Note : Les revendications sont présentées dans la langue officielle dans laquelle elles ont été soumises.
-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.
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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
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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
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<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:
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<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.
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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.