by formula 3:

HO HN

MeOw

3

12. A process of preparing a compound represented by formula 3:

HO HN

MeO b comprising the step of reducing a compound represented by formula 8:

HO N

8, to form the compound of formula 3.

13. The process of item 12, wherein the reducing agent is H2/Pd.

BRIEF DESCRIPTION OF THE FIGURES

15b 17477638_1 (GHMatters) P87639.AU.3

[77] FIGS. 1-10 show the schemes for the synthesis of indolinobenzodiazepine and

oxazolidinobenzodiazepine monomers, the representative linkers and the dimers in the

present invention.

[78] FIG. 11 shows the scheme for the synthesis of the representative B-ring modified

indolinobenzodiazepine monomer.

[79] FIG. 12 shows the scheme for the synthesis of the representative

isoindolinobenzodiazepine monomer.

[80] FIG. 13 shows the scheme for the synthesis of the representative dimer with the linker

directly attached on the indolinobenzodiazepine moiety in the present invention.

[81] FIGS. 14 and 15 show the schemes for the synthesis of the representative dimers

containing (PEG). moieties on the linkers.

[82] FIG. 16 shows the schemes for the synthesis of the representative mixed imine-amine

and imine-amide indolinobenzodiazepine dimers.

[83] FIG. 17 shows the scheme for the synthesis of the representative IBD-poly(N

methylpyrrole-imidazole) conjugates.

15c 17477638_1 (GHMatters) P87639.AU.3 and 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 FIGURES

[77] FIGS. 1-10 show the schemes for the synthesis of indolinobenzodiazepine and

oxazolidinobenzodiazepine monomers, the representative linkers and the dimers in the present

invention.

[78] FIG. 11 shows the scheme for the synthesis of the representative B-ring modified

indolinobenzodiazepine monomer.

[79] FIG. 12 shows the scheme for the synthesis of the representative

isoindolinobenzodiazepine monomer.

[80] FIG. 13 shows the scheme for the synthesis of the representative dimer with the linker

directly attached on the indolinobenzodiazepine moiety in the present invention.

[81] FIGS. 14 and 15 show the schemes for the synthesis of the representative dimers

containing (PEG). moieties on the linkers.

[82] FIG. 16 shows the schemes for the synthesis of the representative mixed imine-amine and

imine-amide indolinobenzodiazepine diners.

[83] FIG. 17 shows the scheme for the synthesis of the representative IBD-poly(N

methylpyrrole-imidazole) conjugates.

[84] FIGS. 18-19 show the synthetic scheme for the preparation of polypyrrolo and

polypyrrolo-imidazolo derivatives of the monomers.

[85] FIG 20 shows a scheme for the synthesis of piperidinylbenzodiazepines bearing a

hydrazone linker.

[86] FIGS. 21-26 show the dose dependent in vitro antiproliferative activity of muB38.1-IGN

03, huN901-IGN-03, huN901-IGN-07, and muB38.1-IGN-10 conjugates on antigen positive and

antigen negative cancer cell lines.

[87] FIG. 27 shows in vivo efficacy of huN901-IGN-07 conjugate in mice bearing Molp-8

tumors.

[88] FIGS. 28-30 show data that demonstrate that IGN-01, IGN-02, and IGN-09 bind and

covalently adduct to double stranded DNA containing guanine residues on opposite strands.

[89] FIG. 31 contains TABLE 1, which shows the IC5 0 values for in vitro antiproliferative

activity of indolinobenzodiazepine dimers and oxazolidinobenzodiazepine dimer on several

cancer cell lines.

[90] FIG. 32 contains TABLE 2, which shows the comparison of the IC5 0 values for in vitro

antiproliferative activity of indolinobenzodiazepine diners with and without linkers.

[91] FIGS. 33-36, 39, 42, 43, 44, 48, 49 and 50 show synthetic schemes for the preparation of

compounds of the present invention.

[92] FIGS. 37, 38, 40 and 41, 45, 46, and 47 show synthetic schemes for the preparation of

likable compounds of the present invention.

[93] Fig. 51 shows the in vitro cytotoxicity of compounds of the present invention.

[94] FIGS. 52, 54, 56, 57 and 58 show the in vitro cytotoxicity and specificity of chB38.1

conjugates.

[95] FIGS. 53 and 55 show the in vitro cytotoxicity and specificity ofhuMy9-6 conjugates.

[96] FIG. 59 shows the in vivo anti-tumor activity of chB38.1 conjugate

DETAILED DESCRIPTION OF THE INVENTION

[97] Reference will now be made in detail to certain embodiments of the invention, examples

of which are illustrated in the accompanying structures and formulas. While the invention will be

described in conjunction with the enumerated embodiments, it will be understood that they are

not intended to limit the invention to those embodiments. On the contrary, the invention is

intended to cover all alternatives, modifications, and equivalents which may be included within

the scope of the present invention as defined by the claims. One skilled in the art will recognize

many methods and materials similar or equivalent to those described herein, which could be used

in the practice of the present invention.

DEFINITIONS

[98] "Linear or branched alkyl" as used herein refers to a saturated linear or branched-chain

monovalent hydrocarbon radical of one to twenty carbon atoms. Examples of alkyl include, but

are not limited to, methyl , ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, -

CH 2CH(CH 3) 2), 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl 3-pentyl, 2-methyl-2-butyl, 3

methyl-2-butyl, 3-methyl-i-butyl, 2-methyl-i-butyl, 1-hexyl), 2-hexyl, 3-hexyl, 2-methyl-2 pentyl,, 3-methyl-2-pentyl,, 4-methyl-2-pentyl,, 3-methyl-3-pentyl,, 2-methyl-3-pentyl, 2,3 dimethyl-2-butyl,, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and the like.

[99] "Linear or branched alkenyl" refers to linear or branched-chain monovalent

hydrocarbon radical of two to twenty carbon atoms with at least one site of unsaturation, i.e., a

carbon-carbon, double bond, wherein the alkenyl radical includes radicals having "cis" and

"trans" orientations, or alternatively, "E" and "Z" orientations. Examples include, but are not

limited to, ethylenyl or vinyl (--CH=CH 2), allyl (--CH 2CH=CH 2), and the like.

[100] "Linear or branched alkynyl" refers to a linear or branched monovalent hydrocarbon

radical of two to twenty carbon atoms with at least one site of unsaturation, i.e., a carbon-carbon,

triple bond. Examples include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1

pentynyl, 2-pentynyl, 3-pentynyl, hexynyl, and the like.

[101] The terms "cyclic alkyl", "cyclic alkenyl", "cyclic alkynyl", "carbocycle",

"carbocyclyl", "carbocyclic ring" and "cycloalkyl" refer to a monovalent non-aromatic,

saturated or partially unsaturated ring having 3 to 12 carbon atoms as a monocyclic ring or 7 to

12 carbon atoms as a bicyclic ring. Bicyclic carbocycles having 7 to 12 atoms can be arranged,

for example, as a bicyclo [4,5], [5,5], [5,6] or [6,6] system, and bicyclic carbocycles having 9 or

ring atoms can be arranged as a bicyclo [5,6] or [6,6] system, or as bridged systems such as

bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and bicyclo[3.2.2]nonane. Examples ofmonocyclic

carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-I

enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-I-enyl, 1-cyclohex-2-enyl,

1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,

cycloundecyl, cyclododecyl, and the like.

[102] "Aryl" means a monovalent aromatic hydrocarbon radical of 6-18 carbon atoms derived

by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring

system. Some aryl groups are represented in the exemplary structures as "Ar". Aryl includes

bicyclic radicals comprising an aromatic ring fused to a saturated, partially unsaturated ring, or

aromatic carbocyclic or heterocyclic ring. Typical aryl groups include, but are not limited to,

radicals derived from benzene (phenyl), substituted benzenes, naphthalene, anthracene, indenyl,

indanyl, 1,2-dihydronapthalene, 1,2,3,4-tetrahydronapthyl, and the like.

[103] The terms "heterocycle," "heterocyclyl" and "heterocyclic ring" are used

interchangeably herein and refer to a saturated or a partially unsaturated (i.e., having one or more

double and/or triple bonds within the ring) carbocyclic radical of 3 to 18 ring atoms in which at

least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus, and sulfur, the

remaining ring atoms being C, where one or more ring atoms is optionally substituted

independently with one or more substituents described below. A heterocycle may be a

monocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected

from N, 0, P, and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 6

heteroatoms selected from N, 0, P,_and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6]

system. Heterocycles are described in Paquette, Leo A.; "Principles of Modem Heterocyclic

Chemistry" (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; "The

Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New

York, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc.

(1960) 82:5566. "Heterocyclyl" also includes radicals where heterocycle radicals are fused with

a saturated, partially unsaturated ring, or aromatic carbocyclic or heterocyclic ring. Examples of

heterocyclic rings include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl,

tetrahydrothienyl, tetrahydropyranyl,_dihydropyranyl, tetrahydrothiopyranyl, piperidino,

morpholino, thiomorpholino, thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl,

thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 2

pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl,

pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,

pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyco[3.1.0]hexanyl, 3

azabicyclo[4.1.0]heptanyl, and azabicyclo[2.2.2]hexanyl. Spiro moieties are also included

within the scope of this definition. Examples of a heterocyclic group wherein ring atoms are

substituted with oxo (=0) moieties are pyrimidinonyl and 1,1-dioxo-thiomorpholinyl.

[104] The term "heteroaryl" refers to a monovalent aromatic radical of 5- or 6-membered

rings, and includes fused ring systems (at least one of which is aromatic) of 5-18 atoms,

containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur.

Examples of heteroaryl groups are pyridinyl (including, for example, 2-hydroxypyridinyl),

imidazolyl, imidazopyridinyl, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl),

pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl,

isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl,

cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, triazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl.

[105] The heterocycle or heteroaryl groups maybe carbon (carbon-linked) or nitrogen

(nitrogen-linked) attached where such is possible. By way of example and not limitation, carbon

bonded heterocycles or heteroaryls are bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3,

4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of a

pyrazine, position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or

tetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4, or 5 of an

isoxazole, pyrazole, or isothiazole, position 2 or 3 of an aziridine, position 2, 3, or 4 of an

azetidine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of an

isoquinoline.

[106] Byway of example and not limitation, nitrogen bonded heterocycles or heteroaryls are

bonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline,

imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3

pyrazoline, piperidine, piperazine, indole, indoline,I H-indazole, position 2 of a isoindole, or

isoindoline, position 4 of a morpholine, and position 9 of a carbazole, or O-carboline.

[107] The heteroatoms present inheteroaryl orheterocyclcyl include the oxidized forms such as

NO, SO, and SO 2 .

[108] The term "halo" or "halogen" refers to F, Cl, Br or I.

[109] The term "compound" or "cytotoxic compound" or "cytotoxic agent" as used herein is

intended to include compounds for which a structure or formula or any derivative thereof has been disclosed in the present invention or a structure or formula or any derivative thereof that has been incorporated by reference. The term also includes, stereoisomers, geometric isomers, tautomers, solvates, metabolites, salts (e.g., pharmaceutically acceptable salts) and prodrugs, and prodrug salts of a compound of all the formulae disclosed in the present invention. The term also includes any solvates, hydrates, and polymorphs of any of the foregoing. The specific recitation of "stereoisomers", "geometric isomers", "tautomers", "solvates", "metabolites", "salt"

"prodrug," "prodrug salt," "conjugates," "conjugates salt," "solvate," "hydrate," or polymorphh"

in certain aspects of the invention described in this application shall not be interpreted as an

intended omission of these forms in other aspects of the invention where the term "compound" is

used without recitation of these other forms.

[110] The term "conjugate" as used herein refers to a compound or a derivative thereof that is

linked to a cell binding agent and is defined by a generic formula: C-L-CBA, wherein C=

compound, L = linker, and CBA = cell binding agent.

[111] The term "linkable to a cell binding agent" as used herein referes to the novel

benzodiazepine compounds (e.g., indolinobenzodiazepine or oxazolidinobenzodiazepine),

derivates thereof or dimers thereof comprising at least one linking group or a precursor thereof

suitable to bond these compounds, derivatives thereof or dimers thereof to a cell binding agent.

[112] The term "precursor"of a given group refers to any group which may lead to that group

by any deprotection, a chemical modification, or a coupling reaction.

[113] The term "linked to a cell binding agent" refers to a conjugate molecule comprising at

least one the novel benzodiazepine compounds (e.g., indolinobenzodiazepine or oxazolidinobenzodiazepine), derivates thereof or dimers thereof bound to a cell binding agent via a suitable linking group or a precursor thereof

[114] The term "chiral" refers to molecules which have the property of non-superimposability

of the mirror image partner, while the term "achiral" refers to molecules which are

superimposable on their mirror image partner.

[115] The term "stereoisomer" refers to compounds which have identical chemical

constitution and connectivity, but different orientations of their atoms in space that cannot be

interconverted by rotation about single bonds.

[116] "Diastereomer" refers to a stereoisomer with two or more centers of chirality and whose

molecules are not mirror images of one another. Diastereomers have different physical

properties, e.g. melting points, boiling points, spectral properties, and reactivities. Mixtures of

diastereomers may separate under high resolution analytical procedures such as crystallization,

electrophoresis and chromatography.

[117] "Enantiomers" refer to two stereoisomers of a compound which are non

superimposable mirror images of one another.

[118] Stereochemical definitions and conventions used herein generally follow S. P. Parker,

Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New

York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley &

Sons, Inc., New York, 1994. The compounds of the invention may contain asymmetric or chiral

centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric

forms of the compounds of the invention, including but not limited to, diastereomers, enantiomers and atropisomers, as well as mixtures thereof such as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and I or (+) and (-)are employed to designate the sign of rotation of plane-polarized light by the compound, with (-)or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.

[119] The term "tautomer" or "tautomeric form" refers to structural isomers of different

energies which are interconvertible via a low energy barrier. For example, proton tautomers

(also known as prototropic tautomers) include interconversions via migration of a proton, such as

keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions by

reorganization of some of the bonding electrons.

[120] A substituent is "substitutable" if it comprises at least one carbon, sulfur, oxygen or

nitrogen atom that is bonded to one or more hydrogen atoms. Thus, for example, hydrogen,

halogen, and cyano do not fall within this definition.

[121] If a substituent is described as being "substituted," a non-hydrogen substituent is in the

place of a hydrogen substituent on a carbon, oxygen, sulfur or nitrogen of the substituent. Thus,

for example, a substituted alkyl substituent is an alkyl substituent wherein at least one non

hydrogen substituent is in the place of a hydrogen substituent on the alkyl substituent. To

illustrate, monofluoroalkyl is alkyl substituted with a fluoro substituent, and difluoroalkyl is

alkyl substituted with two fluoro substituents. It should be recognized that if there is more than

one substitution on a substituent, each non-hydrogen substituent may be identical or different

(unless otherwise stated).

[122] If a substituent is described as being "optionally substituted," the substituent maybe

either (1) not substituted, or (2) substituted. If a carbon of a substituent is described as being

optionally substituted with one or more of a list of substituents, one or more of the hydrogens on

the carbon (to the extent there are any) may separately and/or together be replaced with an

independently selected optional substituent. If a nitrogen of a substituent is described as being

optionally substituted with one or more of a list of substituents, one or more of the hydrogens on

the nitrogen (to the extent there are any) may each be replaced with an independently selected

optional substituent. One exemplary substituent may be depicted as -- NR'R," wherein R' and R"

together with the nitrogen atom to which they are attached, may form a heterocyclic ring. The

heterocyclic ring formed from R' and R" together with the nitrogen atom to which they are

attached may be partially or fully saturated. In one embodiment, the heterocyclic ring consists of

3 to 7 atoms. In another embodiment, the heterocyclic ring is selected from the group consisting

of pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, pyridyl and thiazolyl.

[123] This specification uses the terms "substituent," "radical," and "group"

interchangeably.

[124] If a group of substituents are collectively described as being optionally substituted by one

or more of a list of substituents, the group may include: (1) unsubstitutable substituents, (2)

substitutable substituents that are not substituted by the optional substituents, and/or (3)

substitutable substituents that are substituted by one or more of the optional substituents.

[125] If a substituent is described as being optionally substituted with up to a particular number

of non-hydrogen substituents, that substituent may be either (1) not substituted; or (2) substituted

by up to that particular number of non-hydrogen substituents or by up to the maximum number

of substitutable positions on the substituent, whichever is less. Thus, for example, if a substituent

is described as a heteroaryl optionally substituted with up to 3 non-hydrogen substituents, then

any heteroaryl with less than 3 substitutable positions would be optionally substituted by up to

only as many non-hydrogen substituents as the heteroaryl has substitutable positions. Such

substituents, in non-limiting examples, can be selected from a linear, branched or cyclic alkyl,

alkenyl or alkynyl having from I to 10 carbon atoms, halogen, guanidinium [-NH(C=NH)NH 2],

OR 7 , NRgR 9, NO2, NRCOR', SR1,a sulfoxide represented by SOR', a sulfone represented by

SO2R', a sulfite -SO 3 , a bisulfite -OSO 3 , a sulfonamide represented by SO 2NRR', cyano, an

azido, -COR 1 , OCOR ]or OCONR R12 wherein R 7 , Rs, R 9, Rio, R 1 and R12 are each

independently selected from H, linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1

to 10 carbon atoms, a polyethylene glycol unit (-OCH 2CH 2)n, wherein n is an integer from I to

2000, aryl having from 6 to 10 carbon atoms, heterocyclic ring having from 3 to 10 carbon

atoms.

[126] The term "prodrug" as used in this application refers to a precursor or derivative form

of a compound of the invention that is capable of being enzymatically or hydrolytically activated

or converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer

Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast

(1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed

Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). The prodrugs of this

invention include, but are not limited to, ester-containing prodrugs, phosphate-containing

prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing

prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, .beta.-lactam-containing

prodrugs, optionally substituted phenoxyacetamide-containing prodrugs, optionally substituted

phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs

which can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs

that can be derivatized into a prodrug form for use in this invention include, but are not limited

to, compounds of the invention and chemotherapeutic agents such as described above.

[127] The term "prodrug" is also meant to include a derivative of a compound that can

hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide

a compound of this invention. Prodrugs may only become active upon such reaction under

biological conditions, or they may have activity in their unreacted forms. Examples of prodrugs

contemplated in this invention include, but are not limited to, analogs or derivatives of compounds of any one of the formulae disclosed herein that comprise biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues.

Other examples of prodrugs include derivatives of compounds of any one of the formulae

disclosed herein that comprise --NO, --NO2, --ONO, or -ONO2 moieties. Prodrugs can typically

be prepared using well-known methods, such as those described by Burger's Medicinal

Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff ed., 5th ed); see

also Goodman and Gilman's, The Pharmacological basis of Therapeutics, 8th ed., McGraw-Hill,

Int. Ed. 1992, "Biotransformation of Drugs".

[128] As used herein and unless otherwise indicated, the terms "biohydrolyzable amide",

"biohydrolyzable ester", "biohydrolyzable carbamate", "biohydrolyzable carbonate",

"biohydrolyzable ureide" and "biohydrolyzable phosphate analogue" mean an amide, ester,

carbamate, carbonate, ureide, or phosphate analogue, respectively, that either: 1) does not

destroy the biological activity of the compound and confers upon that compound advantageous

properties in vivo, such as uptake, duration of action, or onset of action; or 2) is itself

biologically inactive but is converted in vivo to a biologically active compound. Examples of

biohydrolyzable amides include, but are not limited to, lower alkyl amides, .alpha.-amino acid

amides, alkoxyacyl amides, and alkylaminoalkylcarbonyl amides. Examples of biohydrolyzable

esters include, but are not limited to, lower alkyl esters, alkoxyacyloxy esters, alkyl acylamino

alkyl esters, and choline esters. Examples of biohydrolyzable carbamates include, but are not

limited to, lower alkylamines, substituted ethylenediamines, amino acids, hydroxyalkylamines, heterocyclic and heteroaromatic amines, and polyether amines. Particularly favored prodrugs and prodrug salts are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal.

[129] The phrase "pharmaceutically acceptable salt" as used herein, refers to

pharmaceutically acceptable organic or inorganic salts of a compound of the invention.

Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride,

bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate,

acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate,

gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate,

methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate

(i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g., sodium and

potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A

pharmaceutically acceptable salt may involve the inclusion of another molecule such as an

acetate ion, a succinate ion or other counter ion. The counter ion may be any organic or inorganic

moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically

acceptable salt may have more than one charged atom in its structure. Instances where multiple

charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions.

Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or

more counter ion.

[130] If the compound of the invention is abase, the desired pharmaceutically acceptable salt

may be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.

[131] If the compound of the invention is an acid, the desired pharmaceutically acceptable salt

may be prepared by any suitable method, for example, treatment of the free acid with an

inorganic or organic base, such as an amine (primary, secondary or tertiary), an alkali metal

hydroxide or alkaline earth metal hydroxide, or the like. Illustrative examples of suitable salts

include, but are not limited to, organic salts derived from amino acids, such as glycine and

arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines, such as

piperidine, morpholine and piperazine, and inorganic salts derived from sodium, calcium,

potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

[132] As used herein, the term "solvate" means a compound which further includes a

stoichiometric or non-stoichiometric amount of solvent such as water, isopropanol, acetone,

ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine dichloromethane, 2

propanol, or the like, bound by non-covalent intermolecular forces. Solvates or hydrates of the

compounds are readily prepared by addition of at least one molar equivalent of a hydroxylic solvent such as methanol, ethanol, 1-propanol, 2-propanol or water to the compound to result in solvation or hydration of the imine moiety.

[133] The terms "abnormal cell growth" and "proliferative disorder" are used

interchangeably in this application. "Abnormal cell growth", as used herein, unless otherwise

indicated, refers to cell growth that is independent of normal regulatory mechanisms (e.g., loss of

contact inhibition). This includes, for example, the abnormal growth of: (1) tumor cells (tumors)

that proliferate by expressing a mutated tyrosine kinase or overexpression of a receptor tyrosine

kinase; (2) benign and malignant cells of other proliferative diseases in which aberrant tyrosine

kinase activation occurs; (3) any tumors that proliferate by receptor tyrosine kinases; (4) any

tumors that proliferate by aberrant serine/threonine kinase activation; and (5) benign and

malignant cells of other proliferative diseases in which aberrant serine/threonine kinase

activation occurs.

[134] The terms "cancer" and "cancerous" refer to or describe the physiological condition in

mammals that is typically characterized by unregulated cell growth. A "tumor" comprises one or

more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma,

blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such

cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer

including small-cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the

lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer,

gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma,

cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, acute leukemia, as well as head/brain and neck cancer.

[135] A "therapeutic agent" encompasses both a biological agent such as an antibody, a

peptide, a protein, an enzyme or a chemotherapeutic agent. A"chemotherapeutic agent" is a

chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents

include Erlotinib (TARCEVA@, Genentech/OSI Pharm.), Bortezomib (VELCADE®,

Millennium Pharm.), Fulvestrant (FASLODEX@, AstraZeneca), Sutent (SUl1248, Pfizer),

Letrozole (FEMARA®, Novartis), Imatinib mesylate (GLEEVEC@, Novartis), PTK787/ZK

222584 (Novartis), Oxaliplatin (Eloxatin@, Sanofi), 5-FU (5-fluorouracil), Leucovorin,

Rapamycin (Sirolimus, RAPAMUNE@, Wyeth), Lapatinib (TYKERB@, GSK572016, Glaxo

Smith Kline), Lonafarnib (SCH 66336), Sorafenib (BAY43-9006, Bayer Labs), and Gefitinib

(IRESSA®, AstraZeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as

thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan

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

ethylenimines and methylamelamines including altretamine, triethylenemelamine,

triethylenephosphoramide, triethylenethiophosphoramide and trimethyomelamine; acetogenins

(especially bullatacin and bullatacinone); a camptothecin (including the synthetic analog

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

synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (Angew Chem. Intl.

Ed. Engl. (1994) 33:183-186); dynemicin, including dynemicin A; bisphosphonates, such as

clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein

enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine,

bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis,

dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN@

(doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin

and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as

mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin,

puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,

zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid

analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as

fludarabine, 6-mercaptopurine, thiamniprine, thioguanine; pyrimidine analogs such as ancitabine,

azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine,

floxuridine; 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; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK@ polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; 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., TAXOL@ (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.),

ABRAXANE@ (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel

(American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE@ (doxetaxel; Rhone

Poulenc Rorer, Antony, France); chloranmbucil; GEMZAR@ (gemcitabine); 6-thioguanine;

mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine;

etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE@ (vinorelbine);

novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA@);

ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO);

retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of

any of the above.

[136] Also included in the definition of "chemotherapeutic agent" are: (i) anti-hormonal

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

selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including

NOLVADEX@; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,

keoxifene, LY117018, onapristone, and FARESTON (toremifine citrate); (ii) aromatase

inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal

glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol

acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR@ (vorozole),

FEMARA@ (letrozole; Novartis), and ARIMIDEX@ (anastrozole; AstraZeneca); (iii) anti

androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as

troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors; (v)

lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit

expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for

example, PKC-alpha, Ralf and H-Ras; (vii) ribozymes such as VEGF expression inhibitors (e.g.,

ANGIOZYME@) and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines,

for example, ALLOVECTIN@, LEUVECTIN@., and VAXID@.; PROLEUKIN@ rlL-2; a

topoisomerase I inhibitor such as LURTOTECAN®; ABARELIX@ rmRH; (ix) anti-angiogenic

agents such as bevacizumab (AVASTIN@, Genentech); and (x) pharmaceutically acceptable

salts, acids and derivatives of any of the above. Other anti-angiogenic agents include MMP-2

(matrix-metalloproteinase 2) inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors, COX-II

(cyclooxygenase II) inhibitors, and VEGF receptor tyrosine kinase inhibitors. Examples of such useful matrix metalloproteinase inhibitors that can be used in combination with the present compounds/compositions are described in WO 96/33172, WO 96/27583, EP 818442, EP

1004578, WO 98/07697, WO 98/03516, WO 98/34918, WO 98/34915, WO 98/33768, WO

98/30566, EP 606,046, EP 931,788, WO 90/05719, WO 99/52910, WO 99/52889, WO

99/29667, WO 99/07675, EP 945864, U.S. Pat. No. 5,863,949, U.S. Pat. No. 5,861,510, and EP

780,386, all of which are incorporated herein in their entireties by reference. Examples of VEGF

receptor tyrosine kinase inhibitors include 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1

methylpiperidin-4-ylmethoxy)qu- inazoline (ZD6474; Example 2 within WO 01/32651), 4-(4

fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)- -quinazoline

(AZD2171; Example 240 within WO 00/47212), vatalanib (PTK787; WO 98/35985) and

SU11248 (sunitinib; WO 01/60814), and compounds such as those disclosed in PCT Publication

Nos. WO 97/22596, WO 97/30035, WO 97/32856, and WO 98/13354).

[137] Other examples of chemotherapeutic agents that can be used in combination with the

present compounds include inhibitors of P13K (phosphoinositide-3 kinase), such as those

reported in Yaguchi et al (2006) Jour. of the Nat. Cancer Inst. 98(8):545-556; U.S. Pat. No.

7,173,029; U.S. Pat. No. 7,037,915; U.S. Pat. No. 6,608,056; U.S. Pat. No. 6,608,053; U.S. Pat.

No. 6,838,457; U.S. Pat. No. 6,770,641; U.S. Pat. No. 6,653,320; U.S. Pat. No. 6,403,588; WO

2006/046031; WO 2006/046035; WO 2006/046040; WO 2007/042806; WO 2007/042810; WO

2004/017950; US 2004/092561; WO 2004/007491; WO 2004/006916; WO 2003/037886; US

2003/149074; WO 2003/035618; WO 2003/034997; US 2003/158212; EP 1417976; US

2004/053946; JP 2001247477; JP 08175990; JP 08176070; U.S. Pat. No. 6,703,414; and WO

97/15658, all of which are incorporated herein in their entireties by reference. Specific examples

of such P13K inhibitors include SF-126 (P13K inhibitor, Semafore Pharmaceuticals), BEZ-235

(P13K inhibitor, Novartis), XL-147 (P13K inhibitor, Exelixis, Inc.).

[138] A "metabolite" is a product produced through metabolism in the body of a specified

compound, a derivative thereof, or a conjugate thereof, or salt thereof. Metabolites of a

compound, a derivative thereof, or a conjugate thereof, may be identified using routine

techniques known in the art and their activities determined using tests such as those described

herein. Such products may result for example from the oxidation,hydroxylation, reduction,

hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, and the

like, of the administered compound. Accordingly, the invention includes metabolites of

compounds, a derivative thereof, or a conjugate thereof, of the invention, including compounds,

a derivative thereof, or a conjugate thereof, produced by a process comprising contacting a

compound, a derivative thereof, or a conjugate thereof, of this invention with a mammal for a

period of time sufficient to yield a metabolic product thereof.

[139] The phrase "pharmaceutically acceptable" indicates that the substance or composition

must be compatible chemically and/or toxicologically, with the other ingredients comprising a

formulation, and/or the mammal being treated therewith.

[140] The term "protecting group" or "protecting moiety" refers to a substituent that is

commonly employed to block or protect a particular functionality while reacting other functional

groups on the compound, a derivative thereof, or a conjugate thereof. For example, an "amino

protecting group" or an "amino-protecting moiety" is a substituent attached to an amino group that blocks or protects the amino functionality in the compound. Suitable amino-protecting groups include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and 9 fluorenylmethylenoxycarbonyl (Fmoc). Similarly, a "hydroxy-protecting group" refers to a substituent of a hydroxy group that blocks or protects the hydroxy functionality. Suitable protecting groups include acetyl and silyl. A "carboxy-protecting group" refers to a substituent of the carboxy group that blocks or protects the carboxy functionality. Common carboxy-protecting groups include phenylsulfonylethyl, cyanoethyl, 2-(trimethylsilyl)ethyl, 2

(trimethylsilyl)ethoxymethyl, 2-(p-toluenesulfonyl)ethyl, 2-(p-nitrophenylsulfenyl)ethyl, 2

(diphenylphosphino)-ethyl, nitroethyl and the like. Common thiol-protecting groups include

those that convert the thiol into a thioester, such as acetyl, benzoyl or trifluoroacetyl, into a

thioether, such as benzyl, t-butyl, triphenylmethyl, 9-fluorenylmetyl, methoxymethyl, 2

tetrahydropyranyl or silyl, into a disulfide, such as methyl, benzyl, t-butyl, pyridyl, nitropyridyl,

phenyl, nitrophenyl or dinitrophenyl, into a thiocarbonate, such as t-butoxycarbonyl, into a

thiocarbamate, such as N-ethyl. For a general description of protecting groups and their use, see

P. G.M. Wuts & T. W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons,

New York, 2007.

[141] For novel benzodiazepines of formula (I) and (II),

S CR1 R2 R5 RI6

R6 W R4 R4 R3

(I) (II)

wherein:

the double line =between N and C represents a single bond or a double bond, provided that

when it is a double bond X is absent and Y is H, and when it is a single bond, X is H, or an

amine protecting moiety that converts the compound into a prodrug that can be transformed into

the free amine in vitro or in vivo;

Y is selected from -OR, an ester represented by -OCOR', a carbonate represented by -OCOOR',

a carbamate represented by -OCONR'R", an amine or a hydroxyl amine represented by NR'R",

amide represented by -NRCOR', a peptide represented by NRCOP, wherein P is an amino acid

or a polypeptide containing between 2 to 20 amino acid units, a thioether represented by SR', a

sulfoxide represented by SOR', a sulfone represented by -SO 2R', asulfite -SO 3, a bisulfite

-OSO3, a halogen, cyano, an azido, or a thiol, wherein R, R' and R" are same or different and

are selected from H, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or

alkynyl having from 1 to 20 carbon atoms a polyethylene glycol unit (-OCH 2CH 2)1 , wherein n is

an integer from 1 to 2000, a 5- or 6-membered heteroaryl ring containing one or more

heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5 to 18 membered fused

ring system, wherein at least one of the rings is aromatic, containing one or more heteroatoms

independently selected from nitrogen, oxygen, and sulfur, aryl having from 6 to 18 carbon atoms,

a 3 to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected from 0, S, N and P

wherein the substituent is selected from halogen, OR, NRsR9 , NO 2, NRCOR', SR1 0 , a sulfoxide

represented by SOR', a sulfone represented by -SO 2R', a sulfite -SO 3, a bisulfite -OSO3, a sulfonamide represented by SO 2NRR', cyano, an azido, -CORI, OCOR Ior OCONRIIR12, wherein R7, Rs, R 9, Rio, Rn and R 12 are each independently selected from H, linear, branched or cyclic alkyl, alkenyl or alkynyl having from I to 10 carbon atoms, a polyethylene glycol unit(

OCH 2CH2) , wherein 1 n is an integer from 1 to 2000, a 5- or 6-membered heteroaryl ring

containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur. a

to 18 membered fused ring system, wherein at least one of the rings is aromatic.containing one

or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, aryl having from

6 to 18 carbon atoms 3 to 18-membered heterocyclic ring having I to 6 heteroatoms selected

from 0, S, N and P and Rio optionally is SR or COR, wherein R13 is selected from linear,

branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene

glycol unit (-OCH 2CH 2) , wherein 1 n is an integer from 1 to 2000, , a 5- or 6-membered

heteroaryl ring containing one or more heteroatoms independently selected from nitrogen,

oxygen, and sulfur, a 5 to 18 membered fused ring system, wherein at least one of the rings is

aromatic, containing one or more heteroatoms independently selected from nitrogen, oxygen, and

sulfur, 3 to 18-membered heterocyclic ring having I to 6 heteroatoms selected from 0, S, N and

P and R1 can also be OR14, wherein R14 is H or has the same definition as R, optionally, R" is

an OH;

W is C=O, C=S, CH2, BH (B=Boron), SO or SO 2;

R 1, R2, R3, R4, are each independently selected from H, substituted or unsubstituted linear,

branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene

glycol unit (-OCH 2CH2)., wherein n is an integer from 1 to 2000, or a substituent selected from a halogen, OR7 , NR8 R 9, NO 2, NRCOR', SR 10, a sulfoxide represented by SOR', a sulfone represented by -SO2 R', a sulfite -SO 3, a bisulfite -OSO 3 , a sulfonamide represented by

SO 2NRR', cyano, an azido, guanidinium [-NH(C=NH)NH 2], -CORI, -OCOR1 or

OCONR 1 R 12 wherein R 7, Rs, R 9, Rio, R 1 and R 12 are as defined above, optionally, any one of

R 1, R 2, R 3, R4 is a linking group that enables linkage to a cell binding agent via a covalent bond

or is selected from a polypyrrolo, poly-indolyl, poly-imidazolyl, polypyrollo-imidazolyl, poly

pyrollo-indolyl or polyimidazolo-indolyl unit optionally bearing a linking group that enables

linkage to a cell binding agent;

R 5 is selected from OR1 5 , CRR'OH, SH, CRR'SH, NHR15 or CRR'NHR1 5 , wherein R15 has the

same definition as R., R and R' have the same definition as given above; optionally, R 5 is a

linking group that enables linkage to a cell binding agent via a covalent bond or is selected from

a polypyrrolo, poly-indolyl, poly-imidazolyl, polypyrollo-imidazolyl, poly-pyrollo-indolyl or

polyimidazolo-indolyl unit optionally bearing a linking group that enables linkage to a cell

binding agent;;

R 6 is OR, SR, NRR', wherein R and R'have the same definition as given above, or optionally

R 6 is a linking group;

Z is selected from (CH 2 )n, wherein n is 1, 2 or 3, CR15 Ri6 , NR 17 , 0 or S, wherein R1 5 , R1 6 and

R 17 are each independently selected from H, linear, branched or cyclic alkyl having from 1 to 10

carbon atoms, a polyethylene glycol unit (-OCH 2CH2),, wherein n is an integer from 1 to 2000;

or their pharmaceutically acceptable solvates, salts, hydrates or hydrated salts, their optical

isomers, racemates, diastereomers, enantiomers of these compounds.

provided that the compound has no more than one linking group that enables linkage to a cell

binding agent via a covalent bond.

[142] In one preferred embodiment, the double line= between N and C represents a double

bond and X is absent and Y is H, or the double line = between N and C represents a single bond

wherein X is H and Y is selected from -OR, a sulfite -SO 3, or an amine protecting moiety that

converts the compound into a prodrug;

W is C=O, CH2 , or SO 2;

R 1, R 2, R 3, R4 , are each H; optionally, independently, any one of R 1, R 2, R3 and R 4 can be a

linking group that enables linkage to a cell binding agent via a covalent bond;

R 5 is selected from ORi 5 , CRR'OH, SH, CRR'SH, NHR1 5 or CRR'NHRi 5 , wherein R 5 is H or

has the same definition as given above for R, or is selected from a polypyrrolo, poly-indolyl,

poly-imidazolyl, polypyrollo-imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolyl unit

optionally bearing a linking group that enables linkage to a cell binding agent, R and R' have the

same definition as given above;

R6 is OCH3 ;

Z is selected from (CH 2 )., wherein n is 1 or 2, NH, NCH 3 or S; or their pharmaceutically

acceptable solvates, salts, hydrates or hydrated salts, their optical isomers, racemates,

diastereomers, enantiomers or the polymorphic crystalline structures of these compounds.

[143] Ina preferred embodiment, compounds of formula (I) and (II) are compounds of

formulae (VII), (VIII) or (IX):

R5 N RH*R Me R2 Me Me 0 R4 R3 0 0

(VII) (VIII) (IX)

wherein the substituents are described as above; or their pharmaceutically acceptable solvates,

salts, hydrates or hydrated salts, their optical isomers, racemates, diastereomers, enantiomers or

the polymorphic crystalline structures of these compounds.

[144] For the novel benzodiazepines of formula (III), in which the diazepine ring (B) is fused

with a heterocyclic ring (C), wherein the heterocyclic ring is monocyclic,

x Y

R5 rZ'

(III)

wherein:

the double line =between Nand Crepresents asingle bond ora double bond, provided that

when itis adouble bond Xis absent and Yis-H, and when it is asingle bond, Xis Hor anamine

protecting moiety that converts the compound into aprodrug;

Y is selected from -OR, an ester represented by -OCOR', acarbonate represented by -OCOOR',

a carbamate represented by -OCONR'R", an amine or ahydroxyl amine represented by NR'R",

amide represented by -NRCOR', apeptide represented by NRCOP, wherein Pis an amino acid

or apolypeptide containing between 2to 20amino acid units, athioether represented by SR', a sulfoxide represented by SOR', a sulfone represented by -SO 2R', a sulfite -SO 3, a bisulfite

OS03, a halogen, cyano, an azido, or a thiol, wherein R, R' and R" are same or different and

selected from H, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl

having from 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH 2CH 2)n, wherein n is an

integer from 1 to 2000, a 5- or 6-membered heteroaryl ring containing one or more heteroatoms

independently selected from nitrogen, oxygen, and sulfur, a 5 to 18 membered fused ring system,

wherein at least one of the rings is aromatic, containing one or more heteroatoms independently

selected from nitrogen, oxygen, and sulfurheteroaryl comprising of 5- or 6-membered rings,

including fused ring systems (at least one of which is aromatic) of 5-18 atoms, containing one or

more heteroatoms independently selected from nitrogen, oxygen, and sulfur, aryl having from 6

to 18 carbon atoms 3 to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected from

, S, N and P, wherein the substituent is selected from halogen, OR, NRR 9, NO 2, NRCOR',

SRio, a sulfoxide represented by SOR', a sulfone represented by -SO 2R', a sulfite -SO 3 , a

bisulfite -OS0 3, a sulfonamide represented by SO 2NRR', cyano, an azido, -COR1 1 , OCOR or

OCONR 11R 12 , wherein R 7 , Rs, R9, Rio, Ra and R 12 are each independently selected from H,

linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a

polyethylene glycol unit (-OCH 2CH 2),, wherein n is an integer from Ito 2000, a 5- or 6

membered heteroaryl ring containing one or more heteroatoms independently selected from

nitrogen, oxygen, and sulfur, a 5 to 18 membered fused ring system, wherein at least one of the

rings is aromatic, containing one or more heteroatoms independently selected from nitrogen,

oxygen, and sulfurheteroaryl comprising of 5- or 6-membered rings, including fused ring systems (at least one of which is aromatic) of 5-18 atoms, containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, aryl having from 6 to 18 carbon atoms

3 to 18-membered heterocyclic ring having Ito 6 heteroatoms selected from 0, S, N and P and

Rio optionally is SR 13 or COR 13 , wherein R 13 is selected from linear, branched or cyclic alkyl,

alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit (-O CH 2) 1

, wherein n is an integer from 1 to 2000, a 5- or 6-membered heteroaryl ring containing one or

more heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5 to 18 membered

fused ring system, wherein at least one of the rings is aromatic, containing one or more

heteroatoms independently selected from nitrogen, oxygen, and sulfurheteroaryl comprising of

- or 6-membered rings, including fused ring systems (at least one of which is aromatic) of 5-18

atoms, containing one or more heteroatoms independently selected from nitrogen, oxygen, and

sulfur, aryl having from 6 to 18 carbon atoms, 3 to 18-membered heterocyclic ring having 1 to 6

heteroatoms selected from 0, S, N and P andRu canalsobe OR 14, wherein R 14 isH orhasthe

same definition as R, optionally R" is OH;

W is C=O, C=S, CH2, BH, SO or S02;

R 5 is selected from OR1 5 , CRR'OH, SH, CRR'SH, NHR1 5 or CRR'NHR 15 , wherein R 15 is H or

has the same definition as R. or is a linking group that enables linkage to a cell binding agent via

a covalent bond or is selected from a polypyrrolo, poly-indolyl, poly-imidazolyl, polypyrollo

imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolyl unit optionally bearing a linking group

that enables linkage to a cell binding agent;is selected from a poly-pyrrolo, poly-indolyl, poly

imidazolyl, polypyrollo-imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolyl unit optionally bearing a linking group that enables linkage to a cell binding agent, optionally, R 5 is a linking group that enables linkage to a cell binding agent via a covalent bond;

R 6 is OR, SR or NRR', wherein R and R' have the same definition as given above, optionally R6

is a linking group;

X' is CH 2, NR, CO, BH, SO or SO 2;

Y' is 0, CH 2, NR or S;

Z' is CH 2 or (CH 2)n, wherein n is 2, 3 or 4; or their pharmaceutically acceptable solvates, salts,

hydrates or hydrated salts, their optical isomers, racemates, diastereomers, enantiomers orthe

polymorphic crystalline structures of these compounds;

provided that the compound has no more than one linking group that enables linkage to a cell

binding agent via a covalent bond.

[145] In one preferred embodiment, the double line = between N and C represents a double

bond and X is absent and Y = H, or the double line = between N and C represents a single bond

wherein X is H and Y is selected from -OR, a sulfite -SO 3, or an amine protecting moiety that

converts the compound into a prodrug;

W is C=O, CH2 , or SO 2 ;

R 5 is selected from OR15 , CRR'OH, SH, CRR'SH, NHR 15 or CRR'NHR1 5 , wherein R1 5 is H or

has the same definition as given above for R, or is selected from a polypyrrolo, poly-indolyl,

poly-imidazolyl, polypyrollo-imidazolyl, poly-pyrolloindolyl or polyimidazoloindolyl unit

optionally bearing a linking group that enables linkage to a cell binding agent;

R 6 is OCH3 ;

X' is selected from CH 2, or C=O;

Y' is 0, CH 2, NR or S;

Z' is (CH2 )n, wherein n is 1 or 2, provided that X', Y' and Z' are not all CH 2 at the same time; or

their pharmaceutically acceptable solvates, salts, hydrates or hydrated salts, their optical isomers,

racemates, diastereomers, enantiomers or the polymorphic crystalline structures of these

compounds.

[146] Ina preferred embodiment, compound of formula III is represented by a compound of

formula (X) or (XI),

R6 N ~ Me:) (

0 0

(X) (XI)

wherein the substituents are described as above; or their pharmaceutically acceptable solvates,

salts, hydrates or hydrated salts, their optical isomers, racemates, diastereomers, enantiomers or

the polymorphic crystalline structures of these compounds.

[147] For the cytotoxic dimers represented by formulas (IV), (V) and (VI)

x Y x Y N- z z N R2' N A-D--L-D'-RA NW R R3 ' R4' (IV) R4 R3

R2 ' R'y X X y R1 R2

R 3' / N A-D-L-D'-A N R3 N-- -aw-N* R4' W RRW

(V) Y X x y

A-D-L-D'-A' N Z

X W R R6 W X (VI)

the double line = between N and C represents a single bond or a double bond, provided that

when it is a double bond X is absent and Y is H, and when it is a single bond, X is H or an amine

protecting moiety that converts the compound into a prodrug;

Y is selected from -OR, an ester represented by -OCOR', a carbonate represented by -OCOOR',

a carbamate represented by -OCONR'R", an amine or a hydroxyl amine represented by NR'R",

amide represented by -NRCOR', a peptide represented by NRCOP, wherein P is an amino acid

or a polypeptide containing between 2 to 20 amino acid units, a thioether represented by SR', a

sulfoxide represented by SOR', a sulfone represented by-SO 2R', a sulfite-SO 3, a bisulfite

OSO3, a halogen, cyano, an azido, or a thiol, wherein R, R' and R" are same or different and are

selected from H, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl

having from I to 10 carbon atoms, a polyethylene glycol unit (-OCH2CH 2 )n, wherein n is an

integer from 1 to 2000, a 5- or 6-membered heteroaryl ring containing one or more heteroatoms

independently selected from nitrogen, oxygen, and sulfur, a 5 to 18 membered fused ring system,

wherein at least one of the rings is aromatic, containing one or more heteroatoms independently

selected from nitrogen, oxygen, and sulfur, aryl having from 6 to 18 carbon atoms, 3 to 18 membered heterocyclic ring having 1to 6 heteroatoms selected from 0, S, N and P wherein the substituent is selected from halogen, OR7 , NRsR9 , NO 2, NRCOR', SRio, a sulfoxide represented by SOR', a sulfone represented by -SO 2R', a sulfite -SO 3, a bisulfite -OSO 3, a sulfonamide represented by SO 2NRR', cyano, an azido, -COR, OCOR Ior OCONRR 12 , wherein R 7, Rs,

R 9, Rio, R 1 and R 12 are each independently selected from H, linear, branched or cyclic alkyl,

alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit (-OH2 C H2)n,

wherein n is an integer from 1 to 2000, a 5- or 6-membered heteroaryl ring containing one or

more heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 5 to 18 membered

fused ring system, wherein at least one of the rings is aromatic, containing one or more

heteroatoms independently selected from nitrogen, oxygen, and sulfur, aryl having from 6 to 18

carbon atoms, 3 toI 0-membered heterocyclic ring having 3 to 18-membered heterocyclic ring

having 1 to 6 heteroatoms selected from 0, S, N and P and RIO is optionally SR 13 or COR 13

, wherein R 13 is selected from linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to

carbon atoms, a 5- or 6-membered heteroaryl ring containing one or more heteroatoms

independently selected from nitrogen, oxygen, and sulfur, a 5 to 18 membered fused ring system,

wherein at least one of the rings is aromatic, containing one or more heteroatoms independently

selected from nitrogen, oxygen, and sulfur, aryl having from 6 to 18 carbon atoms, 3 to 18

membered heterocyclic ring having 1 to 6 heteroatoms selected from 0, S, N and P, optionally

R 1 is OR 14, wherein R 14 has the same definition as R, optionally R" is OH;

W is C=0, C=S, CH2, BH, SO or SO 2;

R 1, R 2, R 3, R4 , R 1', R 2 ', R 3 ' and R4 ' are each independently selected from H, substituted or

unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon

atoms, a polyethylene glycol unit (-OCH 2 CH 2)n, wherein n is an integer from I to 2000, or a

substituent selected from a halogen, guanidinium [-NH(C=NH)NH 2], OR 7, NRsR9 , NO 2

, NRCOR', SR 10, a sulfoxide represented by SOR', a sulfone represented by -SO 2R', a sulfite

SO3, a bisulfite -OSO3, a sulfonamide represented by SO 2NRR', cyano, an azido, -COR 1

, OCOR1 1 or OCONRIR 12 wherein R7 , Rs, R 9, Rio, R 11 and R 12 are as defined above, optionally,

any one of R 1, R 2, R 3, R 4, R 1', R 2', R 3', or R 4 ' is a linking group that enables linkage to a cell

binding agent via a covalent bond or is selected from a polypyrrolo, poly-indolyl, poly

imidazolyl, polypyrollo-imidazolyl, poly-pyrollo-indolyl or polyimidazolo-indolyl unit

optionally bearing a linking group that enables linkage to a cell binding agent,

Z is selected from (CH 2)., wherein n is 1, 2 or 3, CR1 5 R16 , NR 17, 0 or S, wherein R 15, R1 6 and

R 17 are each independently selected from H, linear, branched or cyclic alkyl having from 1 to 10

carbon atoms, a polyethylene glycol unit (-OCH 2CH2)n, wherein n is an integer from 1 to 2000;

R 6 is OR, SR or NRR', wherein R and R'have the same definition as given above, optionally R6

is a linking group;

X' is selected from CH 2, NR, CO, BH, SO or S02 wherein R has the same definition as given

above;

Y' is 0, CH 2, NR or S, wherein R has the same definition as given above;

Z' is CH 2 or (CH 2)n, wherein n is 2, 3 or 4, provided that X', Y' and Z' are not all CH2 at the

same time;

A and A' are the same or different and are selected from 0, -CRR'O, S, -CRR'S, -NR1 5 or

CRR'NHR 1 5, wherein R and R' have the same definition as given above and wherein RI5 has the

same definition as R.

D and D' are same or different and independently selected from linear, branched or cyclic alkyl,

alkenyl or alkynyl having 1 to 10 carbon atoms, optionally substituted with any one of halogen,

OR 7 , NRsR 9, NO2, NRCOR', SRio, a sulfoxide represented by SOR', a sulfone represented by

SO2R', a sulfite -SO 3 , a bisulfite -OSO 3, a sulfonamide represented by SO2NRR', cyano, an

azido, -CORII, OCOR Ior OCONR R12 , wherein the definitions of R7 , RS, R 9 , RIO, R1 and R 12

are as defined above, or a polyethylene glycol unit (-OCH 2CH 2)n, wherein n is an integer from 1

to 2000;

L is an optional phenyl group or 3 to 18-membered heterocyclic ring having 1 to 6 heteroatoms

selected from 0, S, N andP that is optionally substituted, wherein the substituent is a linking

group that enables linkage to a cell binding agent via a covalent bond, or is selected from linear,

branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, optionally

substituted with any one of halogen, OR7 , NRsR9 , NO 2, NRCOR', SRio, a sulfoxide represented

by SOR', a sulfone represented by -SO 2R', a sulfite -SO 3, a bisulfite -OSO3, a sulfonamide

represented by SO 2NRR', cyano, an azido, -COR1 , OCOR or OCONR R 12 , wherein the

definitions of R7, Rs, R 9, Rio, Ru and R 12 are as defined above, a polyethylene glycol unit (

OCH 2CH 2)n, wherein n is an integer from 1 to 2000; optionally, L itself is a linking group that

enables linkage to a cell binding agent via a covalent bond; or their pharmaceutically acceptable

solvates, salts, hydrates or hydrated salts, their optical isomers, racemates, diastereomers, enantiomers or the polymorphic crystalline structures of these compounds; provided that the compound has no more than one linking group that enables linkage to a cell binding agent via a covalent bond.

[148] In one preferred embodiment, the double line= between N and C represents a single

bond or a double bond, provided that when it is a double bond X is absent and Y is H, and when

it is a single bond, X is H or an amine protecting group that converts the compound into a

prodrug;

Y is selected from -OR, NR'R", a sulfite -SO 3, or a bisulfite -OSO 3 , wherein R is selected from

H, linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a

polyethylene glycol unit (-OCH 2CH 2),, wherein n is an integer from 1 to 2000, aryl having from

6 to 10 carbon atoms, heterocyclic ring having from 3 to 10 carbon atoms;

W is C=O, CH2 or SO 2;

R 1, R 2, R 3, R4 , Ri'. R 2 '. R 3 ' and R 4 ' are each independently selected from H, NO 2 or a linking

group that enables linkage to a cell binding agent via a covalent bond;

R 6 is ORis, wherein Ris has the same definition as R;

Z is selected from (CH 2 )., wherein n is 1, 2 or 3, CR15 Ri6 , NR 17 , 0 or S, wherein R15 , R1 6 and

R 1 7 are each independently selected from H, linear, branched or cyclic alkyl having from 1 to 10

carbon atoms, a polyethylene glycol unit (-OCH 2CH2)n, wherein n is an integer from 1 to 2000;

X' is selected from CH 2, or C=O;

Y' is 0, NR, or S, wherein R is defined as above;

Z' is CH 2 or (CH 2)2 ;

A and A' are each 0;

D and D' are same or different and independently selected from linear, branched or cyclic alkyl,

alkenyl or alkynyl having from 1 to 10 carbon atoms;

L is an optional phenyl group or a heterocycle ring having from 3 to 10 carbon atoms that is

optionally substituted, wherein the substituent is a linking group that enables linkage to a cell

binding agent via a covalent bond, or is selected from linear, branched or cyclic alkyl, alkenyl or

alkynyl having from I to 10 carbon atoms, optionally substituted with any one of halogen, OR7

, NRgR9, NO2, NRCOR', SRa,a sulfoxide represented by SOR', a sulfone represented by -SO 2R',

a sulfite -SO 3 , a bisulfite -OS0 3 , a sulfonamide represented by SO 2NRR', cyano, an azido, ,

COR n , OCOR Ior OCONRn R 12, a polyethylene glycol unit (-OCH 2CH 2)n, wherein n is an

integer from I to 2000; optionally, L itself is a linking group that enables linkage to a cell

binding agent via a covalent bond; or their pharmaceutically acceptable solvates, salts, hydrates

or hydrated salts, their optical isomers, racemates, diastereomers, enantiomers or the

polymorphic crystalline structures of these compounds.

[149] In another preferred embodiment, the compound of formula (IV), (V) or (VI) is

represented by compounds of formulae (XII) and (Xli1):

Y X x Y Z AN L A 4

/ N -\ N /\ R2') R R"IR R3 (XII) R3

y x X Y A -A N - IA 0 O (X0 wherein the double line = between N and C represents a single bond or a double bond, provided that when it is a double bond X is absent and Y is H, and when it is a single bond, X is H or an amine protecting group that converts the compound into a prodrug; Y is selected from OH, an ether represented by -OR, NR'R", a sulfite -SO,, or a bisulfite -OSO 3, wherein R, R' and R" are selected from linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms; one of R 2, R 3, R2' and R 3 ' is a linking group that enables linkage to a cell binding agent via a covalent bond and the others are H, NRCOR' or NO 2 ;

R 6 is OR, wherein R has the same definition as above;

Z is CH 2 or NR, wherein R has the same definition as above;

A is 0 or NR1 5 ;

L is (CH 2) 1n, wherein nnis 0 or an integer between 1 and 5, or a substituted or unsubstituted

alkyl or alkenyl having from 2 to 4 carbon atoms, wherein the substituent is selected from

halogen, OR 7 , NRsR, NO2 , NRCOR', SR 1 ,a sulfoxide represented by SOR', a sulfone

represented by -SO2 R', a sulfite -SO 3, a bisulfite -OS03, a sulfonamide represented by

SO2NRR', cyano, an azido,, -CORI,, OCOR or OCONRiR1 2, wherein R7 , Rs, R 9, Rio, Rn, R 12

and RI 5 has the same definition as given above, optionally, L itself is a linking group that enables

linkage to a cell binding agent via a covalent bond;

one of L', L" or L"' is a linking group that enables linkage to a cell binding agent, while the

others are H; preferably L' is the linking group; and

G is CH or N or their pharmaceutically acceptable solvates, salts, hydrates or hydrated salts, their

optical isomers, racemates, diastereomers, enantiomers or the polymorphic crystalline structures

of these compounds.

[150] In yet another preferred embodiment, the compound of formula (IV), (V) or (VI) is

represented by compounds of formulae from formulae (XIV) and (XV):

Y X X Y

R2,# OCH HC R2 0 0 R 31 R3

y x X y

G N OCH3 G H3CON

CXV)

wherein the double line between N and C represents a single bond or a double bond, provided

that when it is a double bond X is absent and Y is H, and when it is a single bond, X is H or an

amine protecting group that converts the compound into a prodrug; Y is selected from OH, an

ether represented by -OR, a sulfite -SO 3 , or a bisulfite -OSO 3, wherein R is selected from linear,

branched or cyclic alkyl, alkenyl or alkynyl having from I to 10 carbon atoms;

nn is 0 or an integer from I to 5;

One of R2 , R 3, R 2' and R' is a linking group that enables linkage to a cell binding agent via a

covalent bond and the others are H, NRCOR', or NO 2 ;

one of L', L" or L"' is a linking group that enables linkage to a cell binding agent, provided that

when one of L', L" or L"' is a linking group others are H (e.g., if L' is a linker, then L" and L"'

are H)

G is CH or N or their pharmaceutically acceptable solvates, salts, hydrates or hydrated salts, their

optical isomers, racemates, diastereomers, enantiomers or the polymorphic crystalline structures

of these compounds.

[151] In order to link the cytotoxic compounds (e.g., indolinobenzodiazepine or

oxazolidinobenzodiazepine), derivatives thereof, or dimers thereof of the present invention to the

cell-binding agent, the cytotoxic compound comprises a linking moiety. While a linker that

connects two moieties is bifunctional, one end of the linker moiety can be first reacted with the

cytotoxic compound to provide the compound bearing a monofunctional linking group, which

can then react with a cell binding agent. Alternatively, one end of the linker moiety can be first

reacted with the cell binding agent to provide the cell binding agent bearing a monofunctional

linking group, which can then react with a cytotoxic compound. The linking moiety contains a

chemical bond that allows for the release of the cytotoxic moiety at a particular site. Suitable

chemical bonds are well known in the art and include disulfide bonds, thioether bonds, acid

labile bonds, photolabile bonds, peptidase labile bonds and esterase labile bonds (see for

example US Patents 5,208,020; 5,475,092; 6,441,163; 6,716,821; 6,913,748; 7,276,497;

7,276,499; 7,368,565; 7,388,026 and 7,414,073). Preferred are disulfide bonds, thioether and peptidase labile bonds. Other linkers that can be used in the present invention include non cleavable linkers, such as those described in are described in detail in U.S. publication number

20050169933, or charged linkers or hydrophilic linkers and are described in provisional patent

applications, 61/049,291, filed April 30, 2008, 61/147,966, filed January 28, 2009, and

61/049,289, filed April 30. 2008, each of which is expressly incorporated herein by reference.

[152] The compounds of formula (1), (II), and (III) (i.e., monomers) can be linked through R1

, R 2 , R 3, R 4 or R 5 . Of these, preferred linkable groups are R2 , R3 , and R5 , and the most preferred

linkable group is R5 . Examples of suitable substituents at R1 , R 2 , R3 , R4 and R5 for compounds

of formula (1), (II) and (111) include, but are not limited to:

-OH,

-O(CR 20 R21 )m(CR22R 23 )(OCH 2CH 2)p(CR 4oR41)p-.Y"(CR 24R 25)q(CO)iX",

-O(CR 20 R21)m.(CR 26=CR27)m'(CR 22R23)(OCH 2CH 2)p(CR4aR4 1)pYY"(CR 24R25)q(CO)tX",

-O(CR 20 R21)m(alkynyl).,(CR 22R 23)(OCH 2CH 2)p(CR 4oR41)p"Y"(CR 24R25)q(CO)tX",

-O(CR 20 R21)m(indolo)p,(CR 22R 23).(OCH 2CH 2)p(CR 4oR 41)p"Y"(CR2 4R 25)q(CO)tX"

-O(CR 20 R 21)m(pyrrolo)q'(CR 22R 23).(OCH 2CH2)p(CR 4oR4 1)p"Y"(CR 24R 25)(CO)tX",

-O(CR 2oR21)m(pyrrolO)q'(imidazolo)q'(CR 22 R23 )n(OCH 2 CH 2 )p(CR 4 oR4 1)p"Y"(CR 2 4 R2 5 )q(CO)tX",

-O(CR 20 R21)m(imidazolo)q(CR 22R23)n(OCH 2CH 2)p(CR4 oR4 1)p'Y"(CR2 4 R2 5 )q(CO)tX',

-O(CR 20 R21 )m(pyrrolo)q'(indolo)q(CR2 2R23)n(OCH 2CH 2)p(CR4oR 41)p,Y"(CR 24R25 )q(CO)tX',

-O(CR 20 R21)m(indolo)q,(imidazolo)q,(CR 22R2 3)n(OCH 2CH 2)p(CR4 oR41)p"Y"(CR 24 R25 )q(CO)tX",

-O(CR20R21)m(piperazino)t,(CR22R23),(OCH2CH2)p(CR4aR41)p"(CR 5 24 R 2 )q(CO)tX",

-O(CR 20 R21)mA"m(CR 22R23)(OCH 2CH 2)p(CR 4oR4 1)p'Y"(CR 24R25)q(CO)tX",

-SH,

-S(CR 2 0R2 ]).(CR 2 2R2 3)(OCH 2CH 2 ) (CR~oR1)p"YY"(CR 2 4R2 5)q(CO)tX",

-S(CR2 0R21).(CR 26 -CR27).'(CR 22 R23 ),(OCH2 CH2 )p(CR4 oR4 )p,Y(CR2 4R2 )q(CO)tX'',

S(CR 2 oR2 1)m,(alkynyl)n',(CR 22 R23)(OCH 2 CH 2)p(CRioR 4 )p,"Y''(CR2 4R2 5)q(CO)iX",

-S(CR 2oR2n),,(indolo)p'(CR 22 R23)1 (OCH 2CH 2)(CR 4OR 4 )"Y''(CR 24 R25 )q(CO)tX'',

S(CR 2 0R21).(PYrrOlO)q'(CR 22 R23 )(OCH 2CH 2)p(CR 4 PI)p"Y " (CR2 4R2 5)q(CO)tX",

-S(CR20 R2])m(imidazolO)q',(CR22R23)n,(OCH2CH2)p(CR4oR41)p"Y"'(CR 24 R25 )q(CO)tX",

S(CR2oR2l)m(PYlrolo)q'(iMidazolo)q'(CR 22R 23 ),(OCH 2CFI2 )p(CR 4 oR4 i)p,"Y''(CR 24 R25 )q(CO)iX'',

S(CR2oR21)m(pyrroO)q'(nflOO)q'(CR 22R 23 )(OCH 2CH 2)(CR 4oR 4 )"Y'(CR 24 R 25)q(CO)tX",

-S(CR20 R21 )m(inldolO)q'(iidazolo)q''(CR 22 R2 3 )(C 2C 2 CRo~)p"Y''(CR 24 R25 )q(CO)tX",

S(CR 2 oR2 1)m(piperazino)t'(CR 22 R23 )n,(OCH 2CH 2 )p(CR4 oR4 I)p"Y''I(CR 24 R25 )q(CO)tX

% -S(CR20R 21).nA"m"(CR 2-2 R'-3 )(OCH 2CH 2)p(CR 4OR 4 )"Y''(CR 24 R25)q(CO)tX"',

-NH 2 ,

-NR 28 (CR2 0R2 1)m(CR 2 2R2 3)(OCH 2 CH)p(CR,4 oR, 4 )p'Y''(CR2 4R2 5)q(CO)tX'',

-NR28 (CR 2 oR2 1)m(CR 2 =CR 27)m'(CR 2 2R 2 3)(OCH 2CH 2 )p(CR4oR 4 l) "Y''(CR2 4R 2 5)q(CO)tX'',

-NR2g(CR2oR 2 i)m(alkynyl)n'(CR 22 R23 )1 (OCH 2CH 2)p(CR4ojR 4 )"Y''(CR 24 R25 )q(CO)tX",

-NR2 S(CR2OR 21)m(ifldolo)p(CR 2 2R23 )(OCH 2CH 2)(CR 4OR 4 )"Y''(CR 24R2 5)q(CO)tX"5

-NR28 (CR20 R2 1)m(PYrrOO)q'(CR 2 R23 )(OCH 2CH 2)p(CR 4 oR4 )"Y''(CR 24R 2 5)q(CO)tX'',

-NR28 (CR2 0R21)ii(imidazole)q'I(CR 22R23 ).(OCH 2 CH2 )p(CR4 oR4 )p "Y''(CR 24R2 5)q(CO)tX'',

-NR28 (CR20 R21)im(pyrrolo)q'(imidazolo)q",(CR 22R23 )i,(OCH 2CH 2)p(CR 4oR 4 i)p"Y''

(CR2 4R 25 )q(CO)tX'',

-NR 2s(CR20 R21)m(pyrrolO)q'(indolO)q(CR22R23)(OCH 2CH2)p(CR4oR 4 1)p"Y"

(CR 24 R2 5)q(CO)X",

-NR 2s(CR 2OR 2 1)m(indolO)q'(imidazolo)q"(CR 2 2 R 2 3 ).(OCH 2CH 2 )p(CR 4 0R 4 1)p"Y

(CR2 4R25)(CO)X",

-NR 28 (CR20 R21)m(piperazino)e(CR 22R 23) (OCH 1 2 CH 2 )p(CR 4oR 4 1)p,Y"(CR 24 R25 )q(CO)tX",

-NR 28 (CR2OR 21)mA"m'(CR 22R23)n(OCH 2CH 2)p(CR 4OR4 1)p"Y"(CR 24R25)(CO)tX",

-(CR 20R 21)m(CR 22R 23)(OCH 2 CH 2 )p(CR 4 oR41),"Y"(CR2 4R2 5)q(CO)tX",

-(CR 20R 2 1)m(CR 26=CR 27 )m'(CR 22R 23)n(OCH 2CH 2)p(CR 4oR 4 1)p"Y"(CR 24R 25)q(CO)tX",

-(CR 20R 21 )m(alkynyl)n,(CR2 2R23),(OCH 2CH2)p(CR4oR 41)p,Y"(CR 24R2 5)q(CO)tX",

-(CR 20 R 21)m(indolo)p,(CR 22R23)n(OCH 2CH 2)p(CR 4oR4 1)pY''(CR 24R25)(CO)tX",

-(CR 2oR 21)m(pyrrolO)q'(CR 22R 23).(OCH 2CH 2)p(CR 4oR4 1)pY"(CR 24R 25 )q(CO)tX',

-(CR 20R 21)m(piperazino)t(CR 22 R 23 ) (OCH2CH 1 2)p(CR 4 R41)p"Y"(CR 24 R25 )q(CO)tX",

-(CR 20R2 1)im(PYrTOl)q(indolO)q'(CR22R 23).(OCH2CH 2)p(CR 4 oR 41)p"Y"-(CR 24R 25)q(CO)iX",

(CR 20R2 I)m(imidazolo)q''(CR 22R23)n(OCH 2CH 2)p(CR 4 oR4 1)pY"-(CR 24R25 )q(CO)tX",

(CR 2 0R2 I)m(pyrrolO)q'(imlidazolo)q"(CR 22R 23),(OCH 2CH2)p(CR4oR4 1), Y"-(CR 24R 25 )q(CO)tX",

(CR 2oR2 1)m(imidazolo)q(indolO)q(CR 2 2R23 ).(OCH 2CH 2)p(CR4oR4 )pY"-(CR 24 R 2 5)q(CO)tX",

-(CR 2 0R2 1 )mA"m"(CR 22 R23 )(OCH 2 CH 2 )p(CR 4 OR4 1)pFY"(CR 24 R 2 5 )(CO)tX",

-(CR2 0R2 1)m(CR 29=N-NR 30)"(CR 22R23)n(OCH 2CH 2)p(CR 4oR4 1)p"Y"(CR 24R25 )q(CO)IX",

-(CR20 R21 )m,(CR 29=N

NR 30)n(CR 26=CR 27)m'(CR22R 23).(OCH 2CH 2)p(CR4oR 41)p,"Y"(CR2 4R25 )q(CO)tX",

-(CR2 oR 2i)m(CR29=N

NR 3 0)n(alkynyl)n,(CR22R 23 )n(OCH 2 CH 2 )p(CR 4 oR 4 1)p"Y "(CR 24 R 2 5 )q(CO)tX",

-(CR20R21)m(CR29=N-NR30),-A"m((C R 2 2 CR4oR41)pY"(CR24R25)(CO)tX",

wherein:

m, n, p, q, in', n', p', q', q", are integer from I to 10 and can be 0;

t, in", n" and p" are 0 or 1;

X" is selected from OR36, SR 37 , NR 8 R3 9, wherein R 36, R 37 , R3 8, R39 are H, or linear, branched or

cyclic alkyl, alkenyl or alkynyl having from I to 20 carbon atoms and, or, a polyethylene glycol

unit -(OCH 2CH 2)n, optionally R,7 is a thiol protecting group, or

when t = 1, COX" forms a reactive ester selected from N-hydroxysuccinimide esters, N

hydroxyphthalimide esters, N-hydroxy sulfo-succinimide esters, para-nitrophenyl esters,

dinitrophenyl esters, pentafluorophenyl esters and their derivatives, wherein said derivatives

facilitate amide formation;

Y" is absent or is selected from 0, S, S-S or NR 2, wherein R32 has the same definition as given

above for R, or

when Y" is not S-S and t = 0, X" is selected from a maleimido group, a haloacetyl group or SR 37 ,

wherein R 3 7 has the same definition as above;

A" is an amino acid selected from glycine, alanine, leucine, valine, lysine, citrulline and

glutamate or a polypeptide containing between 2 to 20 amino acid units;

R 20 , R21, R 22 , R 23 , R 24 , R 25 , R 26 , R 27 are the same or different and are H or a linear or branched

alkyl having from 1 to 5 carbon atoms;

R 28 is H or alkyl;

R 29 and R 3 0 are the same or different and are H or alkyl from 1 to 5 carbon atoms;

optionally, one of R4o and R4 1 is a negatively or positively charged functional group - and the

other is H or alkyl, alkenyl, alkynyl having 1 to 4 carbon atoms.

[153] The compounds of formula (IV), (V), (VI), (VII), (XII) and (XIII) (i.e., dimers) can be

linked through R 1, R 2, R 3, R4 , Ri', R 2 ', R 3 ',R4 ', L', L", L"'. Of these, preferred linkable groups

are R2 ', R 3', R 4', L', L", L"' and most preferred linkable groups are R 2', R 3 ' and L'. Examples

of linking groups for compounds of formula (IV), (V), (VI), (VII), (XII) and (XIII) include, but

are not limited to:

-O(CR 20 R21 )m(CR 22R 23 )(OCH 2 CH 2 )p(CR 4 0 R41 )p"Y"(CR 24R 25 )q(CO)iX",

-O(CR 20 R21)m(CR26=CR 27)m,(CR 22R23)(OCH 2CH 2)p(CR4 oR4 1)p,Y"(CR 24R25)q(CO)X'",

-O(CR 20 R21)m.(alkynyl).,(CR 22R 23).(OCH 2CH 2)p(CR 4oR4 1)p"Y"(CR24R2 5)(CO)tX",

-O(CR 20 R21)m(piperazino)t'(CR 22R 23) 1n(OCH 2CH 2)p(CR4OR 41)p"Y"(CR 24R 25)(CO)tX",

-O(CR 20 R21)m(pyrrolo)t'(CR 22R23)n(OCH 2CH2 )p(CR 4oR4 1)p'Y"(CR 24R25)q(CO)tX",

-O(CR 20 R 21)mA"m(CR 22R23)(OCH 2CH 2)p(CR 4oR 4 1)p"Y "(CR 24 R 2 5 )(CO)tX",

-S(CR 2aR21)m(CR 22R23)(OCH 2CH 2)p(CR4 OR4 1)pY"(CR 24R 2 5)q(CO)tX",

-S(CR 2 aR2 1)m(CR 2 6 =CR27 )m'(CR 22 R23 ),(OCH 2 CH 2 )p(CR 4oR4 1)p,,Y"(CR 2 4 R 2 5 )(CO)tX",

-S(CR 2aR2 1)m(alkynyl)n,(CR 22R 23)(OCH 2CH 2)p(CR4oR41)p"Y"(CR 24 R 25 )q(CO)iX",

-S(CR 2aR21)m,(piperazino) 1 '(CR22R 23)n(OCH 2 CH 2)p(CR 4oR 41)p"Y'(CR 24R 25)(CO)tX",

-S(CR 2aR21)m(pyrrolo)t(CR 22R 23)(OCH 2CH2 )p(CR4 oR4 1)p"Y'(CR 24R 25)q(CO)tX",

-S(CR 20 R21)mA"m"(CR 22R 23),(OCH 2CH2)p(CR 4oR 41)Y"(CR 24R2 5)q(CO)tX",

-NR 33 (C=O)p,(CR 2 R 21)m(CR 22R 23)n(OCH 2CH 2)p(CR 4oR 41)p"Y'(CR 24R25 )q(CO)tX",

-NR 33 (C=O)p,(CR 2 R 2 1)m(CR 26 =CR 27 )m'(CR2 2 R23 ).(OCH 2CH 2 )p(CR 4 OR 4 1 )p"Y"(CR24R25)q

(CO)tX",

-NR 33 (C=O)p-(CR 2 R 21 )m(alkynyl),(CR 22R23)n(OCH2 CH 2)p(CR 4oR 41)p-Y"(CR 24R25 )q(CO)tX',

-NR 33 (C=O)p,(CR 2oR 21)m..(piperazino)t'(CR 22R2 3)1 (OCH 2CH 2)p(CR 4oR 41)p,,Y"(CR24R25)q

(CO)tX",

-NR 33 (C=O)p,(CR 20R2 I)m(pyrrolo),'(CR 22R23)n(OCH 2CH 2)p(CR4oR41)pY"(CR 24R 25)q(CO)tX",

-NR 33 (C=O)p,(CR 2 R 21)mA"m"(CR 22R 23)n(OCH 2CH 2)p(CR 4oR41)p,Y"(CR 24R 25)q(CO)tX",

-(CR 20 R2 1 )m(CR 22 R 23 )(OCH 2 CH 2 )p(CR 4 oR4 1)p,Y"(CR 24 R 25)q(CO)tX",

-(CR 2 0R 21 )m(CR26=CR27)m,(CR22R23).(OCH2CH2)p(CR4oR41)p"Y"(CR 24R 25 )q(CO)tX",

-(CR 20R 21)m(alkynyl)n,(CR22 R23).(OCH 2CH2)p(CR 4 oR 41)p"Y"(CR 24R25)q(CO)tX",

-(CR 20R2 1)m(piperazino)t(CR 22 R 23 ) (OCH2CH 1 2)p(CR 4 oR41)p"Y"(CR 24 R25 )q(CO)tX",

-(CR 20R 21)mA"m"(CR 22R23).(OCH 2CH 2)p(CR 4 OR41)p'Y"(CR 24R25)(CO)tX",

-(CR 20R 21)m(CR 29=N-NR 30),"(CR 22R23)n(OCH 2CH 2)p(CR 4OR4 1)p"Y"(CR 24R25)q(CO)tX",

-(CR 2 oR 21)m(CR 29=N

NR 30)n(CR 26=CR 27)m'(CR22R 23).(OCH 2CH 2)p(CR4 OR 41)p"Y"(CR 24R2 5)q(CO)tX",

-(CR 20R 21 )m(CR 29 =N

NR 3o),"(alkynyl)n,(CR 2 2R 23)n(OCH 2CH 2)p(CR 4 oR41)pYY"(CR 24R 25 )q(CO)iX",

-(CR 20R2 1)m,(CR 29=N-NR 30)n"A"mii(CR 22R 23).(OCH 2CH 2)p(CR 4oR 41)p"Y"1(CR 24R 25)q(CO)L',

wherein:

m, n, p, q, m', n', t' are integer from 1 to 10, or are optionally 0; t, in", n" and p" are 0 or 1;

X" isselected from OR 3 6 , SR 37 , NR3 R39 , wherein R 3 6 , R 37 , R 38, R 3 9 are H, or linear, branched or

cyclic alkyl, alkenyl or alkynyl having from 1 to 20 carbon atoms and, or, a polyethylene glycol

unit -(OCH 2CH 2)n, R 37 , optionally, is a thiol protecting group

when t = 1, COX" forms a reactive ester selected from N-hydroxysuccinimide esters, N

hydroxyphthalimide esters, N-hydroxy sulfo-succinimide esters, para-nitrophenyl esters,

dinitrophenyl esters, pentafluorophenyl esters and their derivatives, wherein said derivatives

facilitate amide bond formation;

Y" is absent or is selected from 0, S, S-S or NR 3 2 , wherein R3 2 has the same definition as given

above for R, or

when Y" is not S-S and t = 0, X" is selected from a maleimido group, a haloacetyl group or SR 37

, wherein R 37 has the same definition as above;

A" is an amino acid selected from glycine, alanine, leucine, valine, lysine, citrulline and

glutamate or a polypeptide containing between 2 to 20 amino acid units;

R 20 , R 2 1 , R 2 ,2 R 23 , R 42, R25 , R 2 6, and R 27 are the same or different and are H or a linear or

branched alkyl having from 1 to 5 carbon atoms;

R 29 and R 30 are the same or different and are H or alkyl from I to 5 carbon atoms;

R 33 is H or linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 12 carbon atoms,

a polyethylene glycol unit -(OCH 2CH2)n, or R 33 is -COR 34 , -CSR3 4, -SOR 34, or -S0 2R 34 ,wherein

R 34 is H or linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 20 carbon atoms

or, a polyethylene glycol unit -(OCH 2CH2)n; and one of R40 and R 41 is optionally a negatively or positively charged functional group and the other is H or alkyl, alkenyl, alkynyl having 1 to 4 carbon atoms.

[154] Further, while the synthesis of cytotoxic compounds (e.g., indolinobenzodiazepine or

oxazolidinobenzodiazepine), derivatives thereof, or dimers thereof bearing a linking moiety is

described below in terms of an amide, thioether or disulfide bond containing linking moieties at

the L' (in the compound of formula XIII) or R3 (in the compound of formula XII) positions, one

of skill in the art will understand that linking moieties at other positions and with other chemical

bonds, as described above, can also be used with the present invention.

[155] The structures of representative compounds, representative conjugates and claimed

compounds in the examples of the present invention are shown in Tables 3~9:

Table 3. Structures of representative compounds of the present invention.

HN ,, 00 HN X"

O0 O

000

00

sz. 0 0

N N ON N

O N 0

0o sz o X

HNyO.)SZ,, 00 HNy0O X' 6 .0 s0 X

01 N N SZNO

Note :cZ; e ISySS-NO, jV; X:" = NHS

0 0

N$

65

HN sz

0 SZ, N X

O --,SZ.X. 0

6 0

O

00

NO O N

N O O? N ONO O O0 N6 00

S SZ" SQ X"4

PO 00 0 0

N "K irOST NNAN..f0N..I

Note:Z"= H,SMe,SPy, SPy-NO 2 ,Ac; X"'= NHS;

N0 Tablec5. Structures of representative compounds of the presentnvention (Continued). 66. NN N 0 0

0O 0 N

-O NN S

0 0 0 06

N 0

0N-= N 0 N-=, 0

N OMe Me NOMe MeO

O 0 00

N 0N- N N NI

N OMe Me \N OMe MeO ' o0 0 0

N- 0 n 0 0 N N 0 L') C. me -;0CY N Me-I e N OMe MeO 00

0 0 0 Ne N-N 0

nO 0 0 0

N N NffN'o~ Meo /~ N N7)aOMe Me. =

O0 0 0

0 0

0 0

N NHk -N

Nm O.) eMeG NCO O~ 0 e 0 0

Tal6.tucueofereettieonuaesfhersntnenin

NSH--- 67

0 0

rN N

/\ N~~IX 0 ~ Me0 N ) \N OMe MeO) N~i~ N901-IGN-03 B38.1-IGN-03 0 NJH,

NIIW0hNO H N0

..N o~ Me - N 6 OMe MeO N 0 0 Nu91IN0 B38.1-IGN-10 J

0,,,~ 1 r y nibd 0 ,s-Gf-Gy-GGly-y-Antbody

7N- OM,% MO:O:rN ~ 6 N > )NMe MO<Nj 0 Dimeri1 0 Diner 2 0

N INlr1'-JAntibody 11 0 Antibody 0

0, '0N N-1

C7,NiIPX 0 ~ MeO ~~Me NrI1~ 0 Dimer 3 00 Dinner 4 0

\/N 0M N GlyGlyGly-GlyAntibody

Dimer 5 0 N - ) -VaI-Arg-Gly-Antibody

NN- N

\/ D =MeMe0 \/N

0 Dirner8 0

Table 7. Structures of compounds from the examples of the present invention. Structure ICompound No. IExample No.

OHC BnO,, N j MeO 0 N2

BnO

MeO 0 N~lb 7

HO b MeO ~Ob C~HO BnO NO2 1 N-J 1 2 MeO 0

BnO jr S N-"O 13 2 MeOx 0

Me) N-' 14 2 0 N o0,-'0 N /\N ~ Ie)(.N- 15 3

-NN

/N '~OMe MeO -Nb~j 1 O 0

N

\,NN OMe MeO K- Co 195

0

N N 34 6 6N I OMe MeO ".N

Table 8. Structures of compounds from the examples of the present invention (Continued). Structure Compound No. ExamplIe No. 0--y OMe

N N--3 6 /\N 0 I 35 0e MeOC,4j 0 0

N II . 6 N OMe Me0 N N~ 0 0 1 NyOMe 0 N

N N

0 e M eOD 0 0 N ~OMe 0 N N 40 6 /\ NaMe MeO N 0 0 0

N N, N= 41 7 N 'Nb O'Me MeO):l 0 0

0 N ~IN== 42 N Oe MeO Ib NKC 0

F= =0 43 7 N Nm NNOe MeO N '

0 0

N I 4 N OMe MeO N0

1NN .- s OH

~ No.. =~45 7 N N O e MeO'O 0 0

Table 9. Structures of compounds from the examples of the resent invention(Continued) Structure Compound No. Example No.

N~4 SN OMe Me(O 0 0 0

N - 48 8 N OMe Me0l INb o 0

N Oj1j. N-N= 49 8 N N 0Me MeOl" 0 0 '

N I N 51 9 OMe Me0 0 0

MeO Me)~ N% 125 10

HO uO N 126 10

0

H HN

N N0127 O 10 MeO ,N

Synthesis of Cytotoxic Compounds

[156] The process of preparation of a representative monomer compound of the present

invention, exemplified by indolinobenzodiazepine compound 8, is shown in Figure 1. Starting

from commercially available indoline-2-carboxylic acid 1, its methyl ester 2 was prepared in

quantitative yield by reaction with thionyl chloride in methanol. Methyl indoline-2-carboxylate

2 was coupled with the acid chloride 4, or directly with acid 3, to furnish the amide 5, which was

further reduced with diisobutylaluminum hydride (DIBAL) to the aldehyde 6. While, many

methods can be used to reduce the nitro functional group of formula 5 to the corresponding

amino group, in this example sodium dithionite was used to conveniently convert to aldehyde 6

to the ring closed compound 7 after further treatment with methanol under acidic conditions.

The benzyl protecting group was removed to furnish monomer 8.

[157] The process of preparation of the oxazolidinobenzodiazepine monomer compound of

formula 14 of the invention is shown in Figure 2. Starting from commercially available

compound 9, its methyl ester 10 was prepared in quantitative yield by treatment with thionyl

chloride in methanol. Compound 10 was deprotected followed by coupling with the acetyl

chloride 4 or directly with acid 3 to furnish the amide 11, which was further converted to the

aldehyde 12. Reduction of the nitro group was accomplished by treatment with sodium

dithionite followed by efficient conversion to the ring closed compound 13 after further

treatment with methanol under acidic conditions. The benzyl protecting group was removed to

furnish monomer 14.

[158] The process of preparation of representative dimer compounds of the present invention is

shown in Figures 3-5 and 7. The dimers were prepared by reacting of the monomers of formula

8 or formula 14 with compounds which possesses two leaving groups such as Br, I, triflate,

mesylate or tosylate.

[159] Dimers which possess linkers that can react with antibodies are prepared by converting

the methyl esters to the corresponding reactive esters of a leaving group such as, but not limited

to, N-hydroxysuccinimide esters, N-hydroxyphtalimide esters, N-hydroxy sulfo-succinimide

esters, para-nitrophenyl esters, dinitrophenyl esters, pentafluorophenyl esters. Representative

examples for the synthesis of the linkable dimers are shown in figures 8. Synthesis of dimers that

bear a thiol or disulfide moiety to enable linkage to cell binding agents via reducible or non

reducible bonds is shown in Figures 9 and 10. The B ring modified monomer 58 devoid of a

carbonyl group is achieved from the benzyl acohol compound 52 by the steps shown in Figure

11. The isoindolino monomer 66 can be prepared from isoindole 59 as outlined in Figure 12.

The linker can also be attached directly to the indolino moiety. Methyl indolino-2-carboxylate

can be converted into the linkable dimer 82 via the synthetic steps shown in Figure 13. The

synthesis of linkable dimers bearing a PEG moiety is shown in Figures 14 and 15.

[160] Thus in one aspect, the invention provides a process for the preparation of the

indolinobenzodiazepine (IBD) monomer of formula (I) (Figure 1), the process comprising the

steps of:

a) coupling compound of formula (1) and compound of formula (2) to give compound of formula

(3); b) converting compound of formula (3) into aldehyde of formula (4); and c) converting compound of formula (4) into compound of formula (I),

R- NO 2 H~ R2z R,'4 R6 W'RN

Rs NW2G + HNR R(. W N R2 (1) R4 )R 3 (3) R4 R3

x y R N Z OHC

W N R2 NR2R5

R4 R3 (4) R4 R3

wherein LG is a leaving group; W' is COOR or CH 2OW",wherein R has the same definition as

above and W" is a protecting group; R 1, R 2, R 3, R 4, R 5, R ,6 W, Z, X, Y and = have the same

definition as described above.

[161] Another aspect of the invention provides a process for the preparation of compound of

formula (II) comprising the steps of:

a) coupling compound of formula (1) and compound of formula (5) to give compound of formula

(6);

b) converting compound of formula (6) into aldehyde of formula (7); and

c) converting compound of formula (7) into compound of formula (II),

R, R2 R, R2

3 R, WLG R3 Rs NO2 AR

(1) R4 W R4 (6)

x y R1 R2 R2 R, \ OHO R R5 R4R 3 6 W

(II) (7) wherein LG is a leaving group; W' is COOR or CH 2OW",wherein R has the same definition as

above and W" is a protecting group; R 1, R 2, R 3, R 4, R 5, R 6, W, X, Y and = have the same

definition as above.

[162] Another aspect of the invention provides a process for the preparation of compound of

formula (III) comprising steps of:

a) coupling compound of formula (1) and compound of formula (8) to give compound of formula

(9);

b) converting compound of formula (9) into aldehyde of formula (10); and

c) converting compound of formula (10) into compound of formula (II),

R 5 (:NO 2 N02 =W R NT LG + XNO X 2

N-.. W Nx (1) (8) (9)

X Y N CNO 2 CHO

. -N 'Y' FR 6 RX:C, (III) (10)

wherein LG is a leaving group; W' is COOR or CH 2OW", wherein R has the same definition as

above and W" is a protecting group; R 5, R 6, W, X, Y, X', Y', Z' and == have the same definition

as above.

[163] Another aspect of the invention provides a process for the preparation of compound of

formula (Another aspect of the invention provides a process for the preparation of compound of

formula (IV) comprising the steps of:

coupling compound of formula (11), compound of formula (11)' and compound of formula (12)

to give compound of formula (IV),

Y X x y R' Z OH LG-D-L-D'-LG HO , R

R2' R2 w W RR ~(12) W~ N R

R 3' R4 ' (11) R4 R3

x yx y Z A-D-L-D' A' N Z R

R2. N W R6 R6 W N R2

R3' R4 ' (IV) R4 R3 wherein LG is a leaving group; R 1, R 2, R 3 , R 4, R ',1 R 2', R', R 4 ', R 6, W, X, Y, Z, A, A', D, D', L and = have the same definition as above.

[164] Another aspect of the invention provides an alternative process for the preparation of

compound of formula (IV) of the present invention comprising steps of:

a) converting compound of formula (15) into aldehyde of formula (16); and

b) converting compound of formula (16) into compound of formula (IV),

R' Z 0 2N A-D-L-D'-A NO2 z R1

R2' W W R R6 iN R2

R3 ' R 4' R4 R3

CHO CHO R1 ' Z..0 2 N A-D-L-D'-A NO 2 Z

R2' WRe 6R6 WN R2 V - (16) R3 ' R4 R4 R3

x Y x y R Z A-D-L-D'-A Z

R2.N W Re R. .N / R

R 3' R4 ' (IV) R4 R3

wherein W' is COOR or CH 2OW", wherein R has the same definition as above and W" is a

protecting group; R 1, R 2 R 3, R4, R ',1 R2, R3, R4', R6, W, X, Y, Z, A, A', D, D', L and = have

the same definition as above.

Another aspect of the invention provides a process for the preparation of compound of formula

(V) comprising the step of coupling compound of formula (13), compound of formula (13)' and

compound of formula (12) to give compound of formula (V),

R 2' R 1'y x x y R1 R2

R3 W OH LG-D-L-D'-LG HO R3 N..(12) W W R6 R :C W4 (13) (13)

R 2' R1 y x X y R1 R2 -NN R3 ' 5 / A-D-L-D-A NW/ R 3 N- - II.: - N R'W R6 R6 WR (V)

wherein LG is a leaving group; R 1, R 2, R3 , R4, R ',1 R 2', R,', R 4 ', R,W, X, Y, A, A', D, D', L

and = have the same definition as above.

[165] Another aspect of the invention provides an alternative process for the preparation of

compound of formula (V) of the invention comprising the steps of:

a) converting compound of formula (17) into aldehyde of formula (18); and

b) converting compound of formula (18) into compound of formula (V),

R2' Rl' R1 R2

R2 R R1 R2 4W (17)

R3 ' / 0N . A-D-L-D-A ~ O2 R3

R 2' Ry R1 R2 - CHO OHC R3 ' 02 N A-D-L-D'-A No 2 R3

NW W _WN (18)

R2 ' R x x R, R2

R3 ' N/ AD-L-D- A' /R 3

R' N-W ) R6 R6 W

wherein W'is COOR or CHOW", wherin Rhas the samecdefinition as abovcand W"is a

protecting group; R,R2 , R,R, R', R2 ', R 3 R',R6 , W, X Y,A,A', D, D', Land ==have the

same definition as above.

[166] Another aspect of the invention provides a process for the preparation of compound of

formula (VI) of the invention comprising the step of coupling compound of formula (14),

compound of formula (14)' and compound of formula (12) to give compound of formula(VI),

Y X x y

ZOH LG-D-L-D'-LG H '. Z'

X'N W R (12) R W X' (14)' (14)

Y X x y

Z A-D-L-D'-A' Z'

N' W R6 R6 W NX' (VI)

wherein LG is a leaving group; R 6,W, X, Y, X', Y', Z' A, A', D, D', L and =have the same

definition as above.

[167] Another aspect of the invention provides a process for the preparation of compound of

formula (VI) of the invention comprising the steps of:

a) converting compound of formula (19) into aldehyde of formula (20); and

b) converting compound of formula (20) into compound of formula (VI),

02N A-D-L-D'-A' N O2 w, I I I.N Y,-NsW R6 R6 W '-N X (19)

CHO OHC 0 2N A-D-L--D'-A' NO 2 Z YKN W R R .. W-N Y W (20) 6

Y X X Y --NN- ., Z'_ A-D-L-D'-A ' Z'

X NW R R6 W X'

(VI)

wherein W' is COOR or CH 2OW", wherein R has the same definition as above and W" is a

protecting group; R ,6 W, X, Y, X', Y', Z' A, A', D, D', L and == have the same definition as

above.

In vitro Cytotoxicity of Compounds

[168] The in vitro cytotoxicityof the cytotoxic compounds (e.g., indolinobenzodiazepine or

oxazolidinobenzodiazepine), derivatives thereof, dimers thereof or conjugates thereof of the

present invention can be evaluated for their ability to suppress proliferation of various cancerous

cell lines in vitro (Tables 1, 2 in FIGS. 31, 32.). For example, cell lines such as the human breast

carcinoma line SK-Br-3, or the human epidermoid carcinoma cell line KB, can be used for the

assessment of cytotoxicity of these new compounds. Cells to be evaluated can be exposed to the

compounds for 72 hours and the surviving fractions of cells measured in direct assays by known

methods. IC5 0 values can then be calculated from the results of the assays.

[169] Examples of in vitro cytotoxicity of compounds of the present invention that were tested

on a panel of cancer cell lines and their data is shown in Table 1. All the indolinobenzodiazepine

dimer compounds tested were highly potent with IC5 0 values in the low picomolar range. IGN

09 retained most of its potency on multi-drug resistant cell lines such as COL0205-MDR (only

4-fold higher IC5 0 than COL0205). Compounds of the invention are 1000 to10,000-fold more

cytotoxic than other DNA interacting drugs used in cancer treatment, such as doxorubicin,

melphalan and cis-platin. In a direct comparison, the potency of the non-linker bearing

compounds IGNi (compound 18) and IGN09 (compound 15) was compared to the linker

bearing compounds IGNO3 (compound 34) and IGN05 (compound 36) was tested towards a

representative cell line Ramos. As shown in Table2, all four compounds are highly potent with

ICso values less than 1 picomolar, demonstrating that the incorporation of linker does not affect

potency.

Cell-binding Agents

[170] The effectiveness of the compounds (e.g., indolinobenzodiazepine or

oxazolidinobenzodiazepine), derivatives thereof, dimers thereof or conjugates thereof of the

invention as therapeutic agents depends on the careful selection of an appropriate cell-binding

agent. Cell-binding agents may be of any kind presently known, or that become known and

includes peptides and non-peptides. Generally, these can be antibodies (especially monoclonal

antibodies), lymphokines, hormones, growth factors, vitamins, nutrient-transport molecules

(such as transferrin), or any other cell-binding molecule or substance.

[171] More specific examples of cell-binding agents that can be used include: polyclonal antibodies; monoclonal antibodies; fragments of antibodies such as Fab, Fab', and F(ab') 2 , Fv (Parham, J. Immunol.

131:2895-2902 (1983); Spring et al. J. Immunol. 113:470-478 (1974); Nisonoff et al. Arch.

Biochem. Biophvs. 89:230-244 (1960));

interferons (e.g. .alpha., .beta., .gamma.);

lymphokines such as IL-2, IL-3, IL-4, IL-6;

hormones such as insulin, TRH (thyrotropin releasing hormone), MSH (melanocyte

stimulating hormone), steroid hormones, such as androgens and estrogens;

growth factors and colony-stimulating factors such as EGF, TGF-alpha, FGF, VEGF, G

CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5:155-158 (1984));

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

vitamins, such as folate.

[172] Monoclonal antibody techniques allow for the production of extremely specific cell

binding agents in the form of specific 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 scFv (single chain variable region), specifically human

scFv (see e.g., Griffiths et al., U.S. Patent Nos. 5,885,793 and 5,969,108; McCafferty et al., WO

92/01047; Liming et al., WO 99/06587). In addition, resurfaced antibodies disclosed in U.S.

Patent No. 5,639,641 may also be used, as may chimeric antiobodies and humanized antibodies.

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 human monoclonal antibodies are

preferred if an appropriate one is available.

[173] For example, the monoclonal antibody MY9 is a murine IgGi antibody that binds

specifically to the CD33 Antigen {J.D. Griffin et al 8 Leukemia Res., 521 (1984)} and can be

used if the target cells express CD33 as in the disease of acute myelogenous leukemia (AML).

Similarly, the monoclonal antibody anti-B4 is a murine IgGi, that binds to the CD19 antigen on

B cells {Nadler et al, 131 J. Immunol. 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 HuB4 is a resurfaced antibody derived from the murine anti-B4 antibody

(Roguska et al., 1994, Proc. Natl. Acad. Sci., 91, pg 969-973). HuN901 is a humanized

antibody that binds to the CD56 antigen expressed on small cell lung cancer, multiple myeloma,

ovarian cancer and other solid tumors including neuroendocrine cancers (Roguska et al., 1994,

Proc. Nat. Acad. Sci., 91, pg 969-973). B38.1 is a chimeric antibody targeting EpCAM. Fully

human antibodies such as panitumumab targeting the EGF receptor expressed on several solid

tumors may also be used (Van Cutsem et al., J Clin Oncol. 2007;25(13):1658-1664). 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), interferons, lymphokines, hormones, growth factors, vitamins, nutrient-transport molecules (such as transferrin), or any other cell-binding molecule or substance.

[174] Where the cell-binding agent is an antibody, 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-i-antitrypsin; insulin A

chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone;

glucagon; clotting factors such as factor vmc, 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; hemopoitic 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-pl, 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, FOLR, mesothelin, cripto, alphabeta6

, 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,

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-I 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 CDIIa,

CD11b, CD11c, CD18, an ICAM, VLA-4 and VCAM; a tumor associated antigen such as

HER2, HER3 or HER4 receptor; and fragments of any of the above-listed polypeptides.

[175] 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 can be used to target the folate receptor expressed

on ovarian and other tumors. Epidermal growth factor can be used to target squamous cancers

such as lung and head and neck. Somatostatin can be used to target neuroblastomas and other

tumor types.

[176] 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.

Production of Cytotoxic Conjugates

[177] The present invention also provides cytotoxic compound-cell-binding agent conjugates

comprising a cell binding agent linked to one or more cytotoxic compounds via a variety of

linkers, including, but not limited to, disulfide linkers, thioether linkers, amide bonded linkers,

peptidase -labile linkers, acid-labile linkers, esterase-labile linkers. Representational cytotoxic

conjugates of the invention are antibody/cytotoxic compound, antibody fragment/cytotoxic

compound, epidermal growth factor (EGF)/ cytotoxic compound, melanocyte stimulating

hormone (MSH)/ cytotoxic compound, thyroid stimulating hormone (TSH)/ cytotoxic

compound, somatostatin/cytotoxic compound, folate/cytotoxic compound, estrogen/cytotoxic

compound, estrogen analogue/cytotoxic compound, androgen/cytotoxic compound, and

androgen analogue/cytotoxic compound.

[178] Ina preferred embodiment, the present invention provides an indolinobenzodiazepine

dimer-cell-binding agent conjugate comprising the cytotoxic agent and the cell binding agent

linked through a covalent bond. The linker can be cleaved at the site of the tumor/unwanted

proliferating cells to deliver the cytotoxic agent to its target in a number of ways. The linker can

be cleaved, for example, by low pH (hydrazone), reductive environment (disulfide), proteolysis

(amide/peptide link), or through an enzymatic reaction (esterase/glycosidase).

[179] Ina preferred aspect, representatative cytotoxic conjugates of the invention are antibody/

indolinobenzodiazepine dimer, antibody fragment/indolinobenzodiazepine dimer, epidermal

growth factor (EGF)/ indolinobenzodiazepine dimer, melanocyte stimulating hormone (MSH)/

indolinobenzodiazepine dimer, thyroid stimulating hormone (TSH)/ indolinobenzodiazepine

dimer, somatostatin/ indolinobenzodiazepine dimer, folate/ indolinobenzodiazepine dimer,

estrogen/ indolinobenzodiazepine dimer, estrogen analogue/ indolinobenzodiazepine dimer,

prostate specific membrane antigen (PSMA) inhibitor/ indolinobenzodiazepine dimer, matriptase

inhibitor/ indolinobenzodiazepine dimer, designed ankyrin repeat proteins (DARPins)/

indolinobenzodiazepine dimer, androgen/ indolinobenzodiazepine dimer, and androgen

analogue/ indolinobenzodiazepine dimer.

[180] Disulfide containing cytotoxic conjugates can be made by reacting a thiol-containing

cytotoxic agent such as 49 with an appropriately modified cell-binding agent. These conjugates

may be purified to remove non-linked cytotoxic agent by using gel-filtration, ion exchange

chromatography, ceramic hydroxyappetite (CHT) chromatography, hydrophobic interaction

chromatography (CHT), tangential flow filtration (TFF), or by HPLC.

[181] A solution of an antibody in aqueous buffer maybe incubated with a molar excess of an

antibody modifying agent such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) or with

N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) to introduce dithiopyridyl groups. The

modified antibody is then reacted with the thiol-containing cytotoxic agent such as compound 49

to produce a disulfide-linked antibody- indolinobenzodiazepine dimer conjugate. The cytotoxic

cell binding conjugate may then be purified using any of the above mentioned methods.

[182] Alternatively, the antibody maybe incubated with a molar excess of an antibody

modifying agent such as 2-iminothiolane, L-homocysteine thiolactone (or derivatives), or N

Succinimidyl-S-acetylthioacetate (SATA) to introduce sulfhydryl groups. The modified

antibody is then reacted with the appropriate disulfide-containing cytotoxic agent, such as,

compound 51 to produce a disulfide-linked antibody-cytotoxic agent conjugate. The antibody

cytotoxic agent conjugate may then be purified by gel-filtration or other methods mentioned

above.

[183] The number of cytotoxic molecules bound per antibody molecule can be determined

spectrophotometrically by measuring the ratio of the absorbance at 280 nm and 330 nm. An

average of 1-10 cytotoxic molecules/antibody molecule(s) can be linked by this method. The

preferred average number of linked cytotoxic molecules per antibody molecule is 2-5, and the

most preferred is 3-4.5.

[184] Alternatively, a solution of an antibody in aqueous buffer maybe incubated with a molar

excess of an antibody-modifying agent such asN-succinimidyl-4-(N-maleimidomethyl)

cyclohexane-1-carboxylate to introduce maleimido groups, or with N-succinimidyl-4

(iodoacetyl)-aminobenzoate (SIAB) to introduce iodoacetyl groups. The modified antibody is

then reacted with the thiol-containing cytotoxic agent to produce a thioether-linked antibody

cytotoxic conjugate. The antibody-cytotoxic conjugate may then be purified by gel-filtration or

other methods mentioned above or by methods known to one of skill in the art.

[185] Cytotoxic agents containing linkers terminating in anN-Hydroxysuccinimidyl (NHS)

ester, such as compounds 43, 44, and 46, can be reacted with the antibody to produce direct

amide linked conjugates such as huN901-IGN-03 and huN901-IGN-07. The antibody-cytotoxic

agent conjugate may then be purified by gel-filtration or other methods mentioned above.

[186] The following cell-binding agent/cytotoxic agent conjugates can be prepared using the

appropriate linkers. Dimer 1 and 2 with peptide cleavable linkers can be prepared from the

corresponding NHS esters, Dimer 3 can be made by reacting the appropriate thiol-containing

cytotoxic agent with SMCC modified cell binding agent, and acid-labile hydrazone Dimer 4 can

be prepared through condensation of a cytotoxic agent containing an alkyl, aryl ketone with a

hydrazide modified cell binding agent.

0 ~Gly-Gly-Gly-GlyAntibody 0 .,s,..YVaI-Arg-GIy-Antibo 0 0 NIN=,N I1 N=,.

\/ ) OM Meo \N - O Me eaIN 00 0 0 Dimer 1 Dimer 2 H 0

0 body 0I SIQ 0 0 Antibody NNF=N Oj 0 N-. N 'eMeO~ \/ P OMe MoO r 000 0 Dimer 3 Dimer 4

[187] Asymmetric indolinobenzodiazepine dimer conjugates such as Dimers 5-8 can also be

prepared using similar methods to those descibed above.

N 0 N-,0 N I % k-GI-GIy-Gy-GIy-Atibod - N ~OMe MeO ~-N _NYY~l 0 Dinner 5 0

N. o, o N ~ 0 N )-VaI-Arg-Gly-Antibody C N OMe MeO \/ N 0 Dinner6 0

~/ 0 0 e / N~"~' Antibody Dinner7 H 0 N 1-- N-, N )I ~Antibody - .OMe MeG' 0 0Dinner8 0I

[188] Conjugates of cell-binding agents with cytotoxic agents of the invention can be evaluated

for their ability to suppress proliferation of various unwanted cell lines in vitro. For example, cell

lines such as the human colon carcinoma line COLO205, the rhabdomyosarcoma cell line RH

, and the multiple myeloma cell line MOLP-8 can be used for the assessment of cytotoxicity of

these conjugates. Cells to be evaluated can be exposed to the compounds for 1-5 days and the

surviving fractions of cells measured in direct assays by known methods. IC50 values can then be

calculated from the results of the assays.

[189] Examples of in vitro potency and target specificity of antibody-cytotoxic agent

conjugates of the present invention are shown in Fig. 21-26. All of the conjugates with cytotoxic

agent/antibody ratios of 1-3 are extremely cytotoxic on the antigen positive cancer cells with an

IC 5 0 in the low picomolar range. Antigen negative cell lines remained viable when exposed to the

same conjugates. The target specificity of conjugates of the indolinobenzodiazepine dimers are

>1000 with the antibodies huN901 (anti-CD56) and muB38.1 (anti-EpCAM). For example, the

B38.1-IGN-3 conjugate killed antigen positive COLO 205 cells with an IC5 0 value of 1.86 pM,

while the antigen negative Namalwa cell line was about 200-fold less sensitive with an IC5 0

value of 336.3 pM, demonstrating antigen specificity. In addition, the conjugate is also highly

potent towards the multidrug resistant COLO 205 MDR cell line with an IC5 o value of 16 pM.

Similarly, the huN901-IGN3 conjugate was highly potent, with an IC 5 0 value of 15 pM for

antigen positive RH30 cells (Fig. 22). Addition of an excess of unconjugated huN901 antibody

abolished this cytotoxic effect (ICso > 3 nM), demonstrating antigen-specificity. Another

huN901-IGN conjugate (huN901-IGN-07) also showed high potency towards antigen expressing

RH-30 cells, with drug load dependent cytotoxicity and IC 5 0 values of 16 pm, 3 pM and 2 pM

respectively for conjugates bearing 1.2, 2.0 and 3.0 linked drugs per antibody molecule (Fig. 23).

Similar results were obtained with huN901-IGN07 and huN901-IGN03 towards antigen-positive

Molp-8 cells. Hu901-IGN07 gave IC 5 0 values of 5 pM, 3 pM and 2 pM respectively for IGN07

loads of 1.2, 2.0 and 3.0 (Fig. 24). The huN901-IGN07 and IGN03 conjugates were much less

potent towards antigen negative Namalwa cells with IC 5 0 values ranging from 1000 pM to >3000

pM(Fig.25). The B38.1-IGN10 conjugate was also specifically potent killing antigen positive

COLO 205 cells, with an IC5 0 of 17 pM, and less potent (170 pM) for antigen-negative Ramos

cells (Fig. 26).

[190] In one example, in vivo efficacy of a cell binding agent/cytotoxic agent conjugate was

measured. Nude mice bearing human MOLP-8 tumors were treated with huN901-IGN-07

conjugate and significant tumor regression was observed compared while untreated mice tumors

grew rapidly (Figure 27).

[191] The indolinobenzodiazepine dimers of the present invention bind and alkylate double

stranded DNA (dsDNA) containing guanine residues on opposite strands spaced 4 base pairs

apart. Figures 28-30 present data from reverse-phase ion pair chromatography assays showing

rate of IGN-01, IGN-02, and IGN-09 binding and crosslinking to dsDNA. The indolino group

(IGN-01) is preferred to the oxazole group (IGN-02) for rapid DNA binding and interstrand

crosslinking (ICL). Initial rate of IGN1-DNA adduct formation is dependent on DNA sequence.

IGN Ibinds faster to DNA containing an internal GATC motif than DNA with a GTAC sequence. DNA probe substituted with deoxylnosine (1) (containing no C-2 amino group) in place of guanine (G) showed no reaction with IGN-1 (Fig. 29).

[192] The IC 5 0 values of various compounds of the present invention towards a panel of cell

lines is listed in Fig. 31. Comparative in vitro potency of linkable and non-linkable compounds

of the present invention are shown in Fig. 32. Incorporation of a linker does not significantly

affect potency of the parent compounds.

Compositions and methods of use

[193] The present invention includes a composition (e.g., apharmaceutical composition)

comprising novel benzodiazepine compounds (e.g., indolinobenzodiazepine or

oxazolidinobenzodiazepine), derivatives thereof, or conjugates 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 novel

benzodiazepine compounds, derivatives thereof,, or conjugates 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). The present compositions

are also useful for treating depression, anxiety, stress, phobias, panic, dysphoria, psychiatric

disorders, pain, and inflammatory diseases in a mammal (e.g., human).

[194] 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 novel benzodiazepine compounds (e.g., indolinobenzodiazepine or oxazolidinobenzodiazepine), derivatives thereof, or conjugates thereof, (and/or solvates and salts thereof) or a composition thereof, alone or in combination with a second therapeutic agent.

[195] The present invention also provides methods of treatment comprising administering to a

subject in need of treatment an effective amount of any of the conjugates described above.

[196] Similarly, the present invention provides a method for inducing cell death in selected cell

populations comprising contacting target cells or tissue containing target cells with an effective

amount of a cytotoxic agent comprising any of the cytotoxic compound-cell-binding agents (e.g.,

indolinobenzodiazepine or oxazolidinobenzodiazepine dimer linked to a cell binding agent) of

the present invention, a salt or solvate thereof. The target cells are cells to which the cell-binding

agent can bind.

[197] If desired, other active agents, such as other anti-tumor agents, maybe administered

along with the conjugate.

[198] Suitable pharmaceutically acceptable carriers, diluents, and excipients are well known

and can be determined by those of ordinary skill in the art as the clinical situation warrants.

[199] Examples of suitable carriers, diluents and/or excipients include: (1) Dulbecco's

phosphate buffered saline, pH about 7.4, containing or not containing about I mg/ml to25 mg/ml

human serum albumin, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose; and may

also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween 20.

[200] The method for inducing cell death in selected cell populations can be practiced in vitro,

in vivo, or ex vivo.

[201] Examples of in vitro uses include treatments of autologous bone marrow prior to their

transplant into the same patient in order to kill diseased or malignant cells: treatments of bone

marrow prior to their transplantation in order to kill competent T cells and prevent graft-versus

host-disease (GVHD); treatments of cell cultures in order to kill all cells except for desired

variants that do not express the target antigen; or to kill variants that express undesired antigen.

[202] The conditions of non-clinical in vitro use are readily determined by one of ordinary skill

in the art.

[203] Examples of clinical ex vivo use are to remove tumor cells or lymphoid cells from bone

marrow prior to autologous transplantation in cancer treatment or in treatment of autoimmune

disease, or to remove T cells and other lymphoid cells from autologous or allogenic bone marrow

or tissue prior to transplant in order to prevent GVHD. Treatment can be carried out as follows.

Bone marrow is harvested from the patient or other individual and then incubated in medium

containing serum to which is added the cytotoxic agent of the invention, concentrations range

from about 10 gM to 1 pM, for about 30 minutes to about 48 hours at about 37°C. The exact

conditions of concentration and time of incubation, i.e., the dose, are readily determined by one

of ordinary skill in the art. After incubation the bone marrow cells are washed with medium

containing serum and returned to the patient intravenously according to known methods. In

circumstances where the patient receives other treatment such as a course of ablative

chemotherapy or total-body irradiation between the time of harvest of the marrow and reinfusion

of the treated cells, the treated marrow cells are stored frozen in liquid nitrogen using standard

medical equipment.

[204] For clinical in vivo use, the cytotoxic agent of the invention will be supplied as a solution

or a lyophilized powder that are tested for sterility and for endotoxin levels. Examples of

suitable protocols of conjugate administration are as follows. Conjugates are given weekly for 4

weeks as an intravenous bolus each week. Bolus doses are given in 50 to 1000 ml of normal

saline to which 5 to 10 ml of human serum albumin can be added. Dosages will be 10 pg to

2000 mg per administration, intravenously (range of 100 ng to 20 mg/kg per day). After four

weeks of treatment, the patient can continue to receive treatment on a weekly basis. Specific

clinical protocols with regard to route of administration, excipients, diluents, dosages, times, etc.,

can be determined by one of ordinary skill in the art as the clinical situation warrants.

[205] Examples of medical conditions that can be treated according to the in vivo or ex vivo

methods of inducing cell death in selected cell populations include malignancy of any type

including, for example, cancer of the lung, breast, colon, prostate, kidney, pancreas, ovary, 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, AIDS, etc.; and

parasite infections, such as giardiasis, amoebiasis, schistosomiasis, and others as determined by

one of ordinary skill in the art.

[206] 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 drugs 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. The contents of the PDR are expressly incorporated herein in its entirety by reference. One of skill in the art can review the PDR, using one or more of the following parameters, to determine dosing regimen and dosages of the chemotherapeutic agents and conjugates that can be used in accordance with the teachings of this invention. These parameters include:

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Analogues and derivatives

[207] 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.

[208] All references cited herein and in the examples that follow are expressly incorporated by

reference in their entireties.

EXAMPLES

[209] The invention will now be illustrated by reference to non-limiting examples. Unless

otherwise stated, all percents, ratios, parts, etc. are by weight. All reagents were purchased from

the Aldrich Chemical Co., New Jersey, or other commercial sources. Nuclear Magnetic

Resonance ('H NMR) spectra were acquired on a Bruker 400 MHz instrument and mass spectra

were acquired on a Bruker Daltonics Esquire 3000 instrument using electrospray ionization.

Example 1

(2S)-1-[5-methoxv-2-nitro-4-(phenvlmethoxv)-benzovl]-2-indolinecarboxylic acid methyl ester

:

HO2 C 4 SOC MeO 2C Me& C* Nt 5h H2 MeO2C 100% 2 TEA/THE BnO N02

C 2 02CI2 MeO):N BnO NO 2 DMF (cat.) BnO NO 2 5 0 MeO .- OH OH 0C H2Cl2/THF MeiC MeO r.t. 2h or Sovernight MeO ~ 3 0 100% 4 0

[210] To a stirred solution of 4-benzyloxy-5-methoxy-2-nitrobenzoic acid 3 (7.01 g, 23.1

mmol) in anhydrous dichloromethane (100 mL) and THF (10 mL) was added oxayl chloride

(4.1 mL, 46.2 mmol) and DMF (30 pL, 0.38 mmol) at room temperature. Large amounts of

bubbles formed after the addition of the DMF. The mixture was stirred overnight (the reaction

usually finished within 3 hours) and then the solvents were removed by rotary evaporation in

vacuo. The residue was co-evaporated one more time by addition of anhydrous dichloromethane

and high vacuumed to give the acetyl chloride 4 as a yellow solid, which was directly used for

the next step.

[211] To a stirred solution of (s)-(-)-Indoline-2-carboxylic acid 1 (3.43 g, 21.0 mmol) in

anhydrous methanol (42 mL) was added thionyl chloride (3.1 mL, 42.0 mmol) dropwise at 0 0 C.

The ice bath was removed after 30 minutes and the mixture continued to be stirred at room temperature for 5 hours. The solvent was removed under reduced pressure and the residue was further dried on high vacuum to give methyl ester 2, which was dissolved in anhydrous THF ((70 mL) in a 500 mL round bottom flask. The solution was cooled to 0 0 C and triethylamine (9.7 mL, 69.3 mmol) was added, followed quickly by addition of freshly prepared acetyl chloride 4 in anhydrous THF (70 mL) via canula at 0 °C. The mixture was stirred at 0~5 °C for another 1.5 hours then at room temperature for 30 minutes. The reaction was quenched by addition of cold

% HCI and then diluted with ethyl acetate and water. The aqueous layer was extracted with

ethyl acetate three times. The combined organic layers were washed subsequently with brine,

saturated sodium bicarbonate and brine, dried over anhydrous sodium sulfate and filtered. The

solvents were evaporated under reduced pressure and the residue was purified via silica gel

chromatography (Hexanes/Ethyl acetate, 2:1, 1.5:1) to give (2S)-1-[5-methoxy-2-nitro-4

(phenylmethoxy)-benzoyl]-2-indolinecarboxylic acid methyl ester 5 as a yellow solid (9.1 g, y =

94%). 'H NMR (400 Hz, CDC 3): the compound appears as three distinct rotomers. 6 8.27 (d, J

= 8.4 Hz, 0.3H), 7.90 (s, 0.1H), 7.82 (s, 0.6H), 7.79 (s, 0.3H), 7.50-7.28 (in, 5.4H), 7.20-7.09 (in,

1.3H), 7.05 (s, 0.6H), 6.97-6.81 (m, 1.6H), 6.76 (s, 0.1H), 5.85 (d, J= 8.0 Hz, 0.1H), 5.70 (d, J =

8.0 Hz, 0.6H), 5.45-5.41 (m, 0.6H), 5.33-5.21 (m, 2.1H), 4.55 (dd, J 1 = 10.8 Hz, J 2 = 2.8 Hz,

0.3H), 3.98 (s, 1.8H), 3.94 (s, 0.9H), 3.83-3.81 (m, 2.4H), 3.62 (dd, Ji= 16.4 Hz, J2 = 11.4 Hz,

1H), 3.56 (s, 0.9H), 3.27-3.13 (m, 1H); "C NMR (400 Hz, CDCl 3): 171.5, 164.7, 155.2, 154.4,

148.6, 148.3, 140.3, 137.4, 135.11, 135.05, 130.5, 129.2, 128.7, 128.4, 127.9, 127.6, 127.5,

126.7, 125.5, 124.8, 124.3, 123.9, 117.6, 112.4, 110.1, 109.2, 108.8, 71.3, 71.2, 61.5, 60.2, 60.1,

56.7, 56.5, 52.5, 52.4, 33.6, 31.4; HRMS(ESI, m/z): calc. 463.1505 (M + H)-, found 463.1516.

(2S)-1-[5-methoxy-2-nitro-4-(phenylmethoxy)-benzoyll-2-indolinealdehyde 6:

MeO 2 C DIBAI-H OHQ BnO , NO 2 Td/CH 2C 2 BnO . NO2 2 -78CQ3h MeO N 8°C 69 MeO N 5 0 6 0

[212] To a stirred solution of the methyl ester 5 (4.4 g, 9.5 mmol) in anhydrous

dichloromethane (11 mL) and toluene (33 mL) was added dibal-H (19 mL, 1.0 M in toluene)

dropwise via a syringe pump in 30 minutes at -78 °C. The mixture continued to be stirred at -78

°C for 3 hours and TLC (hexanes/AcOEt, 1:1.5) showed that the starting material was almost

consumed. The reaction was quenched with methanol (0.4 mL) and 5% HCl (30 mL) at -78 °C. Ethyl acetate (100 mL) was added and the dry ice/acetone bath was removed. The mixture was

stirred at room temperature for 30 minutes and then transferred to a separatory funnel. The

aqueous layer was extracted with AcOEt twice and the combined organic layers were washed

with brine, saturated sodium bicarbonate and brine, and dried over anhydrous sodium sulfate. It

was filtered through celite and the solvents were removed under reduced pressure (temperature <

°C). The residue was purified by flash chromatography (Hexanes/AcOEt, 1.5:1, 1:1, 1:1.5) to

give the aldehyde 6 as a yellow solid (2.85 g, y = 69%). 1H NMR (400 Hz, CDCl 3): the

compound appears as three distinct rotomers. 6 10.02 (s, 0.3H), 9.85 (s, 0.5H), 9.45 (s, 0.2H),

8.32-8.31 (m, 0.2H), 7.93 (s, 0.3H), 7.83 (s, 0.5H), 7.79 (s, 0.2H), 7.53-7.34 (in, 5.2H), 7.26-7.14

(in, 1.3H), 7.08 (s, 0.5H), 7.01-6.94 (in, 1H), 6.91-6.82 (in, 1H), 5.78 (d, J = 8.4 Hz, 0.3 H), 5.71

(d, J = 8.4 Hz, 0.5H), 5.52-5.48 (in, 0.5H), 5.35-5.21 (in, 2.3H), 4.53-4.50 (in, 0.2H), 4.06 (s,

1.5H), 3.98 (s, 0.6H), 3.94 (s, 0.9H), 3.63-3.17 (m, 2H); HRMS (ESI, m/z): calc. 433.1400 (M

+ H), found 433.1387.

Compound 7:

OH Na 2S 2 04 BnO NO2 THF/H 20 BnO N rt, overnight 0 MeO / N then MeOH/AcCI MeO N rt, 30 min O 6 073% 7

[213] To a stirred solution of aldehyde 6 (2.16 g, 5 mmol) in THF (230 mL) was added deioned

water (150 mL) and sodium dithionite (85%, 4.61 g, 22.5 mmol). The obtained slightly cloudy

solution became clear after addition of another 5 mL of deioned water. The clear mixture was

stirred at room temperature for 16 hours and 30 mL of MeOH was added. After stirring for

another 2 hours, the solvents were removed under reduced pressure (bath temperature below 35

°C). The residue was suspended in acetonitrile and evaporated to help remove any remaining

water. The obtained white solid was further completely dried by leaving on a high vacuum for a

few hours. The residue was suspended in dichloromethane/methanol (1:1) and filtered through

celite. The flask and the solid were thoroughly washed with dichloromethane/methanol (1:1).

The filtrate was stripped under reduced pressure. The residue was dissolved in methanol (50

mL) followed by addition of acetyl chloride (1.8 mL, 25 mmol) dropwise. The mixture was

stirred at room temperature for 30 minutes and concentrated under reduced pressure (bath

temperature below 35 °C) to remove half of the methanol. The remainder was quenched with saturated sodium bicarbonate followed by addition of dichloromethane (150 mL) and water (100 mL). The aqueous layer was extracted with dichloromethane (2x100 mL) and the combined organic layers were washed with brine, dried over anhydrous sodium sulfate and filtered. The solvents were removed under reduced pressure and the residue was purified by silica gel chromatography (Hexanes/AcOEt, 1:1, 1:1.3, 1:1.5) to give compound 7 as a yellow solid (1.41 g, y = 73%). 'H NMR (400 Hz, CDCl3 ): 6 8.26 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 4.4 Hz, 1H), 7.57

(s, 1H), 7.46-7.23 (m, 7H), 7.11-7.08 (m, 1H), 6.86 (s, 1H), 5.23 (d, J = 12 Hz, 1H), 5.18 (d, J =

12 Hz, lH), 4.44 (ddd, Ji = 11.2 Hz, J2 = 4.4 Hz, J3 = 4.0 Hz, 1H), 3.97 (s, 3H), 3.67 (dd, Ji =

16.4 Hz, J2 = 11.2 Hz, 1H), 3.46 (dd, Ji= 16.4 Hz, J 2 = 4.0 Hz, 1H); 1C NMR (400 Hz, CDC 3 ):

6 163.8, 163.0, 150.9, 148.3, 141.96, 139.97, 136.0, 129.4, 128.6, 128.1, 128.08, 127.3, 124.7,

124.69, 120.7, 116.8, 111.9, 111.3, 70.8, 56.2, 54.9, 32.5; HRMS(ESI, m/z): calc. 385.1552 (M

+ H)+, found 385.1592.

Indolinobenzodiazepine (IBD) monomer 8:

.N MeSO 3 H HO N BnO BnO ~ >...~ CH2CI 2

MeO -'~ rt,15h e o) e IBD monomer 8

[214] To a stirred solution of the starting material 7 (1.41 g, 3.67 mmol) in dichloromethane (26

mL) was added a freshly mixed solution of methanesulfonic acid (26 mL) in dichloromethane

(52 mL) at room temperature. The mixture was stirred at room temperature for 1.5 hours and

diluted with dichloromethane (100 mL). The mixture was poured on ice (200 g)/MeOH (10

mL). The pH of the obtained solution was adjusted to 7 with saturated NaHC 3, solid NaHCO 3

and water. The mixture was separated and the dichloromethane layer was washed with brine.

The combined aqueous layers were extracted with ethyl acetate (3x80 mL). The ethyl acetate

layers were combined and washed with brine. The dichloromethane and ethyl acetate were

combined, dried over anhydrous sodium sulfate and filtered. The solvents were removed and the

residue (1.26 g) was purified by silica gel chromatography (CH 2Cl 2/MeOH, 20:1, 15:1) to give

the IBD monomer 8 as a yellow solid (1.02 g, y = 95%). 1H NMR (400 Hz, CDCl3): 6 8.29 (d, J

= 8.0 Hz, 1H), 7.91 (d, J = 4.8 Hz, 1H), 7.59 (s, 1H), 7.32-7.28 (in, 2H), 7.13 (t, J = 7.2 Hz, 1H),

6.94 (s, IH), 6.02 (s, -OH), 4.50 (dt, Ji = 10.8 Hz, J2 = 4.4 Hz, 1H), 4.02 (s, 3H), 3.73 (dd, J1 =

16.8 Hz, J2 = 10.8 Hz, 1H), 3.52 (dd, J1 = 16.8 Hz, J2 = 3.6 Hz, 1H); HRMS (ESI, m/z): cale.

295.1083 (M + H)-, found 295.1076.

Example 2

(s)-(-)-3-(Benzyloxycarbonyl)-4-oxazolidinecarboxylic methyl ester 10:

HO2 C, SOC1 2 MeO2CV o MeOH 00 N--/ rt, 4h Cbz 9% Cbz 1 9g 9%10

[215] To a stirred solution of (s)-(-)-3-(Benzyloxycarbonyl)-4-oxazolidinecarboxylic acid 9

(1.75 g, 6.96 mmol) in anhydrous methanol (15 mL) was added thionyl chloride (1.02 mL, 13.9

mmol) at 0 °C. After 30 minutes, the ice/water bath was removed and the reaction mixture

continued to be stirred at room temperature for 3.5 hours. The reaction was quenched by

addition of saturated sodium bicarbonate and diluted with dichloromethane (100 mL) and water

(50 mL). The mixture was separated and the aqueous layer was extracted with dichloromethane

(2x50 mL). The combined organic layers were washed with brine, dried over anhydrous sodium

sulfate and filtered. The solvents were removed under reduced pressure and the residue was

purified by silica gel chromatography (Hexanes/AcOEt, 1.5:1) to give (s)-(-)-3

(Benzyloxycarbonyl)-4-oxazoidinecarboxylic methyl ester 10 as colorless oil (1.84 g, y = 99%).

H NMR (400 Hz, CDC 3 ): the compound appears as a pair of distinct rotomers. 6 7.35 (bs, 5H),

5.22-4.99 (m, 4H), 4.53-4.45 (m, 1H), 4.22-4.09 (m, 2H), 3.76 (s, 1.5H), 3.65 (s, 1.5H); MS

(m/z): found 288.0 (M + Na)+.

Compound 11:

H2 BnO . NO2 CO 2 Me MeO 2C, Pd(OH) 2/C MeO/ C1 Bn NO2 0 TEA/ACOEt then directly filter to 4 NM 4 in THF solution. * MeG N-' Cbz 10 rt, 2h 0-5 OC, 3h 91% 0

[216] To a stirred solution of (s)-(-)-3-(Benzyloxycarbonyl)-4-oxazolidinecarboxylic methyl

ester 10 (1.04 g, 3.92 mmol) in ethyl acetate (16 mL) was added triethyl amine (1.4 mL, 10

mmol) and palladium hydroxide on carbon (20%, 267 mg, 0.337 mmol). The air in the reaction

flask was removed by vacuum, then a hydrogen balloon was applied and the mixture was stirred

under hydrogen atmosphere at room temperature for 2 hours. To a solution of acetyl chloride 4

(prepared from 1.3 g, 4.3 mmol of 4-benzyloxy-5-methoxy-2-nitrobenzoic acid 2 following the

procedures described above) in anhydrous THF (15 mL) was added triethyl amine (1.1 mL, 7.9

mmol) at 0 °C, followed by addition of the above hydrogenation reaction mixture by filtration

through celite. The palladium catalyst/celite was washed with anhydrous THF (15 mL). The

obtained mixture was stirred at 0 0 C for 3 hours. It was diluted with ethyl acetate and saturated

ammonium chloride. The pH of the mixture was adjusted to 6~7 by addition of 5% hydrochloric

acid. The mixture was separated and the aqueous layer was extracted with ethyl acetate (2x80

mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate

and filtered. The solvents were removed under reduced pressure and the residue was purified by

silica gel chromatography (Hexanes/AcOEt, 1:2, 1:3) to give compound 11 as a pale yellow solid

(1.49 g, y = 91%). 'H NMR (400 Hz, CDCl 3 ): the compound appears as a pair of distinct rotomers. 6 7.78 (s, 0.5H), 7.75 (s, 0.5H), 7.48-7.37 (m, 5H), 6.97 (s, 0.5H), 6.91 (s, 0.5H), 5.39

(d, J = 4.8 Hz, 0.5H), 5.26-5.23 (in, 2.51), 4.95 (dd, JI= 7.2 Hz, J2 = 4.4 Hz, 0.5H), 4.81 (d, J =

3.6 Hz, 0.5H), 4.67 (d, J = 3.6 Hz, 0.5H), 4.37-4.30 (in, IH), 4.25-4.11 (in, 1.5H), 4.02 (s, 1.5H),

3.97 (s, 1.5H), 3.87 (s, 1.5H), 3.67 (s, 1.5H); HRMS (ESI, m/z): calc. 417.1298 (M + H)', found

417.1305.

Aldehyde 12:

C0 2 Me DIBAI-H CHO BnO NO 2 ToI/CH CI 0 2 2 BnO N O -78 C, 3h 0 MeO ;N- 70% Meo 0 10 12

[217] To a stirred solution of the methyl ester 11 (1.49 g, 3.6 mmol) in anhydrous

dichloromethane (4 mL) and toluene (12 mL) was added dibal-H (6.5 mL, 1.0 M in toluene)

dropwise via a syringe pump in 30 minutes at -78 0 C. The mixture continued to be stirred at -78

°C for 2 hours. The reaction was quenched with methanol (146 pL, 3.6 mmol) and 5% HCl (30

mL) at -78 °C. Ethyl acetate (100 mL) was added and the dry ice/acetone bath was removed.

The mixture was stirred at room temperature for 30 minutes and then transferred to a separatory

funnel. The aqueous layer was extracted with AcOEt twice. All the organic layers were

combined, washed with brine, saturated sodium bicarbonate and brine. It was dried over

anhydrous sodium sulfate and filtered through celite. The filtrate was evaporated under reduced

pressure and the residue was purified by silica gel chromatography (Hexanes/AcOEt, 1:5, 1:10) to give the aldehyde 12 as a pale yellow solid (980 mg, y = 70%). 'H NMR (400 Hz, CDCl 3): the compound appears as a pair of distinct rotomers. 6 9.83 (s, 0.67H), 9.45 (s, 0.33H), 7.77 (s,

0.67H), 7.72 (s, 0.33H), 7.45-7.37 (in, 5H), 6.90 (s, 1H), 5.31-5.19 (m, 3H), 4.77 (bs, 1H), 4.67

4.56 (in, 1H), 4.36-3.94 (in, 5H); HRMS (ESI, m/z): calc. 387.1192 (M + H)-, found 387.1184.

Compound 13:

CHO F Na 2 S204 (4.5eq) BnO BnO . NO 2 o THF/H 20(0.012 M) Bn

MeO Me ~ N--' rt, overnight N / N then MeH/AcCI MeO

/ O rt, 30 min 0 12 61% 13

[218] To a stirred solution of aldehyde 12 (154 mg, 0.4 mmol) in THF (21 mL) was added

deioned water (14 mL) and sodium dithionite (85%, 369 mg, 1.8 mmol). The clear mixture was

stirred at room temperature for 16 hours and 5 mL of MeOH was added. After being stirred

another 2 hours, the solvents were removed under reduced pressure (bath temperature below 35

°C). The residue was suspended in acetonitrile and evaporated to help remove the remaining

water. The obtained white solid was further completely dried by leaving on a high vacuum for a

few hours. The residue was suspended in dichloromethane/methanol (2:1) and filtered through

celite. The flask and the solid were thoroughly washed with dichloromethane/methanol (1:1).

The filtrate was stripped under reduced pressure. The residue was dissolved in methanol (5 mL)

and a freshly prepared acetyl chloride (0.15 mL)/MeOH (5 mL) solution was added quickly. The

mixture was stirred at room temperature for 30 minutes and quenched by addition of saturated sodium bicarbonate. It was diluted with dichloromethane and water. The two layers were separated and the aqueous layer was extracted with dichloromethane. The combined dichloromethane layers were washed with brine and dried over anhydrous sodium sulfate. The solvents were removed under reduced pressure to give 127 mg crude product. The aqueous layer and the washing solution were combined and acidified to pH 2~3 with KHSO 4. It was concentrated to half under reduced pressure (temperature <40 °C) and extracted with dichloromethane. The combined dichloromethane was washed with saturated sodium bicarbonate and brine, dried over anhydrous sodium sulfate. It was filtered and the filtrate was evaporated under reduced pressure. The residue was combined with above 127 mg crude product and purified by silica gel chromatography (Hexanes/AcOEt, 1:3, 1:5, 1:8) to give compound 13 as a colorless foam (80 mg, y = 610%). 'H NMR (400 Hz, CDCl 3): 6 7.77 (d, J =

4.0 Hz, 1H), 7.52 (s, 1H), 7.46-7.28 (m, 5H), 6.88 (s, 1H), 5.28 (d, J = 5.2 Hz, 1H), 5.23 (d, J =

12 Hz, 1H), 5.17 (d, J = 12 Hz, 1H), 5.05 (d, J = 5.2 Hz, 1H), 4.49 (dd, J1= 9.6 Hz, J2 = 3.2 Hz,

1H), 4.33 (dd, Ji = 9.6 Hz, J2 = 6.4 Hz, 1H), 3.96 (s, 3H), 3.83 (dd, J1= 6.4 Hz, J2 = 3.2 Hz,

IH); MS (m/z): found 361.1 (M + Na)', 379.1 (M + H 2 0 + Na), 339.1 (M + H)+.

Oxazolidinobenzodiazepine (OBD) monomer 14:

Bno Ir 0 HO =sr N-' Pd/C D.M N-' Me M EtOH Me O 82% O 13 OBD monomer 14

[219] A solution of compound 13 (90 mg, 0.27mmol) and Pd/C (10%, 90 mg) in absolute

ethanol (1.5 mL) was bubbled with argon. 1,4-Cyclohexadiene (496 pl, 5.3 mmol) was added

and the argon bubble was continued for 3 hours until the starting material disappeared (TLC,

dichloromethane/methanol 10:1). The mixture was then filtered through celite and the celite was

washed with methanol. The filtrate was evaporated under reduced pressure to give 63 mg of the

crude product as colorless foam, which was purified by silica gel chromatography

(dichloromethane/methanol, 20:1) to give OBD monomer 14 (55 mg, y = 82%) as a white solid.

H NMR (400 Hz, CDC 3): it appears as a mixture of imine and its methyl ethers, C11(R) and

Cl (S) (2:3:1). 6 7.71 (bs, 1H), 7.43 (s, 0.5H), 7.41 (s, 1H), 7.18 (s, 1.5H), 6.83 (s, 1H), 6.36 (s,

1.5H), 6.13 (s, 0.5H), 5.25 (d, J = 4.8 Hz, 0.5H), 5.22-5.20 (m, 1H), 5.14 (d, J = 5.2 Hz, 1.5H),

5.10 (d, J = 4.8 Hz, 0.5H), 5.05 (d, J= 5.2 Hz, 1.5H), 5.00-4.97 (m, 1H), 4.47 (d, J = 8.8 Hz,

1.5H), 4.44-4.41 (m, 1H), 4.32 (apt, J= 8.0 Hz, 0.5H), 4.28-4.25 (m, 1H), 4.18-4.00 (m, 2x1.5H

+ 2x0.5H = 4H), 3.84 (bs, 3x1H + 0.5H = 3.5H), 3.76 (bs, 3x1.5H + 1H = 5.5H), 3.73 (s, 3x0.5H

= 1.5H), 3.56 (dt, JI = 8.8 Hz, J2 = 2.8 Hz, 1.5H), 3.34 (s, 3x1.5H = 4.5H), 3.22 (s, 3x0.5H=

1.5H); MS (m/z): found 303.1 (M + McOH + Na)', 271.1 (M + Na)*.

Example 3

Dimer 15 (IGN-09):

HO N-z HO ~~ DMF N O~ K 2CO 3 N N

MeO 0 N + rt, overnight 15%% -. 6\ N N MeMeO

8 (RP HPLC) 15 (IGN-09)

[220] To a solution ofIBD monomer 8 (147 mg, 0.5 mmol) and 1,3-diiodopropane (23 [, 0.2

mmol) in anhydrous DMF (1.0 mL) was added potassium carbonate (111 mg, 0.8 mmol). The

mixture was stirred at room temperature overnight (16 hours) and diluted with dichloromethane.

It was washed with saturated ammonium chloride and brine, dried over anhydrous sodium sulfate

and filtered. The filtrate was evaporated under reduced pressure and the residue was purified

through preparative reverse phase HPLC (C18 column, acetonitrile/water) to give dimer 15

(IGN-09) (18.9 mg, y = 15%) as a white solid. 1H NMR (400 Hz, CDCI): 6 8.26 (d, J = 8.0 Hz,

2H), 7.87 (d, J = 4.4 Hz, 2H), 7.55 (s, 2H), 7.26 (s, 4H), 7.12-7.08 (m, 2H), 6.88 (s, 2H), 4.45

(ddd, JI = 10.8 Hz, J2 = 4.4 Hz, J3 = 4.0 Hz, 2H), 4.36-4.26 (m, 4H), 3.94 (s, 6H), 3.70 (dd, JI =

16.8 Hz, J2 = 10.8 Hz, 2H), 3.50 (dd, JI = 16.8 Hz, J2 = 4.0 Hz, 2H), 2.45 (p, J = 6.0 Hz, 2H);

HRMS (ESI, m/z): calc. 629.2400 (M + H), found 629.2400.

Example 4

Dimer 18 (IGN-01):

MsCI K 2 00 3 KI TEA N N DOM 71 DMF 0,N -5 0 lh rtrMsO OMsht N Nvernigh HOO 11 -C1h12% NOMe MeO

16 without purification for2steps 17(RP HPLC) 18 (IGN-01)

[221] To a stirred solution of 1,3-Benzenedimethanol 16 (11 mg, 0.08 mmol) in anhydrous

dichloromethane (0.8 mL) was added triethylamine (33 pl, 0.24 mmol) then methanesulfonyl

chloride (16 pL, 0.21 mmol) dropwise in 15 minutes at -5 ~ -10 °C. The solution was stirred at

~ -10 aC for another 60 minutes and was quenched with ice/water, diluted with cold ethyl

acetate. The mixture was separated and the organic layer was washed with cold water, dried

over anhydrous sodium sulfate. It was filtered and the filtrate was evaporated by rotary

evaporation in vacuo (temperature < 35 °C). The residue 17 was high vacuumed for a few hours

before being dissolved in anhydrous DMF (1.5 mL). IBD monomer 7 (94 mg, 0.32 mmol),

anhydrous potassium carbonate (50 mg, 0.36 mmol) and potassium iodide (27 mg, 0.16 mmol)

were added subsequently. The mixture was stirred at room temperature for 17 hours (checked by

mass spectrum) and diluted with dichloromethane. It was washed with brine, dried over

anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure and

the residue was purified by reverse phase HPLC (C18 column, CH3CN/H 20, loaded column with

CH 3CN/H 20, 3:1, stirred for 30 min and centrifuged before injection) to furnish dimer 18 (IGN

01, 6.6 mg) as a white solid. 1H NMR (400 Hz, CDCl3): 6 8.21 (d, J = 8.0 Hz, 2H), 7.79 (d, J =

4.4 Hz, 2H), 7.51 (s, 2H), 7.46 (s, 1H), 7.36 (bs, 3H), 7.23-7.18 (in, 4H), 7.06-7.03 (m, 2H), 6.79

(s, 2H), 5.20 (d, J = 12.4 Hz, 2H), 5.14 (d, J = 12.4 Hz, 2H), 4.41 (ddd, JI = 10.8 Hz, J2 = 4.4

Hz, J3 = 4.0 Hz, 2H), 3.92 (s, 6H), 3.64 (dd, JI = 17.2 Hz, J2 = 11.2 Hz, 2H), 3.42 (dd, JI = 16.8

Hz, J2 = 4.0 Hz, 2H); HRMS (ESI, m/z): calc. 691.2557 (M + H)+, found 691.2570.

Example 5

Dimer 19 (IGN-02):

MsCI K 2CO/KI TEA 14, DMF DMrt, overnight 0j : .- C 0 HO- OH 7 -5O,1h22% MsO v Is KOMs N--.;N without for 2steps 19(IGN-0Me 16 purification 17 (RP HPLC) O 19 (IGN-02)

[222] To a stirred solution of 1,3-Benzenedimethanol 16 (10 mg, 0.074 mmol) in anhydrous

dichloromethane (0.8 mL) was added triethylamine (31 pl, 0.22 mmol) then methanesulfonyl

chloride (15 pL, 0.19 mmol) dropwise in 15 minutes at -5 ~ -10 °C. The solution was stirred at

~ -10 °C for another 60 minutes and was quenched with ice/water, diluted with cold ethyl

acetate. The mixture was separated and the organic layer was washed with cold water, dried

over anhydrous sodium sulfate. It was filtered and the filtrate was evaporated by rotary

0 evaporation in vacuo (temperature < 35 C). The residue 17 was high vacuumed before dissolving in anhydrous DMF (1.5 mL). OBD monomer 14 (70 mg, 0.28 mmol), anhydrous potassium carbonate (51 mg, 0.37 mmol) and potassium iodide (25 mg, 0.15 mmol) were added subsequently. The mixture was stirred at room temperature for 17 hours (checked by mass spectrum) and diluted with dichloromethane. It was washed with brine, dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure and the residue was purified by reverse phase HPLC (C18 column, CH 3CN/H 20, loaded column with

CH 3CN/H 2 0, 3:1, stirred for 30 min and centrifuged before injection) to furnish dimer 19 (IGN

02, 10.0 mg) as a white solid. 1H NMR (400 Hz, CDCl 3): 6 7.75 (d, J= 4.0 Hz, 2H), 7.50-7.48

(bs, 3H), 7.38 (bs, 3H), 6.83 (s, 2H), 5.26 (d, J = 5.2 Hz, 2H), 5.21 (d, J= 14.4 Hz, 2H), 5.15 (d,

J = 14.0 Hz, 2H), 5.03 (d, J = 5.6 Hz, 2H), 4.34-4.30 (m, 2H), 3.94 (s, 6H), 3.86-3.76 (m, 2H);

HRMS (ESI, m/z): calc. 599.2142 (M + H), found 599.2184.

Example 6

Triol 21:

OH OH

MeO OMe 99 HO OH 0 0 20 21

[223] To a stirred solution of dimethyl 5-hydroxyisophthalate 20 (2.1 g, 10 mmol) in anhydrous

THF (50 mL) was added lithium aluminum hydride (2.0 M in THF, 10 mL, 20 mmol) at -20 ~

°C via a syringe pump in 30 minutes. The cooling bath was removed after 30 minutes and the mixture continued to be stirred at room temperature for 4 hours. It was cooled to 0 ~ -10 °C and quenched with saturated sodium sulfate. The mixture was diluted with acetonitrile and 5% hydrochloric acid (20 mL) was added. It was stirred for 30 minutes and dried over anhydrous sodium sulfate. The mixture was filtered through celite and the filtrate was evaporated under reduced pressure. The residue was purified through silica gel chromatography

(Dichloromethane/Methanol, 10:1, 8:1, 5:1) to give triol 21 (1.5 g, y = 99%) as a colorless oil

which became white solid after stocking. 1H NMR (400 Hz, MeOD): 6 6.78, (s, 1H), 6.69 (s,

2H), 4.50 (s, 4H). 3 C NMR (400 Hz, MeOD): 6 158.7, 144.4, 117.8, 113.8, 65.2; MS (m/z):

found 153.0 (M - H).

Compound 22:

0 OHO Br(CH 2 )4 CO 2 Me OMe

HO / OH K2 O CH 3 CN HO ...- OH 21 reflux 22

[224] To a solution of triol 21 (827 mg, 5.37 mmol) and methyl 5-bromovalerate (998 mg, 5.12

mmol) in acetonitrile (40 mL) was added potassium carbonate (3.71 g, 26.9 mmol). The mixture

was put in a 86 °C oil bath and refluxed for 6 hours. The reaction mixture was removed from the

oil bath, cooled to room temperature and the solvents were evaporated under reduced pressure

(temperature < 35 0 C). The residue was diluted with dichloromethane and filtered. The filtrate was washed with brine, dried over anhydrous sodium sulfate and filtered. The filtrate was stripped under reduced pressure and the residue was purified through silica gel chromatography

(Hexanes/Ethyl acetate, 1:2, 1:3) to give compound 22 (1.15 g, y = 84%) as a white solid. 'H

NMR (400 Hz, CDCl3): 6 6.89 (s, 1H), 6.80 (s, 2H), 4.62 (s, 4H), 3.98-3.95 (m, 2H), 3.67 (s,

3H), 2.41-2.37 (m, 2H), 2.23 (bs, -OHx2), 1.84-1.78 (m, 4H); MS (m/z): found 291.1 (M + Na)*.

Compound 23:

OH O'-rOMe HO H O Br(CH 2) 3CO2 Me H

_'Jlj::: CH 3CN HH 21 reflux 23

[225] Following the procedure to prepare compound 22, compound 23 (1.43 g, y = 75%) was

synthesized as a white solid from triol 21 (1.16 g, 7.53 mmol), methyl 4-bromobutyrate (1.52 g,

8.39 mmol) and potassium carbonate (5.2 g, 37.6 mmol). IH NMR (400 Hz, CDCI 3): 6 6.90 (s,

1H), 6.80 (s, 2H), 4.62 (s, 4H), 4.00 (t, J = 6.0 Hz, 2H), 3.68 (s, 3H), 2.51 (t, J = 7.2 Hz, 2H),

2.19 (s, -OHx2), 2.13-2.06 (m, 2H); MS (m/z): found 277.1 (M + Na)+.

Compound 24:

OH O OMe BrCH 2C 2Me O OH K 2C0 3 HOO

21 reflux 24

[226] Following the procedure to prepare compound 22, compound 24 (515 mg, y = 37%) was

synthesized as a white sticky solid from triol 21 (953 mg, 6.19 mmol), methyl bromoacetate (587

ptl, 6.19 mmol) and potassium carbonate (4.3 g, 31 mmol). IH NMR (400 Hz, CDCl 3): 6 6.95 (s,

1H), 6.81 (s, 2H), 4.64 (s, -OHx2), 4.61 (s, 4H), 3.81 (s, 3H), 2.41 (s, 2H); '"C NMR (400 Hz,

CDCI 3 ): 169.4, 158.1, 143.0, 118.5, 112.1, 65.2, 64.8, 52.3; MS (m/z): found 249.0 (M + Na).

Compound 27:

NO 2 Pd/C N2HN'Y ~OMe H 2 , MeOH NH 2 BrCH 2CO 2 Me 5 psi, 2h K2CO3 __0

HO '101 OH 100% HO OH CH 3CN HO OH reflux 25 26 27

[227] To a solution of 5-nitro-m-xylene-a,a'-diol 25 (1.07 g, 5.84 mmol) in methanol (50 mL)

was added Pd/C (10%, 311 mg, 0.29 mmol). Hydrogen was introduced to replace the air then

the mixture was hydrogenated (H2, 5 psi) for 2 hours at room temperature. The solution was

filtered through celite and the filtrate was evaporated by rotary evaporation in vacuo to give

compound 26 as a white solid (900 mg, y = 100%). 1H NMR (400 Hz, McOD): 6 6.71 (s, 1H),

6.66 (s, 2H), 4.51 (s, 4H); "C NMR (400 Hz, MeOD): 6 148.9, 143.8, 116.7, 114.3, 65.5; It was

dissolved in anhydrous acetonitrile (30 mL) and ethyl bromoacetate (443 pl, 4.67 mmol) and

potassium carbonate (807 mg, 5.84 mmol) were added. The mixture was put in a 860 C oil bath

and refluxed for 17 hours. The reaction mixture was removed from the oil bath, cooled to room temperature and diluted with dichloromethane. It was filtered through celite and the solid was washed with dichloromethane. White precipitate appeared in the filtrate. It was collected by filtration to give compound 27 (414 mg, y = 39%) as a white solid. H NMR (400 Hz, MeOD): 6

6.67 (s, 1H), 6.53 (s, 2H), 4.51 (s, 4H), 3.94 (s, 2H), 3.73 (s, 3H); C NMR (400 Hz, MeOD): 6

174.0, 149.7, 143.9, 116.2, 111.6, 65.6, 52.6, 46.5; MS (m/z): found 248.0 (M + Na)+.

Compound 28:

NH 2 H ~ Br(CH 2)3CO 2 Me H o HO OH 2O3 CH HO OH 3CN 26 reflux 28

[228] To a solution of 5-nitro-m-xylene-a,a'-diol 25 (564 mg, 3.08 mmol) in methanol (35 mL)

was added Pd/C (10%, 164 mg, 0.154 mmol). Hydrogen was introduced to replace the air then

the mixture was hydrogenated (H2, 5 psi) for 2 hours at room temperature. The solution was

filtered through celite and the filtrate was evaporated by rotary evaporation in vacuo to give

compound 26, which was dissolved in anhydrous acetonitrile (15 mL) and methyl 4

bromobutyrate (557 mg, 3.08 mmol) and potassium carbonate (426 mg, 3.08 mmol) were added.

The mixture was put in a 86 °C oil bath and refluxed for 18 hours. The reaction mixture was

removed from the oil bath, cooled to room temperature and diluted with dichloromethane. It was

filtered through celite and the solid was washed with dichloromethane/acetonitrile (1:1). The filtrate was evaporated under reduced pressure and the residue was purified through silica gel chromatography (Combiflash, dichloromethane/methanol) to give compound 28 (292 mg, y =

37%) as a white solid. 'H NMR (400 Hz, MeOD): 6 6.62 (s, 1H), 6.55 (s, 2H), 4.50 (s, 4H), 3.65

(s, 3H), 3.13 (d, J = 7.2 Hz, 2H), 2.43 (d, J = 7.2 Hz, 2H), 1.89 (p, J = 7.2 Hz, 2H); "C NMR

(400 Hz, MeOD): 6 175.9, 150.5, 143.7, 115.5, 111.7, 65.7, 52.2, 44.3, 32.5, 25.8; MS (m/z):

found 276.0 (M + Na)v.

Compound 29:

HN OMe CH 3 N OMe

HO OH HO OH reflux 27 29

[229] To a solution of compound 27 (230 mg, 1.02 mmol) in anhydrous acetonitrile (7 mL) was

added methyl iodide (70 pl, 1.12 mmol) and potassium carbonate (155 mg, 1.12 mmol). The

mixture was put in a 86 °C oil bath and refluxed for 17 hours. The reaction mixture was

removed from the oil bath, cooled to room temperature and diluted with dichloromethane. It was

filtered through celite and the solid was washed with dichloromethane/methanol (10:1). The

filtrate was evaporated under reduced pressure and the residue was purified through silica gel

chromatography (Combiflash, dichloromethane/methanol) to give compound 29 (98 mg, y=

%) as a white solid. H NMR (400 Hz, MeOD): 6 6.70 (s, 1H), 6.63 (s, 2H), 4.84 (s, 2x-OH),

4.54 (s, 4H), 4.16 (s, 2H), 3.69 (s, 3H), 3.05 (s, 3H); 3 C NMR (400 Hz, MeOD): 6 173.6, 150.9,

143.8, 115.6, 111.0, 65.7, 54.9, 52.4, 39.8; MS (m/z): found 262.0 (M + Na).

Compound 30:

HN .e NN OMe

HO , OH K2C0 3 HO OH CH 3CN 28 reflux 30

[230] To a solution of compound 28 (151 mg, 0.597 mmol) in anhydrous acetonitrile (4 mL)

was added methyl iodide (74 pl, 1.19 mmol) and potassium carbonate (99 mg, 0.716 mmol).

The mixture was put in an 86 °C oil bath and refluxed for 17 hours. The reaction mixture was

removed from the oil bath, cooled to room temperature and diluted with dichloromethane. It was

filtered through celite and the solid was washed with dichloromethane/methanol (10:1). The

filtrate was evaporated under reduced pressure and the residue was purified through silica gel

chromatography (Combiflash, dichloromethane/methanol) to give compound 30 (63 mg, y =

39%) as a colorless oil. 'H NMR (400 Hz, MeOD): 6 6.67 (s, 2H), 6.65 (s, 1H), 4.54 (s, 4H),

3.65 (s, 3H), 3.36 (t, J = 7.2 Hz, 2H), 2.92 (s, 3H), 2.36 (t, J = 7.2 Hz, 1H), 1.87 (p, J = 7.2 Hz,

2H); "C NMR (400 Hz, MeOD): 6 175.7, 151.3, 143.7, 115.0, 111.4, 65.9, 53.0, 52.2, 38.9,

32.2,23.3; MS (m/z): found 290.0 (M + Na)+.

Compound 34 (IGN-03):

0 0 0 SMsCI KCO3 OOA TEA 0- -NA K2C0 OMe DCM OMe IBD monomer 8 0 I - Ch I..DMFN HO0 1h OH, MO - Os overnight N a o0

22 purification 31 / NOMe MeO 0 34(IGN-03) O

[231] To a stirred solution of compound 22 (80.4 mg, 0.3 mmol) in anhydrous dichloromethane

(2 mL) was added triethylamine (125 pl, 0.9 mmol) then methanesulfonyl chloride (60 pL, 0.78

mmol) dropwise in 15 minutes at -5 ~ -10 °C. The solution was stirred at -5 ~ -10 °C for another minutes and was quenched with ice/water, diluted with cold ethyl acetate. The mixture was

separated and the organic layer was washed with cold water, dried over anhydrous sodium

sulfate. It was filtered and the filtrate was evaporated by rotary evaporation in vacuo

(temperature < 35 °C). The residue 31 was high vacuumed before dissolving in anhydrous DMF

(3 mL). IBD monomer 7 (221 mg, 0.75 mmol) and anhydrous potassium carbonate (207 mg, 1.5

mmol) were added. The mixture was stirred at room temperature for 20 hours (checked by mass

spectrum) and diluted with dichloromethane. It was washed with water and brine, dried over

anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure and

the residue was purified through silica gel chromatography (hexanes/ethyl acetate, 1:3, 1:4, 1:6,

1:10, then ethylacetate/methanol, 10:1) to give compound 34 (169 mg, y = 68%, 86% purity

based on analytical reverse phase HPLC) as a yellowish solid. Fractions that contained

impurities and compound 34 were also collected and the solvents were evaporated to give 70 mg

of yellowish solid. The two yellowish solids were combined and further purified through reverse phase HPLC (C18 column, CH3CN/H 20, loaded column with CH3 CN/H 2 0, 3:1, stirred for 30 min and centrifuged before injection) to furnish dimer 34 (IGN-03, 103 mg, y = 41%) as a white solid. 1H NMR (400 Hz, CDCl 3): 6 8.27 (d, J = 8.0 Hz, 2H), 7.85 (d, J = 3.2 Hz, 2H), 7.58 (s,

2H), 7.29-7.24 (in, 41), 7.12-7.07 (in, 3H), 6.94 (s, 2H), 6.83 (s, 2H), 5.22 (d, J = 12.8 Hz, 2H),

5.16 (d, J = 12.8 Hz, 2H), 4.47 (dt, JI = 11.2 Hz, J2 = 4.4 Hz, 2H), 3.98 (bs, 8H), 3.73-3.64 (in,

2H), 3.68 (s, 3H), 3.48 (dd, J1 = 16.8 Hz, J2 = 3.6 Hz, 2H), 2.42-2.38 (in, 2H), 1.83-1.80 (in,

4H); HRMS (ESI, m/z): calc. 821.3187 (M + H)', found 821.3188.

Compound 35 (IGN-04):

O OMe MsCI O'---y OMe K 2 Co3 OMe 0 TEA IBD monomer 80 DCM 0 MOMEN N HO OH 5Mh MsO OMs ,overnight O O 23 without 32 N N purification 0 35 (IGN-04)0

[232] Following the procedure to prepare compound 34, compound 35 (IGN-04) was

synthesized (151 mg, y = 62%, 88% purity based on analytical reverse phase HPLC) as a

yellowish solid. Part of it was further purified by reverse phase HPLC for IH NMR analysis. I H

NMR (400 Hz, CDCl3): 6 8.17 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 5.2 Hz, 2H), 7.48 (s, 2H), 7.20

7.15 (in, 4H), 7.03-6.99 (in, 3H), 6.85 (s, 2H), 6.75 (s, 2H), 5.12 (d, J = 12.8 Hz, 2H), 5.06 (d, J

= 12.8 Hz, 2H), 4.37 (dt, J1 = 11.2 Hz, J2 = 4.4 Hz, 2H), 3.93 (t, J = 6.0 Hz, 2H), 3.86 (s, 6H),

3.64-3.57 (in, 2H), 3.60 (s, 3H), 3.39 (dd, J1 = 16.8 Hz, J2 = 3.6 Hz, 2H), 2.44 (t, J = 7.2 Hz,

2H), 2.02 (p, J= 6.4 Hz, 2H); HRMS (ESI, m/z): calc. 807.3030 (M + H)+, found 807.3008.

Compound 36 (IGN-05):

OMe K 2 00 3 0O-ThKMe 0-OMe MsCI Oh TEA sCIO e BD monomer 8 O 0 0CM 0O DMF N I HO OH I -5 0Ch MsO I~~. OMs rt, overnight

. - r ni O 24 without 33 '5 N ' OMe MeaO~ purification 036 (IGN-05)

[233] Following the procedure to prepare compound 34, compound 36 (IGN-05) was

synthesized (84.5 mg, y = 18%) as a white solid after preparative reverse phase HPLC. H NMR

(400 Hz, CDCl 3):6 8.24 (d, J = 8.0 Hz, 2H), 7.79 (d, J = 4.4 Hz, 2H), 7.55 (s, 2H), 7.26-7.22 (m,

4H), 7.12-7.07 (m, 3H), 6.96 (s, 2H), 6.81 (s, 2H), 5.18 (d, J = 12.8 Hz, 2H), 5.12 (d, J = 12.8

Hz, 2H), 4.64 (s, 2H), 4.44 (dt, Ji = 10.8 Hz, J2 = 4.4 Hz, 2H), 3.95 (s, 6H), 3.77 (s, 3H), 3.73

3.62 (m, 2H), 3.44 (dd, JI = 16.8 Hz, J2 = 3.6 Hz, 2H); HRMS (ESI, m/z): calc. 779.2717 (M

+ H), found 779.2703.

Compound 39 (IGN-06):

NOMe

HOOMe MsCI hM OMe N 29TEA N< K 2 0 3 8, N IG 0 DCM DMF.- I 0 ~ / '

'NO rt, overnight NN/ 1~ O -5O,1N Ms, OMe MeN

29without 370 3 IN0) 0 29 ~~~purification 3(G-6

[234] Following the procedure to prepare compound 34, compound 39 (IGN-06) was

synthesized in 6% yield as a white solid after preparative reverse phase HPLC. H NMR (400

Hz, CDCl): 6 8.28 (d, J = 8.0 Hz, 2H), 7.86 (d, J = 4.0 Hz, 2H), 7.58 (s, 2H), 7.31-7.26 (m, 4H),

7.12 (t, J = 7.2 Hz, 2H), 6.90-6.86 (in, 3H), 6.72 (s, 2H), 5.22 (d, J = 12.4 Hz, 2H), 5.13 (d, J =

12.4 Hz, 2H), 4.51-4.46 (m, 2H), 3.99 (s, 6H), 3.74-3.68 (m, 2H), 3.71 (s, 3H), 3.49 (dd, J1 =

16.8 Hz, J2 = 3.6 Hz, 2H), 3.09 (s, 3H); HRMS (ESI, m/z): calc. 792.3033 (M + H),found

792.3013.

Compound 40 (IGN-07):

N Ome MsCl O~e N O 5EC T N OeO DC NK2rO7CO1/8, ND0F I rt, overnight HO .- OH -5C,1h MsO OMs v t OMe MeN withoutOe MO 30 purification 38 0 40 (IGN-07)

[235] Following the procedure to prepare compound 34, compound 40 (IGN-07) was

synthesized in 21% yield as a white solid after preparative reverse phase HPLC. 1H NMR (400

Hz, CDC 3): 6 8.27 (d, J= 8.0 Hz, 2H), 7.84 (d, J = 4.4 Hz, 2H), 7.58 (s, 2H), 7.30-7.23 (in, 4H),

7.21-7.02 (in, 3H), 6.88 (s, 2H), 6.74 (s, 2H), 5.23-5.13 (in, 4H), 4.50-4.42 (m, 2H), 3.99 (s, 6H),

3.74-3.70 (m, 2H), 3.67 (s, 3H), 3.51-3.33 (m, 4H), 2.92 (s, 3H), 2.36-2.30 (m, 2H), 1.93-1.84

(in, 2H); HRMS (ESI, m/z): calc. 820.3346 (M + H)+, found 820.3329.

Example 7

Compound 41:

oOme oOH

0 80 0 Mee MeOH N N N N 0 34 (IGN-03) 41 (IGN-03 acid) 0

[236] To a solution of compound 34 (42 mg, 0.051 mmol) in anhydrous 1,2-dichloroethane (1

mL) was added trimethyltin hydroxide (139 mg, 0.77 mmol). The mixture was heated at 78~82

°C (80 °C oil bath) and stirred overnight. The TLC (CH 2Cl 2/MeOH, 10:1) showed the

disappearance of the starting material. The reaction mixture was cooled to room temperature and

diluted with dichloromethane. It was washed with drops of 5% hydrochloric acid in brine,

saturated ammonium chloride and brine, dried over anhydrous sodium sulfate, filtered and

evaporated. The residue was purified by silica gel chromatography (combiflash, CH 2C 2/MeOH,

from 1:0 to 5:1) to give IGN-03 acid 41 (33.8 mg, y = 82%) as a yellowish solid. The residue

can also be used for next step without purification. MS (m/z): found 805.1 (M - H) -, 823.0 (M +

H 20 - H) -, 829.2 (M + Na), 847.2 (M + H 20 + Na)'.

Compound 42:

OH O I /OMe

N IN 0 T / e0 0 N

0 35 (IGN-04) 0 H 20 42 (IGN-04 acid)

[237] To a stirred solution of compound 35 (32 mg, 0.040 mmol) in a mixture of THF (0.4

mL), methanol (0.1 mL) and deioned water (0.1 ml) was added freshly prepared 2N LiOH (24

ptl, 0.048 mmol) at 0 °C. The cooling bath was removed and the mixture was stirred at room

temperature for 8 hours. The reaction mixture was diluted with ethyl acetate and water. The pH

of the mixture was adjusted to 4~5 with 5% hydrochloric acid. It was washed with brine, dried

over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure

and the residue was purified by preparative reverse phase HPLC (C18 column, acetonitrile/H 20)

to give the IGN-04 acid 42 (4.2 mg, y = 13%) as a white solid. MS (m/z): found 791.0 (M - H)-,

809.0 (M + H 2 0 - H) -, 815.2 (M + Na), 833.1 (M + H 2 0 + Na).

Compound 43:

0 O O O-1- OH O O,,,0'N

NHS '1, 0 P-J OEDC=N ji N / Cl N N/ OMe MeC- P OMe MeO 0 41 (IGN-03 acid) 0 0 43 (IGN-03 NHS ester) 0

[238] To a stirred solution of IGN-03 acid 41 (8.9 mg, 0.011 mmol) in anhydrous

dichloromethane (0.2 mL) was added N-hydroxysuccinimide (2.6 mg, 0.022 mmol), N-(3

dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (4.2 mg, 0.022 mmol) and a tiny

particle of dimethylaminopyridine. The mixture was stirred at room temperature overnight and

diluted with dichloromethane. It was washed with saturated ammonium chloride and brine, dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure.

The residue was purified through silica gel chromatography (Combiflash, CH 2Cl 2/MeOH, from

1:0 to 10:1) to give IGN-03 NHS eater 43 (7.9 mg, y = 79%) as a yellowish solid. Reverse phase

preparative HPLC (C18 column, CH3CN/H 20, extracted the product fractions with

dichloromethane) purification gave 3.2 mg white solid for 1 H NMR analysis. 1H NMR (400 Hz,

CDCI 3 ): 8.28 (d, J = 8.0 Hz, 2H), 7.87 (d, J = 4.0 Hz, 2H), 7.59 (s, 2H), 7.31-7.27 (in, 4H),

7.15-7.10 (in, 3H), 6.97 (s, 2H), 6.86 (s, 2H), 5.25 (d, J = 12.4 Hz, 2H), 5.18 (d, J = 12.4 Hz,

2H), 4.49 (dt, Ji = 10.8 Hz, J2 = 4.0 Hz, 2H), 4.04 (t, J= 5.6 Hz, 2H), 4.01 (s, 6H), 3.72 (dd, J1 =

16.8 Hz, J2 = 10.8 Hz, 2H), 3.51 (dd, Ji = 16.8 Hz, J2 = 4.0 Hz, 2H), 2.85 (bs, 4H), 2.72 (t, J =

6.8 Hz, 2H), 1.99-1.91 (in, 4H); HRMS (ESI, m/z): calc. 904.3194 (M + H) , found 904.3182.

Compound 44:

0 O Y OH 0 O-N 0 CN 0 NHS P ' 0 0 O .- EDC N0 N OCHC N - OMe Me '4 rl t 2 OMe MeG

42 (IGN-04 acid) 44 (IGN-04 NHS ester)

[239] Following the procedure to prepare compound 43, compound 44 was synthesized in 86%

yield as a yellowish solid. MS (m/z): found 944.2 (M + MeOH + Na), 976.2 (M + 2MeOH +

Na).

Compound 45 (IGN-07 acid):

N O NN OH

NJ, N 0 I~ -t o e 3 nO H, N N CICH 2 CH CI NN O)e Me - 80 OC - OMe Me 0 0 0 0 40 (IGN-07) 45 (IGN-07 acid)

[240] To a solution of compound 40 (14 mg, 0.017 mmol) in anhydrous 1,2-dichloroethane (0.5

mL) was added trimethyltin hydroxide (62 mg, 0.34 mmol). The mixture was heated at 78~82

°C (80 °C oil bath) and stirred overnight. The TLC (CH 2Cl 2/MeOH, 10:1) showed the

disappearance of the starting material. The reaction mixture was cooled to room temperature and

diluted with dichloromethane. It was washed with saturated ammonium chloride and brine, dried

over anhydrous sodium sulfate, filtered and evaporated to give IGN-07 acid 45 as a pale

yellowish solid (29.2 mg, contaminated with trimethyltin hydroxide). MS (m/z): found 804.1 (M

- H)~, 822.1 (M + H 2 0 - H) -, 828.2 (M + Na), 846.2 (M + H 2 0 + Na). It was used for next

step without purification.

Compound 46:

0

N OH N N0-NI 0 PN 0',N1 N-, NHS N 0 N K- N/\0H 2 01 2 / NN N't O1eMe -OMe MeO 0 0 0 0 45 (IGN-07 acid) 46 (IGN-07 NHS ester)

[241] To a stirred solution ofIGN-07 acid 45 from above reaction (0.017 mmol) in anhydrous

dichloromethane (0.5 mL) was added N-hydroxysuccinimide (6.1 mg, 0.051 mmol), N-(3

dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (9.8 mg, 0.051 mmol) and a tiny

particle of dimethylaminopyridine. The mixture was stirred at room temperature overnight and

diluted with dichloromethane. It was washed with saturated ammonium chloride and brine, dried

over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure.

The residue was purified through silica gel chromatography (Combiflash, CH 2C 2/MeOH, from

1:0 to 10:1) to give IGN-07 NHS eater 46 (9.1 mg, y = 59% for two steps from IGN-07) as a

yellowish solid. 1H NMR (400 Hz, CDCl 3 ): 6 8.25 (d, J = 7.6 Hz, 2H), 7.82 (d, J = 4.4 Hz, 2H),

7.55 (s, 2H), 7.26-7.18 (m, 5H), 7.09 (t, J = 7.6 Hz, 2H), 6.84 (s, 2H), 6.74 (s, 2H), 5.21 (d, J =

12.4 Hz, 2 H), 5.15 (d, J = 12.4 Hz, 2H), 4.46-4.42 (in, 2H), 3.98 (s, 6H), 3.72-3.64 (m, 2H),

3.44-3.37 (m, 4H), 2.95 (s, 3H), 2.74 (bs, 4H), 2.57 (t, J = 7.2 Hz, 2H), 1.95 (t, J = 7.2 Hz, 2H);

HRMS (ESI, m/z): calc. 903.3354 (M + H)+, found 903.3347.

Example 8

Compound 47:

0 11 MeOH- SSMe H N--SH + HC-S-SMeMH H2N SS 3N ai C1 0 47

[242] To a stirred solution ofcysteamine hydrochloride (568 mg, 5 mmol) in anhydrous

methanol (15 mL) was added S-methyl methanethiosulfonate (519 pl, 5.5 mmol) at 0 °C. The mixture was stirred at room temperature overnight. Triethylamine (1.4 mL, 10 mmol) was added and the solvents were removed under reduced pressure. The residue was dissolved in 50 mL of anhydrous dichloromethane and gave a 0.1 M solution of compound 47 in dichloromethane

(assuming 100% yield). An aliquot of the solution (0.2 mL) was used for next step reaction.

The rest of the solution was diluted with dichloromethane, washed with saturated sodium

bicarbonate and brine, dried over anhydrous sodium sulfate and filtered. The filtrate was

evaporated under reduced pressure and the residue was purified through silica gel

chromatography (dichloromethane/methanol, 10:1 with 1% triethylamine) to give compound 47

(82 mg, y = 13%, product lost in the aqueous work up due to its good water solubility) as a

colorless oil. 1H NMR (400 Hz, CDCl3): 6 3.02 (t, J = 6.4 Hz, 2H), 2.77 (t, J = 6.4 Hz, 2H), 2.41

(s, 3H), 1.34 (bs, 2H).

Compound 48 (IGN-08):

0 0

O -'N-,/SSMe oOH 47 H o o EDC o o F-N 0 N- DMAP r N OJ 0 /\N ~ MeO N/\ CH2CI2/\Oe MO

0 41 ° ° 48 (IGN-08)

[243] To a flask containing IGN-03 acid 41 (8.1 mg, 0.01 mmol) was added above 0.1 M

solution of compound 47 in anhydrous dichloromethane (0.2 mL). N-(3-dimethylaminopropyl)

N'-ethylcarbodiimide hydrochloride (3.8 mg, 0.02 mmol), triethylamine (1.4 tl, 0.01 mmol) and

a tiny particle of dimethylaminopyridine were added. The mixture was stirred at room temperature overnight and diluted with dichloromethane. It was washed with saturated ammonium chloride and brine, dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure and the residue was purified through preparative reverse phase HPLC (C18 column, acetonitrile/H 2 ) to give compound 48 (4.0 mg, y = 44%) as a white solid. 1H NMR (400 Hz, CDC 3): 6 8.25 (d, J = 8.0 Hz, 2H), 7.84 (d, J = 4.4 Hz, 2H), 7.57 (s,

2H), 7.29-7.24 (in, 4H), 7.10 (t, J = 7.6 Hz, 2H), 7.06 (s, 1H), 6.92 (s, 2H), 6.82 (s, 2H), 5.22 (d,

J = 12.8 Hz, 2H), 5.17 (d, J = 12.4 Hz, 2H), 4.46 (dt, J= 11.2 Hz, J2 = 4.4 Hz, 2H), 3.98 (bs,

8H), 3.69 (dd, Ji = 16.8 Hz, J2 = 10.8 Hz, 2H), 3.62 (d, J= 6.4 Hz, 1H), 3.58 (d, J= 6.0 Hz, IH),

3.48 (dd, Ji = 17.2 Hz, J 2 = 3.6 Hz, 2H), 2.82 (t, J = 6.4 Hz, 2H), 2.39 (s, 3H), 2.23 (t, J = 6.8 Hz,

2H), 1.80-1.78 (m, 4H); HRMS (ESI, m/z): calc. 912.3101 (M + H), found 912.3118.

Compound 49:

0 0 O H N" SSMe O'' IN/ S H

N 0, I N= OPN

N N~ pH6.5buffer \N '~ome Meo C O 48(IGN-08) 0 O 49 0

[244] To a suspension of tris(2-carboxyethyl) phosphine hydrochloride (TCEP HC, 3.8 mg,

0.013 mmol) in a drop of deioned water (~50 pL) was added saturated sodium bicarbonate

dropwise (25 tL) to adjust the pH to about 6~7, followed by addition of pH 6.5 buffer solution

(0.1 M phosphate buffer, 0.3 mL). The obtained mixture was added to the solution of compound

48 (IGN-08, 4.0 mg, 0.0044 mmol) in methanol (1.0 mL) and acetonitrile (1.0 mL). The solution was stirred at room temperature for 1.5 hours and diluted with pH 6.5 buffer and dichloromethane (the reaction was checked by mass spectra, which showed only the product signals). It was separated and the organic layer was washed with brine, dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure and the residue was purified by silica gel chromatography (Combiflash, dichloromethane/MeOH) to give product 49 as a pale yellow solid (2.7 mg, y = 71%). MS (m/z): found 864.0 (M - H)-, 932.0 (M

+ MeOH + 2H20 - H) -, 888.1 (M + Na), 920.2 (M + MeOH + Na), 952.2 (M + 2MeOH

+ Na)+.

Example 9

Compound 50:

H -NSH + S A) MeOH N

50

[245] To a stirred solution of cysteamine hydrochloride (227 mg, 2 mmol) in anhydrous

methanol (10 mL) was added aldrithiol (661 mg, 3 mmol). Reaction solution became clear

yellow from clear colorless after the addition of aldrithiol. The mixture was stirred at room

temperature for 21 hours. Triethylamine (279 pl, 2 mmol) was added and the solvents were

removed under reduced pressure. The residue was purified through silica gel chromatography

(Combiflash, dichloromethane/methanol, 1:0 to 15:1 with 0.1% triethylamine) to give compound

(301 mg, y = 81%) as a colorless oil. 'H NMR (400 Hz, CDCl 3 ): 6 8.52-8.49 (, 1H), 7.69

7.60 (m, 2H), 7.15-7.10 (m, 1H), 3.04 (t, J = 6.0 Hz, 2H), 2.92 (t, J = 6.0 Hz), 1.92 (bs, 2H).

Compound 51 (IGN-10):

0 0 ON S-S N oOH 50 H N ~EDC NI

/- N-P-,.k0 MeO N\HC 2 / OMe M 0 41 ° ° 51 (IGN-10)

[246] To a solution of IGN-03 acid 41 (from 0.05 mmol of IGN-03 without purification) in

anhydrous dichloromethane (1 mL) was added compound 50 (37 mg, 0.2 mmol), N-(3

dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (38 mg, 0.2 mmol) and a tiny

particle of dimethylaminopyridine. The mixture was stirred at room temperature overnight and

diluted with dichloromethane. It was washed with saturated ammonium chloride and brine, dried

over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure.

The residue was purified through silica gel chromatography (Combiflash,

dichloromethane/methanol, 1:0 to 5:1) to give 51 mg of yellow foam, which was further purified

through preparative reverse phase HPLC (C18 column, acetonitrile/H 2 0) to give compound 51

(7.4 mg, y = 15%) as a yellowish solid. H NMR (400 Hz, CDC 3 ): 6 8.50 (d, J = 4.4 Hz, 1H),

8.28 (d, J = 8.0 Hz, 2H), 7.87 (d, J = 4.4 Hz, 2H), 7.63-7.59 (m, 3H), 7.52 (d, J = 8.0 Hz, 1H),

7.31-7.21 (m, 4H), 7.14-7.09 (m, 4H), 6.96 (s, 2H), 6.85 (s, 2H), 5.23 (d, J= 12.8 Hz, 2H), 5.18

(d, J = 12.4 Hz, 2H), 4.49 (dt, J1 = 11.2 Hz, J2 = 4.4 Hz, 2H), 4.03-4.00 (m, 8H), 3.72 (dd, Ji =

16.8 Hz, J2 = 11.2 Hz, 2H), 3.60 (d, J = 5.6 Hz, 1H), 3.57 (d, J = 5.6 Hz, 1H), 3.50 (dd, J1 = 16.8

Hz, J2 = 3.6 Hz, 2H), 2.95 (t, J= 5.6 Hz, 2H), 2.30 (t, J = 6.4 Hz, 2H), 1.85-1.84 (in, 4H); HRMS

(ESI, m/z): calc. 975.3210 (M + H) , found 975.3190.

Compound 53:

Cu(NO2)2.5H 0 BnO ~ AC 2 , rt 3h 2 nO HN0

en K2CO3, MeO O M OH 52 52 MeOH/THF 52% 53

[247] To a stirred solution of 4-benzyloxy-3-methoxybenzyl alcohol 52 (2.5 g, 10 mmol) in

acetic anhydride (30 mL) was added copper(II) nitrate hydrate (2.7 g, 11 mmol) slowly in

portion at 0 °C. The obtained suspension continued to be stirred at 0 aC for 1 hour and at room

temperature for 3 hours. The reaction mixture was poured on ice/water and stirred for 1 hour. It

was filtered to collect the yellow solid, which was subsequently dissolved in MeOH/THF(1:1,

V/V, 30 mL). Potassium carbonate (2.1 g, 15 mmol) was added and the obtained mixture was

stirred at room temperature for 3 hours. It was concentrated under reduced pressure and the

residue was diluted with dichloromethane, washed with water and brine, dried over anhydrous

sodium sulfate and filtered. The filtrate was evaporated under reduced pressure and the residue

was purified through silica gel chromatography (CH 2Cl2/MeOH, 20:1, 18:1, 15:1) to give

compound 53 (1.50 g, y = 52%) as yellow solid. 1 1 NMR (400 Hz, CDCl 3): 6 7.78 (s, 1H), 7.48

7.33 (in, 5H), 7.20 (s, IH), 5.18 (s, 2H), 4.96 (s, 2H), 4.01 (s, 3H).

Example 10

Compound 123:

H NHBoc H NHBoc

H N N LiOH HN N N N O 1 THF/MeOH/H 2 0 N 0NO i MeO N 55-60 CC HO Yek 122 N 65% 123

[248] To a stirred solution of compound 122 (137 mg, 0.22 mmol) in THF (1.5 mL) and MeOH

(0.5 mL) was added a solution of lithium hydroxide monohydrate (46 mg, 1.1 mmol) in deioned

water (0.5 mL). The mixture was stirred in a 60 °C oil bath for 6 hours. It was cooled to room

temperature and diluted with ethyl acetate and water. The pH was adjusted to 4~5 with 5%

hydrochloric acid. The aqueous layer was extracted with ethyl acetate. The combined organic

layers were washed with saturated sodium bicarbonate and brine, dried over anhydrous sodium

sulfate and filtered. The filtrate was striped to give compound 123 (87.5 mg, y = 65%). MS

(m/z): found 606.1 (M - H)-.

Compound 124:

NHBoc H NHBoc N 0 N)F H \ HMe

H r 7 0N0MeC,,\/,. '0 N N EDCIDMAP 0 N 0 1 DMF0 0 123 70% 124

[249] To a solution of acid 123 (87.5 mg, 0.14 mmol) in anhydrous DMF (1 mL) was added

DMAP (21 mg, 0.17 mmol), methyl 5-aminovalerate hydrochloride (26 mg, 0.15 mmol) and

EDC (40 mg, 0.21 mmol). The mixture was stirred at room temperature overnight and diluted

with ethyl acetate. It was washed with saturated ammonium chloride, brine, saturated sodium

bicarbonate and brine, dried over anhydrous sodium sulfate and filtered. The filtrate was striped

and the residue was purified through silica gel chromatography (Combiflash,

dhchloromethane/MeOH) to give compound 124 (71 mg, y = 70%) as a yellow foam. 1H NMR

(400 Hz, CDCb): 6 9.07 (s, 1H), 8.62 (s, 1H), 8.40 (s, IH), 7.19 (s, IH), 7.17 (s, 1H), 7.09 (s,

1H), 7.00 (s, 1H), 6.74 (s, 1H), 6.62 (s, 3H), 6.46 (s, 1H), 3.94 (s, 3H), 3.85 (bs, 12H), 3.34-3.31

(m, 2H), 2.32 (t, J = 7.2 Hz, 2H), 1.68-1.55 (m, 4H), 1.48 (s, 9H); MS (ESI, m/z): found 721.0

(M + H)-.

Compound 125:

0 HO NMeO Br 0

0err~ K 2 C0 3 DMF MX r' 0 IBD monomer 8 93% 125

[250] To a solution ofIBD monomer 8 (118 mg, 0.4 mmol) and methyl 4-bromobutyrate (109

mg, 0.6 mmol) in anhydrous DMF (1.5 mL) was added potassium carbonate (111 mg, 0.8

mmol). The mixture was stirred at room temperature overnight and diluted with ethyl acetate,

washed with saturated ammonium chloride and brine. It was dried over anhydrous sodium

sulfate and filtered. The filtrate was striped under reduced pressure to give compound 125 (146 mg, y = 93%) as a yellow foam. H NMR (400 Hz, CDC1 3): 6 8.25 (d, J= 8.0 Hz, IH), 7.84 (d, J

= 4.4 Hz, 1H), 7.52 (s, 1H), 7.26-7.22 (m, 2H), 7.10-7.06 (m, 1H), 6.81 (s, 1H), 4.44 (dt, Ji =

10.8 Hz, J2 = 4.0 Hz, 1H), 4.15-4.07 (m, 2H), 3.92 (s, 3H), 3.68 (s, 3H), 3.67-3.64 (m, 1H), 3.46

3.43 (m, 1H), 2.55 (t, J = 7.2 Hz, 2H), 2.22-2.15 (m, 2H); MS (ESI, m/z): found 465.2 (M

+ MeOH + K)+.

Compound 126:

O Me 3SnOH O MeO O)-. CICH 2CH 2CI HO M N 80 0 C HO N ir64%i) 0eO MeO 0

125 64% 126

[251] The mixture of compound 125 (146 mg, 0.37 mmol) and trimethyltin hydroxide (669 mg,

3.7 mmol) in anhydrous 1,2-dichloroethane (2 mL) was heated to 80 °C (oil bath temperature)

and stirred at that temperature for 18 hours. The oil bath was removed and the mixture was

diluted with dichloromethane, washed with brine/5% HCl (0.5 mL), saturated sodium

bicarbonate and brine, dried over anhydrous sodium sulfate and filtered. The filtrate was striped

and the residue was purified through silica gel chromatography (Combiflash,

dichloromethane/MeOH) to give compound 126 (90 mg, y = 64%) as a yellow solid. H NMR

(400 Hz, CDCl 3): 6 8.26 (d, J = 8.0 Hz, 1H), 7.83 (bs, 1H), 7.54 (s, 1H), 7.30-7.25 (m, 2H), 7.11

(t, J= 7.6 Hz, 1H), 6.88 (s, 1H), 4.48 (dt, Ji = 11.2 Hz, J2 = 4.0 Hz, 1H), 4.16-4.13 (m, 2H), 3.94

(s, 3H), 3.71 (dd, Ji = 16 Hz, J2 = 11.2 Hz, 1H), 3.47 (d, J= 16 Hz, 1H), 2.60 (t, J = 6.4 Hz, 2H),

2.22-2.18 (in, 2H).

Compound 127 (IGN-11):

NHBoc O

Y 1. 4N HC1in dioxaneH N H N I 2.126, EDC/TEADMAP H 1 HN )- MeO N N , CHCl22 HNO 0

O MeO0 NN 124 1 0 127(IGN-11)

[252] To a flask containing compound 124 (71 mg, 0.099 mmol) was added 4N HCl in dioxane

(4 mL). The mixture was stirred at room temperature for 2 hours and striped under reduced

pressure. The residue was dissolved in anhydrous dichloromethane (1.5 mL). Compound 126

(42 mg, 0.11 mmol), triethylamine (14 pl, 0.1 mmol), EDC (38 mg, 0.2 mmol) and DMAP (1

mg, 0.0099 mmol) were added subsequently. The reaction mixture was stirred at room

temperature for 22 hours and diluted with dichloromethane, washed with saturated ammonium

chloride and brine. It was dried over anhydrous sodium sulfate and filtered. The filtrate was

striped under reduced pressure and the residue was purified through silica gel chromatography

(Combiflash, dichloromethane/MeOH) to furnish compound 127 (14 mg, y = 49%) as a yellow

solid. HRMS (ESI, m/z): calc. 983.4164 (M + H)-, found 983.4167.

Example 11

Preparation of IGN-03 NHS ester (compound 43) and IGN-07 NHS ester (compound 46) stock

solution:

[253] Solutions of IGN-03 NHS ester and IGN-07 NHS ester are made fresh to a 0.006 M stock

based on a molecular weight of 903.93 g/mole (IGN-03 NHS ester) or 902.95 (IGN-07 NHS ester) in dimethylacetamide (DMA). The stock solution is assayed spectrophotometrically using a reference extinction coefficient determined at 330 nm (330m= 15,231 M-1 cm-1 ).

Example 12

4-(tert-Butoxycarbonylamino)-1-methyl-iH-pyrrole-2-carboxylic acid

NHBOC NHBOC

O NaOH -10- HO \) O THF/H 2 0 O

[254] Methyl 4-(tert-butoxycarbonylamino)-1-methyl-IH- pyrrole-2-carboxylate (Eldon E.

Baird and Peter B. Dervan, J. Am. Chem. Soc. 1996, 118, 6141-6146) (5.0 g, 19.67 mmol) in

120 ml of 1:1 THF/H 20 was added 8 g of NaOH in 30 ml of water. The mixture was stirred

overnight, concentrated, diluted with water, extracted with EtAc/Hexane (1:1). The aqueous

solution was adjusted to pH 4.0 with 20% H 3PO4 and extracted with EtAc (4 x 60 ml). The

organic solutions were combined, dried over MgSO 4, filtered, evaporated and crystallized with

ethanol/EtAc/Hexane to afford 3.81 g (81%) of the title product. H NMR (CD 30D) 12.79 (s,

1H), 10.48 (br, 1H), 7.51 (s, 1H), 6.99 (s, 1H), 3.78 (s, 3H), 1.49 (s, 9H); "C NMR 158.47,

153.82, 123.64, 121.56, 109.58, 79.52, 37.06, 28.42; MS m/z- 239.2 (M-H).

4-(tert-butoxycarbonylamino)-1-methyl-iH-imidazole-2-carboxylic acid

NHBOC H NaOII HO N O 0 N THF/H 0O

[255] Methyl 4-(tert-butoxycarbonylamino)-1-methyl-IH-imidazole-2-carboxylate (5.0 g,

19.59 mmol) in 120 ml of 1:1 THF/H 20 was added 8 g of NaOH in 30 ml of water. The mixture

was stirred overnight, concentrated, diluted with water, extracted with EtAc/Hexane (1:1). The

aqueous solution was adjusted to pH 4.0 with 20% H 3PO4 and extracted with EtAc (4 x 60 ml).

The organic solutions were combined, dried over MgSO 4, filtered, evaporated and crystallized

with ethanol/EtAc/Hexane to afford 3.85 g (81%) of the title product. 'H NMR (DMSO) 9.32 (s,

1H), 7.29 (s, 1H), 3.57 (s, 3H), 1.42 (s, 9H); 13C NMR 172.45, 159.78, 136.93, 135.44, 132.85,

79.50, 35.57, 28.07; MS m/z- 240.8 (M-H).

I-Methyl-4-nitro-1H-pyrrole-2-carboxylic acid

NO2 NO2

O NaOH HO O O

[256] Methyl1-methyl-4-nitro-1H-pyrrole-2-carboxylate(5.0g,27.17mmol)in120mlof1:1

THF/H 20 was added 8 g of NaOH in 30 ml of water. The mixture was stirred overnight,

concentrated, diluted with water, extracted with EtAc/Hexane (1:1). The aqueous solution was

adjusted to pH 3 ~4 with 20% H3PO4 and extracted with EtAc (4 x 60 ml). The organic solutions

were combined, dried over MgSO 4, filtered, evaporated and crystallized with

ethanol/EtAc/Hexane to afford 4.06 g (88%) of the title product. IH NMR (DMSO) 13.12 (s,

1 H), 8.21 (s, 1 H), 7.25 (s, 1 H), 3.91 (s, 3H); 1C NMR 160.97, 134.01, 129.16, 123.81, 111.38,

37.47; MS m/z- 169.1 (M-H).

MethylI-methyl-4-(1-methyl-4-nitro-1IH-pyrrole-2-carboxamido)-1IH-pyrrole-2-carboxylate

NO2 NH2HCl NO 2 H2/Pd/C 0

. -r 'O

" 0 N HC 0 O 0N O EDC/DMA O N

[257] Ina hydrogenation bottle was added methyl 1-methyl-4-nitro-H-pyrrole-2-carboxylate

(3.0 g, 16.30 mmol), 80 ml of THF, 405 mg of 10% Pd/C and 1.3 ml of HC (conc.). After

evacuation under vacuum the bottle was placed under 30 psi hydrogen and shaken for 5 hours.

The mixture was filtered through celites, evaporated to dryness without further purification. To

the dry mixture was added 1-methyl-4-nitro-1H-pyrrole-2-carboxylic acid (2.75 g, 16.18 mmol),

ml of DMA, EDC (8.51 g, 44.27 mmol) and DIPEA (2.80 ml, 16.10 mmol). The mixture was

stirred under Ar overnight, concentrated, diluted with THF/EtAc (1:2, 150 ml), and washed 1M

NaH 2PO4/NaCl(cone) and NaHCO3 (conc) separately. The organic layer was separated and

dried over MgSO 4, filtered concentrated and crystallized with THF/H 20 to afford 3.74 g (75%)

of the title product. I H NMR (DMSO) 10.25 (s, IH), 8.17 (s, IH), 7.25 (s, I H), 6.52 (s, IH), 6.08

(s, 1H), 3.90 (s, 3H), 3.78 (s, 3H), 3.56 (s, 3H); "C NMR 157.87, 156.84, 133.76, 128.16,

123.39, 119.13, 118.18, 111.83, 107.50, 104.17, 51.55, 37.41, 36.03; MS m/z+ 329.1 (M+Na).

Methyl 4-(4-(tert-butoxycarbonylamino)-1-methyl-iH-pyrrole-2-carboxamido)-1 -methyl-1H

imidazole-2-carboxylate

H H0H N 0 NH 2HCH H O

- ' EtAc r)N -0 k 0 N O EDC/DMA O N

[258] Methyl4-(tert-butoxycarbonylamino)-1-methyl-IH-imidazole-2-carboxylate(2.50g,

9.80 mmol) in 30 ml of EtAc was added 6 ml of HCI (conc.). After stirring for 45 min, the

mixture was diluted with ethanol and toluene, concentrated and co-evaporated with

ethanol/toluene (1:1, 3x50 ml) to dryness without further purification. To the dry compound was

added 4-(tert-butoxycarbonylamino)-1-methyl-1H-pyrrole-2-carboxylic acid (2.35 g, 9.8 mmol),

EDC ( 5.60 g, 29.1 mmol), DIPEA (1.70 ml, 9.8 mmol) and 80 ml of DMA. The mixture was

stirred under Ar overnight, concentrated, diluted with THF/EtAc (1:2, 150 ml), and washed IM

NaH 2PO 4/NaCl (conc) and NaHCO3 (conc) separately. The organic solvent layer was separated

and dried over MgSO 4 , filtered, concentrated and purified on SiO2 chromatography eluted with

EtAc/DCM (1:25 to 1:15) to afford 2.72 g (73%) of the title product. H NMR (DMF-d7) 10.27

(s, 1H), 9.08 (s, 1H), 7.41 (s, 1H), 7.32 (s, 1H), 7.07 (s, 1H), 4.10 (s, 3H), 3.93 (s, 3H), 3.84 (s,

3H), 1.47 (s, 9H); 13 C NMR 162.62, 161.20, 153.82, 145.32, 144.12, 132.56, 128.46, 124.39,

119.83, 79.51, 52.75, 36.06, 35.83, 28.88; MS m/z+ 400.2 (M+Na).

Methyl 4-(4-(tert-butoxycarbonylamino)-1-methyl-iH-imidazole-2-carboxamido)-1-methyl-IH

imidazole-2-carboxylate

H H0H N O NH 2HC N N NH O 0 HO 0 O N

0 O EDC/DMA O N

[259] Methyl4-(tert-butoxycarbonylamino)-1-methyl-IH-imidazole-2-carboxylate(2.50g,

9.80 mmol) in 30 ml of EtAc was added 6 ml of HC (cone.). After stirred for 30 min, the

mixture was diluted with ethanol and toluene, concentrated and co-evaporated with

ethanol/toluene (1:1, 3x50 ml) to dryness compound without further purification. To the dryness

compound was added 4-(tert-butoxycarbonylamino)-1-methyl-IH-imidazole-2-carboxylic acid

(2.36 g, 9.8 mmol), EDC (5.90 g, 30.7 mmol), DIPEA (1.70 ml, 9.8 mmol) and 80 ml of DMA.

The mixture was stirred under Ar overnight, concentrated, diluted with THF/EtAc (1:2, 150 ml),

and washed IM NaH 2PO 4/NaC1 (cone) and NaHCO 3 (cone) separately. The organic solvent layer

was separated and dried over MgSO 4 , filtered, concentrated and purified on SiO2

chromatography eluted with EtAc/DCM (1:25 to 1:15) to afford 2.65 g (71.5%) of the title

product. I H NMR (DMSO) 11.17 (s, 1H), 10.48 (s, 1H), 7.58 (s, 1H), 7.32 (s, 1H), 4.01 (s, 3H),

3.94 (s, 3H), 3.92 (s, 3H), 1.45 (s, 9H); C NMR 160.60, 157.30, 135.92, 135.45, 132.86,

126.12, 114.83, 79.50, 52.70, 35.58, 34.92, 28.08;MS m/z+ 401.8 (M+Na).

1-Methyl-4-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)-IH-pyrrole-2-carboxylicacid

H NO2 H NO2 LiOH O A HO~SYQ 0 0

[260] Methyl 1-methyl-4-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)-1H-pyrrole-2

carboxylate (2.0 g, 6.53 mmol) in 50 ml of DMA was added 2 g of LiOH in 30 ml of water. The

mixture was stirred overnight, concentrated, diluted with water, extracted with EtAc/Hexane

(1:1). The aqueous solution was adjusted to pH 4.0 with 20% H 3PO4 to form precipitates. The

precipitates were filtered, washed with water and dried over P20 5 with vaccum to afford 1.4 g

(73%) of the title product. H NMR (DMF-d7) 10.34 (br, 1H), 8.17 (s, 1H), 7.62 (s, 1H), 7.51 (s,

I H), 7.00 (s, 1H), 4.09 (s, 1H), 3.91 (s, ] H); 1C NMR 158.47, 135.61, 129.11, 127.77, 123.65,

121.57, 121.50, 109.48, 108.52, 38.38, 37.05; MS m/z- 291.0 (M-H).

4-(4-(tert-Butoxycarbonylamino)-1-methyl-iH-pyrrole-2-carboxamido)-1-methyl-iH-imidazole

2-carboxylic acid

H H H N OH NVO N LiOH NJ\ 0 DMA HO N O Ori 0 O -1

[261] Methyl4-(4-(tert-butoxycarbonylamino)-1-methyl-iH-pyrrole-2-carboxamido)-1-methyl

1H-imidazole-2-carboxylate (2.0 g, 5.30 mmol) in 50 ml of DMA was added 2 g of LiOH in 30

ml of water. The mixture was stirred overnight, concentrated, diluted with water, extracted with

EtAc/Hexane (1:1). The aqueous solution was adjusted to pH 4.0 with 20% H 3PO 4 to form

precipitates. The precipitates were filtered, washed with water and dried over P205 with vaccum

to afford 1.44 g (75%) of the title product. '1H NMR (DMSO) 10.41 (br, IH), 9.07 (s, IH), 7.48

(s, 1H), 6.97 (s, 1H), 6.88 (s, 1H), 3.92 (s, 1H), 3.81 (s, 1H), 1.47 (s, 9H); 3 C NMR 160.46,

158.42, 152.85, 145.21, 135.81, 129.11, 127.77, 122.39, 121.57, 113.58, 79.81, 36.06, 35.25,

28.17; MS m/z- 362.1 (M-H).

Methyl 4-(4-(4-(4-(tert-butoxycarbonylamino)-1-methyl-IH-pyrrole-2-carboxamido)-1-methyl

1H-imidazole-2-carboxamido)-1-methyl-iH-pyrrole-2-carboxamido)-1-methyl-iH-pyrrole-2

carboxylate

H H NO 2 1). Pd/C/H 2/DMA H N N O

2). EDC/ H HO 0 N O o N

[262] Ina hydrogenation bottle was added methyl 1-methyl-4-(1-methyl-4-nitro-IH-pyrrole-2

carboxamido)-1H-pyrrole-2-carboxylate (1.0 g, 3.27 mmol), 20 ml of THF, 305 mg of 10% Pd/C

(50% wet) and 0.25 ml of HCl (cone.). After evacuation under vacuum the bottle wasplaced

under 50 psi hydrogen and shaken for 4 hours. The mixture was filtered through celite,

evaporated to dryness without further purification. To the dried mixture was added 4-(4-(tert

butoxycarbonylamino)-1-methyl-iH-pyrrole-2-carboxamido)-1-methyl-iH-imidazole-2

carboxylic acid (1.15 g, 3.16 mmol), 10 ml of DMA, EDC (2.0 g, 10.4 mmol) and DIPEA (0.70

ml, 4.02 mmol). The mixture was stirred under Ar overnight, concentrated, diluted with

Hexane/EtAc (1:1, 10 ml) and water 10 ml to form precipitates. The precipitates were filtered,

washed IM NaH 2PO 4, 1 M NaHCO3 and water, dried over P 2 0 5 with vacuum to afford 1.61 g

(82%) of the title product. H NMR (DMF-d7) 10.29 (s, 1H), 10.20 (s, 1H), 10.12 (s, IH), 9.08

(s, 1H), 7.58 (s, 1H), 7.47 (d, 1H, J = 1.7 Hz), 7.26 (d, 1H, J = 1.5 Hz), 7.15 (d, 1H, J = 1.5 Hz),

6.98 (s, 1H), 6.91 (d, 1H, J = 1.8 Hz), 6.86 (s, 1H), 3.97 (s, 3H), 3.82 (s, 3H), 3.73 (s, 3H), 3.56

(s, 3H), 1.45 (s, 9H); 3 C NMR 162.16, 160.05, 159.90, 157.20, 154.31, 137.88, 135.35, 124.56,

124.39, 124.24, 123.09, 120.09, 119.82, 115.32,105.58, 102.27, 79.31, 51.51, 38.13, 36.01,

35.80, 35.08, 28.79; MS m/z+ 644.2 (M+Na).

Methyl 1-methyl-4-(I-methyl-4-(1-methyl-4-(1-methyl-4-nitro-IH-pyrrole-2-carboxamido)-1H

pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxylate

H H H NO2 1). Pd/C/H 2/DMA

\O NN2 )0 N 0 0 0HOn\ 0N

[263] In a hydrogenation bottle was added methyl -ethyl-4-(1-methyl-4-nitro-H-pyrrole-2

carboxamido)-1H-pyrrole-2-carboxylate (2.0 g, 6.53 mmol), 80 ml of DMA, 500 mg of 10%

Pd/C (50% wet) and 0.4 ml of HCl (conc.). After evacuation under vacuum, the bottle was

placed under 50 psi hydrogen and shaken for 4 hours. The mixture was filtered through celite,

evaporated to dryness without further purification. To the dry mixture was added1-methyl-4-(1

methyl-4-nitro-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxylic acid (1.49 g, 5.10 mmol),

ml of DMA, EDC (4.0 g, 20.8 mmol) and DIPEA (1.0 ml, 5.75 mmol). The mixture was stirred under Ar overnight, concentrated, diluted with Hexane/EtAc (1:1, 10 ml) and water 10 ml to form precipitates. The precipitates were filtered, washed IM NaH 2PO 4 , 1 M NaHCO 3 and water, dried over P2 0 5 under vacuum to afford 2.13g (76%) of the title product. 1H NMR

(DMSO) 10.28 (s, 1H), 10.25 (s, 1H), 9.78 (s, 1H), 8.18 (s, 1H), 7.86 (s, 1H), 7.52 (s, 1H), 7.31

(d, 1H, J = 1.7 Hz), 7.25 (s, 1H), 7.23 (s, 1H), 7.17 (d, 1H, J = 1.5 Hz), 6.98 (s, 1H), 6.71 (s, 1H),

4.02 (s, 3H), 3.94 (s, 3H), 3.83 (s, 3H), 3.73 (s, 3H), 3.56 (s, 3H), 1.47 (s, 9H); "C NMR 160.78,

158.93, 158.06, 157.81, 135.25, 127.28, 126.36, 123.78, 122.57, 121.91, 121.40, 120.94, 119.65,

110.73, 108.39, 107.34, 103.75, 80.81, 51.57, 39.74, 38.52, 38.22, 37.08, 28.63; MS m/z+ 573.2

(M+Na).

4-(4-(4-(4-(tert-butoxycarbonylamino)-1-methyl-IH-pyrrole-2-carboxamido)-1-methyl-IH

imidazole-2-carboxamido)-1-methyl-IH-pyrrole-2-carboxamido)-1-methyl-IH-pyrrole-2

carboxylic acid

H H H N o H _Nyo N N NDH N N o -- HO o H I

[264] Methyl4-(4-(4-(4-(tert-butoxycarbonylamino)-1-methyl-iH-pyrrole-2-carboxamido)-1

methyl-iH-imidazole-2-carboxamido)-1-methyl-iH-pyrrole-2-carboxamido)-1-methyl-iH

pyrrole-2-carboxylate (510 mg, 0.82 mmol) in 10 mlofDMA was added 0.8 gof LiOHin10 ml

of water. The mixture was stirred overnight, concentrated, diluted with water, extracted with

EtAc/Hexane (1:1). The aqueous solution was adjusted topH 4.0 with 2000H 3 PO4 to form precipitates. The precipitates were filtered, washed with water and dried over P205 under vaccum to afford 363 mg (73%) of the title product. H NMR (DMF-d7) 10.31 (s, 1H), 10.18 (s, 1H),

10.11 (s, 1H), 9.10 (s, IH), 7.58 (s, IH), 7.54 (s, 1H), 7.41 (s, IH), 7.33 (s, IH), 7.21 (s, IH),

7.10 (s, 1H), 7.06 (s, 1H), 4.10 (s, 1H), 3.98 (s, 1H), 3.95 (s, 1H), 3.93 (s, 1H), 1.47 (s, 9H); 3 C

NMR 162.16, 160.05, 159.90, 157.20, 154.31, 137.88, 135.35, 124.56, 124.39, 123.51, 123.09,

121.76, 120.09, 119.83, 118.96, 115.32, 109.53, 105.58, 102.27, 79.32, 38.13, 36.02, 35.81,

34.88, 28.79; MS m/z- 606.2 (M-H).

S-3-(tert-butoxycarbonylamino)propyl ethanethioate

4 H PPh3 /DIAD _.L H ONOH HSAc/DCM SAc O 212 75% O 213

[265] tert-Butyl 3-hydroxypropylcarbamate (3.22 g, 18.37 mmol) in 100 mlof DCM at0°C was

added thiolacetic acid (2.0 ml, 26.73 mmol) and triphenylphosphine (7.0 g, 26.73 mmol) under

Ar. After stirred at 0C for 15 min, DIAD (6.0 ml, 28.93) was added. The mixture was stirred at

C for 2 h then RT overnight. The mixture was concentrated, diluted with 120 ml of

EtAc/Hexane (1:2), filtered through celite. The solution was washed with NaHCO 3 (conc.)/NaCI

(conc.) and 1 M NaH 2 PO 4 respectively, dried over MgSO 4 , filtered, evaporated and purified on

SiO2 chromatography eluted with EtAc/Hexane (1:7 to 1:6) to afford 3.22 g (75%) of the title

compound. 'H NMR (CDCl3) 3.09 (t, 2H, J= 6.5 Hz), 2.84 (t, 2H, J = 6.9 Hz), 2.27 (s, 3H), 1.69

(dt, 2H, J = 6.8, 13.5 Hz),1.38 (s, 9H); 1 C NMR196.35, 156.16, 79.50, 39.26, 30.79, 30.24,

28.61, 26.44; MS m/z+ 256.0 (M + Na).

S-3-(tert-butoxycarbonyl(methyl)amino)propylethanethioate

H Nal/TIHF ON SAc C I 0 N SAc CHJ, OC Y 213 0% O 214

[266] To a solution ofNaH (0.57 g, 60%, 14.25 mmol) in 20mlof THF at 0C was added S-3

(tert-butoxycarbonylamino)propy ethanethioate (1.25 g, 5.36 mmol) under Ar. After stirring at

C for 30 min, Mel (1.0 ml, 16.06 mmol) was added to the mixture. Stirring was continued at

C for 2 h then RT overnight. The mixture was concentrated, redisolved in 120 ml of

EtAc/Hexane (1:2), washed with 1 M NaH 2PO 4 NaCl (cone.), dried over MgSO 4 , filtered,

evaporated and purified on SiO 2 chromatography eluted with EtAc/Hexane (1:7) to afford 1.121

g (85%) of the title compound. H NMR (CDCl3) 3.69 (t, 2H, J = 7.3 Hz), 2.41 (t, 2H, J= 7.3

Hz), 2.39 (s, 3H), 2.03 (s, 3H), 1.76 (m, 2H),1.47 (s, 9H);1 3 C NMR 173.21, 153.39, 83.28,

43.67, 31.84, 28.26, 28.19, 27.11, 15.65; MS m/z+ 270.0 (M + Na).

S-3-(Methylamino)propyl ethanethioate hydrogen chloride salt

I 20%HCl/EtAc | /0 yNHACNASAC 0 HCl 214 215

[267] S-3-(tert-Butoxycarbonyl(methyl)amino)propyl ethanethioate (206 mg, 0.834 mmol) in 4

ml of EtAc was added 1.0 ml of HCI (conc.) at RT. The mixture was stirred at RT for 1 h, diluted

with ethanol/toluene (6 ml, 1:1), evaporated and co-evaporated with ethanol/toluene (3 x 10 ml),

crystallized with ethanol/EtAc/Hexane, filtered, and dried over a vacuum to afford 135 mg (88%) of the title compound. 1 H NMR (CDCl 3) 9.70 (br, 1H), 8.56 (br, IH), 3.42 (in, 2H), 2.52 (in,

2H), 2.35 (s, 3H), 2.05 (s, 3H), 1.88 (m, 2H); 3 C NMR 174.64,40.57, 31.57,27.69,20.94,

15.62; MS m/z+ 170.0 (M + Na), 148.10 (M + H).

tert-Butyl 2-(pyridin-2-yldisulfanyl)ethylcarbamate (217)

O .'NHICH2CH13SII PYSSPY/f31 H0 II N--Ss S 03 IMNaH 2PO 4 0 0 216 pH1 6 .8 217

[268] To the solution of 2,2'-dithiolpyridine (3.97 g, 18.02 mmol) in 100 ml of methanol and

ml of1 M NaH 2PO 4, pH 6.8 was added tert-Butyl 2-mercaptoethylcarbamate (1.00 g, 5.65

mmol) in 50 ml of methanol. The mixture was stirred under Ar overnight, concentrated,

extracted with dichloromethane, dried over MgSO 4 , filtered, evaporated and purified on SiO 2

chromatography eluted with EtAc/Hexane (1:10 to 1:6) to afford 1.341 g (83%) of the title

compound. 'H NMR (CDCl3) 8.39 (in, 1H), 7.56 (in, 1H), 7.49 (i, 1H), 7.03 (m, 1H), 7.00 (m,

1H), 3.34 (in, 2H), 2.84 (in, 2H),1.37 (s, 9H);1 3 C NMR 160.05, 159.39, 159.07, 149.87, 137.21,

120.78, 79.48, 39.58, 38.96, 28.57; MS m/z+ 309.2 (M + Na).

2-(pyridin-2-yldisulfanyl)ethanamine

+O N-,,S~s N 20%HC/EtAc H2 N,-S-S

217 218

[269] tert-Butyl 2-(pyridin-2-yldisulfanyl)ethylcarbamate (1.06 g, 3.70 mmol) in 16 ml of EtAc

was added 4.0 ml of HCl (conc.) at RT. The mixture was stirred at RT for 0.5 h, diluted with ethanol/toluene (6 ml, 1:1), evaporated and co-evaporated with ethanol/toluene (3 x 10 ml), crystallized with ethanol/EtAc/Hexane, filtered, and dried over a vaccum to afford 135 mg (88%) of the title compound. 'H NMR (CD 3 OD) 7.58 (in, 1H), 7.47 (m, 1H), 7.06 (m, 1H), 6.83 (m,

1H), 3.34 (m, 2H), 3.02 (m, 2H); "C NMR 158.69, 149.07, 137.81, 122.48, 120.98, 39.52,

36.94; MS m/z+ 187.10 (M + H).

Methyl 4-bromobutanoate (223)

Br -s 1 Cl CH 30H, Br OC1 3 o 0 222 223

[270] 4-Bromobutanoyl chloride (3.1 ml, 25.28 mmol) was added to 15 ml of dry metanol at

°C. Stirring was continued at 0Cunder Ar for 2 h then at RT overnight. The mixture was

evaporated, diluted with EtAc/Hexane (1:5), filtered through SiO 2 gel, and evaporated to afford

4.50 g (99%) of the title compound. . 1H NMR (CDC 3) 3.65 (s, 3H), 3.43 (t, 2H, J = 6.5 Hz),

2.47 (t, 2H, J = 7.1 Hz), 2.13 (dt, 2H, J = 6.7, 13.6 Hz); 3 C NMR 173.08, 51.84, 32.82, 32.34,

27.89; MS m/z+ 203.0 (M + Na).

(Z)-methyl4-(7-methoxy-2',3'-benzo[e]-5-oxo-5,11a-dhydro-1H-benzo[e]pyrrolo[1,2

a][1,4]diazepin-8-yloxy)butanoate

O N... OCH3 H3 NH3CO Br 0 CsCO3/Acctonc H3 CO 4N / \ 223 224 0

[271] (Z)-2,3-Benzo-8-hydroxy-7-methoxy-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)

one (60 mg, 0.20 mmol) in 4 ml of acetone was added Cs 2 CO3 (90 mg, 0.28 mmol), followed

added methyl 4-bromobutanoate (50 mg, 0.27 mmol). The mixture was stirred under Ar over

night, evaporated, and purified on Si02 chromatography eluted with EtAc/DCM (1:5 to 1:3) to

afford 50.1 mg (63%) of the title compound. H NMR (CDCl3) 8.19 (d, 1H, J = 7.9 Hz), 7.80 (d,

1H, J= 4.2 Hz), 7.48 (s, 1H), 7.19 (in, 2H), 7.03 (d, 1H, J = 7.4 Hz), 6.77 (s, 1H), 4.41 (in, 1H),

3.88 (s, 3H), 3.64 (in, 2H), 3.62 (s, 3H), 3.42 (dd, 1H, J = 3.4, 13.7 Hz), 2.50 (t, 2H, J = 7.2 Hz),

2.12 (t, 2H, J = 6.8 Hz); 1C NMR, 173.64, 164.12, 163.24, 152.25, 148.41, 142.28, 140.34,

129.69, 128.39, 124.97, 120.85, 117.15, 112.15, 110.68, 68.08, 56.40, 55.18, 51.90, 32.84, 30.64,

24.50; MS m/z+ 187.10 (M + H). MS m/z+ 417.2 (M + Na), 435.2 (M + Na + H 2 0).

4-(7-methoxy-2,3-benzo[e]-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8

yloxy) butanoic acid

0 0 H3CO Me 3 SnOH % 04N / CH2 CICH2 CI HO 3 O]~/ 3 224 0 -2250

[272] (Z)-methyl4-(7-methoxy-2',3'-benzo[e]-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2

a][1,4]diazepin-8-yloxy)butanoate (41 mg, 0.104 mmol) and trimethyltin hydroxide (302 mg,

1.67 mmol) in 15 ml of dichloroethane was refluxed at 85°C under Ar overnight. The mixture

was washed with 1 M NaH 2PO 4, pH 3.5, dried over MgSO 4 , filtered, evaporated and purified on

SiO2 chromatography eluted with EtAc/DCM/HCl (1:25:0.01%) to afford 30 mg (76%) of the

title compound. 1H NMR (CDCl3) 8.18 (d, 1H, J= 7.9 Hz), 7.85 (in, 1H), 7.46 (s, 1H), 7.20 (in,

2H), 7.04 (d, 1H, J = 7.4 Hz), 6.81 (s, 1H), 4.40 (m, IH), 3.86 (s, 3H), 3.63 (m, 2H), 3.23 (dd,

1H, J = 10.2,16.3 Hz), 2.52 (t, 2H, J = 7.2 Hz),2.12 (t, 2H, J = 6.8 Hz); 13C NMR, 173.64,

164.12, 163.24, 152.25, 148.41, 142.28, 140.34, 129.69, 128.39, 125.10, 120.85, 117.19, 112.15,

110.68, 67.94, 56.43, 55.18, 31.81, 30.64, 24.21; MS m/z- 397.0 (M + H 2 0 - H).

4-{[4-({4-[4-(4-(7-methoxy-2',3'-benzo[e]-5-oxo-5,1la-dihydro-1H-benzo[e]pyrrolo[1,2

a][1,4]diazepin-8-yloxy)butyrylamino]-1-methyl-1H-pyrrole-2-carbonyl}amino)-1-methyl-1H

imidazole-2-carbonyl]amino}-1-methyl-IH-pyrrole-2-carbonyl]-amino}-I-methyl-IH-pyrrole-2

carboxylic acid methyl ester (226)

H NHBOC H

NAN 2) 20%HCOEIAc N N O I 2).225/EDC/DMA NH 3 CONpN/

O 181 0 N 226

[273] Tomethyl4-(4-(4-(4-(tert-butoxycarbonylamino)-1-methyl-iH-pyrrole-2-carboxamido)

1-methyl-iH-imidazole-2-carboxamido)-1-methyl-iH-pyrrole-2-carboxamido)-1-methyl-iH

pyrrole-2-carboxylate (15 mg, 0.024 mmol) in 4 ml of EtAc was added 1.0 ml of HC (conc.).

The mixture was stirred at RT for 0.5 h, diluted with ethanol/toluene (6 ml, 1:1), evaporated and

co-evaporated with ethanol/toluene (3 x 10 ml), and dried over a vacuum. The solid compound

was used directly without further purification. To the solid was added 4-(7-methoxy-2',3'

benzo[e]-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)butanoic acid (6

mg, 0.015 mmol), EDC (40 mg, 0.21 mmol), DIPEA (4 ul, 0.023 mmol) and 1 ml of DMA. The

mixture was stirred under Ar over night, evaporated, and purified on HPLC preparative C-18

column (<D10 mm x 200 mm column, flow rate 9 mL/min and a gradient solvent system going from 75:25 solvent A:B at time 0-5 min to 40:60 A:B at 15 min then to 20:80 A:B at 25 min until to 10:90 A:B at 30 min. Solvent A - water, solvent B - acetonitrile/dioxane (1:2)) and lyophilized to afford a white solid (4.2 mg (30%) of the title compound). MS m/z- 900.3 (M

+ H 2 0 - H).

S-3-(4-(4-(4-(4-(tert-butoxycarbonylamino)-1-methyl-iH-pyrrole-2-carboxamido)-1-methyl-iH

imidazole-2-carboxamido)-1-methyl-IH-pyrrole-2-carboxamido)-N,1-dimethyl-1H-pyrrole-2

carboxamido)propyl ethanethioate (227).

NIIBOC H NHBOC H

HN~~ 2). NHS/FDC/DMA IN 1)N 0 N 2. HHC AcS0N N HO 0 197 227

[274] 4-(4-(4-(4-(tert-butoxycarbonylamino)-i-methyl-iH-pyrrole-2-carboxamido)-i-methyl

1H-imidazole-2-carboxamido)-1-methyl-IH-pyrrole-2-carboxamido)-1-methyl-IH-pyrrole-2

carboxylic acid (256 mg, 0.42 mmol), NHS (60 mg, 0.52 mmol) and EDC (500 mg, 2.60 mmol)

in 4 ml of DMA were stirred under Ar for2 h, then S-3-(methylamino)propyl ethanethioate

hydrogen chloride salt (76.5 mg, 0.42 mmol) was added and the mixture was kept stirring for 24

h, evaporated and purified on Si02 chromatography eluted with THF/DCM (1:5 to 1:4) to afford

198 mg (64%) of the title compound. '1HNMR (DMSO) 10.21 (s, 1H), 10.09 (s, 1H), 10.06 (s,

1H), 9.08 (s, 1H), 7.76 (d, 1H, J = 1.7 Hz), 7.52 (s, 1H), 7.28 (s, iH), 7.21 (d, 1H, J = 1.7 Hz),

6.97 (s, 1H), 6.87 (s, 1H), 3.98 (s, IH), 3.86 (s, 3H), 3.75 (s, 3H), 3.73 (s, 3H), 3.66 (in, 2H),

2.85 (s, 3H), 2.60 (m, 2H), 2.01 (s, 3H), 1.45 (s, 9H); 1C NMR 173.31, 162.16, 160.05, 159.90,

157.20, 154.31, 137.88, 135.35, 124.56, 124.39,123.51, 123.09, 121.76, 120.09, 119.83, 118.96,

115.32, 109.53, 105.58, 102.27, 79.32, 43.67, 38.13, 36.02, 35.81, 34.88, 31.84, 28.79, 28.26,

28.21, 27.01; MS m/z+ 759.2 (M + Na).

(Z)-S-3-(4-(4-(4-(4-(4-(7-methoxy-2,3-benzo[e]-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2

a][1,4]diazepin-8-yloxy)butanamido)-1-methyl-iH-pyrrole-2-carboxamido)-1-methyl-iH

imidazole-2-carboxamido)-1-methyl-iH-pyrrole-2-carboxamido)-N,1-dimethyl-1H-pyrrole-2

carboxamido)propyl ethanethioate

H H N O H NN/ N N 1). 20oHC1/EtAc N NI C Ac N 2).225/EDC/DMA AcS N228 o N 1 2' 0 2

[275] S-3-(4-(4-(4-(4-(tert-butoxycarbonylamino)-1-methyl-iH-pyrrole-2-carboxamido)-1

methyl-IH-imidazole-2-carboxamido)-1-methyl-I1-pyrrole-2-carboxamido)-N,1-dimethyl-IH

pyrrole-2-carboxamido)propyl ethanethioate (227) (27 mg, 0.037 mmol) was stirred in 2 ml of

dioxane and 0.5 ml of HCl (cone) for 15 min, diluted with ethanol/toluene (6 ml, 1:1),

evaporated and co-evaporated with ethanol/toluene (4 x 10 ml), crystallized with

EtOHDCM/Hexane and dried over a vacuum to afford 21 mg of solid. The solid compound was

used directly without further purification. To the solid was added 4-(7-methoxy-2,3-benzo[e]-5

oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy) butanoic acid (10 mg,

0.026 mmol), EDC (101 mg, 0.52 mmol), DIPEA (5 ul, 0.028 mmol) and 2 ml of DMA. The

mixture was stirred overnight, evaporated, diluted with DCM, washed with1 M NaH2 PO 4/NaCl

(conc), pH 4.0, dried over MgSO 4 , filtered, evaporated and purified on HPLC preparative C-18

column (<D10 mm x 200 mm column, flow rate 9 mL/min and a gradient solvent system going from 75:25 solvent A:B at time 0-5 min to 40:60 A:B at 15 min then to 20:80 A:B at 25 min until to 10:90 A:B at 30 min. Solvent A - water, solvent B - acetonitrile/dioxane(1:2)) and lyophilized to afford a white solid 8.2 mg (32%) of the title compound. MS m/z- 1015.1 (M

+ H 2 0 - H), UV 8(1=305 nm)= 32800 M- cm-1

. tert-Butyl1-methyl-5-(1-methyl-2-(1-methyl-5-(1-methyl-5-(2-(pyridin-2

yldisulfanyl)ethylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H

imidazol-4-ylcarbamoyl)-1H-pyrrol-3-ylcarbamate(229)

H NHBOC H NIIBOC

H 1). NHS/EDC/DMA H HO O N 2. H ~T\ pys10 HN;A(i\ ngsp 2

1 197 5 ~ PS 1*HN~ 2

[276] 4-(4-(4-(4-(tert-butoxycarbonylamino)-1-methyl-iH-pyrrole-2-carboxamido)-1-methyl

1H-imidazole-2-carboxamido)-1-methyl-iH-pyrrole-2-carboxamido)-1-methyl-iH-pyrrole-2

carboxylic acid (102 mg, 0.17 mmol), 2-(pyridin-2-yldisulfanyl)ethanamine hydrogen chloride

salt (40 mg, 0.18 mmol), DIPEA (30 ul, 0.17 mmol) and EDC (200 mg, 1.04 mmol) in 2 ml of

DMA were stirred under Ar for 24 h, evaporated and purified on SiO 2 chromatography eluted

with THF/DCM (1:5 to 1:4) to afford 90 mg (68%) of the title compound. 1 H NMR (DMSO)

10.93 (s, 1H), 10.19 (s, 1H), 10.06 (s, 1H), 9.03 (s, 1H), 8.81 (m 1H), 8.29 (m, 1H), 8.03 (m,

1H), 7.68 (s, 1H), 7.47 (s, 1H), 7.28 (s, 1H), 7.24 (s, 1H), 7.18 (m, 1H), 6.87 (s, 1H), 3.96 (s,

1H), 3.86 (s, 3H), 3.75 (s, 3H), 3.73 (s, 3H), 3.58 (m, 2H), 2.48 (m, 2H), 1.45 (s, 9H); MS mi/z+

798.0 (M + Na), 776.0 (M + H).

4-(4-(4-(7-methoxy-2,3-benzo[e]-l-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2

a][1,4]diazepin-8-yloxy)butanamido)-1-methyl-iH-pyrrole-2-carboxamido)-1-methyl-N-(1

methyl-5-(1-methyl-5-(methyl(2-(pyridin-2-yldisulfanyl)ethyl)carbamoyl)-1H-pyrrol-3

ylcarbamoyl)-1H-pyrrol-3-yl)-1H-imidazole-2-carboxamide

I, -I N 1) O20%HC,thAc _ \Yf, Hoa N PySS N 229 2). 225/EDC/DMA N'01 NNE3CH A 0 ') NO 0 0I pS 230 1ys-

[277] tert-Butyl 1-methyl-5-(1-methyl-2-(I-methyl-5-(1-methyl-5-(2-(pyridin-2

yldisulfanyl)ethylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H

imidazol-4-ylcarbamoyl)-1H-pyrrol-3-ylcarbamate (229) (30 mg, 0.038 mmol) was stirred in 2

ml of dioxane and 0.5 ml of HCl (cone) for 15 min, diluted with ethanol/toluene (6 ml, 1:1),

evaporated and co-evaporated with ethanol/toluene (4 x 10 ml), crystallized with

EtOH/DCM/Hexane and dried over vacuum to afford 19.5 mg of solid. The solid compound was

used directly without further purification. To the solid was added 4-(7-methoxy-2,3-benzo[e]-5

oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy) butanoic acid (10 mg,

0.026 mmol), EDC (102 mg, 0.52 mmol), DIPEA (5 ul, 0.028 mmol) and 2 ml of DMA. The

mixture was stirred overnight, evaporated, diluted with DCM, washed with1 M NaH 2PO 4/NaCl

(conc), pH 4.0, dried over MgSO 4 , filtered, evaporated and purified on HPLC preparative C-18

column (<D10 mm x 200 mm column, flow rate 9 mL/min and a gradient solvent system going

from 75:25 solvent A:B at time 0-5 min to 40:60 A:B at 15 min then to 20:80 A:B at 25 min

until to 10:90 A:B at 30 min. Solvent A - water, solvent B - acetonitrile/dioxane (1:2)) and lyophilized to afford a white solid 7.5 mg (27%) of the title compound. MS m/z- 1050.0 (M

+ H 2 0 - H), UV (= 305m)= 32855 M-1cm 1

. 1-(4-(2-bromoethoxy)phenyl)ethanone

OH O Br

THF/DIPEA

1rCI 2 CH 2Br(excess) >85% 231 0 232

[278] 1-(4-hydroxyphenyl)ethanone (8.2 g, 60.2 mmol), potassium carbonate (15.2 g, 110.1

mmol), and KI (1.0 g, 6.0 mmol) in 100 DMF was stirred for 5 min, then 1,2-dibromoethane(60

ml, 696.2 mmol)was added. The mixture was stirred overnight, evaporated, diluted with

EtAc/Hexane (1:1), washed with 0.1 M HCl/NaCl (conc), dried over MgSO 4 , filtered, evaporated

and purified by SiO 2 chrmatography eluted with EtAc/Hexane (1:3 to 2:3) to afford 12.41 g

(85.2%) of the title compound. 1H NMR (CDCl) 7.87 (ddd, 2H, J = 2.8, 4.9, 9.7 Hz), 6.88 (ddd,

2H, J = 2.8, 4.9, 9.6 Hz), 4.29 (t, 2H, J = 6.2 Hz), 3.59 (t, 2H, J = 6.2 Hz); 13 C NMR 196.88,

162.11, 131.15, 130.54, 113.80, 68.06, 29.50, 26.62; MS m/z+ 264.80 (M + Na), 266.80 (M + 2

+ Na).

(5-hydroxy-1,3-phenylene)dimethanol

OH OH IijAIH,/THF,

0 O- O- Li8H5/%HF HO >OH 0 0

233 234

[279] To a solution of 100 ml of 2.0 M LiAlH 4 in THF at0C was added dimethyl 5-hydroxy

isophthalate (12.3 g, 58.5 mmol) in 120 ml of THF in 15 mim under Ar. The mixture was stirred

at 0C for 30 min then at RT overnight. The mixture was quenched with 20 ml of methanol at

°C, and the mixture was adjusted to pH 5.0 with addition of H 3 PO 4 , filtered through celite,

evaporated and crystallized with ether/hexane to afford 76.6 (85%) of the title compound. H

NMR (DMSO) 6.68 (s, 1H), 6.61 (s, 2H), 4.69 (s, 4H); MS m/z+ 177.0 (M +Na).

1-(4-(2-(3,5-bis(hydroxymethyl)phenoxy)ethoxy)phenyl)ethanone (235)

O Br OH O

Na 2 CO 3 iDMA HO . OH NaT, 86% HO OH 0

232 234 235

[280] To a stirred solution of (5-hydroxy-1,3-phenylene)dimethanol (3.0, 20.0 mmol), sodium

carbonate (2.5 g, 29.0 mmol) and sodium iodide (0.45 g, 2.9 mmol) in 60 ml of DMA was added

1-(4-(2-bromoethoxy)phenyl)ethanone (5.0, 20.57 mmol). The mixture was stirred overnight,

evaporated and purified on SiO 2 chromatography eluted with EtAc/Hexane (4:1 to 5:1) to afford

5.41 g (86/o) of the title compound. 'H NMR (CD 30D) 7.99 (ddd, 2H, J = 2.8, 4.8, 9.8 Hz), 7.07

(ddd, 211, J = 2.8, 4.7, 9.8 Hz), 6.94 (s, 1 H), 6.89 (s, 2H), 4.58 (s, 4H), 4.42 (dd, 2H, J = 2.2, 6.1

Hz), 4.37 (m, 2H), 2.55 (s, 3H);1 3 C NMR 199.55, 164.66, 160.59, 144.72, 132.03, 131.74,

119.16, 115.64, 113.11, 68.36, 67.87, 65.20, 26.53; MS m/z+ 339.2 (M +Na).

1-(4-(2-(3,5-bis(bromomethyl)phenoxy)ethoxy)phenyl)ethanone(236)

CBr 4/PPh 3/THF >'90% HO OH 0 Br Br 0

235 236

[281] 1-(4-(2-(3,5-bis(hydroxymethyl)phenoxy)ethoxy)phenyl)ethanone (0.216g, 0.68 mmol),

carbon tetrabromide (0.50g, 1.50 mmol) and PPh3 (0.40g, 1.52 mmol) was stirred in 18 ml of

THF under Ar overnight and filtered. The solution was concentrated, purified on SiO 2

chromatography eluted with EtAc/Hexane (1:4) and crystallized with ether/hexane to afford 277

mg (92%) of the title compound. 1H NMR (CDCI3) 7.94 (ddd, 2H, J = 2.7, 4.6, 9.6 Hz), 7.02 (s,

1H), 6.98 (ddd, 2H, J = 2.7, 4.6, 9.6 Hz), 6.91 (d, 2H. J = 1.2 Hz), 4.62 (s, 4H), 4.35 (in, 4H),

2.55 (s, 3H); 'C NMR 197.05, 162.63, 159.14, 139.98, 130.96, 130.85, 122.57, 155.60, 114.52,

66.78, 66.73, 32.88, 26.57; MS m/z+ 462.9 (M +Na), 464.9 (M + 2 + Na).

(R)-Methylpiperidine-2-carboxylate(238)

0 0 HOAI/> MeOIH/SOC1 2 H 3 CO -" HN OC - RT HNO >90% 237 238

[282] To (R)-Piperidine-2-carboxylic acid (5.00 g, 38.73) in 150 ml of dry methanol at 0C was

added thionyl chloride (5.2 ml, 71.28 mmol) under Ar. The mixture was stirred at 0°C for 30

min, then at RT overnight, evaporated and crystallized with EtOwhexane to afford 4.96 g (92%)

of the title product. 'H NMR (CD 30D) 3.67 (s, 3H), 3.57 (m, 1H), 2.79 (m, 1H), 2.69 (m, 1H),

2.01 (m, 1H), 1.98 (m, 1H), 1.73 (m, 1H), 1.55 - 1.45 (m, 4H); "C NMR 171.22, 62.50, 51.35,

45.35, 29.52,28.41, 23.82; MS m/z + 144.0 (M + H).

(R)-Methyl I-(4-(benzyoxy)-5-methoxy-2-nitrobenzoyl)piperidine-2-carboxylate (239)

H 3CO BZO COO EDC/DMA BzO

I': INOD H3 H 800% H 3 C'0

O 239

[283] 4-(benzoyloxy)-5-methoxy-2-nitrobenzoic acid (1.70 g, 5.61 mmol), (R)-methyl

piperidine-2-carboxylate (1.05 g, 5.84 mmol), EDC (3.90 g, 20.31 mmo) and DIPEA (1.0 ml,

5.75 mmol) was stirred in 20 ml of DMA over night. The mixture was evaporated, diluted with

DCM, washed with washed IM NaH2PO 4/NaCI (cone) and 0.1 M NaHCO 3/NaCl (cone)

separately. The organic solvent layer was separated and dried over MgSO 4 , filtered,

concentrated and purified on SiO 2 chromatography eluted with EtAc/DCM (1:15) to afford 1.772

g (74%) of the title product. H NMR (CDC 3) 7.69 (s, IH), 7.40 - 7.38 (in, 2H), 7.35 -7.27 (m,

3H), 6.76 (d, 1H), 5.15 (s, 2H), 3.91 (s, 3H), 3.83 (s, IH), 3.73 (s, 3H), 3.18 (m, 2H), 1.70 (m

2H), 1.47 (in, 4H); 1 3 CNMR 171.89,171.33,155.10,154.78,148.32,135.59,129.05,128.74,

127.80, 109.66, 109.58, 109.41, 71.63, 56.92, 52.70, 52.19, 45.70, 39.92, 27.29, 26.35, 21.63;

MS m/z+ 451.2 (M + Na).

(R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)piperidine-2-carbaldehyde

NO ,z0 BNO 2 o Bz H3CO ~ DIBAL H q

CH0 CI 2/TOI ,ON H 3 CO 2 -78C,H 0%CO

O 239 240

[284] (R)-Methyl 1-(4-(benzoyloxy)-5-methoxy-2-nitrobenzoyl)piperidine-2-carboxylate (1.50

g, 3.50 mmol) in 30 ml of 1:1 DCM/benzene at -78°C was added 7.5 ml of 1.0 M DIBAL in

toluene under Ar in 10 min. The mixture was stirred at -78 0C for 1 hr and the reaction was

quenched with 0.5 ml of methanol. The mixture was diluted with 150 ml of EtAc and 100 ml of

0.2 M HCL. The organic solvent layer was separated and was separated and the aqueous layer

was extracted with EtOAc (3 x 80 ml). The organics were combined, dried over MgSO 4 , filtered,

concentrated and purified on Si02 chromatography eluted with EtAc/hexane (3:2) to afford 1.52

g (90%) of the title product. 'H NMR (CDCl3 ), 9.60 (s, 1H), 7.70 (s, 1H), 7.65 - 7.28 (m, 5H),

6.78 (m, 1H), 5.16 (s, 2H), 3.92 (s, 3H), 3.22, (m, 1H), 3.01 (m, 1H), 2.20 (m, 1H), 1.84 (m, 1H),

3 1.65 - 1.40 (in, 4H); C NMR 200.24, 171.31, 155.13, 154.78, 148.41, 146.20, 137.57, 135.47,

129.03, 128.73, 127.31, 109.83, 109.41, 71.61, 64.50, 56.96, 45.98, 25.25, 23.42, 18.70; MS

m/z- 421.1 (M + Na).

(R,Z)-3-(benzyloxy)-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-12(6aH)

one

13z0 NO2 0 Bzo,:

H O THF/H 20, H CO

240 241

[285] To (R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)piperidine-2-carbaldehyde (1.0 g,

2.51 mmol) in a mixture solution of 25 ml of THF and 15 ml of water was added Na 2 S 2 04 (3.0 g,

17.25 mmol). The mixture was stirred for 4 h, diluted with methanol and dioxane, evaporated

and co-evaporated with dioxane (3 x 60 ml) to dryness. The solid was sonicated with a mixture

of CH 30H/CH 2C2 (1:1, 80 ml), filtered and evaporated to solid. The yield solid was dissolved in

CH 30H (100 ml) and 0.4 ml of HCl (cone) was added. The mixture was stirred for I h,

neutralized to pH 3.0 with 0.1 M NaHCO3 , concentrated, and extracted with CH2Cl2 (4 x 60 ml),

The organic layers were combined, washed with IM NaHCO /NaCl 3 (cone.), dried over Na 2 SO 4

, filtered, evaporated and purified on SiO 2 chromatography eluted with EtAc/CH 2 Cl 2 (1:3) to

afford 615 mg (70%) of the title product. 1 H NMR (CDCl 3), 7.81 (d, 1H, J= 5.7 Hz), 7.38 ~ 7.23

(in, 6H), 6.74 (s, 1H), 5.12 (dd, 2H, J = 2.3, 21.8 Hz), 4.18 (in, 1H), 3.88 (d, 3H), 3.69 (in, 1H), 3.15 (m, IH), 1.99 (m, 1 H), 1. 87 (m, I H), 1.79 ~ 1.65 (m, 4H); 13C NMR 167.76,163.31,

150.72, 148.48, 140.09, 136.46, 128.87, 128.28, 127.53, 121.77, 111.01, 71.02, 56.41, 49.84,

39.93, 24.76, 23.21, 18.62; MS m/z+ 373.2 (M + Na), 391.2 (M + Na + H 2 0), 405.3 (M + Na +

CH 30H).

(R,Z)-3-Hydroxy-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-I2(6aH)

one (242)

H CO NCH 2C1 2, >70% H3co 0 0) 241 242

[286] To (R,Z)-3-(benzyloxy)-2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2

a][1,4]diazepin-12(6aH)-one (241) (215 mg, 0.614 mmol) in 25 ml of CH2 Cl 2 at 0C was added

ml of CH 2SO 3H. The mixture was stirred at 0Cfor 10 min and then at RT for 2 h, diluted

with CH 2 C2, neutralized with cold 1.0 M NaHCO 3, extracted with CH2C 2 , dried over Na 2 SO 4

, filtered, evaporated and purified on SiO 2 chromatography eluted with CH 30H/CH 2Cl 2 (1:15) to

afford 122 mg (70%) of the title product. H NMR (CDCl 3), 7.75 (d, 1H, J= 5.7 Hz), 7.28 (s,

1H), 6.70 (s, 1H), 4.08 (m, 1H), 3.83 (d, 3H), 3.61 (i, 1H), 3.08 (m, 1H), 1.91 (m, 1H), 1.81 (m, 3 1H), 1.71 ~ 1.55 (in, 4H); C NMR 167.81, 163.46, 148.53, 145.71, 140.84, 121.23, 111.89,

111.39, 56.45, 49.83, 39.96, 24.71, 23.22, 18.60; MS i/z+ 283.7 (M + Na).

(5Z,5'Z,6aR,6a'R)-3,3'-(5-(2-(4-Acetylphenoxy)ethoxy)-1,3

phenylene)bis(methylene)bis(oxy)bis(2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2

a][1,4]diazepin-12(6aH)-one) (243)

HO N-2-36 o~ 0

H 3CO Cs 2 CO 3/Acetone N 0 242 0 243

[287] To a stirring solution of (R,Z)-3-hydroxy-2-methoxy-7,8,9,10

tetrahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-12(6aH)-one (242) (42 mg, 0.16 mmol), Cs2CO3

(100 mg, 0.307 mmol), KI (3.2 mg, 0.018 mmol) in 5 ml of acetone was added 1-(4-(2-(3,5

bis(bromomethyl)phenoxy)ethoxy)phenyl)ethanone (236) (36 mg, 0.081 mmol). The mixture

was stirred over night, evaporated and purified on HPLC preparative C-18 column (<D10 mm x

200 mm column, flow rate 9 mL/min and a gradient solvent system going from 80:20 solvent

A:B at time 0-5 min to 50:50 A:B at 15 min then to 30:70 A:B at 25 min until to 10:90 A:B at

min. Solvent A - water, solvent B - dioxane) and lyophilized to afford a white solid 39.1 mg

(61%) of the title compound. 1H NMR (DMF-d), 8.30 (in, 2H), 7.75 (d, 2H, J= 5.7 Hz), 7.30 (s,

2H), 7.01 (m, 2H), 6.71 (s, 2H), 6.68 (s, 1H), 6.63 (s, 2H), 5.21 (s, 4H), 4.43 (in, 2H), 4.32 (in,

2H), 4.08 (in, 2H), 3.83 (s, 6H), 3.61 (in, 2H), 3.08 (m, 2H), 2.56 (s, 3H), 1.91 (in, 2H), 1.81 (in,

2H), 1.71 ~ 1.55 (in, 8H); MS m/z+ 823.2 (M + Na), 839.3 (M + K), 857.3 (M + K+ H 2 0); MS

m/z- 799.2 (M - H).

tert-Butyl 2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl)hydrazinecarboxylate (245)

0 0 OH NH 2 NTHBOC NHNHBOC 0 EDC/DCM 0 0 0 245 244

[288] 4-Maleimidobutyric acid (245 mg, 1.33 mmol), tert-butyl hydrazinecarboxylate (201 mg,

1.52 mmol) and EDC (400 mg, 2.08 mmol) in 5 ml of CH2C2, were stirred overnight under Ar, washed with 1 M NaH 2PO 4 ,/NaCI(conc), dried over MgSO 4 , filtered, evaporated and purified on

SiO2 chromatography eluted with MeOH/DCM (1:25) to afford 335 mg (85%) of the title

compound. 1H NMR (CDCl), 7.83 (br, 1H), 6.65 (s, 2H), 6.50 (br, IH), 3.58 (t, 2H, J= 6.3 Hz),

2.15 (t, 2H, J = 7.0 Hz), 1.90 (dt, 2H, J = 6.8, 13.4 Hz), 1.40 (s, 9H); "C NMR 171.30, 155.61,

134.41, 82.00, 37.13, 31.38, 28.36, 24.95; MS m/z+ 320.2 (M + Na).

4-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanehydrazidetrifluroaceticacidsalt(246)

N-r NHNHBOC 20%TIF N NHNH 2 *TFA O DCM 0 0 245 0 246

[289] Totert-Butyl2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl)

hydrazinecarboxylate (245) (200 mg, 0.673 mmol) in 8 ml of DCM was added 2 ml of TFA. The

mixture was stirred for 45 min, diluted with ethanol/toluene (8 ml, 1:1), evaporated and co

evaporated with ethanol/toluene (3 x 10 ml), crystallized with ethanol/EtAc/Hexane, filtered, and

dried under vaccum to afford 188 mg (90%) of the title compound. 1H NMR (CD 30D) 6.72 (s,

2H), 5.39 (s, 0.6H), 3.47 (t, 2H, J = 6.6 Hz), 2.20 (in, 2H), 1.85 (m, 2H); 13 C NMR 172.72,

135.56, 54.93, 39.20, 37.99, 25.20; MS mz+ 197.9 (M + H).

(E)-N'-(1-(4-(2-(3,5-bis(((S,Z)-2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2

a][1,4]diazepin-3-yloxy)methyl)phenoxy)ethoxy)phenyl)ethylidene)-4-(2,5-dioxo-2,5-dihydro

IH-pyrrol-1-yl)butanehydrazide (247)

0 ^~ \ 0 0 0" H, ~~O N--

' H 5% HCl/DCM NN

O 246 243, 4A MS C H 3CO N CN 0-H3 3CO247 0 0

[290] 4-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanehydrazidetrifluroacetic acid salt(246) (3

mg, 0.0096 mmol), (5Z,5'Z,6aR,6a'R)-3,3'-(5-(2-(4-Acetylphenoxy)ethoxy)-1,3

phenylene)bis(methylcne)bis(oxy)bis(2-methoxy-7,8,9,10-tetrahydrobenzo[e]pyrido[1,2

a][1,4]diazepin-12(6aH)-one) (243) (7.5 mg, 0.0093 mmol) and 50 mg 4 Amolecular sieves was

stirred in 2 ml of dry 5% HAc in DCM (one day earlier dried by 4 A molecular sieves) for 2 h,

neutralized with 0.5 ml of DIPEA, evaporated and purified on HPLC preparative C-18 column

(CD10 mmx 200 mm column, flow rate 9 mL/min and a gradient solvent system going from

:20 solvent A:B at time 0-5 min to 50:50 A:B at 15 min then to 30:70 A:B at 25 min until to

:90 A:B at 30 min. Solvent A - water, solvent B - methanol/dioxane (2:1)) and lyophilized to

afford a white solid 5.6 mg (61%) of the title compound. MS m/z+ 1066.3 (M + 2CH 30H + Na).

Example 13

Preparation of huN901-IGN-07 conjugate:

[291] huN901 antibody that binds to the CD56 antigen was selected for conjugation of IGN

derivatives. A solution of huN901 antibody at a concentration of 3 mg/mL in an aqueous buffer containing 0.05 M N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid (HEPES) and 2 mM ethylenediaminetetra-acetic acid (EDTA), pH 8 was treated with a 20-fold molar excess of a solution of IGN-07 NHS ester in dimethylacetamide (DMA) such that the final concentration of

DMA in the buffer was 10% v/v. The reaction mixture was stirred at room temperature for 120

min and then loaded onto a Sephadex G25 gel filtration column (HiPrepTM 26/10 Desalting

Column GE# 17-5087-01) that has been previously equilibrated into an aqueous buffer

containing 0.01 M sodium citrate, 0.135 M sodium chloride, pH 5.5. The conjugated antibody

containing fractions are collected and pooled to yield product. The pooled sample was dialyzed

overnight against the same elution buffer (0.01 M sodium citrate, 0.135 M sodium chloride, pH

5.5) to further purify the product.

[292] The final conjugate was assayed spectrophotometrically using the extinction coefficients

that were determined for IGN-07 (8 3 3 0 m = 15,231 M-1 cm-1 and 8280unm = 26,864 M-1 cm-1) and

huN901 antibody (F280 im = 225,000 M-lcm-1). An average of 3.1 IGN molecules per molecule

of antibody were linked.

H N-huN901 N

;-a-O~ Meo 0 0 huN901-IGN-07

Preparation of IGN-10 (compound 51) stock solution:

[293] A solution of IGN-10 was made fresh to a 0.004 M stock based on a molecular weight of

975.14 g/mole in dimethylacetamide (DMA). The stock solution was assayed

spectrophotometrically using a reference extinction coefficient determined at 330nm (F330 nm

,500 M-1 cm-1).

Example 14

Preparation of muB38.1-IGN-10 coniugate:

[294] muB38.1 antibody that binds to the EpCAM antigen was selected for conjugation ofIGN

derivatives through a disulfide bond. A solution of muB38.1 antibody at a concentration of 2.5

mg/mL in an aqueous buffer containing phosphate buffered saline (PBS) pH 7.4 was treated with

120 molar excess of 1-homocysteine thiolactone for 12 hr at 37 °C. The reaction mixture was

loaded onto a Sephadex G25 gel filtration column (HiPrepTM 26/10 Desalting Column GE# 17

5087-01) that was previously equilibrated in PBS pH 7.4. Fractions containing antibody are

collected and pooled and assayed for reactive thiol content using the Ellman's assay. The

modified antibody was then treated with a 4-fold molar excess of IGN-10 (in DMA) per free

thiol and allowed to react at room temperature for 8 hr. The reaction mixture was loaded onto a

Sephadex G25 gel filtration column (HiPrepTM 26/10 Desalting Column GE# 17-5087-01) that

has been previously equilibrated into an aqueous buffer containing 0.01 M sodium citrate, 0.135

M sodium chloride, pH 5.5. The conjugated antibody-containing fractions are collected and

pooled to yield product. The pooled sample was dialyzed overnight against the same elution

buffer(0.01 M sodium citrate, 0.135 M sodium chloride, pH 5.5) to further purify the product.

[295] The final conjugate was assayed spectrophotometrically using the extinction coefficients

that were determined for IGN-10 (V33nm = 15,500 M-1 cm-1 and 28Onm = 26,864 M-1 cm-1) and

muB38.1 antibody (F280.= 215,525 M-icm-1). An average of 0.7 IGN molecules per molecule

of antibody was linked.

0 I H3 H O NNS NamuB38.' H o ,N O /1O N

N 0 OMe MeO0 ;N

muB38.1-IGN-10

Example 15

DNA probe assay for measuring IGN dimer binding and alkylation to double stranded DNA

(dsDNA):

[296] Reaction conditions: dsDNA (25 pM final concentration) in 100 mM TRIS, 1 mM

EDTA, pH 8 was mixed with 3.7 molar equivalents of IGN-01 (compound 18), IGN-02

(compound 19), or IGN-09 (compound 15) dissolved in acetonitrile (final acetonitrile

concentration <2% by volume). The reaction was incubated at 15 C (below TM of the dsDNA)

and 10 pl aliquots are injected on reverse phase-HPLC at various time points after mixing

[297] HPLC conditions: Waters Xbridge C8 2.1 x 50 mm column, Buffer A: 100 mM

hexafluoroisopropanol, 16.3 mM triethylamine, in water, Buffer B: Methanol; 98% A -> 100% B over 32 min, 0.25ml/min flow, 60 °C column heat, 260 nm detection. Areas under the curve

(AUC) for the probe DNA peak and the resulting IGN/DNA adduct peak are used to determine

the % crosslinking at each time point of incubation.

[298] DNA annealing: single stranded DNA (Invitrogen) was annealed into dsDNA using a

Peltier thermal cycler (PTC-200, MJ Research). 1 mM DNA in 100 mM TRIS, 1 mM EDTA pH

8 was heated to 80 C and then gradually cooled to 4 C over 90 min in 15 degree steps. The

resulting dsDNA was kept at 4 °C until used in the assay. IGN-01, IGN-02, and IGN-09 did not

form covalent adducts with single stranded DNA (ssDNA) in control experiments.

Example 16

TsCI TEA MeO 0O M0 OH DMAP MeOO -O ,OOTs CH 2Cl 2 249a y = 99%

2-(2-(2-methoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate:

[299] To a stirred solution of 2-(2-(2-methoxyethoxy)ethoxy)ethanol (1.64 g, 10 mmol) in

anhydrous dichloromethane (30 mL) was added triethylamine (2.53 g, 25 mmol), tosyl chloride

(3.81 g, 20 mmol) and DMAP (0.061 g, 0.5 mmol) subsequently at room temperature. The

mixture continued to be stirred overnight and worked up by diluted with ethyl acetate and

filtered to remove the triethylamine hydrochloride solid. The solid was washed with ethyl

acetate and the filtrate was evaporated. The residue was diluted with ethyl acetate and filtered to

remove the additional precipitate. The filtrate was evaporated to give the crude product as liquid. It was purified by silica gel chromatography (dichloromethane/methanol) to give compound 249a as an oil (3.16 g, yield = 99%). 'H NMR (400 Hz, CDCl3): 6 7.81 (d, J= 8.0 Hz,

2H), 7.35 (d, J = 8.0 Hz, 2H), 4.17 (t, J = 3.2 Hz, 2H), 3.70 (t, J = 4.8 Hz, 2H) , 3.64-3.60 (m,

6H), 3.54 (t, J = 4.8 Hz, 2H), 3.38 (s, 3H), 2.46 (s, 3H); 3 C NMR (400 Hz, CDCl 3 ): 6 144.7,

133.0, 129.8, 127.9, 71.9, 70.7, 70.52, 70.50, 69.2, 68.6, 59.0, 21.6; MS (m/z): found 341.1 (M

+ Na).

NH 2

HO /: OH MeOO -O-'NH MeO 0 oO 26 ,

K 2CO3/DMF 249b HO OH 249a y = 48%

(5-(2-(2-(2-methoxyethoxy)ethoxylethylamino)-1,3-phenylene)dimethanol:

[300] To the mixture of the tosylate 249a (1.85 g, 5.81 mmol) and aniline compound 26 (1.78g,

11.6 mmol) in anhydrous DMF (9 mL) was added anhydrous potassium carbonate (1.61 g, 11.6

mmol). The mixture was heated to 85 °C and stirred at that temperature overnight. The solution

was cooled to room temperature and diluted with dichloromethane. It was filtered through celite

and the solid was washed with dichloromethane. The filtrate was evaporated and the residue was

diluted with dichloromethane and filtered again to remove the additional solid. The filtrate was

evaporated and the residue was purified by silica gel chromatography

(dichloromethane/methanol) to give compound 249b as a light yellowish oil (835 mg, yield =

48%). 1H NMR (400 Hz, CDCl): 6 6.60 (s, 1H), 6.47 (s, 2H), 4.48 (s, 4H), 4.31 (bs, 1H), 3.66

3.59 (in, 8H), 3.55-3.52 (in, 2H), 3.36 (s, 3H), 3.24 (t, J 4.8 Hz, 2H); 13 = C NMR (400 Hz,

CDC 3 ): 6 148.5, 142.4, 114.6, 110.7, 71.8, 70.4, 70.3, 70.1, 69.4, 64.9, 58.9, 43.5; MS (m/z):

found 322.2 (M + Na).

MeOHO Of H Br OMe MeO'-'''O'OM OMe

HO -b H K2CO/CH 3CN H '- H 249b y = 58% 249c

Compound 249c (IGN-14 linker):

[301] To the solution of compound 249b (319 mg, 1.07 mmol) and methyl 4-bromobutyrate

(248 mg, 1.37 mmol) in anhydrous acetonitrile (5 mL) was added anhydrous potassium

carbonate (177 mg, 1.28 mmol). The mixture was stirred and heated at reflux (86 °C oil bath) overnight. It was cooled to room temperature and diluted with dichloromethane. The mixture

was filtered through celite and the filtrate was evaporated. The residue was purified by silica gel

chromatography (dichloromethane/methanol) to give compound 249c (IGN-14 linker) as

colorless oil (246 mg, yield = 58%). 1H NMR(400 Hz, CDC 3 ): 6 6.69 (s, 2H), 6.66 (s, 1H), 4.64

(s, 4H), 3.71 (s, 3H), 3.64-3.62 (in, 8H), 3.57-3.54 (in, 4H), 3.40-3.38 (in, 5H), 2.38 (t, J = 7.2

Hz, 2H), 1.93 (p, J = 7.2 Hz, 2H); MS (m/z): found 422.3 (M + Na)+.

MeO~'^'ON .N OMe 1. MsCI/TEA/CH 2C1 2 0 2. K 2 CO/DMF HO -~ H HO N I 0 249c MeO N /\N*<& OMe MeO 8 0 0 0 y = 34% 249d (IGN-14-OMe)

Compound 249d (IGN-14-OMe):

[302] To a stirred solution of compound 249c (120 mg, 0.3 mmol) in anhydrous

dichloromethane (3 mL) was added triethylamine (146 pl, 1.05 mmol). The mixture was cooled

to -10 °C and methanesulfonyl chloride (70 pl, 0.9 mmol) was added slowly in 15 minutes. The

solution continued to be stirred between -10 °C to -5 °C for 60 minutes and quenched by addition

of ice/water. It was diluted with ethyl acetate and washed with cold water. The organic layer

was dried over anhydrous sodium sulfate, filtered, evaporated and high vacuumed to give the

mesylate as colorless oil. The mesylate was transferred to a 10 mL round bottom flask with ethyl

acetate, evaporated and high vacuumed. Compound 8 (221 mg, 0.75 mmol) was added followed

by addition of anhydrous DMF (2 mL) and anhydrous potassium carbonate (207 mg, 1.5 mmol).

The mixture was stirred at room temperature overnight. It was diluted with dichloromethane and

washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered and

evaporated. The residue was purified by preparative reverse phase HPLC (C18 column, eluted

wit CH 3CN/H 2 0) to give compound 249d (IGN-14-OMe) as a light yellowish solid (98 mg,

yield = 34%). 1H NMR (400 Hz, CDCl 3 ): 6 8.29 (d, J = 8.0 Hz, 2H), 7.86 (d, J = 4.4 Hz, 2H),

7.58 (s, 2H), 7.31-7.26 (m, 4H), 7.12 (t, J = 8.0 Hz, 2H), 6.88 (s, 2H), 6.83 (s, 1H), 6.76 (s, 2H),

5.18 (dd, Ji= 23.2 Hz, J2 = 12.4 Hz, 4H), 4.49 (dt, J1 = 10.8 Hz, J2 = 4.4 Hz, 2H), 3.99 (s, 6H),

3.73-3.52 (m, 19H), 3.40-3.37 (m, 5H), 2.35 (t, J = 7.2 Hz, 2H), 1.90 (p, J = 7.2 Hz, 2H); "C

NMR (400 Hz, CDCl 3): 6 173.7, 164.9, 163.2, 151.1, 148.5, 148.4, 142.1, 140.2, 137.8, 129.7,

128.2, 124.9, 120.7, 117.0, 113.8, 112.0, 111.4, 110.4, 72.0, 71.3, 70.7, 70.6, 68.6, 59.1, 56.3,

55.0, 51.7, 50.7, 32.7, 31.3, 22.4; MS (m/z): found 974.6 (M + Na), 992.7 (M + H 2 0 + Na)+,

1010.7 (M + 2H 20 + Na)j, 950.3 (M - H)-, 1022.3 (M + 4H 20 - H)-.

0 MeO -O -N OMe Me 3SnOH MeO O N OH NHS/EDC MeO O N 0-N

O NH CICHdCH2C OC N 0 0 0 0 = 1&fO Me0'Ib 249d (IGN-14-OMe) 249e (IGN-14-acid) 0 249f(IGN-14-NHS) 0

Compound 249f (IGN-14-NHS):

[303] To the solution of compound 249d (105 mg, 0.11 mmol) in anhydrous 1,2 dichloroethane

(2 mL) was added trimethyltin hydroxide (299 mg, 1.65 mmol). The mixture was heated to 80

°C and stirred overnight. It was cooled to room temperature, diluted with dichloromethane and

washed with mixed solution of saturated sodium chloride and 5% hydrochloric acid (~1 mL),

then brine. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated.

The residue was passed a short silica gel column and eluted with dichloromethane/methanol to

remove the extra trimethyltin hydroxide. The product fractions were evaporated and high

vacuumed to give the acid 249e as a yellowish solid (102 mg, yield = 99%). MS (m/z): found

936.1 (M - H)-, 960.3 (M + Na)+. Compound 249e was then dissolved in anhydrous

dichloromethane (1 mL). N-hydroxysuccinimide (NHS, 37.5 mg, 0.326 mmol) and N-(3

dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, 62.5 mg, 0.326 mmol) was

added subsequently. The mixture was stirred at room temperature overnight and diluted with

dichloromethane, washed with brine and dried over anhydrous sodium sulfate. It was filtered,

evaporated and the residue was purified by preparative reverse phase HPLC (C18 column, eluted with acetonitrile/water). The product fractions were combined and extracted with dichloromethane. The organic layers were dried over anhydrous sodium sulfate, filtered, evaporated and high vacuumed to give compound 249f (IGN-14-NHS) as a light yellowish solid

(57.8 mg, yield = 51%). ' 1H NMR (400 Hz, CDC 3): 6 8.28 (d, J = 7.6 Hz, 2H), 7.86 (d, J = 4.4

Hz, 2H), 7.58 (s, 2H), 7.31-7.27 (m, 4H), 7.12 (t, J = 7.6 Hz, 2H), 6.87 (s, 2H), 6.81 (s, 1H), 6.74

(s, 2H), 5.23 (dd, J 1 = 26.4 Hz, J2 = 12.4 Hz, 4H), 4.49 (dt, Ji = 10.8 Hz, J2 = 4.4 Hz, 2H), 4.00

(s, 6H), 3.73-3.47 (m, 18H), 3.37 (s, 3H), 2.79-2.74 (m, 4H), 2.59 (t, J = 7.2 Hz, 2H), 1.97 (p, J

= 7.2 Hz, 2H); MS (m/z): found 1057.4 (M + Na).

Example 17

OMe OMe -1-lOMe NaBH4

Me MeOEtOH N Me MeO n M eO

34 (IGN-03-OMe) 250a (IGN-16-OMe) 250b(IGN-18-OMe)

Compounds 250a (IGN-16-OMe) and 250b (IGN-18-OMe):

[304] To a stirred solution of compound 34 (111 mg, 0.135 mmol) in absolute ethanol (1.0 mL)

and anhydrous dichloromethane (0.5 mL) was added sodium borohydride (1.0 mg, 0.027 mmol)

at 0 °C. After 30 minutes, the ice/water bath was removed and the reaction mixture continued to

be stirred at room temperature for 3 hours. The reaction was quenched by addition of saturated

ammonium chloride and diluted with dichloromethane. The mixture was separated and the

organic layer was washed with brine, dried over anhydrous sodium sulfate and filtered. The solvents were removed under reduced pressure and the residue was purified by preparative reverse phase HPLC (C18 column, eluted with acetonitrile/water) to give compounds 250a

(IGN-16-OMe, 6.6 mg) and 250b (8.0 mg) as white solid. 250a: MS (m/z), found 845.3 (M

+ Na)f, 863.3 (M + H 2 0 + Na)*. 250b: 'H NMR (400 Hz, CDCI3 ), 6 8.34 (d, J = 8.0 Hz, 2H), 7.49

(s, 2H), 7.27-7.03 (m, 6H), 6.89-6.87 (m, 3H), 6.05 (s, 2H), 4.96 (dd, J, = 20.8 Hz, J2 = 12.8 Hz,

4H), 4.40-4.34 (in, 2H), 3.94-3.91 (in, 2H), 3.87 (s, 6H), 3.67 (s, 3H), 3.53-3.42 (in, 6H), 2.78

(dd, J, = 16.8 Hz, J2 = 4.0 Hz, 2H), 2.38-2.37 (m, 2H), 1.79-1.77 (in, 4H); MS (n/z), found

847.3 (M + Na)+.

Example 18

H 0 PO 2 Me BnO ,NO 2' H 2/AcOEt HO N

MeO / N /Y \ Pd/C MeO /

5 0 y=91% 251a

Compound 251a:

[305] To a stirred solution of compound 5 (840 mg, 1.82 mmol) in ethyl acetate (10 mL) was

added palladium on charcoal (10%, 193 mg, 0.182 mmol). The flask was briefly vacuumed and

replaced with H 2 in a balloon. The mixture continued to be hydrogenated for overnight and

filtered through celite. The solid was washed with methanol and the filtrate was treated with 5%

hydrochloric acid (0.1 mL). The solution was stripped under reduce pressure and the residue

was purified by silica gel chromatography (dichloromethane/methanol) to give compound 251a as a white solid (512 mg, yield = 91 %). H NMR (400 Hz, CDCl), 6 8.21 (d, J = 8.0 Hz, IH),

8.09 (bs, NH), 7.53 (s, 1H), 7.31-7.25 (in, 2H), 7.12 (t, J = 7.6 Hz, 1H), 6.61 (s, 1H), 6.22 (bs,

IH), 4.73 (dd, Ji = 10.4 Hz, J2 = 2.8 Hz, 1H), 4.07 (dd, Ji = 16.4 Hz, J2 = 2.4 Hz, 1H), 3.98 (s,

3H), 3.34 (dd, Ji = 16.4 Hz, J2 = 10.4 Hz, 1H); MS (m/z), found 333.1 (M + Na), 308.9 (M - H)

HO MsO MOMe N N OMe H 0

251a y 28% 251b (IGN-17-OMe)

Compound 251b (IGN-17-OMe):

[306] To a solution of compound 38 (0.165 mmol, prepared from 44 mg of compound 30

following the procedure described in example 6) and 251a (128 mg, 0.413 mmol) in anhydrous

DMF (1.5 mL) was added anhydrous potassium carbonate (114 mg, 0.825 mmol). The mixture

was stirred at room temperature overnight and diluted with dichloromethane, washed with brine

and dried over anhydrous sodium sulfate and magnesium sulfate. It was filtered, evaporated and

part of the residue was purified by preparative reverse phase HPLC (Cl8 column, eluted with

acetonitrile/water) to give 1.9 mg of compound 251b as a white solid. The rest of the residue

was purified by preparative thin layer chromatography (dichloromethane/methanol, 12:1) to give

36.8 mg of product as a white solid. Total 38.7 mg of compound 251b (IGN-17-OMe) was

isolated (yield = 28%). 'H NMR (400 Hz, CDCl3 ): 6 8.61 (s, 2H), 8.15 (d, J = 8.0 Hz, 2H), 7.48

(s, 2H), 7.25 (d, J = 7.6 Hz, 2H), 7.20 (t, J = 7.6 Hz, 2H), 7.07 (t, J = 7.6 Hz, 2H), 6.73 (s, 1H),

6.69 (s, 2H), 6.58 (s, 2H), 5.02 (dd, Ji = 17.6 Hz, J2 = 13.2 Hz, 4H), 4.66 (dd, Ji = 10.4 Hz, J2 =

2.8 Hz, 2H), 4.00 (dd, Ji = 16.4 Hz, J 2 = 2.4 Hz, 2H), 3.90 (s, 6H), 3.68 (s, 3H), 3.39-3.23 (in,

4H), 2.89 (s, 3H), 2.44-2.30 (in, 2H), 1.91-1.92 (m, 2H); 1 C NMR (400 Hz, CDC1 3 ): 6 174.5,

169.1, 164.2, 151.6, 149.6, 146.9, 141.2, 137.3, 130.6, 129.4, 127.5, 124.9, 124.8, 119.6, 117.1,

114.2, 112.5, 110.9, 106.0, 71.4, 58.0, 56.2, 51.9, 51.7, 38.3, 31.1, 28.2, 21.8; MS (m/z), found

874.3 (M + Na)v, 850.2 (M - H)-.

Example 19

0 0 Br + P ,COOMe THF Br OMe BrB+Ph 3P' y =60% Br". C Br

252a

Compound 252a:

[307] The mixture of 1,3-dibromoaceton (0.863 g, purity 75%, 3.0 mmol) and methyl

(triphenylphosphoranyliden)acetate (1.505 g, 4.5 mmol) in anhydrous THF (15 mL) was heated

to reflux for 4.5 hours. The solution was cooled to room temperature and evaporated. The

residue was purified by silica gel chromatography (hexanes/ethyl acetate) to give compound

252a as colorless liquid (485 mg, yield = 60%). 'H NMR (400 Hz, CDC 3): 6 6.06 (s, 1H), 4.76

(s, 2H), 4.19 (s, 2H), 3.79 (s, 3H);13 C NMR (400 Hz, CDC 3): 6 165.1, 150.4, 121.3, 51.8, 33.6,

25.5.

HO N= OMe

OMe0 N N ~ OMe MeO Br K 2CO3/DMF O 0 -/ 252a 252b (IGN-19-OMe)

Compound 252b (IGN-19-OMe):

[308] The mixture of compound 252a (32 mg, 0.118 mmol), monomer 8 (87 mg, 0.294 mmol)

and anhydrous potassium carbonate (49 mg, 0.353 mmol) in anhydrous DMF (1 mL) was stirred

at room temperature overnight. It was diluted with dichloromethane, washed with brine and

dried over anhydrous sodium sulfate. The solution was filtered, evaporated and purified by silca

gel chromatography (dichloromethane/methanol) to give 105 mg of compound 252b mixed with

side products as yellowish foam. Part of the produxts was further purified by preparative reverse

phase HPLC (C18 column, eluted with acetonitrile/water) to give 10 mg of compound 252b

(IGN-19-OMe) as a white solid. MS (m/z): found 721.2 (M + Na)t, 739.2 (M + H 2 0 + Na)+,

757.2 (M + 2H20 + Na)t, 697.1 (M - H)-, 769.1 (M + 4H20 - H)-.

Example 20

H SSMe + CH 3NH 2 EtOH then NaBH4 0 +y=65% SSMe 253a

Compound 253a:

[309] To a solution of 2-(methyldithio)-isobutyraldehyde (690 mg, 4.59 mmol) in absolute

ethanol (15 mL) was added methylamine (629 pl, 33%wt, 5.05 mmol). The mixture was stirred at room temperature for four hours and cooled to 0 °C followed by addition of sodium borohydride (174 mg, 4.59 mmol). After one hour, the reaction was quenched with a few drops of cold 5% hydrochloric acid and then basified with saturated sodium bicarbonate. The mixture was diluted with dichloromethane and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated under reduce pressure. The residue was purified by silica gel chromatography (0.2% triethylamine in dichloromethane/methanol) to give volatile compound 253a as light yellowish liquid (491 mg, yield = 65%). 1H NMR (400 Hz,

CDCl 3 ): 6 2.61 (s, 2H), 2.45 (s, 3H), 2.39 (s, 3H), 1.32 (s, 6H), 1.20 (s, NH);1 3 C NMR (400 Hz,

CDC 3 ): 6 61.2, 51.7, 37.2, 26.5, 25.3; MS (m/z): found 166.0 (M + H)-.

MeO O - NyOMe MeSnOH MeO- 0 'ON OH 1,2 dichloroethane HO OH y=83% HO / OH 249c 253b

Compound 253b:

[310] The mixture of compound 249c (117 mg, 0.293 mmol) and trimethyltin hydroxide (794

mg, 4.39 mmol) in anhydrous 1,2-dichloroethane (1.5 mL) was heated to 80 aC and stirred

overnight. It was cooled to room temperature, diluted with dichloromethane and washed with

mixed solution of saturated sodium chloride and 5% hydrochloric acid (~1 mL), then brine. The

organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was

purified by silica gel chromatography (dichloromethane/methanol) to give the acid 253b as a

colorless oil (93.9 mg, yield = 99%). 1H NMR (400 Hz, CDC 3): 6 6.62 (s, 2H), 6.57 (s, 1H),

4.50 (s, 4H), 3.63-3.54 (m, 8H), 3.53-3.46 (m, 4H), 3.36-3.31 (m, 5H), 2.29 (t, J = 6.8 Hz, 2H),

1.83 (p, J = 6.8 Hz, 2H); C NMR (400 Hz, CDC 3): 6 177.0, 148.2, 142.4, 113.8, 110.1, 71.9,

70.7, 70.6, 70.4, 68.8, 65.2, 59.0, 50.8, 50.7, 31.4, 22.3; MS (m/z): found 384.2 (M - H)-, 408.4

(M + Na)'.

H \/'O--O"- 0 S MeAo EDC/DMAP eO NSM SSMe + CH 2 C1 2 H N Oe 25aHO /- OH y 3'HO / OH 253b 253c

Compound 253c:

[311] To a solution of amine 253a (44.3 mg, 0.268 mmol) and carboxylic acid 253b (93.3,

0.244 mmol) in anhydrous dichloromethane (1.5 mL) was added N-hydroxysuccinimide (NHS,

70.1 mg, 0.365 mmol) and DMAP (5.95 mg, 0.049 mmol). The mixture was stirred at room

temperature overnight and diluted with dichloromethane, washed with saturated ammonium

chloride and brine, dried over anhydrous sodium sulfate, filtered and evaporated. The residue

was purified by silica gel chromatography (dichloromethane/methanol) to give compound 253c

as colorless oil (69.1 mg, yield = 53%). 1H NMR (400 Hz, CDCl 3 ): 6 6.71 (s, 2H), 6.64 (s, 1H),

4.57 (s, 4H), 3.63-3.59 (in,8H + 20H), 3.54-3.51 (in, 4H), 3.38-3.34 (in,5H), 3.08 (s, 2.35H),

3.00 (s, 0.65H), 2.86 (bs, 2H), 2.43 (s, 3H), 2.34 (t, J = 6.8 Hz, 2H), 1.95-1.88 (m, 2H), 1.36 (s,

1.3H), 1.31 (s, 4.7H); 1 3C NMR (400 Hz, CDCI 3 ): 6 173.7, 148.5, 142.7, 113.2, 109.8, 72.0, 70.8,

70.7, 70.6, 68.9, 65.6, 59.1, 56.5, 53.0, 52.2, 51.0, 50.8, 38.8, 30.6, 26.6, 25.6, 22.3; MS (m/z):

found 555.5 (M + Na)+.

1. MsCI/TEA MeO O /N SSMe MeOG O NN N SSMe 2. K 2CO 3/DMF 0 s 0 NWkO

/ HO 2 OH MHO N N OMe MeO N 253c 80 0. 253d0 y = 36%

Compound 253d:

[312] To a stirred solution of compound 253c (69.1 mg, 0.13 mmol) in anhydrous

dichloromethane (1.5 mL) was added triethylamine (63 pl, 0.454 mmol). The mixture was

cooled to -10 °C and methanesulfonyl chloride (30 pl, 0.389 mmol) was added slowly in 15

minutes. The solution continued to be stirred between -10 °C to -5 °C for 60 minutes and

quenched by addition of ice/water. It was diluted with ethyl acetate and washed with cold water.

The organic layer was dried over anhydrous sodium sulfate, filtered, evaporated and high

vacuumed to give the mesylate as colorless oil. The mesylate was transferred to a 10 mL round

bottom flask with ethyl acetate, evaporated and high vacuumed. Compound 8 (99 mg, 0.338

mmol) was added followed by addition of anhydrous DMF (1 mL) and anhydrous potassium

carbonate (90 mg, 0.65 mmol). The mixture was stirred at room temperature overnight. It was

diluted with dichloromethane and washed with brine. The organic layer was dried over

anhydrous sodium sulfate, filtered and evaporated. The residue was purified by silica gel

chromatography (dichloromethane/methanol) to give 150 mg yellowish foam, which was further

purified by preparative reverse phase HPLC (C18 column, eluted wit CH 3CN/H 20) to give

compound 253d as a light yellowish solid (50.7 mg, yield = 36%). MS (m/z): found 1107.7 (M +

Na)+, 1125.7 (M + H 2 0 + Na), 1143.7 (M + 2H 20 + Na)+, 1083.4 (M - H)-, 1155.5 (M + 41120

-H)-.

Me ^-OSSMe 1. TCEP/MeOH/CH 3CN M ,- - OH 0 O pH 6.5 buffer O N 11-00.-1.O: = N --O0 N ~JI~r~y~2. MeOH/TEACH 2CI 2 -': NX)OeMe Nf7 CN 0 Me MeS 0 N~ SA- j OH C Me2253ee y=36% 253d y= 30% Compound 253e:

[313] To a small vial containing tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 19.1 mg,

0.067 mmol) was added a few drops of deioned water. Saturated sodium bicarbonate was added

dropwise until pH is about 7 indicated by a pH test paper. It was then diluted with pH 6.5

phosphate buffer (0.4 mL) to give a fresh TCEP solution. To a stirred solution of compound

253d (24.1 mg, 0.022 mmol) in methanol (3 mL) and acetonitrile (1 mL) was added the TCEP

solution and stirred at room temperature for 1.5 hours. It was diluted with dichloromethane and

washed with brine, dried over anhydrous sodium sulfate, filtered and evaporated to give the thiol

as a yellowish solid (21.9 mg) which was directly used for next step (the thiol is not able to be

purified due to aggregation). To a solution of the thiol (21.9 mg, 0.021 mmol) in anhydrous

dichloromethane (0.1 mL) and methanol (0.4 mL) was added 4-(2-pyridyldithio)butanoic acid

(24.2 mg, 0.105 mmol) and triethyl amine (29 pl, 0.211 mmol). The mixture was stirred at room

temperature for five hours and quenched by saturated ammonium chloride. It was diluted with

dichloromethane, separated and the organic layer was washed with brine, dried over anhydrous

sodium sulfate, filtered and evaporated. The residue was purified by preparative reverse phase

HPLC (C18 column, eluted with acetonitrile/water) to give compound 253e as a white solid (7.3

mg,yield= 30%). 'HNMR(400Hz,CDCl):68.28(d,J= 7.6Hz,2H),7.89(bs, 2H),7.60(bs,

2H), 7.31-7.26 (m, 4H), 7.12 (t, J = 7.6 Hz, 2H), 6.91-6.78 (in, 5H), 5.22-5.13 (in, 4H), 4.54-4.49

(in, 2H), 3.99 (s, 6H), 3.68-3.41 (in, 20H), 3.38 (s, 3H), 3.07 (s, 3H), 2.78-2.77 (in, 2H), 2.47 (bs,

2H), 2.35 (bs, 2H), 2.01-1.95 (in, 4H), 1.31 (s, 6H); MS (m/z): found 1179.7 (M + Na), 1197.7

(M + H 2 0 + Na)-, 1073.6 (M + H20 - H)-, 1191.5 (M + 2H 20 - H)-.

Me~go--O- - N - OH NHS/EDC MeO O N YS A O-N O CH 2 C1 2 ov - 0 O N. ~ N O <0N=- 0 OMe MeO y=45% Me MeO 253e 253f (IGN-20-NHS)

Compound 253f:

[314] To a solution of compound 253e (9.0 mg, 0.00778 mmol) in anhydrous dichloromethane

(0.5 mL) was added N-hydroxysuccinimide (NHS, 2.68 mg, 0.023 mmol) and N-(3

dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, 4.47 mg, 0.023 mmol)

subsequently. The mixture was stirred at room temperature overnight and diluted with

dichloromethane, washed with brine and dried over anhydrous sodium sulfate. It was filtered,

evaporated and the residue was purified by preparative reverse phase HPLC (C18 column, eluted

with acetonitrile/water). The product fractions were combined and extracted with

dichloromethane. The organic layers were dried over anhydrous sodium sulfate, filtered, evaporated and high vacuumed to give compound 253f (IGN-20-NHS) as a yellowish foam (4.4 mg, yield = 45%). MS (m/z): found 1276.7 (M + Na).

Example 21

MeOs-^g 0 N--COMe Me~oO^''O N Me MeO-' O-0--N-- OCMe

ONaBH. H H O H N D 0 N, 0 c-' NN=% + N,-0~ O N Me MeO EOH/CI 2CI 2 MeMee OMe MeO N

249d (IGN-14-OMe) 254a (IGN-23-OMe) 254b (IGN-24-OMe) y = 24.5% y = 26.6%

Compound 254a (IGN-23-OMe) and 254b (IGN-24-OMe):

[315] To a stirred solution of compound 249d (91.8 mg, 0.103 mmol) in absolute ethanol (1.0

mL) and anhydrous dichloromethane (0.4 mL) was added sodium borohydride (0.4 mg, 0.0106

mmol) at 0 °C. After 30 minutes, the ice/water bath was removed and the reaction mixture

continued to be stirred at room temperature for 3 hours. The reaction was quenched by addition

of saturated sodium ammonium chloride and diluted with dichloromethane. The mixture was

separated and the organic layer was washed with brine, dried over anhydrous sodium sulfate and

filtered. The solvents were removed under reduced pressure and the residue was purified by

preparative reverse phase HPLC (C18 column, eluted with acetonitrile/water) to give compounds

254a (IGN-23-OMe, 24.2 mg, yield = 24.5%) and 254b (IGN-24-OMe, 26.3 mg, yield =

26.6%) as a yellowish solid. 254a: 'H NMR (400 Hz, CDC 3): 6 8.34 (d, J = 8.0 Hz, 1H), 8.27

(d, J = 7.6 Hz, IH), 7.83 (d, J = 4.4 Hz, 1H), 7.57 (s, 1H), 7.46 (s, IH), 7.29-7.02 (m, 6H), 6.87

(s, 1H), 6.75 (s, 1H), 6.70-6.66 (m, 2H), 6.10 (s, 1H), 5.21-5.02 (m, 4H), 4.49-4.39 (in,2H), 3.99

(s, 3H), 3.89 (s, 3H), 3.66 (s, 3H), 3.64-3.41 (m, 19H), 3.39-3.34 (m, 4H), 2.78 (dd, Ji = 16.4 Hz,

J2 = 3.6 Hz, 1H), 2.33 (t, J = 7.2 Hz, 2H), 1.90-1.84 (m, 2H); "C NMR (400 Hz, CDCl3 ): 6

173.8, 166.8, 164.0, 163.5, 152.3, 151.2, 148.7, 148.5, 143.0, 142.1, 140.7, 140.2, 138.5, 137.8,

129.8, 129.7, 128.3, 127.9, 125.0, 124.7, 123.9, 120.9, 117.5, 117.0, 114.6, 113.4, 113.2, 112.1,

111.6, 110.2, 110.1, 104.2, 72.1, 71.4, 71.2, 70.80, 70.76, 70.70, 68.7, 59.2, 57.3, 56.5, 56.4,

55.1, 54.8, 51.8, 50.9, 50.7, 33.3, 32.7, 31.3, 22.4; MS (m/z), found 976.7 (M + Na), 994.6 (M

+ H 2 0 + Na)r; 254b: MS (m/z), found 978.7 (M + Na)'.

MeO'-> 0 N' OMe MeO O'''>O N OH Me O-O N 0-N 0 Me 3 SnOH 0 NHS CICH 2CH 2 CI H EDC H O 6 6 N OMeMeO M N1 M N >>OMeMeO Je` N 66% N 43% -Nr-;:OQMeMeo- N C 00> 0 254a 254c 254d (IGN-23-NHS)

Compound 254c and 254d (IGN-23-NHS):

[316] To the solution of compound 254a (31.8 mg, 0.033 mmol) in anhydrous 1,2

dichloroethane (1 mL) was added trimethyltin hydroxide (90 mg, 0.5 mmol). The mixture was

heated to 80 °C and stirred overnight. It was cooled to room temperature, diluted with

dichloromethane and washed with mixed solution of saturated sodium chloride and 5%

hydrochloric acid (-1 mL), then brine. The organic layer was dried over anhydrous sodium

sulfate, filtered and evaporated. The residue was passed a short silica gel column and eluted with

dichloromethane/methanol to remove the extra trimethyltin hydroxide. The product fractions

were evaporated and high vacuumed to give the acid 254c as a yellowish solid (20.8 mg, yield=

66%). MS (m/z): found 938.2 (M - H)-, 962.3 (M + Na). Compound 254c (20.8 mg, 0.022

mmol) was then dissolved in anhydrous dichloromethane (1 mL). The reaction flask was briefly

vacuumed and replaced with argon. N-hydroxysuccinimide (NHS, 5.09 mg, 0.044 mmol) and N

(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, 8.48 mg, 0.044 mmol) was

added subsequently. The mixture was stirred at room temperature overnight and diluted with

dichloromethane, washed with brine and dried over anhydrous sodium sulfate. It was filtered,

evaporated and the residue was purified by preparative reverse phase HPLC (C18 column, eluted

with acetonitrile/water). The product fractions were combined and extracted with

dichloromethane. The organic layers were dried over anhydrous sodium sulfate, filtered,

evaporated and high vacuumed to give compound 254d (IGN-23-NHS) as a light yellowish solid

(9.8 mg, yield =43%). MS (m/z): found 1059.6 (M + Na), 1077.6 (M + H2 0 + Na).

Meo O OMe MeO OHSOH S 0 1) NH MeO _CN -IrO N

CICHCH2CI H H N n '~OM O e M M N- Nr -- 1 ~MeOSO MeO TN- D C CH N- 0 N 2 2 O 0 100% 0 0 -%OMe MeO_ 254b 2 54e00 254f (IGN-24-NHS)

Compound 254e and 254f (IGN-24-NHS):

[317] To the solution of compound 254b (26.3 mg, 0.028 mmol) in anhydrous 1,2

dichloroethane (1 mL) was added trimethyltin hydroxide (74.6 mg, 0.413 mmol). The mixture

was heated to 80 °C and stirred overnight. It was cooled to room temperature, diluted with

dichloromethane and washed with mixed solution of saturated sodium chloride and 5% hydrochloric acid (-1 mL), then brine. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was passed a short silica gel column and eluted with dichloromethane/methanol to remove the extra trimethyltin hydroxide. The product fractions were evaporated and high vacuumed to give the acid 254e as a yellowish solid (26 mg, yield =

100%). MS (m/z): found 940.5 (M - H)-, 964.6 (M + Na). Compound 2542 (26 mg, 0.028

mmol) was then dissolved in anhydrous dichloromethane (1 mL). N-hydroxysuccinimide (NHS,

9.57 mg, 0.083 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC,

15.9 mg, 0.083 mmol) and DMAP (0.34 mg, 0.0028 mmol) was added subsequently. The

mixture was stirred at room temperature overnight and diluted with dichloromethane, washed

with saturated ammonium chloride and brine, dried over anhydrous sodium sulfate. It was

filtered, evaporated and the residue was purified by preparative reverse phase HPLC (C18

column, eluted with acetonitrile/water). The product fractions were combined and extracted with

dichloromethane. The organic layers were dried over anhydrous sodium sulfate, filtered,

evaporated and high vacuumed to give compound 254f (IGN-24-NHS) as a light yellowish solid

(3.0 mg, yield = 10%). MS (m/z): found 1061.7 (M + Na)'. Note: DMAP should not have been

added and it may be the cause of the low yield.

Example 22

TsCI/TEA CH 2012 MeO{ o)OH C MeO O0 OTs 97% 255a

Compound 255a:

[318] To a stirred solution of O-methyl-undecaethylene glycol (500 mg, 0.968 mmol) in

anhydrous dichloromethane (3 mL) was added triethylamine (270 pl, 1.94 mmol), tosyl chloride

(277 mg, 1.45 mmol) and DMAP (5.91 mg, 0.048 mmol) subsequently at room temperature.

The mixture continued to be stirred overnight and worked up by diluted with ethyl acetate and

filtered to remove the triethylamine hydrochloride solid. The solid was washed with ethyl

acetate and the filtrate was evaporated. The residue was diluted with ethyl acetate and filtered to

remove the additional precipitate. The filtrate was evaporated to give the crude product as

liquid. It was purified by silica gel chromatography (dichloromethane/methanol) to give

compound 255a as a light yellowish oil (630 mg, yield = 97%). 1H NMR (400 Hz, CDCl,): 6

7.81 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 4.17 (t, J = 4.8 Hz, 2H), 3.72-3.54 (m, 42H),

3.39 (s, 3H), 2.46 (s, 3H); MS (m/z): found 693.6 (M + Na).

HN TOMe Me -OY N OMe f, 0W 1 1 K 2C03 /Kl 10 a MeO OTs + HO OH MHO OH 255a 28 255b

Compound 255b:

[319] To the mixture of the tosylate 255a (630 mg, 0.939 mmol) and aniline 28 (238 mg, 0.939

mmol) in anhydrous DMF (3 mL) was added anhydrous potassium carbonate (195 mg, 1.409

mmol) and potassium iodide (31.2 mg, 0.188 mmol). The mixture was heated to 85 °C and

stirred at that temperature overnight. The solution was cooled to room temperature and diluted

with dichloromethane. It was filtered through celite and the solid was washed with dichloromethane. The filtrate was evaporated and the residue was diluted with dichloromethane and filtered again to remove the additional solid. The filtrate was evaporated and the residue was purified by silica gel chromatography (hexanes/10% methanol in THF) to give compound 255b as a colorless oil (41.8 mg, yield = 5.9%). H NMR (400 Hz, CDC 3): 6 6.66 (s, 2H), 6.65 (s,

1H), 4.60 (s, 4H), 3.69 (s, 3H), 3.66-3.58 (m, 42H), 3.56-3.53 (m, 2H), 3.39-3.36 (m, 5H), 2.52

(broad s, 20H), 2.36 (t, J = 7.2 Hz, 2H), 1.91 (p, J = 7.2 Hz, 2H); 1 3 C NMR (400 Hz, CDC 3 ): 6

173.9, 148.5, 142.8, 113.4, 109.9, 72.1, 70.8, 70.7, 68.9, 65.7, 59.2, 51.8, 50.9, 50.7, 31.3, 22.4;

MS (m/z): found 774.3 (M + Na)t.

1. MsCIFEA/CH 2 C1 2 MeO W>N' <OMe 2. K 2C0 3/DMF 10 0 MeO<- YN>< 1 0_L 0 Me -N O<~->1-O HOMO HO «<OHHO N=i. N OMe MeO> 255b 8 O 255c (IGN-26-OMe)

Compound 255c (IGN-26-OMe):

[320] To a stirred solution of compound 255b (41.8 mg, 0.056 mmol) in anhydrous

dichloromethane (1 mL) was added triethylamine (27 pl, 0.196 mmol). The mixture was cooled

to -10 °C and methanesulfonyl chloride (12.9 pl, 0.167 mmol) was added slowly in 15 minutes.

The solution continued to be stirred between -10 °C to -5 °C for 60 minutes and quenched by

addition of ice/water. It was diluted with ethyl acetate and washed with cold water. The organic

layer was dried over anhydrous sodium sulfate, filtered, evaporated and high vacuumed to give

the mesylates as colorless oil. MS (m/z): found 930.3 (M + Na)<. The mesylates (30 mg, 0.033

mmol) was transferred to a 5 mL round bottom flask with ethyl acetate, evaporated and high vacuumed. Compound 8 (29.2 mg, 0.099 mmol) was added followed by addition of anhydrous

DMF (0.5 mL), anhydrous potassium carbonate (22.8 mg, 0.165 mmol) and potassium iodide

(5.5 mg, 0.033 mmol). The mixture was stirred at room temperature overnight. It was diluted

with dichloromethane and washed with brine. The organic layer was dried over anhydrous

sodium sulfate, filtered and evaporated. The residue was purified by silica gel chromatography

(hexanes/10% methanol in THF) to give 20.5 mg of a mixture which contained compound 255c.

It was dissolved in anhydrous dichloromethane (0.3 mL). Triethylamine (4 pl, 0.03 mmol),

tosyl chloride (3.8 mg, 0.02 mmol) and DMAP (0.2 mg, 0.002 mmol) were added subsequently

at room temperature. The mixture continued to be stirred at room temperature overnight and

then was evaporated. The residue was purified by silica gel chromatography

(dichloromethane/methanol) to give 11 mg of light yellowish foam. It was further purified by

preparative reverse phase HPLC (C18 column, eluted with CH 3CN/H 20) to give compound 255c

(IGN-26-OMe) as colorless foam (1.6 mg, yield = 2.2%). MS (ml/z): found 1326.5 (M + Na),

1344.6 (M + H 2 0 + Na), 1362.5 (M + 2H 20 + Na).

Example 23

BnO N=' NaBH 4 BnO HN- NeOXIO4NC>b MeO 96% MeO 7 256a

Compound 256a:

[321] To a stirred solution of compound 7 (384 mg, 1.0 mmol) in absolute ethanol (6 mL) and

anhydrous dichloromethane (2 mL) was added sodium borohydride (37.8 mg, 1.0 mmol) at 0 °C. After 30 minutes, the ice/water bath was removed and the reaction mixture continued to be

stirred at room temperature overnight. The reaction was quenched by addition of saturated

ammonium chloride and diluted with dichloromethane. The mixture was separated and the

organic layer was washed with brine, dried over anhydrous sodium sulfate and filtered. The

solvents were removed under reduced pressure to give compound 256a as a white solid (369 mg,

yield 96%). 1H NMR (400 Hz, CDCl 3 ): 6 8.37 (d, J = = 8.0 Hz, IH), 7.50 (s, 1 H), 7.40-7.24 (in,

6H), 7.18 (d, J = 7.2 Hz, 1H), 7.05 (t, J = 7.2 Hz, 1H), 6.12 (s, 1H), 5.06 (s, 2H), 4.40 (tt, Ji =

10.0 Hz, J2 = 3.6 Hz, 1H), 3.87 (s, 3H), 3.52-3.41 (m, 3H), 2.78 (dd, Ji= 16.8 Hz, J2 = 3.6 Hz,

1H); 3C NMR (400 Hz, CDCl 3): 6 166.5, 152.1, 142.73, 142.70, 140.4, 136.3, 129.5, 128.5,

127.9, 127.7, 127.1, 124.5, 123.8, 117.2, 114.5, 112.7, 103.4, 70.5, 57.1, 56.2, 54.5, 33.1; MS

(m/z), found 409.2 (M + Na)+.

CH 3 1 BnO H _N~- KCO_ BnO N-

MeO r N 51% MeO- Y 256a 256b

Compound 256b:

[322] To a solution of compound 256a (369 mg, 0.955 mmol) in anhydrous acetonitrile (9 mL)

was added iodomethane (65 pl, 1.05 minol) and potassium carbonate (158 mg, 1.15 mmol). The

mixture was stirred, heated to 82 aC and refluxed overnight. The reaction mixture was removed from the oil bath, cooled to room temperature and diluted with dichloromethane. It was filtered through celite and the filtrate was evaporated under reduced pressure. The residue was purified through silica gel chromatography (hexanes/ethyl acetate) to give compound 256b as a colorless foam (195 mg, yield = 51%). Also 123 mg of starting material 256a was recovered. 'H NMR

(400 Hz, CDCl3 ): 6 8.29 (d, J = 8.0 Hz, 1H), 7.46 (s, 1H), 7.44 (s, 1H), 7.39-7.24 (m, 5H), 7.16

(d, J = 7.2 Hz, 1H), 7.04 (t, J = 7.6 Hz, 1H), 6.46 (s, 1H), 5.19 (dd, Ji = 15.2 Hz, J2 = 12.4 Hz,

2H), 4.36-4.29 (m, 1H), 3.89 (s, 3H), 3.38-3.31 (in, 2H), 3.02 (dd, J1 = 10.8 Hz, J2 = 4.0 Hz, 1H),

2.70 (dd, J 1 = 16.8 Hz, J2 3 = 2.8 Hz, I H), 2.65 (s, 3H); C NMR (400 Hz, CDCl 3): 6 166.9,151.2,

144.2, 142.1, 141.9, 136.7, 129.8, 128.6, 128.0, 127.8, 127.3, 125.1, 123.9, 121.7, 117.1, 113.5,

104.7, 71.1, 64.2, 57.2, 56.3, 40.2, 32.0; MS (m/z): found 423.2 (M + Na)*.

BnO N -- H 2 /Pd/C/AcOEt HO N

MeO ) N 8 3% MeO

256b 256c

Compound 256c:

[323] To a stirred solution of compound 256b (195 mg, 0.487 mmol) in ethyl acetate (2.5 mL)

was added palladium on charcoal (10%, 25.9 mg, 0.024 mmol). The flask was briefly vacuumed

and replaced with H 2 in a balloon. The mixture continued to be hydrogenated for ovemight and

filtered through celite. The filtrate was stripped under reduce pressure and the residue was

purified by silica gel chromatography (dichloromethane/methanol) to give compound 256c as a

white solid (126 mg, yield = 83%). 1H NMR (400 Hz, MeOD): 6 8.14 (d, J = 8.0 Hz, 1H), 7.30

7.23 (m, 2H), 7.22 (s, 1H), 7.10 (t, J = 7.6 Hz, 1H), 6.56 (s, 1H), 4.46-4.38 (m, 1H), 3.88 (s, 3H),

3.48-3.37 (in, 2H), 3.12 (dd, J1 = 10.8 Hz, J2 = 4.4 Hz, 1H), 2.84 (dd, Ji= 16.8 Hz, J2 = 2.8 Hz,

1H), 2.80 (s, 3H).

MeoOe O-"N O^ e MeO <O N- OMe MeO-rgO>O N OMe 0 1. MsCI/TEA/CH 2Cl 2 0 0 HO - OH 2. monomers 8 and 256c - N 0 0 N- + 0» O N

N ' OMe MeO N OMe MeO 249c K 2CO3/DMF 256d (IGN-29-OMe) 256e

Compound 256d (IGN-29-OMe):

[324] To a stirred solution of compound 249c (136 mg, 0.34 mmol) in anhydrous

dichloromethane (2 mL) was added triethylamine (142 Il, 1.02 mmol). The mixture was cooled

to -10 °C and methanesulfonyl chloride (66 pl, 0.85 mmol) was added slowly in 15 minutes. The

solution continued to be stirred between -10 °C to -5 °C for 60 minutes and quenched by addition

of ice/water. It was diluted with ethyl acetate and washed with cold water. The organic layer

was dried over anhydrous sodium sulfate, filtered, evaporated and high vacuumed to give the

mesylate as colorless oil. The mesylate was transferred to a 10 mL round bottom flask with ethyl

acetate, evaporated and high vacuumed. Compound 8 (120 mg, 0.41 mmol) and 256c (106 mg,

0.34 mmol) were added to it followed by addition of anhydrous DMF (1.5 mL), anhydrous

potassium carbonate (235 mg, 1.7 mmol). The mixture was stirred at room temperature

overnight. It was diluted with dichloromethane and washed with brine. The organic layer was

dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by

preparative reverse phase HPLC (C18 column, eluted wit CH3 CN/H20) to give compound 256d

(IGN-29-OMe) as a light yellowish solid (46 mg, yield = 14%) and compound 256e. 256d: 'H

NMR (400 Hz, CDCl 3 ), 6 8.27 (d, J = 8.0 Hz, 2H), 7.84 (d, J = 4.8 Hz, 1H), 7.57 (s, 1H), 7.32

7.04 (m, 7H), 6.87 (s, IH), 6.82 (s, 1H), 6.76-6.70 (in,2H), 6.50 (s,1H)5.18-5.12 )m, 4H), 4.49

4.43 (m, 1H), 4.40-4.35 (m, 1H), 3.98 (s, 3H), 3.89 (s, 3H), 3.68-3.52 (m, 18H), 3.41-3.36 (m,

6H), 3.08 (dd, Ji= 10.8 Hz, J2 = 4.4 Hz, 1H), 2.56 (dd, Ji= 16.8 Hz, J2 = 2.8 Hz, 1H), 2.70 (s,

3H), 2.34 (t, J = 7.2 Hz, 2H), 1.92-1.85 (m, 2H); MS (m/z): found 990.6 (M + Na) ,1008.6 (M

+ H 2 0 + Na). 256e: MS (m/z): found 1006.6 (M + Na)

. 0

MeaQ oO-<>N<"OMe 0 MeO O N OH Me0--0- N0- -N 0 N Me3SnOH O NHS/EDC 0 0 N~<>~ 0 ' CICH 2CH2CI ! DMAP N ff~ y=8lN CH 2CI 2 Me>r , <N- MeO N =OMe HOMe MeO 6Me

256d (IGN-29-OMe) 256f 256g (IGN-29-NHS)

Compound 256f and Compound 256% (IGN-29-NHS):

[325] To the solution of compound 256d (46 mg, 0.048 mmol) in anhydrous 1,2 dichloroethane

(1.5 mL) was added trimethyltin hydroxide (129 mg, 0.71 mmol). The mixture was heated to 80

°C and stirred overnight. It was cooled to room temperature, diluted with dichloromethane and

washed with mixed solution of saturated sodium chloride and 5% hydrochloric acid (~1 mL),

then brine. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated.

The residue was passed a short silica gel column and eluted with dichloromethane/methanol to

remove the extra trimethyltin hydroxide. The product fractions were evaporated and high

vacuumed to give the acid 256f as a yellowish solid (36.9 mg, yield = 81%). MS (m/z): found

952.8 (M - H)-. Compound 256f (36.9 mg, 0.039 mmol) was then dissolved in anhydrous dichloromethane (0.8 mL). N-hydroxysuccinimide (NHS, 13.4 mg, 0.12 mmol) and N-(3 dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, 22.2 mg, 0.12 mmol) was added subsequently. The mixture was stirred at room temperature overnight and diluted with dichloromethane, washed with brine and dried over anhydrous sodium sulfate. It was filtered, evaporated and the residue was purified by preparative reverse phase HPLC (C18 column, eluted with acetonitrile/water). The fractions containing product were combined and extracted with dichloromethane. The organic layers were dried over anhydrous sodium sulfate, filtered, evaporated and high vacuumed to give compound 2 56g (IGN-29-NHS) as a light yellowish solid

(25.4 mg, yield = 62%). MS (m/z): found 1073.4 (M + Na), 1091.4 (M + H20 + Na)*, 1103.3

(M + 3H 20 - H)-.

Example 24

MeO 0 OMe O NO 2 TEA O NO 2 0 2N H N0 CI O N

258a O O 4 258b NO2

methyl 1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-nitroindoline-2-carboxylate (258b):

[326] Methyl 6-nitroindoline-2-carboxylate (258a) (0.233 g, 1.048 mmol) was dissolved in

anhydrous tetrahydrofuran (4 ml) in a separate flask and cooled to 0 °C an ice bath. In another

flask 4-(benzyloxy)-5-methoxy-2-nitrobenzoyl chloride (4) (0.371 g, 1.153 mmol) was dissolved in anhydrous tetrahydrofuran (4 ml) and cooled to 0 °C in an ice bath. To the flask containing the indoline was added triethylamine (0.438 ml, 3.15 mmol) via syringe and the acetyl chloride 4 was added quickly to the reaction mixture via cannula at 0 °C. The reaction was stirred for 90 minutes at 0 °C and then at room temperature for an additional 1 hour. The reaction was then quenched with 5% HCI and extracted with ethyl acetate (3x). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by silica gel chromatography using 30% Acetone in hexane to give methyl 1-(4

(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-nitroindoline-2-carboxylate (258b) (0.220 g, 0.434

mmol, 41.4 % yield) as a yellowish foam. 'H NMR (400 Hz, CDCl 3 ): 6 3.30 (m, 1H), 3.60 (s,

3H), 3.69 (in, 1H), 3.86 (s, 3H), 4.64 (dd, 1 H, J= 2.4 Hz, 10.8), 5.23 (s, 2H), 7.31 (m, 1H), 7.46

(m, 6H), 7.99 (dd, 1H, J = 2.0, 8.0 Hz), 9.04 (d, IH, J = 2.0 Hz). MS (m/z), found 530.1

([M]*+Na).

MeO O DIBAL-H H 0

o NO 0 -NNO

N58 00 NN

258b N2 258c NO 2

1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-nitroindoline-2-carbaldehyde (258c):

[327] Methyl 1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-nitroindoline-2-carboxylate

(258b) (1.023 g, 2.016 mmol) was dissolved in a mixture of anhydrous dichloromethane (2.5

mL) and toluene (7.5 mL) and cooled to -78 °C in a dry ice and acetone bath. After 15 minutes

DIBAL-H (1.OM in THF) (4.03 mL, 4.03 mmol) was added via a syringe pump over about a 20 minute period. The resulting solution was stirred for 2 hrs at -78 °C after which methanol (Iml) was added dropwise to quench the reaction at -78 °C. The reaction was then diluted with 5%

HCI and ethyl acetate and warmed to room temperature. The aqueous layer was washed with

additional ethyl acetate and the combined organic layers were washed with brine and dried over

anhydrous sodium sulfate. The reaction mixture was passed through a layer of celite and

concentrated in vacuo. The crude residue was purified by silica gel chromatography using 40%

acetone in hexane to give 1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-nitroindoline-2

carbaldehyde (258c) (621 mg, 1.301 mmol, 64.5 % yield). 1H NMR (400 Hz, CDCI 3 ): 6 3.15

3.60 (m, 2H), 3.90 (s, 0.6H), 3.92 (s, 1.2H), 3.97 (s, 1.2H), 4.57 (d, 0.2H, J = 4.8 Hz), 5.21 (m,

2.4H), 5.5 (m, O.4H), 6.39 (s, 0.4H), 6.46 (s, 0.2H), 6.76 (s, 0.2H), 6.89 (s, 0.4H), 7.01 (s, 0.4H),

7.19-7.41 (m, 5.6H), 7.60-7.77 (m, 1.6H), 7.86-7.91 (m, 0.8H), 8.94 (s, 0.4H), 9.34 (s, 0.4H),

9.74 (s, 0.4H), 9.90 (s, 0.2H). MS (m/z), found 500.1 ([M]++Na).

cx-0 N

0 ::,-NOEDC, 0 0O N2 Na 2S 204 DMAP N 0~ 0~ NN 0 0I1 - N

NO 2 HN S 258c 258d NH 2 258e

Compound 258e:

[328] 1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-6-nitroindoline-2-carbaldehyde (258c)

(0.125 g, 0.262 mmol) was dissolved in tetrahydrofuran (8 mL) and water (5.33 mL). To this solution was added sodium hydrosulfite (0.456 g, 2.62 mmol) and the reaction was capped with a septa and vented with a needle (no nitrogen/argon needed) and stirred overnight. Methanol was added to the reaction mixture and stirred an additional 30 minutes at which point the reaction was concentrated in vacuo to remove all solvents. The residue was dissolved in a 1:1 mixture of methanol and dichloromethane (20 mL) which left a residue which did not dissolve. The mixture was passed through a short pad of silica on top of a short pad of celite and rinsed thoroughly with the 1:1 mixture of methanol and dichloromethane. The filtrate was filtered again through celite and then a solution of HCI in dioxane (4M) was added with stirring until a pH of ~3-4 was obtained. The reaction was then stirred for an additional 30 minutes and then aqueous sodium bicarbonate was added until the reaction became basic (~pH 8-9) at which time additional dichloromethane was added and the organic layer removed. The aqueous layer was washed with additional dichloromethane and the resulting organic layers were combined and washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue containing compound 258d (0.105 g, 0.263 mmol, 100 % yield) was used in the next step without further treatment. MS (m/z), found 454.2 ([M]'+Na+CH30H).

To a small vial containing 4-methyl-4-(methyldisulfanyl)pentanoic acid (0.061 g, 0.313 mmol),

EDC (0.060 g, 0.313 mmol), and DMAP (0.038 g, 0.313 mmol) were dissolved in

dichloromethane (1 mL) with stirring. To this mixture compound 258d (0.125 g, 0.313 mmol)

dissolved in dichloromethane (1.5 mL) was added and the mixture was stirred at room

temperature overnight. Water was added and the layers were seperated. The organic layer was

washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified on a silica gel column using 50% ethyl acetate in hexane to give compound 258e

(0.037 g, 0.064 mmol, 20.54 % yield). 'H NMR (400 Hz, CDC 3 ): 6 1.27 (s, 6H), 1.97 (t, 2H, J

= 8.0 Hz), 2.06 (t, 2H, J = 8.0 Hz), 2.45 (s, 3H), 3.48 (in, 1H), 3.67 (m, 1H), 3.99 (s, 3H), 4.49

(m, 1H), 5.24 (q, 2H, J = 8.4 Hz), 6.90 (s, 1H), 7.22 (d, 1H, J = 8.0 Hz), 7.39 (m, 5H), 7.55 (s,

1H), 7.82 (d, 1H, J = 8.0 Hz), 7.87 (d, 1H, J = 4.0 Hz), 8.07 (s, 1H). MS (m/z), found 630.3

([M]++Na+MeOH).

HO N 0 N -0 N. Il MeSO 3H O N

HN 0s 258e HN 258f H-SN O 0

Compound 258f:

[329] Compound 258e (0.0185 g, 0.032 mmol) was dissolved in anhydrous dichloromethane

(0.5 ml) and to this solution was added methanesulfonic acid (0.021 ml, 0.321 mmol) dissolved

in anhydrous dichloromethane (0.500 ml) and the resulting mixture was stirred at room

temperature for three hours. The reaction was poured over a mixture of ice and methanol and

neutralized to pH 7 with aqueous sodium bicarbonate. The reaction was then diluted with

dichloromethane and the layers were separated. The organic layer was washed with brine, dried

over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by silica

ptle using 3% methanol in dichloromethane to give NH(4-methyl-4-methyldithio-pentanoate)

indole IGN monomer (0.007 g, 0.014 mmol, 44.9 % yield). MS (m/z), found 484.0 ([M]--1).

HOK2CO

0 0Nb 8 258g

Compound 258g:

[330] In a small vial dissolved Compound 8 (0.033 g, 0.112 mmol) in DMF (1.5 ml) with

stirring at room temperature. 1,3-diiodopropane (0.065 ml, 0.561 mmol) was added followed by

the addition of potassium carbonate (0.023 g, 0.168 mmol). The reaction was covered in foil and

stirred at room temperature overnight. The reaction was diluted with dichloromethane and

washed with aqueous ammonium chloride and brine. The organic layer was dried over

anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica

2 ptlc using 50% ethyl acetate in hexane to give Compound 58g (0.018 g, 0.039 mmol, 34.7

% yield). MS (m/z), found 533.0 ([M]+K).

HO N , -N ,IO N

N K2C0 3 N N o IN 0hNi 0N

NN -I N 258f HN - .Ss 258g 258h HN S

0 0

Compound 258 h (IGN-15-SMe):

[331] In a small vial dissolved Compound 258f (0.007 g, 0.014 mmol) in dimethylformamide

(1 ml) with stirring at room temperature. Compound 8 (6.66 mg, 0.014 mmol) was added

followed by the addition of potassium carbonate (1.992 mg, 0.014 mmol). The reaction was covered in foil and stirred at room temperature overnight. Reaction was diluted with dichloromethane and washed with aqueous ammonium chloride and brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica ptlc plate in 5% methanol in dichloromethane to give Compound 258h (IGN

-SMe) (0.005 g, 7.32 pmol, 50.8 % yield). MS (m/z), found 906.3 ([M]+Na+2CH30H).

Example 25

N O N HO

O 259a N

Compound 259a:

[332] Ina small vial dissolved Compound 8 (0.100 g, 0.340 mmol) inDMF (5 ml) with stirring

at room temperature. 1,5-diiodopentane (0.506 ml, 3.40 mmol) was added followed by the

addition of potassium carbonate (0.070 g, 0.510 mmol). The reaction was covered in foil and

stirred at room temperature overnight. The reaction was diluted with dichloromethane and

washed with aqueous ammonium chloride and brine. The organic layer was dried over

anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica

ptle using 50% ethyl acetate in hexane to give Compound 259a (0.045 g, 7.32 mol, 27 %

yield). 1H NMR (400 Hz, CDC 3): 6 1.64 (m, 2H), 1.94 (M, 4H), 3.24 (t, 2H, J = 6.5 Hz), 3.52

(dd, 1H, J = 4.0, 16.6 Hz), 3.73 (dd, 1H, J = 10.5, 16.6 Hz), 3.98 (s, 3H), 4.12 (m, 2H), 4.50 (dt,

1H, J = 4.0, 11.2 Hz), 6.84 (s, 1H), 7.13 (t, 1H, J = 6.0 Hz), 7.29 (m, 2H), 7.57 (s, 1H), 7.90 (d,

1H, J = 4.4 Hz), 8.29 (d, 1H, J= 8.0 Hz). MS (m/z), found 533.3 ([M]'+K).

HO N- N- -0 N 0 K2C0 3 N 0 Nb 0 1 N 259b 259a HN 258f HN 0 0

Compound 259b (IGN-21-SMe):

[333] Ina small vial dissolved Compound 258f (15 mg, 0.031 mmol) in dimethylforniamide (1

ml) with stirring at room temperature. Compound 259a (17.42 mg, 0.036 mmol) was added

followed by the addition of potassium carbonate (4.27 mg, 0.031 mmol). The reaction was

covered in foil and stirred at room temperature overnight. Reaction was diluted with

dichloromethane and washed with aqueous ammonium chloride and brine. The organic layer

was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was

purified by silica ptle plate in 5% methanol in dichloromethane to give Compound 259a (IGN

-SMe) (0.006 g, 7.32 pmol, 22 % yield). MS (m/z), found 934.1 ([M]+Na+2CH30H).

Example 26

H D 0 BnO N-O 0 EDC I DMAP N 0n HO D BnO 0 )a 0

256a 0 260a

Compound 260a:

[334] Compound 256a (55 mg, 0.142 mmol) was dissolved in anhydrous dichloromethane and

then 4-methoxy-4-oxobutanoic acid (76mg, 0.575 mmol), EDC (70mg, 0.365 mmol), and DMAP

(8.69 mg, 0.071 mmol) were added sequentially. The mixture was stirred overnight at room

temperature and was checked by TLC to ensure no starting material remained. The reaction was

then diluted with water and ethyl acetate. After further extraction with ethyl acetate, the organic

was washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The crude

residue was purified by silica gel chromatography using 50% ethyl acetate in hexane to give

compound 260a (54 mg, yield = 76%). 'H NMR (400 Hz, CDCl 3): 6 8.21 (d, J = 8.0 Hz, 1H),

7.45-7.25 (m, 7H), 7.20 (d, J = 7.2 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H), 6.825 (s, 1H), 5.27 (q, J =

15.1 Hz, 2H), 4.56 (t, J = 12.6 Hz, 1H), 4.35-4.29 (m, 1H), 3.99 (s, 3H), 3.65 (s, 3H), 3.44

3.38(m, 2H), 2.88 (dd, Ji = 16.4 Hz, J2 = 2 Hz, 1H ), 2.58-2.50 (m, 1H), 2.40-2.33 (m, 1H), 2.26

2.18 (m, 1H), 1.99-1.92 (m, 1H); MS (m/z), found 523.1 (M + Na)+.

0 0

O MeSO 3H BnO N.. HO N

N O N

260a 260b

Compound 260b:

[335] To a solution of compound 260a (50mg, 0.100 mmol) in anhydrous dichloromethane

(11.5 ml) was added drop wise methanesulfonic acid (0.389 ml, 5.99 mmol) resulting in a yellow

solution. The reaction stirred at room temperature and was monitored by TLC until completion

beginning at 30 minutes. It was diluted with water and methanol then neutralized to pH 7 using

saturated sodium bicarbonate. The aqueous layer was extracted with dichloromethane and the

organic layer dried over sodium sulfate. The crude product was purified by silica gel

chromatography using 6% methanol in dichloromethane to give compound 260b (40mg, yield =

98%). 1H NMR (400 Hz, CDC 3): 6 8.22 (d, J = 8.0 Hz, 1H), 7.35 (s, 1H), 7.28 (t, J = 7.8 Hz,

IH), 7.22 (d, J = 7.2 Hz, 1H), 7.09 (t, J = 7.4 Hz, 1H), 6.90 (s, 1 H), 6.06 (s, 1H), 4.63 (t, J = 12.6

Hz, 1H), 4.38-4.30 (m, 1H), 4.00 (s, 3H), 3.66 (s, 3H), 3.47-3.39 (m, 2H), 2.90 (dd, Ji= 16.2

Hz, J2 = 2.2 Hz, 1H), 2.69-2.59 (m, 2H), 2.52-2.45 (m, 1H), 2.22-2.14 (m, IH); MS (m/z),found

433 (M +Na).

0 0 o-N ,

HO N- O N K 2CO 3 N 0 O N

0 259a N O O 260c O N

Compound 260c:

[336] Compound 260b (20mg, 0.049 mmol) and compound 259a (30mg, 0.061 mmol) were

dissolved in anhydrous N,N-dimethylformamide (1 ml). Potassium carbonate (20.20 mg, 0.146

mmol) was added and the reaction stirred overnight at room temperature. It was quenched with

water and extracted with dichloromethane. The organic was washed with brine and dried over

sodium sulfate. The crude product was purified by silica gel chromatography using 5%

methanol in dichloromethane to give compound 260c (25 mg, yield = 66%). MS (n/z), found

813.5 (M + Na + H 2 0)+.

Example 27

O O0 MeO 0

HO MO OQ O NO2 TEA BnO NO2Kr S HN-&S ' HN -S + -7 'CI C N. N O O

4 0261a

Compound 261a:

[337] The commercially available starting material, thiazolidine-4-carboxylic acid (1.3g, 9.59

mmol) was dissolved in anhydrous methanol (19.18 mL) and cooled to 0°C in an ice bath.

Thionyl chloride (1.40 mL, 19.18 mmol) was added drop wise and the reaction stirred for 30

minutes. The ice bath was removed and stirring continued either for 4-5 hours or overnight. The

solvent was stripped and the product placed on the high vacuum to give 4

(methoxycarbonyl)thiazolidin-3-ium chloride. Without further purification and assuming 100%

yield, the 4-(methoxycarbonyl)thiazolidin-3-ium chloride (1.761 g, 9.59 mmol) and compound 4

(3.39 g, 10.55 mmol) were each dissolved separately in tetrahydrofuran (32.0 mL) and cooled to

°C. Triethylamine (4.41 mL, 31.6 mmol) was added to the solution with 4

(methoxycarbonyl)thiazolidin-3-ium chloride and then compound 4 was added quickly via

canula. After 20 minutes, the pH of the solution was checked to ensure it was basic. The

reaction stirred at 0°C for 1.5 hours and then at room temperature for 30 minutes and was

checked by MS. It was quenched with cold 5% hydrochloric acid and diluted with cold ethyl

acetate and water. The solution was extracted with ethyl acetate three times and the combined

organic washed with brine, saturated sodium bicarbonate and then brine again. It was dried over sodium sulfate, filtered and stripped. The crude material was purified by silica gel chromatography using a gradient of 50% to 75% ethyl acetate in hexanes to give compound 261a

(4.lg, yield = 99%). 1H NMR (400 Hz, CDCI3): the compound appears as a pair of distinct

rotomers. 6 7.78 (s, 0.6H), 7.74 (s, 0.4H), 7.48-7.35 (in, 5H), 6.96 (s, 0.4H), 6.92 (s, 0.6H), 5.40

(dd, J1 = 7.0Hz, J2 = 3.4 Hz, 0.6H), 5.31-5.22 (i, 2H), 5.13 (d, 9.6Hz, 0.4H), 4.60 (d, J= 9.6 Hz,

O.4H), 4.46 (dd, Ji= 4.4Hz, J2 = 3.2 Hz, 0.4 H), 4.36 (d, J = 8.4 Hz, 0.6 H), 4.26 (d, J= 8.4Hz,

0.6H), 4.02 (s, 1.8H), 3.96 (s, 1.2 H), 3.86 (s, 1.8H), 3.71 (s, 1.2H), 3.48-3.43 (m, 0.6H), 3.36

3.29 (in, 1.4H); MS (m/z), found 455.3 (M + Na)t.

MeON .O OHC BnO NO 2 DIBAL BnO NNO 2 SI. N,/ N-_ 0 0 261a 261b

Compound 261b:

[338] Compound 261a (4.1 g, 9.48 mmol) was dissolved in dichloromethane (11 mL) and

toluene (33 mL) then cooled to -78°C in an acetone/dry ice bath. Diisobutylaluminium hydride

(18.96 mL, 18.96 mnol) was added very slowly, over at least 30 minutes, using a syringe pump.

The reaction stirred at -78°C for 3 hours and was quenched with methanol (0.4mL) and then 5%

hydrochloric acid (30mL). Ethyl acetate (100ml) was added and the ice bath removed. The

mixture continued to stir at room temperature for 30 minutes. It was extracted using ethyl

acetate and the combined organic washed with brine, saturated sodium bicarbonate, and then

brine again. It was dried over anhydrous sodium sulfate and filtered through celite. The crude material was purified by silica gel chromatography using 75% ethyl acetate in hexanes to give compound 261b (2.3g, yield = 60%). 'H NMR (400 Hz, CDCl 3 ): the compound appears as a pair of rotomers. 6 9.80 (s, 0.8H), 9.41 (s, 0.2H), 7.80 (s, 0.8H), 7.73 (s, 0.2H), 7.49-7.36 (m, 5H),

6.91 (s, 0.2H), 6.84 (s, 0.8H), 5.25-5.22 (in, 2H), 4.85-4.73 (in, 1H), 4.35-4.30 (in, 1H), 4.22

4.17 (m, 1H), 4.04-3.97 (m, 3H), 3.40-3.26 (m, 2H); MS (m/z), found 425.0 (M + Na)f.

OHC BnO Ng

OaS24 0): 0 0 261b 261c

Compound 261c:

[339] Compound 261b was dissolved in tetrahydrofuran (230 mL) then water (150 mL).

Sodium hydrosulfite (5.27 g, 25.7 mmol) was added slowly, in small portions. If the solution

remained cloudy, additional water was added drop wise until the solution cleared. The reaction

was capped with a septa and needle to allow release of the SO 2 gas and was stirred overnight.

The solution changed from a yellow to very pale, almost colorless solution. The following

morning, water was added until the solution cleared and then methanol (30 mL) was added. It

stirred for an additional 2 hours and the solvents were then evaporated and the residue re

evaporated with acetonitrile at least twice. The white residue was placed on the high vacuum for

a few hours. It was re-dissolved in methanol:dichloromethane [1:1], filtered through celite, and

stripped. The filter step was repeated until dilution in methanol appeared clear with no particles.

The intermediate was placed on the high vacuum until completely dry then dissolved in anhydrous methanol (50ml). Acetyl chloride (1.9m, 26.7 mmol) was added drop wise at room temperature, causing a yellow precipitate to form. It stirred at room temperature for 30 minutes and was quenched with saturated sodium bicarbonate. The mixture was diluted with dichloromethane and water (130mL/85mL) and extracted with dichloromethane. The aqueous layer was acidified with sodium hydrogensulfate, concentrated to a reduced volume, and then re extracted. The combined organic was washed with saturated sodium bicarbonate and brine and dried over sodium sulfate. The stripped residue was purified by silica gel chromatography using

% ethyl acetate in hexanes to give compound 261c (1. 2 g, yield = 59 %). 1 H NMR (400 Hz,

CDC 3 ): 6 7.69 (d, J = 4.4Hz, 1 H), 7.52-7.28 (in, 6H), 6.87 (s, 1H), 5.22 (q, J = 12.3 Hz, 2H),

4.85, (d, J = 10.4Hz, IH), 4.58 (d, J = 10.4 Hz, 1H), 4.03-4.02 (in, IH), 3.98 (s, 3H), 3.51-3.47

(in, 1H), 3.45-3.23 (in, 1H); MS (m/z), found 377.3 (M + Na).

BnO N HO N

O N~ TFA O N j 0 0 261c 261d

Compound 261d:

[340] Compound 261c (75mg, 0.212 mmol) was dissolved in neat trifluoroacetic acid (0.4 ml,

5.19 mmol) . It refluxed for approximately I hour at 50°C and then the temperature was

increased to 80°C. After 3 hours total, the solvent was evaporated. The residue was directly

purified by PTLC using 5 % methanol in dichloromethane to give compound 261d (19.4 mg,

%). '1H NMR (400 Hz, CDCl 3 ): 67.72 (d, J = 4.4 Hz, I H), 7.51 (s, I H), 6.91(s, 1H), 6.18 (s,

1H), 4.85 (d, J= 10.4Hz, 1H), 4.58 (J = 10.4 Hz, 1H), 4.05-4.02 (m, 1H), 3.99 (s, 3H), 3.50 (dd,

Ji = 12.4Hz, J2 = 6 Hz, 1H), 3.32, (dd, Ji= 12.4 H, J 2 = 2 Hz, 1H); MS (m/z), found 319.0 (M

+ Na+ MeOH).

Example 28

N1. MsCI, TEA O 0 2. 261d, K2CO3 HO OH N O N

249c SN O 62 -N 0 0

Compound 262:

[341] Compound 249c (18 mg, 0.045 mmol) was dissolved in anhydrous dichloromethane

(0.45mL) and then cooled in an ice/brine bath. First, triethylamine (0.022 ml, 0.158 mmol) and

then methanesulfonyl chloride (10.46 1, 0.135 mmol) were added; the second very slowly. The

mixture continued to stir in the bath for 1 hour. The reaction was quenched with ice/water and

diluted with cold ethyl acetate. After separation, the organic layer was washed again with cold

water and dried over sodium sulfate. It was filtered and evaporated under reduced pressure,

keeping the temperature below 20°C, and then placed on the high vacuum to be used directly.

Once completely dry, the product, and compound 261d (28.5 mg, 0.108 mmol) were dissolved in

anhydrous N,N-dimethylformamide (350 pL). Potassium carbonate (29.8 mg, 0.216 mmol) was added. After stirring overnight at room temperature, the reaction was diluted with dichloromethane, washed with brine, dried over sodium sulfate, filtered and stripped.

The crude product was first purified by silica gel chromatography using 4% methanol in

dichloromethane to remove baseline residue. The recovered material was then purified using

reverse phase HPLC ( C18 column, CHCN/H 20, loaded column with 3:1, centrifuged before

injection) to give compound 262 as a solid. 'H NMR (400 Hz, CDCl 3 ): 6 7.68 (dd, Ji = 4.4 Hz, J2

= 1.6 Hz, 2H), 7.51 (s, 2H), 6.86 (s, 2H), 6.78 (s, 1H), 6.71 (s, 2H), 5.16 (dq, Ji= 8.4 Hz, J2 =2.2,

4H), 4.85 (d, J = 10.4 Hz, 2H), 4.58 (J = 10.4 Hz, 2H), 4.04-3.97 (i, 7H), 3.68-3.38 (in, 18 H),

3.40-3.29 (in. 7H), 2.33 (t, 7.2 Hz, 2H), 1.89-1.35 (in, 2 H) MS (m/z), found 914.1 (M + Na).

Example 29 (IGN-13)

OH

HO -N OH O 0 21 TsO OO A HO 1 21 "O OH 263a 0 K2CO3, DMF HO '

26% 263b

methyl 3-(2-(2-(2-(3,5-bis(hydroxymethvl)phenoxy)ethoxy)ethoxy)ethoxy) propanoate

(263b):

[342] To a stirred mixture of methyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate

(263a) (1.504 g, 3.85 mmol) and (5-hydroxy-1,3-phenylene)dimethanol (21) (0.54 g, 3.50 mmol)

in anhydrous DMF (7.8 ml) was added potassium carbonate (0.726 g, 5.25 mmol). The reaction

was stirred at room temperature for 18 hours at 75 aC. The mixture was allowed to cool to room temperature, quenched with water, and extracted with ethyl acetate. The organic extracts were washed with brine, dried over anhydrous magnesium sulfate, filtered and concentrated.

Purification by silica gel chromatography (5% MeOH/CH 2C 2) yielded methyl 3-(2-(2-(2-(3,5

bis(hydroxymethyl)phenoxy)ethoxy) ethoxy)ethoxy)propanoate (263b)(340mg, 26%). 'H NMR

(400 Hz, CDC 3 ): 66.83 (s, 1H), 6.75 (s, 2H), 4.52 (s, 4H), 4.05 (t, J = 4.8 Hz, 2H), 3.79 (t, J =

4.8 Hz, 2H), 3.70 (t, J = 6.4 Hz, 2H), 3.65 (s, 3H), 3.70-3.56 (in, 8H), 3.26 (s, 2H), 2.55 (t, J =

6.4 Hz, 2H); 1 3 C NMR (400 Hz, CDCI3): 6 172.31, 159.1, 143.0, 117.7, 112.1, 70.8, 70.7, 70.5,

70.4, 69.8, 67.5, 66.6, 64.7, 51.8, 34.9; MS (m/z), found 395.2 (M + Na).

-,,-O-O,-o 0 0 1. MsCI, Et3 N N o~

HO 3 EDMF OMe 'OH MeO 263b 263c MeO ANb 263c 8 30%

Compound 263c:

[343] To a stirred solution of methyl 3-(2-(2-(2-(3,5-bis(hydroxymethyl)phenoxy)ethoxy)

ethoxy)ethoxy)propanoate (263b) (145 mg, 0.389 mmol) in anhydrous dichloromethane (5.5 ml)

was added triethylamine (0.163 ml, 1.168 mmol). The mixture was cooled to -5 0 C and

methanesulfonyl chloride (0.076 ml, 0.973 mmol) was added slowly. After stirring for one hour

at -5 °C the reaction was quenched with cold water and extracted with cold ethyl acetate. The

organic extracts were washed with cold water, dried over anhydrous sodium sulfate, filtered and

concentrated under reduced pressure to give methyl 3-(2-(2-(2-(3,5 bis((methylsulfonyloxy)methyl) phenoxy)ethoxy)ethoxy)ethoxy) propanoate. MS (m/z), found

551.1 (M + Na)+. To a stirred mixture of methyl 3-(2-(2-(2-(3,5-bis((methylsulfonyloxy)methyl)

phenoxy)ethoxy)ethoxy)ethoxy)propanoate (206 mg, 0.390 mmol) and compound 8 (287mg,

0.974 mmol) in anhydrous DMF (3.9 ml) was added potassium carbonate (269 mg, 1.949 mmol).

The reaction was allowed to stir at room temperature for 18 hours. The mixture was quenched

with water and extracted three times with dichloromethane. The organic extracts were washed

with water and brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo.

Purification by flash silica gel chromatography (5% MeOH/CH 2C 2) followed by preparative

reverse phase HPLC (C18 column, eluted with CH 3CN/H 20) gave compound 263c (110mg,

%) as a white solid. H NMR (400 Hz, CDC 3 ): 6 8.18 (d, J = 8.0 Hz, 2H), 7.77 (m, 2H), 7.49

(s, 2H), 7.19 (m, 4H), 7.02 (m, 2H), 6.89 (s, 2H), 6.87 (s, 1H), 6.75 (s, 2H), 5.10 (m, 4H), 4.39

(m, 2H), 4.05 (m, 2H), 3.90 (s, 6H), 3.77 (m, 2H), 3.67 (t, J = 6.4 Hz, 2H), 3.64 (m, 2H), 3.59 (s,

3H), 3.70-3.54 (in, 8H), 3.40 (in, 2H), 2.51 (t, J = 6.4 Hz, 2H); MS (m/z), found 965.3 (M

+ H 20+Na)+, 983.3 (M +2H 20+ Na).

0--0 0--- O-OsOO--0-,O OH

Me3SnOH N OQ: O N,

70% O5e263dMeO N Oe MeO 1. ~~ 0 J I263d 263c

Compound 263d:

[344] To a solution of compound 263c (51 mg, 0.055 mmol) in 1,2-Dichloroethane (2.2 ml)

was added trimethyl tin hydroxide (199 mg, 1.103 mmol). The reaction was stirred for 18 hours at 80 °C, then cooled to room temperature, and quenched with saturated ammonium chloride.

The mixture was extracted with dichloromethane. The organic layer was washed with brine,

dried over anhydrous sodium sulfate, filtered and concentrated. Purification by silica gel

chromatography (10% MeOH/CH 2C 2) yielded compound 263d (35mg, 70%). 'H NMR (400

Hz, CDC 3 ): 6 8.26 (d, J = 8.0 Hz, 2H), 7.88 (m, 2H), 7.58 (s, 2H), 7.28 (m, 4H), 7.11 (m, 3H),

7.00 (s, 2H), 6.88 (s, 2H), 5.21 (m, 4H), 4.49 (m, 2H), 4.18 (in, 2H), 4.00 (s, 6H), 3.89 (in, 2H),

3.79 (in, 2H), 3.70 (m,1OH), 3.51 (in, 2H), 2.62 (in, 2H); MS (m/z), found 909.2 (M -1)-, 927.2

(M -1+ H 20)-, 945.2 (M -1+2H 2 0)-.

0

O -O,-,aO,-HO-NO'N N~N0 O O O N 0_,O -N OO O- , Ng

OMe Me EDCDMAP N OMe Me0 ' N Ne 13% 1. 0 0 (N, 263d 263e

Compound 263e:

[345] To a solution of compound 263d (30 mg, 0.033 mmol) in anhydrous dichloromethane

(2.5 mL) was added N-hydroxy succinimide (9.77 mg, 0.082 mmol), EDC (15.78 mg, 0.082

mmol), and DMAP (0.406 mg, 3.29 ptmol). The reaction was stirred for 18 hours at room

temperature and then diluted with dichloromethane. The mixture was washed with saturated

ammonium chloride and brine. The organic layer was dried over anhydrous sodium sulfate,

filtered and concentrated in vacuo. The crude material was purified by preparative reverse phase

HPLC (C18 column, eluted with CH 3CN/H 20). Fractions containing product were extracted

with dichloromethane, dried over anhydrous sodium sulfate, filtered and co-evaporated with acetonitrile under reduced pressure to give compound 263e (4.5mg, 13%) as a white solid; MS

(m/z), found 1030.4 (M + Na)', 1046.3 (M +K)+.

Example 30 (IGN-27)

NH2 HO OH HN O O.

TsO- O O"'O O 26 HO OH 3 0 263a 0 K 2CO 3 , DMF 25% 264a

methyl 3-(2-(2-(2-(3,5-bis(hydroxymethyl)phenylamino)ethoxy)ethoxy) ethoxy)propanoate

(264a):

[346] To a mixture of methyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate (263a)

(250 mg, 0.640 mmol) and (5-amino-1,3-phenylene)dimethanol (26)(108 mg, 0.704 mmol) in

anhydrous DMF (1.4 ml) was added potassium carbonate (133 mg, 0.960 mmol). The reaction

stirred for 18 hours at 80 °C and then was allowed to cool to room temperature. The mixture was

quenched with water and extracted two times with ethyl acetate. The organic extracts were

washed with brine, dried over anhydrous magnesium sulfate, filtered and concentrated.

Purification by silica gel chromatography (5% Methanol/methylene chloride) yielded methyl 3

(2-(2-(2-(3,5-bis(hydroxymethyl)phenylamino)ethoxy)ethoxy) ethoxy)propanoate (264a) (61mg,

%); 1H NMR (400 Hz, CDC 3): 6 6.58 (s, 1H), 6.47 (s, 2H), 4.49 (s, 4H), 3.67 (t, J = 6.4 Hz,

2H), 3.62 (s, 3H), 3.64-3.54 (m, 10H), 3.21 (t, J = 5.2 Hz, 2H), 2.51 (t, J = 6.4 Hz, 2H); MS

(m/z), found 394.3 (M + Na)+.

HN Mel, K 2CO 3 N OO

HO OH 3 CH 3CN HOJi OH

264a 56% 264b

Compound 264b:

[347] To a solution of methyl 3-(2-(2-(2-(3,5-bis(hydroxymethyl)phenylamino)ethoxy)

ethoxy)ethoxy)propanoate (264a)(60 mg, 0.162 mmol) in acetonitrile (1.6 ml) was added

iodomethane (0.013 ml, 0.210 mmol) and potassium carbonate (26.8 mg, 0.194 mmol). The

reaction stirred at 82 aC for 18 hours. The mixture was cooled to room temperature and then the

solvent was removed under reduced pressure. The crude material was diluted with 3:1

CH 2 C 2 /MeOH and filtered through Celite. The filtrate was concentrated and purified by silica

gel chromatography eluting with 5% Methanol/dichloromethane to give Compound 264b (35mg,

56%). H NMR (400 Hz, CDC 3 ): 6 6.58 (s, 3H), 4.52 (s, 4H), 3.64 (t, J = 6.4 Hz, 2H), 3.60 (s,

3H), 3.53 (in, 12H), 2.91 (s, 3H), 2.51 (t, J = 6.4 Hz, 2H), 2.28 (s, 2H); 1C NMR (400 Hz,

CDC 3 ): 6 172.1, 149.8, 142.4, 113.4, 109.9, 70.7, 70.6, 70.4, 70.3, 68.6, 66.5, 65.6, 52.3, 51.7,

38.9, 34.8; MS (m/z), found 408.4 (M + Na).

'N ~ ~ >1. MsCI, Et 3N N3

3 HOIk.HHO HOOH 2.K2 C 3, DMF O0e MeO IN 0 00 N 264b MeO N 264c

19%

Compound 264c:

[348] To a stirred solution of compound 246b (60 mg, 0.156 mmol) in anhydrous

dichloromethane (2.8 mL) was added triethylamine (0.065 mL, 0.467 mmol). The mixture was

cooled to -5 ° C and methanesulfonyl chloride (0.030 mL, 0.389 mmol) was added slowly. After

stirring for one hour at -5 °C the reaction was quenched with cold water and extracted with cold

ethyl acetate. The organic layer was washed with cold water, dried over anhydrous sodium

sulfate, filtered and concentrated under reduced pressure to give the dimesylate intermediate. MS

(m/z), found 564.0 (M + Na). To a mixture of the dimesylate linker (49 mg, 0.090 mmol) and

compound 8 (66.6 mg, 0.226 mmol) in anhydrous DMF (0.9 mL) was added potassium

carbonate (62.5 mg, 0.452 mmol). The reaction was stirred for 18 hours at room temperature,

quenched reaction with water and extracted three times with dichloromethane. The organic

extracts were washed with water and brine, dried over anhydrous sodium sulfate, filtered and

concentrated in vacuo. Purification by flash silica gel chromatography (5% MeOH/CH 2CI 2

) followed by preparative reverse phase HPLC (C18 column, eluted with CH3CN/H 20) gave

compound 264c (16mg, 19%) as a white solid. 1H NMR (400 Hz, CDCl3 ): 6 8.18 (d, J = 8.0 Hz,

2H), 7.76 (m, 2H), 7.48 (s, 2H), 7.18 (m, 4H), 7.02 (t, J = 7.2 Hz, 2H), 6.79 (m, 2H), 6.74 (s,

1H), 6.65 (s, 2H), 5.08 (m, 4H), 4.39 (in, 2H), 3.89 (s, 6H), 3.66 (t, J = 6.4 Hz, 2H), 3.62 (m,

2H), 3.60 (s, 3H), 3.53 (m, 12H), 3.40 (in, 2H), 2.91 (s, 3H), 2.51 (t, J= 6.4 Hz, 2H); MS (m/z),

found 978.3 (M + H 2 0 + Na)f, 996.3 (M + 2H 20 + Na).

1N O" 0 NN'O "- OH

eO N O 0 Me 3SnOH N N *, OMN Me ks.e-I rN'~ 55% 6 NCk'Me Me0IC)r N 264c 264d 264d

Compound 264d :

[349] To a solution of Compound 264c (26 mg, 0.028 mmol) in anhydrous 1,2-Dichloroethane

(1.1 ml) was added trimethyl tin hydroxide (100 mg, 0.554 mmol). The reaction was stirred for

18 hours at 80 °C. The mixture was allowed to cool to room temperature and extracted with

dichloromethane and saturated ammonium chloride. The organic extracts were washed with

brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by

preparative TLC in 5% Methanol/methylene chloride yielded compound 264d (14mg, 55%). MS

(m/z), found 922.1 (M-1)-, 940.0 (M -1+ H 2 O)-, 958.1 (M -1+ 2H 2 0)-.

0

N( O" OH HO-N N O3 O--N 0 N0 0 N 0 0 N 0

OM9% eOEDO DMAP OM MeO N

(5 MeON 264e 264d

Compound 264e :

[350] To a stirred solution of compound 264d (13 mg, 0.014 mmol) in anhydrous

dichloromethane (1.0 mL) was added N-hydroxysuccinimide (5.01 mg, 0.042 mmol), EDC (8.09

mg, 0.042 mmol), and DMAP (0.172 mg, 1.407 pmol). The reaction stirred for 18 hours at room

temperature. The mixture was extracted with dichloromethane and saturated ammonium chloride. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude material was purified by preparative reverse phase HPLC

(C18 column, eluted with CH 3CN/H 20). Fractions containing product were combined and

extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered and co-evaporated

with acetonitrile under reduced pressure to obtain compound 264e (4.1mg, 29%). MS (m/z),

found 1021.3 (M + H), 1043.2 (M + Na)v, 1061.2 (M + H 2 0 + Na), 1079.2 (M + 2H 20 + Na).

Example 31 (IGN-28)

0 0

o 0'- "O- ' TsCI, Et3 N 0 o O e -'O'DMAP H O O 67% TsO0, o0

265a 265b

methyl 1-(tosyloxy)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oate

(265b):

[351] To a stirred solution of methyl 1-hydroxy-3,6,9,12,15,18,21,24,27,30,33,36

dodecaoxanonatriacontan-39-oate (265a)(1.2 g, 1.897 mmol) in dichloromethane (9.48 mL) at 0

°C was added triethylamine (0.529 mL, 3.79 mmol), toluene sufonylchloride (0.542 g, 2.84

mmol) and DMAP (0.023 g, 0.190 mmol). The mixture was stirred for one hour at 0 °C and then three hours at ambient temperature, after which it was quenched with water and extracted twice

with dichloromethane. The organic extracts were washed with brine, dried over anhydrous

magnesium sulfate, filtered and concentrated in vacuo. Purification by silica gel chromatography

(5% MeOH/CH 2Cl 2) gave methyl 1-(tosyloxy)-3,6,9,12,15,18,21,24,27,30,33,36

dodecaoxanonatriacontan-39-oate (265b)(1.0g, 67%) as a light yellow oil. 1H NMR (400 Hz,

CDC 3): 6 7.80 (d, J= 8.4Hz, 2H), 7.35 (d, J = 8.0Hz, 2H), 4.16 (t, J= 4.8Hz, 2H), 3.75 (t, J=

6.4Hz, 2H), 3.69 (s, 3H), 3.64 (in, 46H), 2.60 (t, J = 6.4 Hz, 2H), 2.45 (s, 3H).

NH2

0 HOJ i1 OH o 26 HN ' O oo 12 \ .o K2 CO 3 , DMF HO OH 42% 265c 265b

methyl 1-(3,5-bis(hydroxymethyl)phenylamino)-3,6,9,12,15,18,21,24,27,30,33,36

dodecaoxanonatriacontan-39-oate(265c):

[352] To a stirred mixture of methyl 1-(tosyloxy)-3,6,9,12,15,18,21,24,27,30,33,36

dodecaoxanonatriacontan-39-oate (265b)(700 mg, 0.890 mmol) and (5-amino-1,3

phenylen)dimethanol (26) (150 mg, 0.978 mmol) in anhydrous DMF (2.0 ml) was added

potassium carbonate (184 mg, 1.334 mmol). The reaction was stirred at 800 C overnight. The

mixture was cooled to room temperature, quenched with water and extracted with 10%

Methanol/Methylene chloride. The organic layer was washed with brine, dried over anhydrous

magnesium sulfate, filtered and concentrated in vacuo. The crude product was purified by silica

gel chromatography (eluted with 5 ->)15% MeOH/CH2CI2) to give methyl 1-(3,5

bis(hydroxymethyl) phenylamino)-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan

39-oate (265c) (285mg, 42%). 1H NMR (400 Hz, CDC 3 ): 6 6.62 (s, 1H), 6.51 (s, 2H), 4.52 (s,

4H), 3.72 (t, J = 6.4 Hz, 2H), 3.65 (s, 3H), 3.61 (m, 48H), 2.94 (s, 2H), 2.63 (s, 1H), 2.57 (t, J=

6.4 Hz, 2H); MS (m/z), found 790.4 (M +Na).

HN O- Mel, K2CO3 N

CHCN HO OH HO OH HO OH92%

265c 265d

methyl 2-(3,5-bis(hydroxymethyl)phenyl)-5,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-2

azahentetracontan-41-oate(265d):

[353] To a stirred solution of methyl 1-(3,5-bis(hydroxymethyl)phenylamino)

3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oate (265c) (67 mg, 0.087 mmol)

in anhydrous DMF (1.0 ml) was added iodomethane (7.06 pl, 0.113 mmol) and potassium

carbonate (14.47 mg, 0.105 mmol). The reaction was stirred at 82 °C for 18 hours. Themixture

was cooled to room temperature, diluted with water and extracted with dichloromethane. The

organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo.

Purification by preparative TLC (10% MeOH/ CH 2 CI 2 ) gave methyl 2-(3,5

bis(hydroxymethyl)phenyl)-5,8,11,14,17,20,23,26,29, 32,35,38-dodecaoxa-2-azahentetracontan

41-oate (265d) (62mg, 92%). 1 H NMR (400 Hz, CDCl 3): 6 6.65 (s, 3H), 4.59 (d, J = 5.6 Hz,

4H), 3.74 (t, J = 6.4 Hz, 2H), 3.67 (s, 3H), 3.61 (m, 46H), 3.54 (t, J = 6.0 Hz, 2H) 2.98 (s, 3H),

2.59 (t, J= 6.4 Hz, 2H), 2.55 (m, 2H); MS (m/z), found 820.5 (M +K)+.

1. MsCI, Et3 N N O 0 O N O* N O O12N0 N-- 1 2. K2 G0 3 , DMF O$1.. 0

HO OH 12 2M NOMe MeO](rNb

265d 8 O 265e

21%

Compound 265e:

[354] To a stirred solution of methyl 2-(3,5-bis(hydroxymethyl)phenyl)-5,8,11,14,17,20,23,

26,29,32,35,38-dodecaoxa-2-azahentetracontan-41-oate (265d) (71 mg, 0.091 mmol) in

anhydrous dichloromethane (1.4 mL) was added triethylamine (0.038 mL, 0.272 mmol). The

mixture was cooled to -5 0 C and methanesulfonyl chloride (0.018 mL, 0.227 mmol) was added

slowly. After stirring for one hour at -5 °C the reaction was quenched with cold water and

extracted with cold ethyl acetate. The organic extracts were washed with cold water, dried over

anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give methyl 2

(3,5-bis((methylsulfonyloxy)methyl) phenyl)-5,8,11,14,17,20, 23,26,29,32, 35,38-dodecaoxa-2

azahentetracontan-41-oate. MS (m/z), found 960.2 (M + Na)+. To a mixture of methyl 2-(3,5

bis((methylsulfonyloxy)methyl)phenyl)-5,8,11,14,17,20,23,26,29,32,35,38-dodecaoxa-2

azahentetracontan-41-oate (69 mg, 0.074 mmol) and compound 8 (54.1 mg, 0.184 mmol) in

anhydrous DMF (0.8 mL) was added potassium carbonate (50.8 mg, 0.368 mmol). The reaction

was allowed to stir for 18 hours at room temperature. The reaction was quenched with water and

extracted twice with dichloromethane. The remaining aqueous layer was extracted twice with

% MeOH/CH 2 Cl 2. The combined organic extracts were washed with brine, dried over

anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by flash silica gel

chromatography (5% MeOH/CH 2C 2) followed by preparative reverse phase HPLC (C18

column, eluted with CH 3CN/H 20) gave compound 265e (23mg, 23%). MS (m/z), found 1375.4

(M +Na + H 20)+,1393.4 (M + Na + 2H2 0).

N O12 0 OM e Me 3SnOH 12 N,

N)C e M ONb 349% [{Q OMe MeOC( N

265e 265f

Compound 265f:

[355] To a stirred solution of compound 265e (22 mg, 0.016 mmol) in anhydrous 1,2

dichloroethane (300 pL) was added trimethyl tin hydroxide (44.7 mg, 0.247 mmol). The reaction

stirred at 90 °C for 18 hours. The mixture was allowed to cool to room temperature and then

diluted with dichloromethane. The organic layer was washed with brine containing a few drops

% concentrated hydrochloric acid and then with brine alone, dried over anhydrous sodium

sulfate, filtered and concentrated in vacuo. Purification by preparative TLC (2x 5%

MeOH/CH2C2) gave compound 265f (7.5mg, 34%). MS (m/z), found 1318.4 (M -1)-, 1336.4

(M -1+ H 20)-, 1354.4 (M -1+2H 20)-.

O " O N OH HO-NNO

IN O l IN ~ 0 O~1~~o N

OMe eO . N EDC,DMAP jN.i O ke MeoOr.Ni

265g Il5 60

265f

Compound 265g:

[356] To a stirred solution of compound 265f (7.5 mg, 5.68 pmol) in anhydrous

dichloromethane (400 pL) was added N-hydroxy succinimide (1.961 mg, 0.017 mmol), EDC

(3.27 mg, 0.017 mmol), and DMAP (0.069 mg, 0.568 pmol). The reaction stirred for 18 hours at

room temperature. The mixture was extracted with dichloromethane and saturated ammonium

chloride. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered

and the solvent was removed under reduced pressure. The crude material was purified by

preparative reverse phase HPLC (C18 column, eluted with CH 3CN/H 20). Fractions containing

product were extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered and

co-evaporated with acetonitrile to give compound 2 65g (1.5mg, 19%). MS (m/z), found 1439.9

(M +Na)+, 1457.9 (M + Na + H 2 0) .

Example 32 (IGN-22)

0~ N- N-HO o-/ O~ N N

'O' N O ,O"' N O EDC,DMAP O O 58% 266a HN O NH2 258d 0

Compound 266a:

[357] To a solution of compound 258d (20mg, 0.050mmol) in dichloromethane (1.OmL) was

added mono-methyl succinate (13.23 mg, 0.100 mmol), EDC (19.20 mg, 0.100 mmol), and

DMAP (3.06 mg, 0.025 mmol) was added. The reaction stirred at room temperature for 18 hours.

The mixture was diluted with water and extracted with ethyl acetate. The organic extracts were

washed with brine, filtered and concentrated under reduced pressure. Purification by silica gel chromatography (3% MeOH/CH2C 2) gave compound 266a (15mg, 58%). MS (m/z), found

568.4(M + Na + MeOH)

. HO N

MeSO 3 H O N O1 N 0 93% 0 0 HN 266a HN O 266b 0 0

Compound 266b:

[358] To a solution of compound 266a (15 mg, 0.029 mmol) in dichloromethane (3.5 ml) was

added methanesulfonic acid (0.114 ml, 1.753 mmol). The reaction was stirred for one hour at

room temperature then diluted with methanol and water. The mixture was neutralized with

saturated sodium bicarbonate to pH=7 and extracted three times with dichloromethane. The

organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo.

Purification by preparative TLC (2x 5% MeOH/CH 2 C 2) gave compound 266b (11.5mg, 93%).

MS (m/z), found 446.4(M +Na)', 478.4 (M +Na+MeOH).

H N- N-N 0

O 259a N 0 N

0 K2CO 3, DMF HN HN 18% 266c 0 266b 0

Compound 266c:

[359] To a mixture of compound 266b (11.5 mg, 0.027 mmol) and compound 259a (19.98 mg,

0.041 mmol) in anhydrous DMF (0.5 ml) was added potassium carbonate (11.26 mg, 0.081

mmol). The reaction was stirred for 18 hours at room temperature. The mixture was quenched

with water and extracted three times with dichloromethane. The organic layer was washed with

brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.

Purification by preparative TLC (5% MeOH/CH 2 CI 2 ) followed by preparative reverse phase

HPLC (C18 column, eluted with CH 3CN, H 2 0) yielded compound 266c (4mg, 18%). 1H NMR

(400 Hz, CDCl 3): 6 8.27 (d, J = 8.0Hz, 1H), 8.06 (s, 1H), 7.87 (m, 2H), 7.74 (in, 1H), 7.55 (s,

1H), 7.52 (s, 1H), 7.49 (m, 1H), 7.26 (m, 1H) 7.19 (d, J = 8.8Hz, 1H), 7.10 (m, 1H), 6.82 (m,

2H), 4.49 (m, 2H), 4.12 (m, 4H), 3.95 (s, 6H), 3.71 (s, 3H), 3.48 (m, 4H), 2.75 (m, 2H), 2.66 (m,

2H), 1.98 (m, 4H), 1.70 (m, 2H); MS (m/z), found 824.1(M +K).

Example 33 (IGN-31)

MeO--Cr----- NH TBDMSCI MeO,-''ONO,-NH

TBDMSO ' OTBDMS HO J I OH Imidazole

249b 85% 267a

3,5-bis((tert-butvldimethylsilyloxymethyl)-N-(2-(2-(2-methoxyethoxy)ethoxy) ethyl)aniline

(267a):

[360] To a solution of (5-(2-(2-(2-methoxyethoxy)ethoxy)ethylamino)-1,3

phenylene)dimethanol (249b)(0.4 g, 1.336 mmol) in dichloromethane (6.68 mL) was added t

butyldimethylsilyl chloride (0.604 g, 4.01 mmol) and imidazole (0.318 g, 4.68 mmol). The reaction stirred at room temperature for 90 minutes. The mixture was diluted with dichloromethane and filtered through Celite. The filtrated was concentrated and purified by silica gel chromatography eluting with 20% Ethyl acetate/Hexanes to yield 3,5-bis((tert butyldimethylsilyloxy)methyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl) aniline (267a) (600mg,

%). MS (m/z), found 550.3 (M + Na).

MeO -- ON.O,-NH HO 33 MeO---OO-'^'N SS

TBDMSO - OTBDMS : TBDMSO J I OTBDMS EDC,ODMAP 267a 48% 267b

N-(3,5-bis((tert-butyldimethylsilyloxy)methyl)phenvl)-N-(2-(2-(2

methoxyethoxy)ethoxy)ethvl)-4-methyl-4-(methyldisulfanyl)pentanamide (267b):

[361] To a mixture of 3,5-bis((tert-butyldimethylsilyloxy)methyl)-N-(2-(2-(2

methoxyethoxy)ethoxy)ethyl)aniline (267a) (525 mg, 0.995 mmol) and 4-methyl-4

(methyldisulfanyl)pentanoic acid (232 mg, 1.193 mmol) in anhydrous dichloromethane (9.0 mL)

was added EDC (229 mg, 1.193 mmol) and DMAP (12.15 mg, 0.099 mmol). The reaction was

stirred at room temperature for five hours. The mixture was diluted with dichloromethane and

water. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered

and concentrated under reduced pressure. Purification by silica gel chromatography (30% Ethyl

acetate/Hexanes) gave N-(3,5-bis((tert-butyldimethylsilyloxy) methyl)phenyl)-N-(2-(2-(2

methoxyethoxy)ethoxy)ethyl)-4-methyl-4-(methyldisulfanyl)pentanamide (267b) (335mg, 48%).

M S HF.Pyr MeO-'^OO'-O--N S

TBDMSO ,I OTBDMS 89% HO , OH

267b 267c

N-(3,5-bis(hydroxvmethyl)phenyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-4-methyl-4

(methyldisulfanyl)pentanamide (267c):

[362] To a stirred solution of N-(3,5-bis((tert-butyldimethylsilyloxy)methyl)phenyl)-N-(2-(2

(2-methoxyethoxy)ethoxy)ethyl)-4-methyl-4-(methyldisulfanyl)pentanamide (267b)(315 mg,

0.447 mmol) in anhydrous acetonitrile (7.0 mL) at 0'°C was added anhydrous pyridine (7.00 mL)

followed by dropwise addition of HF.Pyridine (3.mL, ImL/100mg). The reaction stirred at 0

°C for two hours. It was diluted with ethyl acetate and slowly quenched with saturated sodium

bicarbonate. The mixture was extracted three times with ethyl acetate. The organic layer was

washed with water and brine, dried over sodium sulfate, filtered and concentrated. Purification

by silica gel chromatography, eluting with 5% MeOH/CH 2Cl 2, yielded N-(3,5

bis(hydroxymethyl)phenyl)-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-4-methyl-4

(methyldisulfanyl)pentanamide (267c)(190mg, 89%). 1H NMR (400 Hz, CDCl3): 6 7.21 (s, 1H),

7.16 (s, 2H), 4.63 (s, 4H), 3.79 (t, J = 5.2,5.6 Hz, 2H), 3.53 (in, 6H), 3.48 (in, 4H), 3.29 (s, 3H),

2.53 (s, 2H), 2.27 (s, 3H), 2.07 (in, 2H), 1.84 (in, 2H), 1.08 (s, 6H); MS (m/z), found 498.2 (M +

Na)'.

/ MeO --- O-'^'N ss 1. MsCI, Et 3 N

HO , OH 2. K 2CO 3, DMF N O 0 Me N 267c HOO N N OMe Me N 267d Me 8 N 18%

Compound 267d:

[363] To a stirred solution of N-(3,5-bis(hydroxymethyl)phenyl)-N-(2-(2-(2

methoxyethoxy)ethoxy)ethyl)-4-methyl-4-(methyldisulfanyl)pentanamide (267c)(72 mg, 0.151

mmol) in anhydrous dichloromethane (3.0 mL) was added triethylamine (0.063 mL, 0.454

mmol). The mixture was cooled to -5 0 C and methanesulfonyl chloride (0.029 mL, 0.378 mmol)

was added slowly. After stirring for one hour at -5 °C the reaction was quenched with cold water

and extracted with cold ethyl acetate. The organic layer was washed with cold water, dried over

anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give (5-(N-(2-(2

(2-methoxyethoxy)ethoxy) ethyl)-4-methyl-4-(methyldisulfanyl)pentanamido)-1,3

phenylene)bis(methylene) dimethanesulfonate. MS (m/z), found 654.1 (M + Na)'. To a mixture

of (5-(N-(2-(2-(2-methoxycthoxy)ethoxy)ethyl)-4-methyl-4-(methyldisulfanyl)pentanamido)

1,3-phenylene)bis(methylene) dimethanesulfonate (89 mg, 0.141 mmol) and compound 8 (83

mg, 0.282 mmol) in anhydrous DMF (1.5 mL) was added potassium carbonate (97 mg, 0.704

mmol). The reaction stirred for 18 hours at room temperature. The mixture was quenched with

water and extracted twice with dichloromethane. The organic layer was washed with brine, dried

over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification by silica gel

chromatography (5 % MeOH/CH 2C 2) and preparative reverse phase HPLC (C18 column, eluted with CH 3 CN/ H20) yielded compound 267d (27mg, 18%). 1H NMR (400 Hz, CDCl 3 ): 6 8.28 (d,

J = 4.8 Hz, 2H), 7.87 (in, 2H), 7.61 (s, 2H), 7.37-7.27 (in, 7H), 7.13 (t, J = 7.2, 7.6 Hz, 2H), 6.88

(s, 2H), 5.25 (m, 4H), 4.50 (m, 2H), 4.00 (s, 6H), 3.90 (m, 2H), 3.73 (m, 2H), 3.60 (m, 6H), 3.51

(in, 6H), 3.30 (s, 3H), 2.32 (s, 3H), 2.15 (in, 2H), 1.90 (m, 2H), 1.13 (s, 6H); MS (m/z), found

1050.3 (M + Na), 1068.3 (M+H 20+Na), 1086.3 (M+2H 20+Na).

Example 34 (IGN-32)

H o H O O MeO<0 ^.0- N OH H 0 H MeO O ON^ r N O O EDC,DMAP OOH O H H2N''Y Nf'YN HOJ.»-OH HO 00 64% HO> OH 253b 268a

Compound 268a:

[364] To a mixture of compound 253b (150 mg, 0.389 mmol) and tert-butyl 3-(2-(2-(2

aminoacetamido)acetamido)acetamido)propanoate (148 mg, 0.467 mmol) in anhydrous DMF

(1.5 ml) was added EDC (90 mg, 0.467 mmol) and DMAP (4.75 mg, 0.039 mmol). The reaction

stirred for 18 hours at room temperature. The mixture was directly purified by preparative

reverse phase HPLC (C18 column, eluted with CH3 CN/H 2 0 + 0.1 % formic acid). Further

purification by preparative TLC (15% MeOH/CH 2Cl2) yielded compound 268a (170mg, 64%).

1H NMR (400 Hz, CDCl 3): 6 7.62 (in, 1H), 7.56 (in, 1H), 7.38 (in, 1H), 7.11 (in, 1H), 6.55 (s,

2H), 6.52 (s, 1H), 4.45 (s, 4H), 4.17 (s, 2H), 3.63 (in,6H), 3.55-3.40 (in, 12H), 3.28 (in, 7H),

2.33 (t, J = 6.4 Hz, 2H), 2.16 (m, 2H), 1.79 (m, 2H), 1.36 (s, 9H); MS (m/z), found 706.3 (M +

Na)'.

H 0 H o 1. MsCI, EtN Me H 0 HO o 0 MeOre-,-O N l'Y N< < 2. K 2CO3, DMF N O O 0 O HO

' N OMe MeO -N HO OH MeOr N 0 0 0 268a 8 28 268b 21%

Compound 268b:

[365] To a stirred solution of compound 268a (59 mg, 0.086 mmol) in anhydrous

dichloromethane (1.75 ml) was added triethylamine (0.036 ml, 0.259 mmol). The mixture was

cooled to -5 °C and methanesulfonyl chloride (0.017 ml, 0.216 mmol) was added slowly. After

stirring for one hour at -5 °C the reaction was quenched with cold water and extracted with cold

ethyl acetate. The organic extracts were washed with cold water, dried over anhydrous sodium

sulfate, filtered and concentrated under reduced pressure to give the desired dimesylate

intermediate. MS (m/z), found 862.3 (M + Na) .

To a solution of the dimesylate intermediate (65 mg, 0.077 mmol) and compound 8 (114 mg,

0.387 mmol) in anhydrous DMF (1.0 mL) was added potassium carbonate (86 mg, 0.619 mmol).

The reaction was stirred for 18 hours at room temperature, then quenched with water and

extracted three times with dichloromethane. The organic layer was washed with brine, dried

over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Purification by

silica gel chromatography (2%->10% MeOH/CH 2Cl 2) yielded compound 268b (22 mg, 21%).

1H NMR (400 Hz, CDC 3): 6 8.26 (d, J = 8.0 Hz, 2H), 7.88 (in, 2H), 7.58 (s, 2H), 7.28 (m, 4H),

7.13 (t, J = 7.2 Hz, 2H), 6.89 (s, 2H), 6.81 (s, 1H), 6.73 (s, 2H), 5.19 (in, 4H), 4.48 in, 2H), 3.99

(s, 6H), 3.7-3.4 (in, 26H), 3.34 (s, 3H), 2.45 (t, J= 6.4 Hz, 2H), 2.30 (in, 2H), 1.81 (in, 2H), 1.44

(s, 9H).

Example 35

Preparation of chB38.1-IGN14 coniugate:

[366] A solution of chB38.1 antibody at a concentration of 2 mg/mL in an aqueous buffer

containing 0.05 M N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid (HEPES) and 2 mM

ethylenediaminetetra-acetic acid (EDTA), pH 8 was treated with a 10-fold molar excess of a

solution of IGN14-NHS in dimethylacetamide (DMA) such that the final concentration of DMA

in the buffer was 10% v/v. The reaction mixture was stirred at room temperature for 120 min and

then loaded onto a Sephadex G25 gel filtration column (HiPrepTM 26/10 Desalting Column GE#

17-5087-01) that had been previously equilibrated into an aqueous buffer containing 10 mM

histidine, 250 mM glycine, 1% sucrose pH 5.5. The conjugated antibody-containing fractions

were collected and pooled to yield product. The pooled sample was dialyzed overnight against

the same clution buffer to further purify the product. The final conjugate was assayed

spectrophotometrically using the extinction coefficients that were determined for IGN-14 (33 0 =

,231 M- cm- and - o=26,864 M-1 cm-1) and chB38.1 antibody (F28nm = 204,000 M-em-1). An

average of 3.3 IGN14 molecules per molecule of antibody were linked.

Example 36

Preparation of huMy9-6-IGN23 conjugate

[367] A solution of huMy9-6 antibody at a concentration of 2 mg/mL in an aqueous buffer

containing 0.05 M N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid (HEPES) and 2 mM

ethylenediaminetetra-acetic acid (EDTA), pH 8.5 was treated with a 12.5-fold molar excess of a

solution of IGN23-NHS in dimethylacetamide (DMA), glycerol, and sucrose. The final

concentration of DMA, glycerol and sucrose in the buffer was 15%, 5% and 5% (v/v)

respectively. The reaction mixture was stirred at room temperature for 120 min and then loaded

onto a Sephadex G25 gel filtration column (HiPrepT'M 26/10 Desalting Column GE# 17-5087-01)

that had been previously equilibrated into an aqueous buffer containing 10 mM histidine, 250

mM\ glycine, 1% sucrose, pH 5.5. The conjugated antibody-containing fractions were collected

and pooled to yield product. The pooled sample was concentrated using Millipore centrifugal

filter devices, and then dialyzed overnight against the same elution buffer to further purify the

product.

[368] The final conjugate was assayed spectrophotometrically using the extinction coefficients

that were determined for IGN-23 (e330 = 15,231 M-1 cm-1 and e 280 = 26,864 M-1 cm- 1) and

huMy9-6 (e 280,m = 206,460 M-1cm-1). An average of 2.2 IGN23 molecules per molecule of

antibody were linked.

Example 37

Preparation of chB38.1-IGN27 conjugate

[369] A solution of chB38.1 antibody at a concentration of 2 mg/mL in an aqueous buffer

containing 0.05 M N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid (HEPES) and 2 mM

ethylenediaminetetra-acetic acid (EDTA), pH 8.5 was treated with a 12-fold molar excess of a

solution of IGN27-NHS in dimethylacetamide (DMA, 5 mM stock) such that the final

concentration of DMA in the buffer was 15% v/v. The reaction mixture was stirred at room

temperature for 4 hr and then loaded on to a Sephadex G25 gel filtration column (HiPrepTM

26/10 Desalting Column GE# 17-5087-01) that had been previously equilibrated into an aqueous

buffer containing PBS pH 7.4. The conjugated antibody-containing fractions were collected and

pooled to yield product. The pooled sample was dialyzed overnight against the same elution

buffer to further purify the product.

[370] The final conjugate was assayed spectrophotometrically using the extinction coefficients

that were determined for IGN-27 (e330 = 15,231 M-1 cm-1 and e280 = 26,864 M-1 cm-1) and

chB38.1 antibody (e28om = 204,000 M-lcm-1). An average of 2.9 IGN27 molecules per

molecule of antibody were linked.

Example 38

In Vitro Potency IGN Free Drugs and IGN Conjugates:

[371] General Procedure Used: Samples of IGN Free Drugs or IGN Conjugates were added to

96-well flat bottomed tissue culture plates and titrated using serial dilutions to cover the desired

molar range. Antigen positive (Ag+) or Antigen negative (Ag-) cells were added to the wells in specific cell densities in such a way that there were triplicate samples for each drug concentration for each corresponding cell line. The plates were then incubated at 37°C in an atmosphere of 5% CO2 for 4-5 days depending on the cell line. COLO 205 (1,000 cells/well),

Namalwa (3,000 cells/well)- 4 days; RH30(1,000 cells/well), Ramos (10,000 cells/well), KB

(1,000 cells/well) - 5 days)

[372] At the end of the incubation period cytotoxic potencies were then assessed using a WST

based cell viability assay and surviving cells were measured by developing with WST (2-7

hours). The absorbance in each well was measured and the surviving fraction of cells at each

concentration was plotted to reveal the cytotoxicity and antigen specificity (of the conjugates).

[373] The cytotoxicity of the IGN Free Drugs and the potency and specificity of the IGN

conjugates were measured against a panel of human cancer cell lines selected from COLO 205,

NB-4, LOVO, Namalwa, RH30, Ramos, KB, and/or LOVO. Results are illustrated in Figures 51

-58.

[374] Figure 51: Table which demonstrates the high potency (in nM) of the IGN Free Drugs

against multiple cell lines. In general the IGN Free Drugs are found to be potent in the low

picomolar range against this panel of cell lines.

[375] Figure 52: (A) chB38.1-IGN13 conjugate (3.8 IGN/Ab) was found to be potent at sub

picomolar levels against COLO 205 (Ag+) cells and the activity was significantly diminished

(0.26 nM) when the antigen binding sites were blocked with 1 PM unconjugated chB38.1

antibody indicating the high specificity of this conjugate (>260 fold). (B) chB38.1-IGN13 conjugate (3.8 IGN/Ab) was found to be potent picomolar levels (0.002 pM) against LOVO

(Ag+) cells in a clonogenic assay.

[376] Figure 53: huMy9-6-IGN13 conjugate (3.4 IGN/Ab) was found to be potent at picomolar

levels against NB-4 (Ag+) cells (0.077 nM) and the activity was significantly diminished (1.0

nM) when the antigen binding sites were blocked with1I M huMy9-6 antibody indicating that

this conjugate is specific.

[377] Figure 54: (A) chB38.1-IGN14 conjugate (3.1 IGN/Ab) was found to be potent at sub

picomolar levels against COLO 205 (Ag+) cells and the activity was significantly less towards

Namalwa (Ag-) cells (0.9 nM) indicating the high specificity of this conjugate (>900 fold). (B)

chB38.1-IGN14 conjugate (2.6 IGN/Ab) was found to be very potent towards LOVO (Ag+) cells

(0.012 nM) and the activity was significantly less towards Namalwa (Ag-) cells (>3.0 nM)

indicating the high specificity of this conjugate (>250 fold).

[378] Figure 55: huMy9-6-IGN14 conjugate (3.3 IGN/Ab) was found to be highly potent

against NB-4 (Ag+) cells (0.033 nM) and the activity was significantly less towards Namalwa

(Ag-) cells (0.6 nM) indicating the high specificity of this conjugate.

[379] Figure 56: (A) chB38.1-IGN23 conjugate (2.5 IGN/Ab) was found to be potent at

picomolar levels against LOVO (Ag+) cells (0.063 nM) and the activity was significantly less

towards Namalwa (Ag-) cells (>3.0 nM) indicating the high specificity of this conjugate. (B) chB38.1-IGN23 conjugate (2.0 IGN/Ab) was found to be potent at picomolar levels against

COLO 205 (Ag+) cells (0.006 nM) and the activity was significantly diminished (2.5 nM) when

the antigen binding sites were blocked with 1 M chB38.1 indicating that this conjugate is

specific.

[380] Figure 57: chB38.1-IGN29 conjugate (2.8 IGN/Ab) was found to be potent at sub

nanomolar levels against COLO 205 (Ag+) cells (0.410 nM) and the activity was significantly

diminished (18 nM) when the antigen binding sites were blocked with 1 M chB38.1 indicating

that this conjugate is specific.

Example 39

In vivo efficacy of chB38.1-IGN14 coniugate in COLO 205 tumor bearing nude mice:

[381] In this study, the anti-tumor activity of chB38.1-IGN14 was investigated in female nude

mice bearing COLO 205 tumors, a human colon carcinoma model. COLO 205 tumor cells, 2 x

106 cells/mouse were subcutaneously inoculated at a volume of 0.1 mL/mouse in the area over

the right shoulder of female athymic nude mice, 5 weeks of age. Eight days after tumor cell

inoculation mice were randomized into groups (n = 6 per group) by tumor volume. Treatment

was initiated the day of randomization, and groups included a control group dosed with PBS

(200 pL/injection), naked chB38.1 antibody (2.8 mg/kg), non-targeting chKTI-IGN14 (50 pg/kg)

conjugate and chB38.1-IGN14 (50 pg/kg IGN14 dose; 2.5 mg/kg antibody dose). All treatments

were administered twice on a weekly schedule (day 8 and 15, post-cell inoculation). Arrows

indicate dosing times post inoculation. All treatments were well tolerated with the mean body weight losses comparable to loss seen in PBS control mice. Median tumor volume vs time is shown (Figure 58) with the data demonstrating the anti-tumor activity of the chB38.1-IGN14 conjugate. Both the non-targeting and the naked antibody show no activity beyond that seen with the vehicle control, suggesting that the anti-tumor activity observed with the chB38.1-IGN

14 conjugate is antigen-specific.

Example 40

[382] Figure 59 shows the mass spectrum of chB38.1-IGN14 (deglycosylated antibody). Peaks

are labeled D1-D7 to indicate the number of IGN14 molecules attached per antibody. The

average number of IGN14 molecules per antibody was calculated to be 3.5 (matching drug load

calculated by UV-vis).

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

243a 17477638_1 (GHMatters) P87639.AU.3

Claims (1)

1. CLAIMS We claim: 1. A process for the preparation of compound of formula (8):

   H

   MeO 8 comprising the steps of: a) converting a compound of formula (6):

   OHQ BnO NO

   60O

   into a compound of formula (7):

   Bn Cb MoOC( 0 7 ;and b) deprotecting the benzyl protecting group of the compound of formula (7) to form a compound of formula (8).

   2. The process of claim 1, further comprising the steps of: a) coupling a compound of formula (2): MeO 2C

   H 2O

   and a compound of formula (4): BnO NO 2 C1 MeO CI (4)

   to give a compound of formula (5):

   MeO 2C Bn N

   ; and b) reducing the compound of formula (5) to form an aldehyde of formula (6):

   OHC BnO NO NOb MeQU0 N 60

   3. The method of claim 1, wherein the compound of formula (6) is reacted with sodium dithionite to the compound of formula (7).

   4. A compound represented by formula (1):

   BnO HN-.

   MeO N/\

   1

   5. A process for preparing a compound represented by the following formula:

   BnO HN

   MeO C

   comprising the step of reducing a compound represented by formula 7:

   BnO N

   MeO

   7 to form the compound of formula:

   BnO HN

   MeO N/\

   6. The process of claim 5, wherein the step of reducing is carried out by using NaBH4.

   7. The process of claim 6, wherein the reduction reaction is carried out in a mixture of ethanol and dichloromethane.

   8. The process of claim 7, wherein the reaction is carried out at a temperature between 0 °C and room temperature.

   9. The process of claim 7, wherein the reaction is quenched by addition of saturated ammonium chloride.

   10. A compound represented by formula 7:

   BnO N

   MeO _

   7

   11. A compound represented by formula 3:

   HO HN

   MeO N

   3

   12. A process of preparing a compound represented by formula 3:

   HO HN

   MeO3.

   comprising the step of reducing a compound represented by formula 8:

   HO N

   MeO b

   to form the compound of formula 3.

   13. The process of claim 12, wherein the reducing agent is H2/Pd.

   246 17477638_1 (GHMatters) P87639.AU.3