TARGETED FORMS OF METHYLTRITHIO ANTITUMOR AGENTS

FORMES CIBLEES D'AGENTS ANTITUMORAUX METHYLTRITHIO

English Abstract

This disclosure describes a method for constructing
carrier-drug conjugates from the family of methyltrithio
antibacterial and antitumor agents.

Claims

-36-
THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A carrier-drug conjugate of the formula
<IMG>
prepared from a compound of formula CH3SSS-W wherein CH3SSS-W
is an antitumor antibiotic designated as LL-E33288.alpha.1Br, .alpha.1I,
.alpha.2Br, .alpha.2I, .alpha.3Br, .alpha.3I, .alpha.4Br, .beta.1Br, .beta.1I,
.beta.2Br, .beta.2I, .gamma.1Br, .gamma.1I,
61I, BBM-1675, FR-900405, FR-900406, PD 114759, PD 115028, CL-
1577A, CL-1577B, CL-1577D, CL-1577E, or CL-1724
<IMGS>

-36a-
<IMGS>
R5 is CH3, C2H5, or (CH3)2CH; R8 is OH and R9 is H, or R8 and
R9 together is a carbonyl group; X is an iodine or bromine
atom, R5 is a hydrogen or the group RCO, wherein R is
hydrogen, CH3 or a mono-substituted C6-C11 aryl group
comprising:
reacting CH3SSS-W with a compound of general formula Q-
Sp-SH, wherein Sp is a straight or branched-chain divalent
(C1-C18) radical, divalent aryl or heteroaryl radical,
divalent (C3-C18) cycloalkyl or heterocycloalkyl radical,
divalent aryl- or heteroaryl-alkyl (C1-C18) radicals, divalent
cycloalkyl- or heterocycloalkyl-alkyl (C1-C18) radical or
divalent (C2-C18) unsaturated alkyl radical, and Q is, or can
be subsequently converted to, halogen, amino, alkylamino,
carboxyl, carboxaldehyde, hydroxy, thiol, .alpha.-haloacetyloxy,
lower alkyldicarboxyl, -CONHNH2, -NHCONHNH2, -NHCSNHNH2,
-ONH2, -CON3,

-37-
<IMGS>
to produce an intermediate of formula Q-Sp-SS-W, wherein Q,
Sp, and W are as hereinbefore defined,
reacting Q-Sp-SS-W with a molecule of the formula
Tu-(Y)n wherein Tu is defined as a mono- or polyclonal anti-
body, its fragments, its chemically or genetically manipulat-
ed counterparts, growth factors, or steroids, Y is a side-
chain amino, carboxy, or thiol group of a protein, an alde-
hyde derived from carbohydrate residues, or an amidoalkylthio
group; and n is an integer of from 1 to 100, to produce a
compound of the formula:
<IMG>
wherein Tu, Y, Sp, W, and n are as hereinbefore defined, and
Z is formed from covalent reaction of the groups Q and Y
directly or after subsequent reduction, and Z is -CONH-,
-CONHN=CH-, -CONHNHCH2-, -NHCONHN=CH-, -NHCONHNHCH2-,

38
-NHCSNHN=CH-, -NHCSNHNHCH2-, -ON=CH-, -NH-, -NHCH2-, -N=CH-,
-CO2-, -NHCH2CO2-, -SS-,
<IMGS>
and m is 0.1 to 15.
2. A protein-drug conjugate of the formula
<IMG>
prepared from the antitumor antibiotic designated LL-E33288.UPSILON.1I
(CH3SSS-W) wherein W is as defined in claim 1 having
(a) ultraviolet spectrum as shown in Figure 1;
(b) a proton magnetic resonance spectrum as shown in
Figure 2; and
(c) an infrared spectrum as shown in Figure 3;
comprising:
displacing the diothiomethyl moiety with a compound
of formula Q-Sp-SH, wherein Sp is straight or branched-chain
divalent (C2-C5) radicals or divalent aryl- or heteroaryl-alkyl
(C2-C5) radicals, and Q is, or can be subsequently converted to,
carboxyl, lower alkyldicarboxylanhydride, -CONHNH2, or
<IMG>
to produce an intermediate of general formula Q-Sp-SS-W,
wherein Q, Sp, and W are as hereinbefore defined,

39
reacting Q-Sp-SS-W with a molecule of the formula
Tu-(Y)n wherein Tu is a monoclonal antibody which exhibits
preferential reactivity with a human tumor-associated antigen,
Y is a side-chain amino group on the antibody, or an aldehyde
generated by oxidation of the carbohydrate groups of the
antibody, and n is an integer of from 1 to 100, to produce a
compound of the formula:
<IMG>
wherein Tu, Y, Sp, W, and n are as hereinbefore defined, and Z
is formed from covalent reaction of the groups Q and Y directly
or after subsequent reduction, and Z is -CONH-, -CONHN=CH-,
-CONHNHCH2-, OR~~~
<IMG>
and m is 0.1 to 15.
3. A carrier-drug conjugate according to claim 1 wherein
CH3SSSW is LL-E33288~1I, Q is the 4-nitrophenyl ester of a
carboxyl group, Sp is -CH2CH2-, Tu is a monoclonal antibody
which is CT-M-01, Y is -NH2, Z is -CONH-, and m is 0.5 to 15.
4. A carrier-drug conjugate according to claim 1 wherein
CH3SSSW is LL-E33288~1I, Q is the hydroxysuccinimide ester of a
carboxyl group, Sp is -CH2CH2-, Tu is a monoclonal antibody
which is MAC-68, Y is -NH2, Z is -CONH-, and m is 0.5 to 15.
5. A carrier-drug conjugate according to claim 1 wherein
CH3SSSW is LL-E33288~1I, Q is -CONHNH2, Sp is -CH2CH2-, Tu is a
monoclonal antibody which is Lym 1, Y is -CHO, Z is -CONHNHCH2-,
and m is 0.1 to 10.

40
6. A carrier-drug conjugate according to claim 1 wherein
CH3SSSW is LL-E33288.UPSILON.1I, Sp is
<IMG>
Tu is a monoclonal antibody which is B72.3, Y is -CHO, Z is
-CONHNHCH2-, and m is 0.1 to 10.
7. A carrier-drug conjugate according to claim 1 wherein
CH3SSSW is LL-E33288.UPSILON.1I, Sp is
<IMG>
Tu is a monoclonal antibody which is Lym 2, Y is -CHO, Z is
-CONHNHCH2-, and m is 0.1 to 10.

-41-
8. A process for preparing a targeted derivative
<IMG>
of a compound of formula CH3SSS-W wherein CH3SSS-W is an
antitumor antibiotic LL-E33288.alpha.1Br, .alpha.1I, .alpha.2Br, .alpha.2I,
.alpha.3Br, .alpha.3I,
.alpha.4Br, .beta.1Br, .beta.1I, .beta.2Br, .beta.2I,.gamma.1Br, .gamma.1I,
.sigma.1I, BBM-1675, FR-
900405, FR-900406, PD 114759, PD 115028, CL-1577A, CL-1577B,
CL-1577D, CL-1577E, or CL-1724 and W is as defined in claim 1
comprising:
reacting CH3SSS-W with a compound of formula Q-Sp-SH,
wherein Sp is a straight or branched-chain divalent (C1-C18)
radical, divalent aryl or heteroaryl radical, divalent
(C3-C18) cycloalkyl or heterocycloalkyl radical, divalent
aryl- or heteroaryl-alkyl (C1-C18) radicals, divalent
cycloalkyl- or heterocycloalkyl-alkyl (C1-C18) radical or
divalent (C2-C18) unsaturated alkyl radical, and Q is halogen,
amino, alkylamino, carboxyl, carboxaldehyde, hydroxy, lower
alkyldicarboxyl anhydride, -CONHNH2, -NHCONHNH2, -NHCSNHNH2,
-ONH2, or
<IMG>
in acetonitrile in the presence of one equivalent of
triethylamine or one equivalent of acetic acid at -10° to
-30°C for 1-48 hours,
isolating the intermediate of formula Q-Sp-SS-W, wherein

-42-
Q, Sp, and W are as hereinbefore defined, then
reacting the compound of formula Q-Sp-SS-W, wherein Sp
and W are as hereinbefore defined and Q is halogen, amino,
alkylamino, carboxyl, carboxaldehyde, hydroxy or lower
alkyldicarboxylic anhydride with a molecule of the formula Tu-
(Y)n wherein Tu is a mono- or polyclonal antibody, its
fragments, its chemically or genetically manipulated
counterparts, growth factors, or steroids; Y is a side-chain
amino or carboxyl functionality; n is 1-100, in aqueous buffer
at a pH of between 6.5 and 9, at 4° to 40°C either directly or
in the presence of a water-soluble carbodiimide, to generate
the compound
<IMG>
wherein Tu, Sp, W, n, and Y are as hereinbefore defined, m is
1-15 and Z is formed from covalent react ion of the groups Q
and Y and is -CONH-, -NH-,
<IMG>
-N=CH-, or -CO2-
or
reacting the compound of formula Q-Sp-SS-W, wherein Sp
and W are as hereinbefore defined and Q is a carboxylic acid,
with N-hydroxysuccinimide, 2, 3, 5, F, -tetrafluorophenol,
pentafluorophenol, or 4-nitrophenol in the presence of a

43
carbodiimide activating agent to generate a compound of formula
Q-Sp-SS-W wherein Sp and W are as hereinbefore defined and Q is
<IMGS>
and reacting the resulting compound with a molecule of formula
Tu(Y)n,
where Tu and n are as hereinbefore defined, and Y is
a side-chain amino group, in an aqueous buffered solution at a
pH between 6.5 and 9, at a temperature of between 4° and 40°C,
inclusive, to generate compounds of the formula:
<IMGS>
wherein Tu, Sp, Y, and n are as hereinbefore defined, m is
1-15, and Z is formed from covalent reaction between Q and Y
and is defined as -CONH-, or
reacting a compound of formula Q-Sp-SS-W, wherein Sp
and W are as hereinbefore defined and Q is -CONHNH2 with nitrous
acid in aqueous actonitrile to generate a compound of formula
Q-Sp-SS-W, wherein Sp and W are as hereinabove defined and Q is
-CON3, then reacting Q-Sp-SS-W with a compound of formula Tu-
(Y)n, wherein, Tu, Y and N are as hereinabove defined to produce
a compound of the formula

44
<IMG>
wherein Tu, Z, Sp, W, m, Y, and n are as hereinabove defined;
or
reacting a compound of formula Q-Sp-SS-W wherein Sp
and W are as hereinbefore defined and Q is hydroxy, with an
alpha-haloacetic anhydride to produce a compound wherein Q is
.alpha.-haloacetyloxy, and reacting the a-haloacetyloxy-Sp-SS-W or
a compound of formula Q-Sp-SS-W, wherein Sp and W are as here-
inbefore defined and Q is
<IMGS>
with a molecule of the formula Tu-(Y)n wherein Tu is as here-
inbefore defined, Y is a side-chain thiol of a protein, or
an amidoalkylthio group introduced on an amine of Tu using
3-(2-dithiopyridyl)propionic acid hydroxy-
succinimide ester followed by reduction with
dithiothreitol, or an amidoalkylthio group introduced on an
amine of Tu using 2-iminothiolane, and n is 1-10, under aque-
ous buffered conditions at a pH between 4.5 and 7, at a tem-
perature between 4° and 40°C, inclusive, to produce a com-
pound of formula:

45
<IMG>
wherein Tu, Sp, W, and n are as hereinbefore defined, and Z
is formed from covalent reaction of the groups Q and Y and Z
is
-SS-, <IMGS>
and m is 0.1 to 10;
or
reacting a compound of the formula Q-Sp-SS-W where-
in Sp and W are as hereinbefore defined and Q is -NH2,
-CONHNH2, -NHCONHNH2, -NHCSNHNH2, or -ONH2 with a molecule of
formula Tu-(Y)n wherein Tu is as hereinbefore defined, Y is
an aldehyde generated from carbohydrate residues on Tu by
oxidation in the presence of an alkaline earth periodate, in
an aqueous buffer at a pH between 4.0 and 6.5, at 4° to 40°C,
inclusive, and n is 1 to 20 to generate a compound of formu-
la:

-46-
<IMG>
wherein Tu, Sp, W, Y, and n are as hereinbefore defined and Z
is formed from the covalent reaction of Q and Y and is
-ON=CH-, -N=CH-, -CONHN=CH-, -NHCONHN=CH-, or -NHCSNHN=CH-,
and m is 0.1 to 15; or treating the compound immediately here-
inabove of formula:
<IMG>
wherein Tu, Z, Sp, W, Y, n, and m are as immediately herein-
above defined with acetylhydrazine or tyrosine hydrazine in
an aqueous buffer at a pH between 4.0 and 6.5, at 4° to 40°C,
inclusive, to generate a compound of formula:
<IMG>
wherein Tu, Z, Sp, W, n, and m are as immediately hereinabove
defined and Y is -CH=NNHCOCH3 or

-47-
<IMG>
and
reacting this compound with sodium cyanoborohydride
or sodium borohydride, in an aqueous buffer at a pH of 4.0 to
6.5, at a temperature of 4o to 40°C, inclusive, to generate a
compound of formula:
<IMG>
wherein Tu, Sp, W, m, and n are as hereinabove defined, Z is
-NH-CH2-, -CONHNHCH2-, -NHCONHNHCH2-, or -NHCSNHNHCH2-, and Y
is -CH2NHNHCOCH3 or
<IMG>
9. Use of an oncolytic amount of a product
<IMG>

48
prepared from a compound of general formula CH3SSS-W wherein
CH3SSS-W is an antitumor antibiotic designated as LL-E33288.alpha.1Br,
.alpha.1I, .alpha.2Br, .alpha.2I, .alpha.3Br, .alpha.3I, .alpha.4Br,
.beta.1Br, .beta.1I, .beta.2Br, .beta.2I, .gamma.1Br, .gamma.1I, .delta.1I,
BBM-1675, FR-900405, FR-900406, PD 114759, PD 115028, CL-1577A,
CL-1577B, CL-1577D, CL-1577E, or CL-1724, wherein W is as
defined in claim 1, comprising:
reacting CH3SSS-W with a compound of general formula
Q-Sp-SH, wherein Sp is a straight or branched-chain divalent
(C1-C18) radical, divalent aryl or heteroaryl radical, divalent
(C3-C18) cycloalkyl or heterocycloalkyl radical, divalent aryl-
or heteroaryl-alkyl (C1-C18) radical, divalent cycloalkyl- or
heterocycloalkyl-alkyl (C1-C18) radical or divalent (C2-C18)
unsaturated alkyl radical, and Q is, or can be subsequently
converted to, halogen, amino, alkylamino, carboxyl,
carboxaldehyde, hydroxy, thiol, .alpha.-haloacetyloxy, lower
alkyldicarboxyl, -CONHNH2, -NHCONHNH2, -NHCSNHNH2, -ONH2, -CON3,
<IMGS>
to produce an intermediate of formula Q-Sp-SS-W, wherein Q, Sp,
and W are as hereinbefore defined,
reacting Q-Sp-SS-W with a molecule of the formula
Tu-(Y)n wherein Tu is defined as a mono- or polyclonal

-49-
antibody, its fragments, its chemically or genetically manipu-
lated counterparts, growth factors, or steroids; Y is a side-
chain amino, carboxy, or thiol group of a protein, an alde-
hyde derived from carbohydrate residues, ar en amidoalkylthio
group; and n is an integer of from 1 to 100, to produce a
compound of the formula:
<IMG>
wherein Tu, Y, Sp, W, and n are as hereinbefore defined, and
Z is formed from covalent reaction of the groups Q and Y
directly or after subsequent reduction, and Z is -CONH-,
-CONHN=CH-, -CONHNHCH2-, -NHCONHN=CH-, -NHCONHNHCH2-,
-NHCSNNN=CH-, -NHCSNHNHCH2-, -ON=CH-, -NH-, -NHCH2-, -N=CH-,
-CO2-, -NHCH2CO2-, -SS-,
<IMGS>
and m is 0.1 to 15 to inhibit growth of a tumor in a
mammal.
10. Use of an oncolytic effective amount of a
protein-drug conjugate of the formula

50
<IMG>
prepared from the antitumor antibiotic designated LL-E33288.UPSILON.1I
(CH3SSS-W) wherein W is as defined in claim 1 having
(a) ultraviolet spectrum as shown in Figure 1;
(b) a proton magnetic resonance spectrum as shown in
Figure 2; and
(c) an infrared spectrum as shown in Figure 3;
comprising
displacing the dithiomethyl moiety with a compound of
formula Q-Sp-SH, wherein Sp is straight or branched-chain
divalent (C2-C5) radicals or divalent aryl- or heteroarylalkyl
(C2-C5) radicals, and Q is, or can be subsequently converted to,
carboxyl, lower alkyldicarboxylanhydride, -CONHNH2, or
<IMG>
to produce an intermediate of general formula Q-Sp-SS-W,
wherein Q, Sp, and W are as hereinbefore defined,
reacting Q-Sp-SS-W with a molecule of the formula
Tu-(.UPSILON.)n wherein Tu is a monoclonal antibody which exhibits
preferential reactivity with a human tumor-associated antigen,
.UPSILON. is a side-chain amino group on the antibody, or an aldehyde
generated by oxidation of the carbohydrate groups of the
antibody, and n is an integer of from 1 to 100, to produce a
compound of the formula:

-51-
<IMG>
wherein Tu, Y, Sp, W, and n are as hereinbefore defined, and
Z is formed from covalent reaction of the groups Q and Y
directly or after subsequent reduction, and Z is -CONH-,
-CONHN=CH-, -CONHNHCH2-, or
<IMG>
and m is 0.1.to 15 to deliver a compound to an antigenic
site in a mammal.

-52-
11. A carrier-drug conjugate of the formula
<IMG>
wherein SS-W has the meaning ascribed to it in a compound of
formula CH3SSS-W wherein CH3SSS-W is an antitumor antibiotic
designated as LL-E33288.alpha.1Br, .alpha.1I, .alpha.2Br, .alpha.2I,
.alpha.3Br, .alpha.3I, .alpha.4Br,
.beta.1Br, .beta.1I, .beta.2Br, .beta.2I, .gamma.1Br, .gamma.1I, .delta.1I,
BBM-1675, FR-900405, FR-
900406, PD 114759, PD 115028, CL-1577A, CL-1577B, CL-1577D,
CL-1577E, or CL-1724, Sp is a straight or branched-chain
divalent (C1-C18) radical, divalent aryl or heteroaryl
radical, divalent (C3-C18) cycloalkyl or heterocycloalkyl
radical, divalent aryl- or heteroaryl-alkyl (C1-C18) radicals,
divalent cycloalkyl- or heterocycloalkyl-alkyl (C1-C18)
radical or divalent (C2-C18) unsaturated alkyl radical and W
is as defined in claim 1,
Tu is defined as a mono- or polyclonal antibody its
fragments, its chemically or genetically manipulated
counterparts, growth factors, or steroids; .gamma. is a side-chain
amino, carboxy, or thiol group of a protein, an aldehyde
derived from carbohydrate residues, or an amidoalkylthio
group; and n is an integer of from 1 to 100,
Z is -CONH-, -CONHN=CH-, -CONHNHCH2-, -NHCONHN=CH-,
-NHCONHNHCH2-, -NHCSNHN=CH-, -NHCSNHNHCH2-, -ON=CH-, -NH-,
-NHCH2-, -N=CH-, -CO2-, -NHCH2CO2-, -SS-,

53
<IMGS>
and m is 0.1 to 15.
12. Use of a pharmaceutically effective amount of a
carrier-drug conjugate according to any one of claims 3 to 7 to
prepare a conjugate to inhibit growth of a tumor in a mammal.
13. A commercial package comprising a pharmaceutically
effective amount of a conjugate prepared from a carrier-drug
conjugate according to any one of claims 3 to 7 together with
instructions for use thereof to inhibit growth of a tumor in a
mammal.

Description

1341315
1
TARGETED FORMS OF METHYLTRITHIO
ANTITUMOR AGENTS
SUMMARY OF THE INVENTION
The invention describes carrier-drug conjugates of
(3 Yl and b
the disulfide analogs of the Ocl, Ocz, Oc3, Oc4, (31, z.
components of the LL-E33288 complex as well as the disulfide
analogs of BBM-1675, FR-900405, FR-900406, PD 114759, PD
115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E and CL-1724
antitumor antibiotics. The carrier portion of the conjugate is
a mono- or polyclonal antibody, their fragments, chemically or
genetically manipulated counterparts, growth factors or
steroids. The invention includes the method of using the
carrier-drug conjugates as well as their process of
manufacture. Conjugates of the invention can be used to
inhibit growth of tumors in mammals. Commercial packages of
pharmaceutically effective amounts of such conjugates together
with instructions for such a use are also part of the
invention.
DESCRIPTION OF THE DRAWINGS
Figure 1: The ultraviolet spectrum of the antitumor
antibiotic designated as LL-E33288Y1i.
Figure 2: The proton magnetic resonance spectrum of
the antitumor antibiotic designated as LL-E33288Y1I.
Figure 3: The infrared spectrum of the antitumor
antibiotic designated as LL-E33288Y1i.
DETAILED DESCRIPTION OF THE INVENTION
The family of antibacterial and arrtitumor agents,
known collectively as the LL-E33288 complex are described and
claimed in U.S. Patent 4,970,198 and are used to prepare

-2- 1341315
the disulfur antitumor agents which are starting materials
for targeted forms of the antitumor agents of our invention.
U.S. Patent 4,970,198 describes the
LL-E33288 complex, the components thereof, namely, LL-
E33288a1Sr, LL-E33288a1I, LL-E33288a2Br, LL-E33288a I
, LL-
E33288a3Br, LL-E33288a3I, LL-E33288a4$r, LL-E33288~~Br, LL-
E33288~ , LL-E33288 H Sr
1 R2 , LL-E33288~2 , LL-E33288~1 r, LL-
E33288~1I and LL-E33288d1I, and methods for their production
by aerobic fermentation utilizing a new strain of Micromono-
spora echinospora ssp calichensi.s or natural or derived
mutants thereof. U.5. Patent 4,970,198 also discloses proposed
structures for some of the above named components. These
proposed structures are reproduced in Table 1.
20
30
tB I

_3_ 1341315
Table 1
Proposed Structures for CH3-SSS-W
(wherein W is the substituent attached
to CH3-SSS- below)
s
O
HO~",
H ,....- \ N H O
_ CHI O CH~SSS.~ /~,,
R'S O C ~CH~
\ ~ o o.... ''~,,~
OH HN~~~ H
ORz
CHI O CH~O CO-
X
Are ~ Ars ~ C ~O ~ N~.H
R, O O C 1"!~ ~ O O C hip
OCH~
C H=
. '/~/~ R 'O 'iw'
R2, = N O R'~ ~ ORS R,, = C H~ O
~~ H
Rs OCh4~ CHI 'O "'y OCH~ OH
~O
I
Deslgnatlon R~ R2 R3 R4 Rg Rg R~ X
LL-E33288a21 Ark R2, H H C2H5 ~ I
LL-E33288a31 Ar1 H H R4, ~ I
LL-E33288~i~ Ark R2, H R4, (CH3)2C H I
I
LLE33288y~ Ar1 R2, H R4, C2H5 i
I
LLE332885~1 Ar1 R2. H R4, CH3 I
LL-E33288p~8r Ari R2, H R4, (CN3)2CH er
LL-E33288y1 Ark R2, H R4, C2H5 Br
B r
LL-E33288a2B Ar1 R2, H H C2H6 Br
r
LL-E33288a3B Ark H H Ra, Br
r
Esperamicin CH3 R2, R3, (CH3)2CH H Ar2
A1
Esperamlcln CH3 R2, R3, (CH3)2C H Ar2 H
A2
Esperamicln CH3 R2, R3, CH3CH2 H Ar2
A1 b

-4 1341315
Certain other antibiotics are useful in our inven-
tion, namely:
1) Esperamicin BBM-1675, a novel class of potent antitumor
antibiotics. I. Physico-chemical data and partial structure.
M. Konishi, et. al., J. Antibiotics, 38, 1605 (1985). A new
antitumor antibiotic complex, M. Konishi, et. al., U.S.
Patent 4.675,187.
2) New antitumor antibiotics, FR-900405 and FR-900406. I.
Taxonomy of the producing strain. M. Iwami, et. a~., J. Anti-
biotics 38, 835 (1985). New antitumor antibiotics FR-900405
and FR-900406.II. Production, isolation, characterization and
antitumor activity. S. Kiyoto, et. al., J. Antibiotics, 38,
340 (1985).
3) PD 114759 and PD 115028, novel antitumor antibiotics with
phenomenal potency. I. Isolation and characterization. R.H.
Bunge, et. al., J. Antibiotics, 37, 1566 (1984). Biological
and biochemical activities of the novel antitumor antibiotic
PD 114759 and related derivatives. D.W. Fry et. al.,
Investigational New Drugs, 4, 3 (1986).
4) New antibiotic complex CL-1577A, CL-1577B produced by
Streptomyces sp. ATCC 39363. European Patent Application
0,132,082, A2.
5) CL-1577D and CL-1577E Antibiotic antitumor compounds,
their production and use. U.S. Patent 4,539,203.
6) CL-1724 Antibiotic compounds, their production and use.
U.S. Patent 4,554,162.
The complete structures of esperamicins A1. A2'
and Alb (the BBM-1675 complex) have been reported, and
these are included in Table 1. Tne physical characteristics
of the above-named antitumor antibiotics indicate that they
all are identical or very similar in structure to the
esperamicins, and all contain a methyltrithio functional
group.

-5-
1341315
As can be seen from the structures disclosed above,
the a1, a2, a3, a4. ~l' ~2' 71 and a components of the LL-
E33288 complex, as well as the BBM-1675, FR-900405, FR-900406,
PD 114759, PD 115028, CL-1577A, CL-1577B, CL-1577D, CL-1577E
and CL-1724 antibiotics each contain a methyltrithio group in
their structure. The methyltrithio moiety of the above-named
antibiotics is subject to displacement by a variety of thiol-
containing organic molecules resulting in the formation of a
new class of anticancer and antibacterial agents.
It has now been discovered that the displacement of
the methyltrithio unit of the compounds listed in Table 1 and
as depicted in Scheme I can be used to introduce a spacer
(Sp), the judicious choice of which enable the introduction
of targeting units into the compounds of the above-named pat-
ents and applications.
Q-Sp-SH
CH3SSS-W ~ Q-Sp-SS-W
Cnhcmc T
wherein Sp is a straight or branched-chain divalent (C1-C18)
radical, divalent aryl or heteroaryl radicals, divalent (C3-
C18) cycloalkyl or heterocycloalkyl radicals, divalent aryl-
or heteroaryl-alkyl (C1-C18) radicals, divalent cycloalkyl-
or heterocycloalkyl-alkyl (C1-C18) radicals, or divalent (C2-
C18) unsaturated alkyl radicals; Q is, or can be subsequently
converted to, halogen, amino, alkylamino, carboxyl, carboxal-
dehyde, hydroxy, thiol, a-haloacetyloxy, lower alkyldicar-
boxyl, -CONHNH2, -NHCONHNH2, -NHCSNHNH2, -ONH2, -CONS,

1341 315
- 6 -
O O F F
-SS
N
CO 2
-C02N ~ ~ ~ N-.
\O
O F F
F F -CHZ CH3
C02 N I O _ O
-C02 O F. -C02 O -N02.
F F
fZ~ f~a
110..-_
Cli ~ f-~ --- ~/ Nt~ O
O
fl~S ~O CI13 . \ OCRs
and W is ~ ~ O _
011 tiN-10
' OF~z
C 1-y O
X
. fi, is I \ ~~ or Cty;
f~40 ~ OCty
OC11~
JWVN/V'
1
fig is NS O or H;
R5 OCI-13
R6OOR7
R~ is or ti;
CH3 0....
1
61109-7670

13+1315
_ ~a _
Fed is ~Oa ~ or II;
oct~~ oti
cE-i~o ~ ,co-
t~s or R~ is If or
Clyp
~~OCH~
'C[llz
R5 is CH3, C2H5, or (CH3)2CH; R8 is OH and R9 is H, or R8 and
R9 together is a carbonyl group; X is an iodine or bromine
atom, R5 is a hydrogen or the group RCO, wherein R is
hydrogen, CH3 or a mono-substituted C6-C11 aryl group.
As long as the product from Scheme I contains at
least one functional group which can be converted to, or is
directly reactive with a targeting unit (Tu), targeted forms
of the antitumor antibiotics of the above-named patents and
applications can be generated, as shown in Scheme II below:
Tu-(Y)n
Q-Sp-SS-W '-~ ~u-(Z-Sp-SS-w)m
(Y)n_m
Scheme II
wherein Q, Sp and W are as hereinbefore defined, Tu is a mono-
or polyclonal antibody, its fragments, its chemically or
genetically manipulated counterparts, growth factors, or
steroids; Y is a side-chain amino, carboxyl, or thiol group of
a protein, an aldehyde derived from carbohydrate residues, or
an amidoalkylthio group; n is an integer of from 1 to 100; Z
61109-7670

1341315
is formed from covalent reaction of the groups Q and Y di-
rectly or after subsequent reduction and Z is -CONH-,
-CONHN=CH-, -CONHNHCH2-, -NHCONHN=CH-, -NHCONHNHCH2-,
-NHCSNHN=CH-, -NHCSNHNHCH2-, -ON=CH-, -NH-, -NHCH2-, -N=CH-,
-C02-, -NHCH2C02-, -SS-,
O 02C_ O I
-S S N -S ~ CH3 -NHCOCH2-CH
N-~ . or (CH2)0 i
. CH2 ~ .
0 0 C02H
and m is 0.1 to 15.
As an example, and with reference to Scheme II,
above, the 3-mercaptopropionic acid derivative of E-33288711
(Q=C02H, Sp=-CH2CH2-), when converted to its activated
hydroxysuccinimide form (Q=C02Su, Sp=-CH2-CH2-) can be used
to react with some of the e-amino groups of lysine residues
(e. g., Tu=monoclonal antibody, Y=-NH2 wherein n=50-100 from
available lysine residues), at a pH between 7.0 and 9.5 in
aqueous buffered solutions at temperatures between 4°C to
40°C to produce targeted forms of the antibiotics attached at
random sites along the protein backbone (Tu=monoclonal anti-
body, Z=-NHCO-, Sp=-CH2CH2-, m=1-10). Only a fraction of the
available lysine residues are substituted in this manner,
since high loading is generally not considered compatible
with preserving the antibody immunoreactivity. The same
randomly-substituted immunoconjugates can also be prepared
from the 3-mercaptopropionic acid derivative using other car-
boxyl group activating agents such as a variety of carbodi-
imides, or the corresponding acyl azide. Alternatively, a
3-mercaptopropionyl hydrazide derivative of E-3328871I

-8-
1341315
(Q=H2NNHC0-, Sp=-CH2CH2-), when reacted with a periodate-
oxidized antibody (Tu=monoclonal antibody, Y=-CHO, n=1-15) as
described in U.S. Patent No. 4,671,958 at a pH between 4 and
7, in a buffered aqueous solution at a temperature of between
4°C and 40°C, reacts only at the aldehyde functionality
(derived from cleavage of vic-diols of carbohydrate residues
situated on the Fc portion of the antibodies) to generate
monoclonal antibody conjugates containing the drug substi-
tuted at specific sites along the backbone of the protein
(Tu=monoclonal antibody, Z=-CH=NNHCO-, Sp=-CH2CH2-, m=0.5-
10). In order to block unreacted aldehyde groups on the
. antibody and thus avoid crosslinking, as well as stabilize
the hydrolytically labile Schiff's base linkages, it is pref-
erable (though not essential) to react the latter conjugate
first with a compound such as acetyl hydrazide or tyrosine
hydrazide, then reduce with sodium cyanoborohydride or sodium
borohydride to produce the stabilized constructs of this
invention (Tu=monoclonal antibody, Z=-CH2NHNHCO-, Sp=-CH2CH2-,
m=0.5-10). Other aldehyde-reactive groups as part of the
drug construct are within our invention to generate the prod-
ucts of~Scheme II. Such functional groups are preferably,
though not limited to, those which react with aldehydes under
acidic aqueous conditions. The reactivity of protein lysines
under basic conditions is sufficiently great such that their
amines compete with the products of Scheme II for available
aldehydes of the monoclonal antibody. Alternative aldehyde-
reactive groups are, for example, the semicarbazide, the
thiosemicarbazide, and the O-substituted hydroxylamine func-
tionalities.
Assembly of targeted forms of the compounds listed
in Table 1 is not restricted to the sequence outlined in
Scheme II. The targeting unit (Tu) can be first modified to
contain a thiol group, which is then reacted with the com-
pounds of Table 1, in accordance with Scheme III below:

-g_
1341315
Tu(Y)n + Q-Sp-S-P ~ Tu-(Z-Sp-SH)m
( Y ) n-m
CH3-SSS-W
Tu-(Z-Sp-SS-W)m
( Y ) n-m
Scheme III
wherein Tu, Y, Q, Sp, W, n, and m are as hereinbefore defined,
and P is hydrogen or 2-(pyridylthio), with the proviso that
when Y is a thiol derived from a backbone amino acid residue
of Tu, Z-Sp taken together is a covalent. bond.
As an example, and with references to Scheme III,
above, a monoclonal antibody can be reacted with 3-(2-dithio-
pyridyl)propionic acid hydroxysuccinimide ester to modify the
protein through lysine residues (Tu=monoclonal antibody,
Y=NH2, n=50-100, Q=-C02Su, Sp=-CH2-CH2-, P=2-pyridylthio).
Following reduction with, for example, dithiothreitol, an in-
termediate is generated (Tu=monoclonal antibody, Z=-NHCO-,
Sp=-CH2CH2-, m=1 to 15) which can be reacted with the com-
pounds of Table 1 to generate the subject immunoconjugates.
Similarly, 2-iminothiolane can be reacted with a monoclonal
antibody to introduce thiol groups onto the surface of the
protein directly, without requiring a reduction step
(Tu=monoclonal antibody, Z=-NHCO-, Sp=-(CH2)3-, m=1 to 15),
and this intermediate can be reacted with the compounds of
Table 1 as before. Alternatively, sulfhydryl groups inherent
within the structure of monoclonal antibodies in dimeric form
as cystine residues can be used to participate in the reac-
tion of Scheme III directly. Such sulfhydryls are tradition-
ally exposed by a combination of enzymatic digestion and re-
duction of native monoclonal antibodies (Tu=Fab' fragment,
Z-Sp=bond, Y=SH), but the use of genetically-altered

-10-
1341315
constructs of monoclonal antibodies containing unpaired
cystine residues is likewise contemplated.
A preferred embodiment of this invention is a
protein-drug conjugate of the formula:
Tu- ( Z-Sp-SS-W) m
( Y ) n-m
prepared from the antitumor antibiotic designated LL-
E3328871I (CH3SSS-W) having
a) ultraviolet spectrum as shown in Figure I;
b) a proton magnetic resonance spectrum as
shown in Figure II;
c) an infrared spectrum as shown in Figure III;
and
displacing the dithiomethyl moiety with a compound
of formula Q-Sp-SH, wherein Sp is straight or branched-chain
divalent (C2-C5) radicals or divalent (C2-C5) arylalkyl or
heteroarylalkyl radicals, and Q is carboxyl, lower alkyldi-
carboxyl anhydride, -C02Su, -CONHNH2, or
_C02 O N02
to produce an intermediate of general formula Q-Sp-SS-W,
wherein Q, Sp, and W are as hereinbefore defined,
reacting Q-Sp-SS-W with a molecule of the formula
Tu-(Y)n wherein Tu is a monoclonal antibody which exhibits
preferential reactivity with a human tumor-associated anti-
gen, Y is a side-chain amino group on the antibody, or an
aldehyde generated by oxidation of the carbohydrate groups of
the antibody, and n is an integer of from 1 to 100, to produce
a compound of the formula:
Tu-(Z-Sp-SS-W)m
(Y) n-m

-11- 1341315
wherein Tu, Y, Sp, W, and n are as hereinbefore defined, and
Z is formed from covalent reaction of the groups Q and Y
directly or after subsequent reduction, and Z is -CONH-,
CONHN=CH-, -CONHNHCH2-, or
-NHCOGH2 -CH
(CII2)0 1
,. CO 2 I-I
and m is 1 to 15.
A number of different monoclonal antibodies
(MoAb's) are used to exemplify targeting of the methyltrithio
anticancer compounds. MoAb's Lym 1 and Lym 2 recognize dif-
ferent antigens on mature B-lymphocytes and their product
lymphomas. The production and characteristics of these
MoAb's are described by A.L. Epstein, et. al., "Cancer
Research" ~, 830 (1987). MoAb 872.3 targets primarily to
carcinomas of the breast and colon, through reactivity with
z0 pancreatic, ovarian, and lung carcinomas has also been noted.
The ant~.body has been described by T.L. Klug, et. al., "Int.
J. Cancer" 38, 661 (1986). MoAb CT-M-O1, which recognizes
primarily breast tmnors is described in Canadian patent application
513,110 filed July 4th, 1986 and MAiC-68 is produced by a
sub-clone of the hybridoma which produces CT-M-O1, and recog-
nizes both breast and colon carcinomas. Intermediates of tha
subject compounds useful for, and conjugates with these anti-
bodies, are described in the experimental section. It should
not, however, be construed that this patent is limited to or
restricted by the aforementioned antibodies. Instead, the
methodology is sufficiently general that it can be applied to
all antibodies regardless of their class or isotype, their
enzymatically-derived fragments, their chemically manipulated
and stabilized fragments, as well as their respective chim-
eric and humanized counterparts. Nor are the targeting units
restricted only to monoclonal antibodies. Other proteins, as
well as small molecules for which receptors exist on target

1341315
12
tissues, are within the purview of our discovery as targeting
entities.
The methods of this invention used to produce mono-
clonal antibody conjugates from the compounds of Table 1
yield constructs which retain good immunoreactivity with tar-
get cell lines, as determined by the following in vitro
assays:
Tarcret Cells
All target cells were maintained in RPMI 1640 media
supplemented with 5% Fetal Calf Serum (FCS), ITS (Collabora-
tive Research, Cat# 40351), streptomycin (50 ~g/ml), peni-
cillin (50 units/ml), gentamycin sulfate (50 ~g/ml) and glu-
tamine (.03%). The cells were maintained in a humidified 5%
C02 incubator at 37°C.
I. Immunoreactivity Assays
Procedure I - Elisa
Appropriate target cells were harvested, counted
and suspended in Dulbecco's Phosphate Buffered Saline (DPBS)
at an optimal concentration for monoclonal antibody (MoAb)
being tested. 0.1 ml of cells was aliquoted in each well of
a sterile tissue culture polystyrene 96-well plate. The
plates were centrifuged for 5 minutes at 1,000 RPM's and the
supernatant was flicked off. Plates were air-dried overnight
and may be stored at 4°C for up to 3 months.
Non-specific binding sites were blocked by adding
200 ~1 of 1% gelatin in DPBS per well and incubating the
plate for 1 hour at 37°C in a humid incubator. (All subse-
quent incubations are done under similar.canditions). The
plates were washed once with 250 ~cl of 0.05% TWEEN-20*in DPBS
(washing solution) using the automated ELISA washing system
from Dynatech (Ultrawash Ii). Samples to be tested were
diluted to make a final concentration of 3 ~g/ml MoAb equiva-
lents in 0.1% gelatin-DPBS. Six additional threefold serial
dilutions were prepared from each 3 ~g/ml sample and 100 ~cl
3b was added to appropriate wells in triplicate. The bottom row
of wells only received 100 ~1 of 0.1% gelatin as background.
Plates were incubated for 45 minutes and then washed three
*Trade-mark

-13- 1341315
times. Alkaline phosphatase conjugated affinity purified
goat anti-mouse immunoglobulins (Cappel Cat# 8611-0231) was
diluted 1:125 in 0.1% gelatin and 100 ul was added to each
well. Plates were incubated for 45 minutes and then washed
three times. 200 ul of p-nitrophenyl phosphate substrate
solution (see below) was added to each well. After 45 min-
utes at room temperature the reaction was stopped by the
addition of 50 ul of 3M NaOH. The absorbance of the contents
of each Well was read at 405 nm in the automated spectropho-
tometer from Dynatech (EIA Autoreader # EL-310).
Substrate Diethanolamine Buffer (10%Z
- 97 ml diethanolamine
800 ml water
0.2 grams NaN3
100 mg MgCl2'6H20
The reagents were dissolved by continuous stirring
and 1M HC1 was added until the pH was 9.8. The total volume
was made up to 1 liter with water and filter sterilized with
a 0.2 ~ filter. The buffer was stored in the dark at 4°C.
Immediately before use, p-nitrophenyl phosphate (Sigma,
Cat# 104-40) was dissolved in the 10% diethanolamine buffer
(must be at room temperature) to give a final concentration
of 1 mg/ml.
Calculation of O. D. Values
The percentage binding of each sample was calculated
by the following equation:
A-B
x 100 - % Binding
C-B
A = Average O.D. of test sample
8 = Average O.D. of background
C = Average O.D. of 3 ~g/ml unmanipulated MoAb control
The % binding was plotted on the non-log scale of a
semi-log graph and the MoAb concentration was plotted on the
log scale. The BD50 (i.e. dose of antibody needed to give
50% binding) of each test sample was derived from the graph

-14- 1 3 41 3 1 5
and the amount of retention of immunoreactivity was calculated
by the following equation:
BD50 of MoAb control
x 100 = % Immunoreactivity retained
BD50 of test sample
Procedure 2 - Indirect RIA
Appropriate amounts of target cells in 0.2 ml of
10% FCS media were aliquoted into 4 ml polystyrene tubes.
Samples to be tested were diluted to a concentration of
2 ug/ml MoAb equivalents in l0% FCS media. Five three-fold
serial dilutions were prepared from each 2 ~g/ml sample and
0.2 ml was added to each tube in duplicate. Background sam-
ples received only cells and media. Cells were incubated at
4°C for 1 hour, then washed 2 times (all RIA washes were done
with a 3 ml volume) with 2% FCS media. 0.05 ml of sheep
F(ab')2 anti-mouse IgG [125I] (puPont, Cat# NEX 162-0142)
containing approximately 500,000 CPM's was added to each tube;
cells were incubated an additional hour at 4°C, washed once
with 2% FCS and twice with PBS. 0.5 ml of PBS was added to
each tube, cells were vortexed, transferred to clean tubes
and counted for 1 minute in a Packard Gamma 500.
The % binding of each value was determined and
graphed like the preceding ELISA equation, except CPM's were
substituted for O.D. units and C = Average CPM's of 1 ug/ml
unmanipulated MoAb control. The % immunoreactivity retained
of each sample was calculated as previously discussed.
Procedure 3 - Direct RIA
Appropriate amounts of target cells in 1 ml of 10%
FCS media were aliquoted into 4 ml polystyrene test tubes,
centrifuged and supernatant was discarded. ~ Samples to be
tested were diluted to make a concentration of 200 ug/ml MoAb
equivalents in 10% FCS media. Five additional five-fold
serial dilutions were re ared from each 200
p p ug/ml sample and
0.05 ml was added to each tube in duplicate. 0.05 ml of
1251-MoAb was added to each tube (optimal amount is indivi-

-15-
1341315
dually determined for each MoAb and batch). Positive control
samples contained. cells, media and 1251-MoAb. Background
samples contained non-specific cells, media and 1251-MoAb.
Cells were incubated for 1 hour at 4°C, washed once with 2%
FCS media, twice with PBS, transferred and counted as previ-
ously mentioned.
The % 1251-MoAb binding inhibition of each sample
was calculated by the following formula:
A-B
x 100 _ % 1251-MoAb Binding inhibition
C-B
A = Average CPM's of sample
B = Average CPM's of background
C = Average CPM's of positive control
The plot and % immunoreactivity retained by each
sample was calculated as previously'discussed except the BD50
is actually BID50 (Dose of MoAb needed to give 50% inhibition
of the binding of 1251-MoAb).
Notes:
1) Tubes were always vigorously vortexed immediately after
the addition of every reagent in the RIA's.
2) An internal control sample equalling 50% of the unmani
pulated MoAb control was included in each set of assays
to confirm whether each procedure was quantitative in
predicting the conjugates' retention of immunoreactivity.
The results from these assays are tabulated below in Table 2.
35

-16- '~ 3 41 3 1 5
Table 2
Immunoreactivity of MoAb Con~ucrates
Non-specific conjugates Immunoreactivitv
using the product of ~ of unmodified MoAb
example 3 with: Preparation control
Lym 1 #1 15
B72.3 #1 70
#2 10
Hydrazide of 3-mercap-
topropionic acid di-
sulfide analog of
E3328871I (example 4)
conLgated to
Lym 1 #1 100
#2 ° 87
#3 64
#4 80
#5 100
30

-17-
1341315
Table 2 i(continued~
Hydrazide of 3-mercapto-
propionic acid disulfide Immunoreactivity
analog of E3328871I % of unmodified
example 4 conjugated to): Preparation MoAb control
Lym 2 #1 57
#2 85
#3 39
#4 70
B72.3 #1 100
- #2 90
CT-M-O1 #1 60
MAC-68 #1 40
#2 ° 28
Hydrazide conjugates
prepared using the
product of example 5
with:
Lym 1 #1 100
#2 100
The monoclonal antibody conjugates of this inven-
tion are active as anticancer agents. The assay described
below for assessing in vitro cytotoxicity shows the dramatic
30.
preference of the constructs for target cell lines as opposed
to non-target cells, and provides a measure of utility of
targeted forms of the compounds compared to their non-target-
ed counterparts.
Cytotoxicity Assays
In Vitro
Samples to be tested were diluted to a concentra-
tion of 0.2 or 0.02 ug/ml of LL-E3328871I equivalents

-18- 1 3 41 3 1 5
(starting concentration is dependent on cell line to be
tested). Three additional five-fold dilutions were prepared
from each original sample dilution and 0.2 ml was added to
sterile 15 ml polystyrene tubes. At least one similar conju-
gate consisting of LL-E3328871I and an irrelevant MoAb was
included in each assay to determine specificity of the rele-
vant conjugate. 105 appropriate target cells in 0.2 ml of
10% FCS media were aliquoted into the tubes and vortexed. In
addition,~ an identical test was performed utilizing irrelevant
cells as targets to further confirm specificity of relevant
conjugate. MoAb controls received only equivalent amounts of
- MoAb and positive control samples received only 10% FCS media.
Cells were incubated at 37°C for 7 minutes then
washed 4 times with 8 ml of 2% FCS media. 0.1 ml of 10% FCS
was added to each tube, cells were vortexed and 0.2 ml was
aliquoted to each well of a sterile 96-well polystyrene tis-
sue culture plate. '
Plates were incubated for 2 days in a humidified
37°C incubator with 5% C02. One half of the media was re
moved and replaced with fresh media containing 2 ~Ci/ml 3H
thymidine (DuPont, NEN, Cat# NET-027). Incubation was con-
tinued for 24 hours, cells were frozen, thawed and harvested
by a PHD cell harvester (Cambridge Technology, Inc.). Each
sample was counted for 1 minute in a Beckman LS 5800 scintil-
lation counter on Channel 1.
The % growth inhibition was calculated as follows:
Average CPM of test value
x 100 - % Growth
Average CPM of media control
100 - % Growth - % Inhibition
The % inhibition was plotted on the non-log scale
of a semi-log graph and the LL-E3328871I concentration was
plotted on the log scale. The IC50 (concentration of LL-
E3328871I needed to give 50% inhibition) of each test sample

-19-
1341315
was derived from the graph and the amount of retention of
cytotoxicity was calculated by the following equation:
IC50 of LL-E3328871I
x 100 - % Cytotoxicity Retained
IC50 of test sample
The results from the in vitro cytotoxicity assay
are tabulated below in Table 3.
- Table 3
In Vitro Cytotoxicity of MoAb ConZu ads
MoAb Preparation Cytotoxicity
- % product of
%E3328871I Example 1
Non-specific con-
jugates prepared
using product of
Example 3 with:
Lym 1 #1 .9 11.3
872.3 #1 .001
#2 1.4 3.8
% product of
%E3328871I Example
4
Hydrazide conju-
gates prepared
using product of
Example 4 with:
Lym 1 #1 80
#2 56 191
#3 40 60

-20-
1341315
Table 3 ~(continuedl
MoAb Preparation Cytotoxicity
% product of
%E3328871I Example 4
Hydrazide conju-
gates prepared
using product of
Example 4 with:
Lym 1 (#3 Against 0 0
non-tar-
geted cells)
Lym 2 #1 29
#2 2 100
#3 2 55
B72.3 #1 ~ 0 0
#2 0 0
MAC-68 #1 90
CTM-O1 #1 111 830
%E3328871I
Hydrazide conju-
gates prepared
using product of
Example 5 with:
Lym 1 #1 300
#2 100

-21 1341315
The following assay system was used to measure the
in vivo activity of the conjugates of this invention.
In vivo tests for antitumor activity on drug-
monoclonal antibody conjugates were done using human tumor
xenographs in athymic (nude) mice.
Burkitt lymphoma (Raji) and myeloma (HS Sultan)
cells were harvested from culture flasks and inoculated subcu-
taneously (> 80 x 106 Raji cells or 40 x 106 HS Sultan cells)
into test mice. Solid tumors, ovarian carcinomas (CA73,
Ovcar-3) and breast carcinoma (MX-1) were propagated in
athymic mice, removed, cut into 2 mm3 fragments and implanted
- subcutaneously into test mice (5-8 fragments per mouse).
Drugs, monoclonal antibodies and drug-monoclonal
antibody conjugates were administered intraperitoneally once
each 3 days for 3 or 5 total injections starting on day 2, 3,
4, 6, 7 or 8 days after tumor implantation. Tumor measure-
ments (the length and width of the tumor) were made by means
of a Fowler ultra CAL II electronic caliper each 7 days for 4
or 5 weeks post tumor implantation. Tumor mass in mg was
estimated from the formula:
Length(mm) x Width(mm)
2
Tumor growth inhibition was calculated for each
test group on a percent of control [mean mg of treated (T)
divided by mean mg of control (C) x 100]. A T/C value < 42%
in groups with > 65% surviving animals is considered neces-
sary to demonstrate activity.
The results from this assay appear in Table 4.

-22-
1341315
Table 4
In Vivo Antitumor Testinc,~ Results
Dosag e(mcq) Tumor Size S
T
MoAb Drucr (TIC) %control
Hydrazide of 3-mercaptopro- 14.5 0.26 12 5/6
pionic acid disulfide analog
of E3328871I conjugated to
Lym 2
Hydrazide alone - 0.26 34 4/6
- MoAb Lym 2 alone 14.5 - 32 6/6
Mixture, hydrazide +
MoAb Lym 2 14.5 0.26 20 5/6
ip treatment against human melanoma'cell line H.S. Sultan,
3 injections starting on day 5 after tumor implantation,
measurements given made on day 35 post-implantation
25
35

-, , -23- 1 3 41 3 1
5
Table 4 [continued)
Dosage(mc~ Tumor Size S T
MoAb Drucr (T~) %control
Hydrazide of 3-mercapto- 15.5 0.25 39 7/7
propionic acid disulfide
analog of E3328871I
conjugated to MAC-68
Hydrazide-alone - 0.25 - 0/6
MoAb MAC-68 alone 31 - 78 6/6
Mixture, hydrazide + 15.5 0.25 - 0/6
MoAb MAC-68
Melphalan (as positive - 10 43 6/6
control)
ip treatment against human ovarian cancer line CA73,
three injections started 3 days after tumor implantation,
measurements given made on day 35 post-implantation
30

'. , -24- 1 3 4 1 3 1 5
Table 4 (continued)
Dosacre (mcqZ Tumor Size S T
MoAb Drucx ~(TJC) %control
Hydrazide of 3-rnercapto- 8.75 0.25 14 4/6
propionic acid disulfide
analog of E3328871I con-
jugated to CT-M-O1
Hydrazide alone - 0.25 - 0/6
MoAb CT-M-01 alone 8.75 - 75 5/6
Mixture, hydrazide + 8.75 0.2.5 - 0/6
MoAb CT-M-Ol
Vincristine (positive - 1.0 0 4/4
control)
ip treatment against human breast cancer cell line MX-1,'
three injections started on day 2 following tumor implanta-
tinn_ maact~rPmPnts given made on day 35 host-implantation
30

-25-
1341315
Table 4 (continued)
Dosaqe(mca) Tumor Size S
T
MoAb Drua (T/C)%control
Hydrazide of 3-mercapto- 6.2 0.125 62 6/6
propionic acid disulfide
analog of E3328871I con-
jugated to B72.3
Hydrazide alone - 0.125 85 6/6
MoAb B72.3 alone 6.2 - 96 6/6
Mixture, hydrazide + 6.2 0.125 105 5/6
MoAb B72.3
E3328871I - 0.005 141 5/6
(3 treatments)
Cis platinum (positive - 3.0 6 6/6
control, 3 treatments)
ip treatments against human ovarian cell line OVCAR-3,
five injections starting on day 4 after
tumor implantation
(unless otherwise noted), measurements ven made on
gi day
35

-26-
1341315
Table 4 (continued)
Dosaae(mcqZ
Tumor Size
S T
MoAb Drua ~T /C)%control
Hydrazide of 3-mercapto- 27 0.26 6 3/6
propionic acid disulfide
analog of E3328871I con-
jugated t o Lym 1
Hydrazid~ alone - 0.26 72 6/6
MoAb Lym 1 alone 27 - 72 6/6
Mixture, hydrazide + 13 0.13 61 4/6
MoAb Lym 1
ip treatm ent against human cell line Raji
Burkitt lymphoma
TC, three injections started on day'7 after tumor implanta-
tion, mea surements given madeon day 28 post -implantation
25
35

-27-
1341315
The invention will be further described in conjunc-
tion with the following non-limiting examples.
ExamQle 1
3-Mercaptopropionic Acid Disulfide Analoq of
LL-E3328871I
To a solution of 90 mg of LL-E3328871I in 90 ml of
acetonitrile was added 10.6 mg of 3-mercaptopropionic acid in
1 ml of acetonitrile. The solution was vortexed and then
stored at -20°C for 6 days. The solvent was removed in vacuo
and the residue chromatographed over 10 ml of silica gel in
methylene chloride. The column was developed with 50 ml of
methylene chloride, 50 ml of 4% methanol in methylene chloride
and finally 100 ml of 8% methanol in methylene chloride.
Evaporation of this last fraction gave a residue which was
taken up in ethyl acetate with the aid of a little acetone
and added dropwise to an excess of hexane. The precipitate
was collected and dried, giving 39 mg of the desired product
(FABMS, M+H 1394). Retention time on C18 reverse phase HPLC:
18 minutes with 50% acetonitrile/0.1 M aqueous ammonium chlo-
ride. (,~L-E3328871I. 8.0 minutes, ester hydrolysis product:
1.5 minutes)
Example 2
Reaction of LL-E3328871I with the p-nitrophenyl
ester of 3-mercaptopropionic acid
(A) Preparation of p-nitrophenyl ester of 3-mercaptopro-
pionic acid
Commercial 3-mercaptopropionic.acid in methylene
chloride containing a catalytic amount of concentrated sul-
furic acid was treated with isobutylene for 20 minutes. The
solution was then extracted with 1N sodium bicarbonate solu-
tion after which the methylene chloride solution was dried
using anhydrous magnesium sulfate. The solution was then
evaporated to a colorless mobile liquid which NMR and mass
spectral data indicated was the S-t-butylmercaptopropionic
acid, t-butyl ester.

1341315
28
An aliquot of this ester was refluxed with 6N hy-
drochloric acid in dioxane for 2.5 hours. The solvent was
evaporated, ethyl acetate was added and this solution was
extracted with sodium carbonate. The sodium carbonate ex-
tract was treated with 6N hydrochloric acid until the pH of
the suspension was 2Ø The suspension was then extracted
with ethyl acetate, the extract dried over anhydrous magne-
sium sulfate and the solvent evaporated to a colorless liquid
which 1H-NMR -and mass spectral data indicated was S-t-butyl-
mercaptopropionic acid.
This compound was converted to the p-nitrophenyl
ester by treatment with equimolar amounts of g-nitrophenol
and dicyclohexylcarbodiimide in tetrahydrofuran for 4 hours.
The dicyclohexyl urea by-product was removed by filtration
and the filtrate was evaporated to an oil which was purified
by passage over neutral silica gel using the solvent system
hexane:methylene chloride (50:50). 'The pure p-nitrophenyl
ester derivative was a faintly yellow, mobile oil.
The free mercaptan was unmasked by the following
procedure. The S-t-butylmercaptopropionic acid p-nitrophenyl
ester was dissolved in trifluoroacetic acid and a slight molar
excess (10%) of mercuric acetate was added. The mixture was
stirred for 30 minutes, then the trifluoroacetic acid was
evaporated and the residue taken up in dimethylformamide.
This solution was treated with hydrogen sulfide gas for 15
minutes, then the black mercuric sulfide was filtered off and
the filtrate evaporated under reduced pressure to eliminate
up to 99% of the dimethylformamide. The. resultant slightly
brownish mobile liquid was purified over neutral silica gel
using hexane:methylene chloride (50:50). The major component
was shown by 1H -NMR to contain a small amount of the t-butyl
mercapto derivative. Analytical HPLC over two Perkin-Elmer
Pecosphere~ClB columns in tandem [4.6 x 33 mm and 4.6 x
83 mm] using a gradient system of 37.5/62.5 to 47.5/52.5 of
acetonitrile and O.1M ammonium acetate buffer at pH 6.5
(acetic acid) over a 12 minute span indicated that the prod-
uct was 88% of the p-nitrophenyl ester of 3-mercaptopropionic
*Trade-mark

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1341315
acid and 10% of the less polar S-t-butylmercaptopropionic
acid p-nitrophenyl ester. There was also a small amount of
free p-nitrophenol present.
(B) Reaction of p-nitrophenyl ester of 3-mercaptopro-
pionic acid with LL-E3328871I
A 100 mg portion of LL-E3328871I was dissolved in
50 ml of acetonitrile. To this was added a solution of
25.7 mg _of p-nitrophenyl ester of 3-mercaptopropionic acid in
1 ml of acetonitrile. The reaction was left at -20oC for 48
hours. HPLC indicated the reaction was complete. The solu-
tion was evaporated to dryness and the residue taken up in
4-5 ml of ethyl acetate using sonication to effect solution.
The mixture was filtered and the filtrate dripped into 45 ml
of stirred hexane. The resultant faintly yellow solid was
collected and dried under reduced pressure, giving 93 mg of
the p,-nitrophenyl ester of propionip acid derivative of LL-
E3328871I as established by 1H NMR. By FABMS the [M+H] ion
appeared at M/Z=1515.
Example 3
N_~YdroxYsucc ~,nimidyl 3-mercaptopropionate disulfide
analog of LL-E3328871I
To a solution of 5 mg of the 3-mercaptopropionic
acid disulfide analog of LL-E3328871I from Example 1 in
0.5 ml of tetrahydrofuran was added 0.45 mg of N-hydroxysuc-
cinimide in 0.1 ml of tetrahydrofuran and then 1.8 mg of
dicyclohexylcarbodiimide in 0.2 ml of tetrahydrofuran. The
reaction was allowed to stir at room temperature for 4 hours
and was then quenched with a large excess of hexanes. The
solid was isolated by filtration and dissolved in ethyl ace-
tate. The resulting solution was washed three times with
brine, dried with magnesium sulfate, and evaporated to 5 mg
of the desired product as a tan powder which was used without
further purification. Retention time on reverse phase C18
HPLC: 15 minutes with 40% acetonitrile/0.1 M aqueous ammonium
chloride (starting material: 6.0 minutes).

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x341315
Example 4
3-Mercaptopropionyl hydrazide disulfide analog
of LL-E3328871I
To 5.4 ml (3 eq) of anhydrous hydrazine in 100 ml
of refluxing tetrahydrofuran under argon was added dropwise
9.2 ml (83 mmol) of methyl 3-mercaptopropionate in 50 ml
tetrahydrofuran over 2 hours. The solution was refluxed an
additional two hours, evaporated, and then diluted and evapo-
rated twice from 300 ml of toluene. The product was applied
lg to a plug of silica gel with 5% ethyl acetate/chloroform and
eluted from the plug with 20% methanol/chloroform. The re-
' sultant 3-mercaptopropionyl hydrazide was a faintly pink oil
which solidified when cooled but melted at room temperature.
To 50 mg of LL-E3328871I in 50 ml of acetonitrile
at -15°C was added 6.6 mg of 3-mercaptopropionyl hydrazide in
1 ml tetrahydrofuran. One equivalent of triethylamine and/or
one equivalent of acetic acid was added as catalyst. The _
reaction was allowed to stir at 0°C for one hour and the sol-
vent was then evaporated. The residue was chromatographed on
silica gel with a 10-15% methanol in chloroform gradient to
yield 26 mg of the desired product. Retention time on
reverse phase C18 HPLC: 5.0 minutes in 41% acetonitrile/0.1 M
aqueous ammonium chloride.
Example 5
N- ~j~(4-Methyl-coumarin-7-yl)amino]acetyl)cysteine hydrazide
disulfide analog of LL-E3328871I
A mixture of 1.0 g (5.7 mmol) of 4-methyl-7-amino-
coumarin, 3.0 ml of ethyl bromoacetate (5 eq), 90 mg (0.1 eq)
of sodium iodide, and 30 ml dimethylformamide was heated under
argon at 80°C for 5 hours. The mixture was cooled, diluted
with ethyl ether, washed three times with 50% brine, dried
with magnesium sulfate, and evaporated to dryness. The crude
product was dissolved in chloroform containing 1% ethyl ace-
tate and filtered through a plug of silica gel. Recrystal-
lization from diethyl ether containing a trace of chloroform
yielded pure ethyl N-[(4-methyl-coumarin-7-yl)amino]acetate.

. ' -31- 1 3 41 3 1 5
To 1.96 g (7.5 mmol) of the above ester in 15 ml of
methanol and 15 ml of tetrahydrofuran was added 10 ml of 1N
aqueous sodium hydroxide. After 30 minutes, 4 ml of 10%
aqueous hydrochloric acid was added. The organic solvents
were evaporated and the resultant crystalline product was
filtered and washed with cold ethanol and then ether. This
material was dissolved in 20 ml of tetrahydrofuran and 4 ml
of dimethylformamide. Dicyclohexylcarbonyldiimidazole (1.3 g,
2.2 eq) was added and the reaction allowed to stir for 15
minutes. Cysteine ethyl ester hydrochloride (1.6 g, 2.5 eq)
and triethylamine (1.2 ml) were then added. After a further
three hours, the reaction was diluted with ethyl ether con-
taining 5% methylene chloride and washed once with 10% aqueous
hydrochloric acid and twice with brine. After drying with
magnesium sulfate and evaporating the solvents, the crude
product was crystallized by dissolving in chloroform con-
taining a minimal amount of ethanol and then adding an excess
of ether. The crystals were filtered and dried to give pure
N-[[(4-methyl-coumarin-7-yl)amino]acetyl]cysteine ethyl ester.
A mixture of 5 ml of chloroform, 20 ml of methanol,
and 0.4~m1 of hydrazine hydrate were heated to reflux under
argon. To this was added 550 mg of N-[[(4-methyl-coumarin-
7-yl)amino]acetyl]cysteine ethyl ester. After refluxing for
9 hours the mixture was cooled and the solid product was fil-
tered and washed with chloroform and then ethyl ether. The
crude product (which contained thiol and disulfide) was dis-
solved in dimethylformamide containing dithiothreitol and
triethyl amine. After 30 minutes the product was precipitated
with excess ethyl ether and collected by filtration. This
material was purified further by recrystallization from de-
gassed acetonitrile containing dithiothreitol and a trace of
triethyl amine to give pure N-[[(4-methyl-coumarin-7-yl)-
amino]acetyl]cysteine hydrazide.
To 12 mg of 70% pure LL-E3328871I in 12 ml aceto-
nitrile at 0°C was added 4 mg of N-[[(4-methyl-coumarin-7-
yl)amino]acetyl]cysteine hydrazide in 1.2 ml dimethylform-
amide. After stirring overnight another 2 mg of N-[[(4-

-32- 1 3 4 1 3 1 5
methyl-coumarin-7-yl)amino]acetyl]cysteine hydrazide in
0.6 ml dimethylformamide was added. The reaction was stirred
for 3 days at 0°C and filtered. The acetonitrile was evapo-
rated and the resultant dimethylformamide solution was diluted
with an excess of 1:1 hexanes/ether. The product was isolated
by filtration and further purified by chromatography on silica
gel with a 15-20% gradient of methanol in chloroform to yield
3 mg of the desired product. Retention time on reverse phase
C18 HPLC: 3.5 minutes using 45% acetonitrile/0.1 M aqueous
ammonium chloride.
Example 6
3-Mercaptopropionyl hYdrazide disulfide analocr of
LL-E33288a3I
To 10 mg of LL-E33288a3I in 9 ml of acetonitrile at
-15°C was added 6.6 mg of 3-mercaptopropionyl hydrazide in
1 ml acetonitrile. One equivalent of triethylamine and/or
one equivalent of acetic acid were added as a catalyst. The.
reaction was allowed to stir at 0°C for one hour and the sol-
vent was then evaporated. The residue was chromatographed on
silica gel with a 10-15% methanol in chloroform gradient to
give the desired product. Retention time on reverse phase
C18 HPLC: 3.5 minutes in the system 45% acetonitrile/0.1 M
aqueous ammonium chloride.
Example 7
Non-specific con~ju_qation to proteins
The hydroxysuccinimide ester described in Example 3
was covalently attached to antibodies under slightly alkaline
conditions. The following is a general procedure used to
make the antibody conjugates listed in Table 5. Antibody at
a concentration of 3-5 mg/ml in phosphate buffer containing
O.1M sodium chloride, pH 7.5 was reacted with a 5-20-fold
molar excess of the product from Example 3 with stirring, at
room temperature for from 1-4 hours. The conjugated protein
was desalted chromatographically and aggregated protein was
separated from monomeric material by gel filtration HPLC.
Monomeric fractions were pooled and concentrated.

'. -33-
1341315
Table 5
Non-specific coniuqates prepared usinc~the product
of Example 3
MoAb Drug Loading
Lym 1 5.2
B72.3 6.0
B72.3 2.9
Example 8
Site-specific conjugate preparation
The general method for attaching hydrazide deriva-
tives of drugs to oxidized antibodies is described in T. J.
McKearn, et al., in U.S. Patent No. 4,671,958. The procedure
has been applied to preparing antibody conjugates from the
products of Examples 4 and 5 with specific modifications as
described below. The products from'these reactions and their
characteristics are summarized in Table 6.
(A) Antibody Oxidation Antibody at a concentration of 5 to
10 mg/ml was dialyzed overnight against a 200 fold volume of
50mM sodium acetate buffer, pH 5.5 containing O.1M sodium
chloride (Buffer A). After dialysis, the MoAb was oxidized
with lSmM to 200mM periodic acid in 0.2M sodium acetate. The
oxidation was allowed to proceed in the dark, with stirring,
at 4°C for 45 minutes after which time the oxidized MoAb was
desalted on a >5 bed volume Sephadex G-25 column. The degree
of oxidation of the antibody was assessed by reaction with
p-nitrophenylhydrazine and comparing absorbance of the pro-
tein at 280mm vs. p-nitrophenylhydrazine at 395mm.
(B) Druq Hydrazide Conjugation The oxidized MoAb was react-
ed with 25 to 200-fold molar excess of drug hydrazide. The
hydrazides were dissolved into dimethylformamide and added to
the aqueous solution of MoAb. To avoid precipitation of MoAb,
the final volume of dimethylformamide added did not exceed
lOx of the total reaction volume. Reaction was allowed to
proceed for 3 hours at room temperature, with stirring. To
prevent crosslinking of unreacted aldehydes and subsequent

' -34-
1341315
aggregation, a blocking agent, acetyl hydrazide was added in
100-fold molar excess three hours after addition of the drug
hydrazide. To stabilize the Schiff's base linkage between
aldehyde and drug hydrazide (a hydrazone), the product gen-
erally was reduced to an alkyl hydrazine by the addition of
lOmM sodium cyanoborohydride, allowing the reaction to pro-
ceed for one more hour (total conjugation time - 4 hours).
The conjugate was chromatographically desalted and exhaus-
tively dialyzed (minimum time 48 hours) into pH 6.5 phosphate
buffer for storage and testing.
Conjugates were analyzed for the presence of aggre-
- gates by gel filtration HPLC and for free drug by reverse
phase HPLC. Drug loading was determined spectroscopically
using the extinction coefficients of both the antibody and
the drug to estimate molar concentrations of drug in conju-
gates.
25
35

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1341315
Table 6
Hydrazide conZuqates prepared from the product of
Example 4
MoAb Preparation Druq Loadin~/M
Lym 1 #1 1.4
#2 2.4
#3 1.0
#4 6.7
#5 3.3
Lym 2 #1 2.9
#2 1.9
#3 2.0
#4 2.8
B72.3 #1 ' 2.3
#2 ' 1.3
CTM-O1 3.1
MAC-68 1.7
Hvdrazide Conjugates prepared from the product of Example
5
Lym 1 #1 0.15
#2 0.76
35