Note: Descriptions are shown in the official language in which they were submitted.
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BRANCHED HYDRAZONE LINKERS
Bifunctional compounds which link cytotoxic
reagents to antibodies (i.e., "linkers") are known in the
art. These compounds have been particularly useful in
the formation of immunoconjugates directed against tumor
associated antigens. Such immunoconjugates allow the
selective delivery of toxic drugs to tumor cells. (See
e.g., Hermentin and Seller, "Investigations With
Monoclonal Antibody Drug Conjugates," Behringer Insti.
Mitl. 82:197-215 (1988); Gallego et al., "Preparation of
Four Daunomycin-Monoclonal Antibody 791T/36 Conjugates
With Anti-Tumor Activity," Int. J. Cancer 33:7737-44
(1984); Arnon et al., "In Vitro and In Vivo Efficacy of
Conjugates ofDaunomycin With Anti-Tumor Antibodies,"
Immunological Rev. 62:5-27 (1982}.
Greenfield et al. have described the formation of
acid-sensitive immunoconjugates containing the
acylhydrazine compound, 3-(2-pyridyl-dithio)propionyl
hydrazide conjugated via an acylhydrazone bond to the 13-
keto position of an anthracycline molecule, and
conjugation of this anthracycline derivative to an
antibody molecule (Greenfield ,fit al., European Patent
Publication EP 0 328 147, published August 16, 1989,
which corresponds to pending U.S. Serial No. 07/270,509,
filed November 16, 1988 and U.S. Serial No. 07/155,181,
filed February 11, 1988, now abandoned). This latter
reference also discloses specific thioether-containing
linkers and conjugates, including hydrazone thioether
containing immunoconjugates.
Kaneko et al. (U.S. Serial No. 07/522,996, filed May
14, 1990, which is equivalent to European Patent
Publication, EP A 0 457 250, published November 21, 1991)
have also described the formation of conjugates
containing anthracycline antibiotics attached to a
bifunctional linker by an acylhydrazone bond at the C-13
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position of an anthracycline molecule. In their
invention the linkers contain a reactive pyridinyldithio-
or an ortho-nitrophenyldithio- group, by which the linker
reacts with a suitable gzoup attached to a cell reactive
ligand, to form the completed conjugate. An important
consideration for immunoconjugates is that the
relationship between drug potency and antigen expression
must be appropriate in order to effect cytotoxicity on a
broad range of malignant cells. Alterations in the
potency of various immunoconjugates can be affected by
changing the monoclonal antibody (MAb) utilized and/or
the potency of the unconjugated drug. It is also
possible to effect the potency of immunoconjugates by
changes in the linker, both in terms of stability in
circulation (Koizumi,M.,K., Kunimatsu,M.,
Sakahara,H.,Nakashima,T.,Kawamura,Y.,
Watanabe,Y.,Ohmomo,Y.,Arano,Y.,Yokoyama,A. and
Torizuka,K. (1987), Preparation of 67Ga-labeled
antibodies using deferoxamine as a bifunctional chelate.
~,T Tmmt~nc~l Methods 104, 93-102; Thorpe, P.E. ,
Wallace,P.M.,Knowless,P.P.,ReIf,M G., Brown,A.N.F.,
Watson,G.J.Knyba,R.E.,Wawrzynczak,E.J. and
Blakey,D.C.(1987), New coupling agents for the synthesis
of immunotoxins containing a hindered disulfide bo3a.d with
improved stability in vivo. Cancer Res. 47,5924-5931;
Trail,P.A.,Wilner,D., Lasch,S.J., Henderson,A.J.,
Greenfield,R.S.,King,D., Zoeckler,M.E. and
Braslawsky,G.R.(1992), Antigen specific activity of
carcinoma reactive BR64-adriamycin conjugates evaluated
in vitro and in human tumor xenograft modelsk, Cancer
Research 52, 5693-5700; Trail,P.A.,Willner,D.,Lasch,S.J.,
Henderson,A.J.,Hofstead,S.J.,Casazza,A.M.,Firestone
R.A.,Hellstrbm,K.E.(1993), Cure of xenografted human
carcinomas by BR96-Doxorubicin Immuno-conjugates, Science
261,212-215; Trail,P.A., Willner,D. and Hellstrom, ,
-2-
CA 02239183 1998-06-O1
WO 97123243 ~CTNS9b/205I3
K.E.(1995), Site-directed delivery of anthracyclines for
cancer therapy. ~7rua Development Research 34, 196-209)
and in terms of drug/MAb molar ratio (Shih,L.B.,
Goldenberg,D.M., Xuan,H.,Lu,H.,Sharkey,R.M. and
~ 5 Hall,T.C.(1991), Anthracycline immunoconjugates prepared
by a site specific linkage via an aminodextran
intermediate carrier. International Journal of Cancer 41,
8320839; Trail et a1.1992; Trail et a1.,1995).
In particular, the in vitro potency of doxorubicin
conjugates prepared with the internalizing anticarcinoma
MAb BR64 and an acid labile hydrazone bond, was shown to
increase as drug/MAb molar ratios increased from 1-8
(Trail et a1.,1992; Trail et a1.,1995). However, in
these studies the increase in drug/MAb molar ratios was
based on increasing the number of conjugation sites on
the MAb which is self-limiting and has other drawbacks
such as reduced antibody binding affinity.
In view of the above, it is clear that one of the
problems in prior art immunoconjugates is the relatively
low ratio of drug to targeting ligand (e. g.,
immunoglobulin) achievable. Tt would be highly desirable
to have immunoconjugates which provide a higher ratio of
drug to targeting ligand.
The present invention provides novel branched
hydrazone linkers. The novel linkers are used to prepare
novel drug/linker molecules and biologically active
conjugates composed of a targeting ligand, a
therapeutically active drug , and a branched linker
capable of recognizing a selected target cell population
(e. g., tumor cells) via the targeting ligand.
As used herein the term "drug/linker" or
"linker/drug" molecule refers to the linker molecule
coupled to two or more therapeutically active drug
molecules, and the term "conjugate" refers to the
drug/linker molecule coupled to the targeting ligand.
The linkers are branched so that more than one drug
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molecule per linker are coupled to the ligand. The
number of drugs attached to each linker varies by a
factor of 2 for each generation of branching. Thus, the
number of drug molecules per molecule of linker can be 2,
4, 8, 16, 32, 64, etc. The factor of branching can be
expressed mathematically as 2n wherein n is a positive
integer. Thus, a singly branched linker will have a .
first generation of branching or 21, i.e., contains two
drug molecules per linker. A doubly branched linker will
have a second generation of branching or 22, i.e.,
contains four drug molecules per linker.
Thus, the present invention is directed to a
branched linker for linking a thiol group derived from a
targeting ligand to two or more drug moieties which
comprises a compound having a terminus containing a thiol
acceptor for binding to a thiol group (also called a
sulfhydryl group) derived from a targeting ligand, at
least one point of branching which is a polyvalent atom
allowing for a level of branching of 2n wherein n is a
positive integer, and at least two other termini
containing acylhydrazide groups capable of forming
acylhyrdazone bonds with aldehyde or keto groups derived
from a drug moiety. It is preferred that n is 1,2, 3, or
4; more preferably 1, 2 or 3; most preferably 1 or 2. It
is also preferred that the polyvalent atom is carbon or
nitrogen, and the targeting ligand is an antibody or
fragment thereof.
As used in the preceeding paragraphf the phrase
"thiol group derived from the targeting ligand" means
that the thiol group is already present on the targeting
ligand or that the targeting ligand is chemically
modified to contain a thiol group, which modification
optionally includes a thiol spacer group between the
targeting ligand and the thiol group. Likewise, the
phrase "an aldehyde or keto group derived from a drug
moiety" means that the aldehyde or keto group is already
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present on the drug or the drug is chemically modified to
contain an aldehyde or keto group.
Also provided by the invention are intermediates for
preparing the linkers, drug/linkers and/or conjugates;
and a method for treating or preventing a selected
disease state which comprises administering to a patient
a conjugate of the invention.
Figure 1 - In vitro potency of BR96 straight chains
hydrazone and branched hydrazone conjugates following
various exposure times as described in Example 62.
--/-- represents BR96 MCDOXHZN and "'~-' represents
BR96 MB-Glu-(DOX)2.
Figures 2 - In vitro potency of IgG straight chain
hydrazone and branched hydrazone conjugates following
various exposure times as described in Example 62.
-Q-- represents IgG MCDOXHZN and ~ represents
TgG MB-Glu-(DOX)2.
According to the present invention the drug
molecules are linked to the targeting ligand via the
linker of the invention_ The drug is attached to the
linker through an acylhydrazone bond. The targeting
ligand is attached to the linker through a thioether
bond. The thioether bond is created by reaction of a
sulfhydryl (thiol) group on the ligand, or on a short
"thiol spacer" moiety attached to the ligand, with a
thiol acceptor. The thiol acceptor can be a Michael
Addition acceptor which becomes, after the reaction, a
Michael Addition adduct. In a preferred embodiment, the
targeting ligand is attached directly to the linker
through a covalent thioether bond without a thiol spacer.
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In a preferred embodiment the novel linker molecule
of the invention has the formula
O
II
O H yCH2)a (NH)b-""C-(VV)rri X
v
A- Q-C-N-C' I
(NH)b it-(W)m x
O
wherein
A is a thiol acceptor;
Q is a bridging group;
b is an integer of 0 or 1;
W is a spacer moiety;
m is an integer of 0 or 1;
a is an integer of 2, 3 or 4; and
X is a moiety of the formula -NH-NH2 or
O
-NHNHC-NHNH2
or a moiety of the formula
1
E-I ~(C~"~2)a (NH)e C-(W)rri X
-N-CH
\(NH)e ~ (W)m'Xi
O
wherein
W, a, b and m are as defined
hereinbefore, and
X1 is a moiety of the formula -NH-NH2 or
O
-NHNHC-NHNH2
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or a moiety of the formula
H ~~CH2)a (NH)e WW~m'X2
-N-CH
'(NH)b- (W)m-X2
O
wherein
W, a, b, and m are defined hereinbefore, and
X2 is a moiety of the formula NH-NH2 or
O
II
-NHNHC-NHNH2
or a moiety of the formula
H yC~"~2)a (NH)e WW~m-X3
-N-CH
'(NH)b- (W)m'X3
O
wherein
W, a, b, and m are as defined hereinbefore,
and
X3 is a moiety of the formula
W-~2 or
O
II
-NHNHC-NHNH2
or a moiety of the formula
H yC~"~2)a (NH)b C--'(W)m-X4
-N-CH
'(NH)b- (W)m-X4
O
wherein
. W, a, b and m are as defined hereinbefore, and
X4 is a moiety of the formula
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-NH-NH2 or
O
li
-NHNHC-NHNH2 .
In another preferred embodiment the novel branched
linker of the invention has the formula
H
0
A- CH C C > [N-(CH2)~]~ -'T
( 2)n a !
wherein
n is an integer of 1 to 6
a is an integer of 0 or 1,
j is an integer of 2 to 6,
c is an integer of 0 or 1,
provided that when a is 0, c must also be 0;
is a thiol acceptor;
T is of the formula
O
II
N-' [(CH2)m - (NH)b C X~2 , or
O O
-N[(CH2) - (NH)b C
-N/(CH2)d f g O
O
C,-N[(CH2) - (NH)~ C
HC
(C 2)d f 9
wherein
d is an integer of 2 to 6,
m is an integer of 1 or 2,
f is an integer of 0 or 1,
b is an integer of 0 or 1,
g is an integer of 1 or 2, and
X is a moiety of the formula -NH-NH2 or
O
ii
-NHNHC-NHNH2
Preferred branched linkers of formula II are where d
is 2, f is 0, g is l, and/or b is 0.
_g_
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Specific preferred compounds of formula II have the
following formulae
O
N-(CH2)n N[(CH2)mC0-X)2
O
O N[(CH2)mC0'X~2
N-(CH2)~ N
O
N[(CH2)mC0-X~2
N[(CH2)mC0-X]2
O O
N-(Cf"12)n~N
O
N[(CH2)mC0-X]2
N[(CH2)mC0-X~2
O O
~ N
N-(CH2)n- _N~
1 H
O N[(CH2)mC0-X~2
_g_
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N~~CHz)mC0-X~2
O
'N-WHz)n-N
O
O NI~CH2)mC0-X~2
Preferred drug/linker molecules (alternatively
referred to herein as "linker/drug" molecules) of the
invention are when the X moieties of the compounds of
formula I or II are of the formula -NH-N=Drug or
O
-NHN H-C-NHN=Drug
Preferred linker/drug molecules of the invention
within the scope of formula I have the formulae
H
N N- N=XS
fpm
O O
0 tCHz)a H H
N~ ~C~ N N-N=X5
~CHz)n N ~ ~ m
O H O O
wherein
a is an integer of 0, 1, 2, or 3,
n is an integer of 1 to 6,
m is an integer of 0 or 1, and
X5 is an anthracycline antibiotic;
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H _
O N / ~ N- X5
m
(CFip)a H
N N- N. X5
O
IIII [[ m
~m O O
O O
~ ~ O (CHI H H O O
Nw ~ N N
(CH~n H ~~ v
O O O m (CH~~HN ~ N-N-X5
O
i H
O 1H ~ m N- N= X5
wherein
n is an integer of 1 to 6,
a is an integer of 0, 1, 2, or 3,
m is an integer of 0 or 1, and
X~ is an anthracycline antibiotic;
Preferred novel conjugates prepared from the
drug/linker molecules of the invention have the formula
O
H Y OH II
I II II I ~ (CH~a - (NH) b C-(Vl~m X
(N-G(CH2)p)Z S-A-Q-C-N-CH
\(NH)b iI-N~mX III
O
q
wherein
A is a thiol adduct,
W is a spacer moiety,
Q is a bridging group,
m is an integer of 0 or 2,
a is an integer of 2, 3, or 4,
~ b is an integer of 0 or 1,
p is an integer of 1 to 6,
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Y is O or NH2+Cl-,
z is an integer of 0 or l,
q is an integer of 1 to 10,
G is a targeting ligand, and
X is a moiety of the formula -NH-N=Drug or '
O
-N HN H-C-N H N=Drug
or a moiety of the formula
1
H ~(CH2)a (NH)~ C-(W)m X
-N-CH
~(NH)e -(W)m-Xi
wherein W, a, b and m are as defined hereinbefore,
and X1 is a moiety of the formula -NH-N=Drug, or
O
-NHNH-C-NHN=Drug
or a moiety of the formula
2
H ~(C~"i2)a (NH)e C,--tW)m_X
-N-CH
'(NH)b- (W)m-X2
wherein W, a, b and m are as defined hereinbefore,
and X2 is a moiety of the formula -NH-N=Drug, or
O
-N H N H-C-N H N=D rug
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or a moiety of the formula
H ~~CH2)a (NH)~i ~~W)m'X3
-N-C'H
\(NH)b- -lW)m-X3
O
wherein
W, a, and m are defined hereinbefore, and
X3 is a moiety of the formula -NH-N=Drug, or
O
-N HN H-C-NH N=Drug
or a moiety of the formula
H yC~'-~2)a (NH)b C--(W)m-X4
-N-CH
~(NH)b- (W)m-X4
O
wherein W a, b, and m are defined
hereinbefore, and
X4 is a moiety of the formula -NH-N=Drug or
O
-NHNH-C-NHN=Drug
Other preferred novel conjugates of the invention
have the formula
G (N-C-(CH~p)z S-A-(CH2)n (C)a-IN-(CH2)jlc'T
IV
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wherein
A is a thiol adduct,
n is an integer of 1 to 6,
a is an integer of 0 or 1,
j is an integer of 2 to 5,
c is an integer of 0 or 1,
p is an integer of 1 to 6, .
Y is O or NH2+C1-,
z is an integer of 0 or 1,
q is an integer of 1 to 10,
G is a targeting ligand, and
T is of the formula -
O
I I
N'-[(CH2)rr, - (NH)b - C-X~2 , or
O O
CH C C,-N[(CH2) -(NH)b C.-X~2
/( 2)d f J
- N~ O - O
CH ~ C ~-N[(CH2) (NH)e C-Xl2
( 2)d f 9
wherein
d is an integer of 2 to 6,
m is an integer of 1 or 2,
f is an integer of 0 or 1,
b is an integer of 0 or 1,
g is an integer of 1 or 2, and
X is a moiety of the formula
-NH-N=Drug or
O
-NHNH-C-NHN=Drug
In one embodiment the drug moiety is an
anthracycline antibiotic and the ligand is an antibody.
In a preferred embodiment the anthracycline is bound
to the linker through an acylhydrazone bond at the 13-
keto position of the anthracycline compound. The ,
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targeting ligand, preferably an antibody or fragment
thereof, then is bound, through the linker, to the
anthracycline compound. In an especially preferred
embodiment, this linkage occurs through a reduced
disulfide group (~.~e. a free sulfhydryl or thiol group {-
SH)) on an antibody).
In a most preferred embodiment the anthracycline
drug moiety is adriamycin, the thiol acceptor is
Michael Addition acceptor, from which the Michael
Addition adduct is derived, especially a maleimido-
group, and the antibody moiety is a chimeric or humanized
antibody.
The conjugates of the invention retain both
specificity and therapeutic drug activity for the
l5 treatment of a selected target cell population. They may
be used in a pharmaceutical composition, such as one
comprising a pharmaceutically effective amount of a
compound of Formula III or IV associated with a
pharmaceutically acceptable carrier, diluent or
excipient.
The present invention provides novel branched
linker/drug molecules composed of a drug, and a
thioether-containing linker having at least two drug
molecules which can be joined to a ligand capable of
targeting a selected cell population. The drugs are
joined to the linker through an acylhydrazone bond. The
point of branching is a polyvalent atom, preferably a
carbon atom or nitrogen atom. In a preferred embodiment,
the ligand is joined directly to the linker through a
thioether bond. Normally, this bond will be created by
reaction of a reactive sulfhydryl (-SH) group on the
ligand, or on a spacer moiety (e.g., one derived from the
SPDP or iminothiolane chemistry described below), with a
thiol acceptor such as a Michael Addition acceptor.
The invention also provides methods for the
production of these drug conjugates and pharmaceutical
WO 97/Z3243 CA 02239183 2005-02-22 p~,~sg~p~l3
compositions and methods for delivering the conjugates to
target cells in which a modification in biological
process is desired, such as in the treatment of diseases
such as cancer, viral or other pathogenic infections,
autoimmune disorders, or other disease states.
The conjugates comprise at least two drug molecules
connected by a linker of the invention to a targeting
ligand molecule that is reactive with the desired target
cell population. The targeting ligand molecule can be an
immunoreactive protein such as an antibody, or fragment
thereof, a non-immunoreactive protein or peptide ligand
such as bombesin or, a binding ligand recognizing a cell
associated receptor such as a lectin or steroid molecule.
For a better understanding of the invention, the
Drugs, the ligands and various components of the
hydrazone linkers will be discussed~individually.
The Sflacer ( "~T" )
As used herein, the term "spacer" refers to a
bifunctional chemical moiety which is capable of
covalenting linking together two spaced chemical moieties
into a stable tripartate molecule. Specifically, the "W"
spacer links a keto group to a nitrogen atom. ales
of spacer molecules are described in S.S. Wong, ~emistrv
~f protein Conjuaation and Crogslinkina CRC Press,
Florida, (1991); and G.E. Means and R.E. Feeney,
BioconZuaate Che~mistr~r, vol. 1, pp.2-12, (1990).
Preferred spacers have the formula
H O
n
-N-(CH2)g C-
wherein g is an integer of 1 to 6, preferably 2 to
4, more preferably 2.
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The most preferred spacer has the formula
H
--N
0
The Bridginc Groan ("0")
The bridging group is a bifunctional chemical moiety
which is capable of covalenting linking together two
spaced chemical moieties into a stable tripartate
molecule. Examples of bridging groups are described in
S.S. Wong, Chemistry of Protein Coniugat~on and
c~rossl?nkincr, CRC Press, Florida, (1991); and G.E. Means
and R.E. Feeney, Bioconiuaate Chemistry, vol. 1, pp.2-12,
(1990).
Specifically, the bridging group "Qn
covalently links the thiol acceptor to a keto moiety. An
example of a bridging group has the formula
'(CH2)f t~g ~ ~CH2)h
wherein
f is an integer of 0 to 10,
h is an integer of 0 to 10,
g is an integer of 0 or 1,
provided that when g is 0, then f + h is
1 to 10,
Z is 5, 0, NH, 502, phenyl, naphthyl, a
cycloaliphatic hydrocarbon ring containing 3
to 10 carbon atoms, or a heteroaromatic
hydrocarbon ring containing 3 to 6 carbon
atoms and 1 or 2 heteroatoms selected from 0,
N, or S.
Preferred cycloaliphatic moieties include
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the
like. Preferred heteroaromatic moieties include pyridyl,
furanyl, pyranyl, pyrimidinyl, pyrazinyl, pyridazinyl,
. oxazinyl, pyrrolyl, thiazolyl, morpholinyl, and the like.
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In the bridging group it is preferred that when g is
0, f + h is an integer of 2 to 6 preferably 2 to 4 and
more preferably 2. When g is 1, it is preferred that f
is 0, 1 or 2, and that h is 0, 1 or 2.
mhe Tha.olAcceptor
In the molecules of Formulas I, II, III, and IV, the ,
thiol acceptor "A" is linked to the ligand via a sulfur
atom derived from the ligand. The thiol acceptor becomes
a thiol adduct after bonding to the ligand through a
thiol group via a thioester bond. The thiol acceptor can
be , for example, an alpha-substitited acetyl group.
Such a group has the formula-
O
II
Y-CH2 C-
wherein Y is a leaving group. Examples of leaving
groups include Cl, Br, I, meaylate, tosylate, and the
like. If the thiol acceptor is an alpha-substituted
acetyl group, the thiol adduct after linkage to the
ligand forms the bond -S-CH2-
Preferably, the thiol acceptor is a Michael Addition
acceptor. A representative Michael Addition acceptor of
this invention has the formula
O
N-
O
After linkage to the ligand, the Michael Addition
acceptor becomes a Michael Addition adduct, such as of
the formula A
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A
The Drug
The drug of the drug/linker molecule and conjugates
of the present invention are effective for the usual
purposes for which the corresponding drugs are effective,
and have superior efficacy because of the ability,
inherent in the ligand, to transport the drug to the
desired cell where it is of particular benefit. Further,
because the conjugates of the invention can be used for
modifying a given biological response, the drug moiety is
not to be construed as limited to classical chemical
therapeutic agents.
The preferred drugs for use in the present invention
are cytotoxic drugs, particularly those which are used
for cancer therapy. Such drugs include, in general, DNA
damaging agents, anti-metabolites, natural products and
their analogs. Preferred classes of cytotoxic agents
include the anthracycline family of drugs. Particularly
useful members of that class include, for example,
daunorubicin, doxorubicin, carminomycin, morpholino
doxorubicin, diacetylpentyl doxorubicin and their
analogues.
As noted previously, one skilled in the art may make
chemical modifications to the desired compound in order
to make reactions of that compound more convenient for
purposes of preparing conjugates of the invention.
In the conjugate of Formula II, D is a drug moiety
having pendant to the backbone thereof a chemically
reactive functional group by means of which the drug
backbone is bonded to the linker, said functional group
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selected from the group consisting of an aldehyde or a
ketone.
A highly preferred group of cytotoxic agents for use
as drugs in the present invention include drugs of the
following formula:
ThP Anthracvclines Antibiotics Of Formula (V)- ,
C, O
~ R1
OH
Ft' O OH
O
CH3
Rs Rs
R4
(V)
wherein
R1 is -CH3, -CH20H, -CH20C0(CHZ)3CH3 or -
CH20COCH(OC2H5)2
R2 is -OCH3, -OH or -H _ .
R3 is -NH2, -NHCOCF3, 4-morpholinyl, 3-cya.no-4-
morpholinyl, 2-piperidinyl, 4-methoxy-1-piperidinyl,
benzylamine, dibenzylamine, cyanomethylamine, 1-cyano-2-
methoxyethyl amine, or NH-(CH2)4-CH(OAc)2;
R4 is -OH, -OTHP, or -H; and
R5 is -OH or -H provided that R5 is not -OH when R4
is -OH or -OTHP.
One skilled in the art understands that structural
Formula (V) includes compounds which are drugs, or are
derivatives of drugs, which have acquired in the art
different generic or trivial names. Table I, which
follows, represents a number of anthracycline drugs and
-20-
CA 02239183 1998-06-O1
WO 97/23243 PCT/US96/ZOSI3
their generic or trivial names and which are especially
preferred for use in the present invention.
Of the compounds shown in Table I, the most highly
preferred drug is Doxorubicin. Doxorubicin (also
- 5 referred to herein as "DOX").is that anthracycline shown
on Table I in which R1 is -CH20H, R3 is -OCH3, R4 is -NH2,
~ R5 is -OH, and R6 is -H.
-21 -
CA 02239183 1998-06-O1
WO 97/23243 PCT/US96/205I3
'°a~ xxxxxoxxx x x x~
a o 0 0 0
oooooxx
o
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U
G~ _O
N N N N N N N NC~M
~
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fi i
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nl N ~
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F~ ~3 ~3
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~ O d I~f 'O
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ooouH
-22-
CA 02239183 1998-06-O1
WO 97/23243 PCT/US96/20513
the Tarsretiag Lis~and
The "ligand" includes within its scope any molecule
that specifically binds or reactively associates or
complexes with a receptor or other receptive moiety
associated with a given target cell population. This
cell reactive molecule, to which the drug reagent is
linked via the linker in the conjugate, can be any
molecule that binds to, complexes with or reacts with the
cell population sought to be therapeutically or otherwise
biologically modified and, which possesses a free
reactive sulfhydryl (-SH) group or can be modified to
contain such a sulfhydryl group. The cell reactive
molecule acts to deliver the therapeutically active drug
moiety to the particular target cell population with
which the ligand reacts. Such molecules include, but are
not limited to, large molecular weight proteins such as,
for example, antibodies, smaller molecular weight
proteins, polypeptides or peptide ligands, and non-
peptidyl ligands.
The non-immunoreactive protein, polypeptide, or
peptide ligands which can be used to form the conjugates
of this invention may include, but are not limited to,
transferrin, epidermal growth factors ("EGF"), bombesin,
gastrin, gastrin-releasing peptide, platelet-derived
growth factor, IL-2, IL-6, tumor growth factors ("TGF").
such as TGF-a and TGF-b, vaccinia growth factor ("VGF"),
insulin and insulin-like growth factors I and II. Non-
peptidyl ligands may include, for example, carbohydrates,
lectins, and apoprotein from low density lipoprotein.
The immunoreactive ligands comprise in antigen-
recognizing immunoglobulin (also referred to as
"antibody"), or an antigen-recognizing fragment thereof.
Particularly preferred immunoglobulins are those
immunoglobulins which can recognize a tumor-associated
antigen. As used, "immunoglobulin" may refer to any
recognized class or subclass of immunoglobulins such as
-23-
CA 02239183 1998-06-O1
WO 97/23243 PCT/US96/20513
IgG, IgA, IgM, IgD, or IgE. Preferred are those
immunoglobulins which fall-within the IgG class of
immunoglobulins. The immunoglobuin can be derived from
any species. Preferably, however, the immunoglobulin is
of human, murine, or rabbit origin. Furthermore, the -
immunoglobulin may be polyclonal or monoclonal,
preferably monoclonal.
As noted, one skilled in the art will appreciate
that the invention also encompasses the use of antigen
recognizing immunoglobulin fragments. Such
immunoglobulin fragments may include, for example, the
Fab', F(ab')2, Fv or Fab fragments, or other antigen
recognizing immunoglobulin fragments. Such
immunoglobulin fragments can be prepared, for example, by
proteolytic enzyme digestion, for example, by pepsin or
papain digestion, reductive alkylation, or recombinant
techniques. The materials and methods for preparing such
immunoglobulin fragments are well-known to those skilled
in the art. See aenerallv, Parham, J. Immunology, 131,
2895 {1983); Lamoyi et al., J. Immunoloaical Methods,
235 (1983); Parham, ice.., 53, 133 (1982); and Matthew et
al., ia., 50, 239 (1982).
The immunoglobulin can be a "chimeric antibody" as
that term is recognized in the art. Also the
immunoglobulin may be a "bifunctional" or "hybrid°
antibody, that is, an antibody which may have one arm
having a specificity for one antigenic site, such as a
tumor associated antigen while the other arm recognizes a
different target, for example, a hapten which is, or to
which is bound, an agent lethal to the antigen-bearing
tumor cell. Alternatively, the bifunctional antibody may
be one in which each arm has specificity for a different
epitope of a tumor associated antigen of the cell to be
therapeutically or biologically modified. In any case,
the hybrid antibodies have a dual specificity, preferably
with one or more binding sites specific for the hapten of
-24-
WO 9'1~Z3243 CA 02239183 2005-02-22 p~NSg~p513
choice or more or more binding sites specific for a
target antigen, for example, an antigen associated with a
tumor, an infectious organism, or other disease state.
Biological bifunctional antibodies are described,
for example, in European Patent Publication, EPA 0 105
360, to which those skilled in the art are referred.
Such hybrid or bifunctional antibodies may be derived, as
noted, either biologically, by cell fusion techniques, or
chemically, especially with cross-linking agents or
disulfide bridge-forming reagents, and may be comprised
of whole antibodies and/or fragments thereof. Methods
for obtaining such hybrid antibodies are disclosed, for
example, in PCT Application W083/03679, published
October 27, 1983, and published European Application
EPA 0 217 577, published April 8, 1987.
Particularly preferred
bifunctional antibodies are those biologically prepared
from a "polydoma" or "quadroma" or which are
synthetically prepared with cross-linking agents such as
bis-(maleimido)-methyl ether ("B1~"), or with other.
cross-linking agents familiar to those skilled in the
art.
In addition the immunoglobulin may be a single chain
antibody ("SCA"). These may consist of single chain Fv
fragments ("scFv") in which the variable light ("V~,") and
variable heavy ("Vs") domains are linked by a peptide
bridge or by disulfide bonds. Also, the immunoglobulin
may consist of single VH domains (dAbs) which possess
antigen-binding activity. See, e-,cr., G. Winter and C.
Milstein, Nature, 349, 295 (1991); R. Glockshuber ~ ~,
Biochp~istrv ~., 1362 (1990); and E. S. Ward et al.,
at a ~,4 ,. 544 ( 1989 ) .
Especially preferred for use in the present
invention are chimeric monoclonal antibodies, preferably
those chimeric antibodies having specificity toward a
tumor associated antigen. As used herein, the term
-25-
CA 02239183 1998-06-O1
WO 97/23243 PCT/US96/20513
"chimeric antibody" refers to a monoclonal antibody
comprising a variable region, ie., binding region,from
one source or species and at least a portion of a
constant region derived from a difference source of
species, usually prepared by recombinant DNAtechniques. .
Chimeric antibodies comprising a murine variable region
and a human constant region are especially preferred in -
certain applications of the invention, particularly human
therapy, because such antibodies are readily prepared and
may be less immunogenic than purely murine monoclonal
antibodies. Such murine/human chimeric antibodies are
the product of expressed immunoglobulin genes comprising
DNA segments encoding murine immungobulin constant
regions. Other forms of chimeric antibodies encompassed
by the invention are those in which the class or subclass
has been modified or changed from that of the original
antibody. Such "chimeric" antibodies are also referred
to as "class-switched antibodies". Methods for producing
chimeric antibodies involve conventional recombinant DNA
and gene transfection techniques now well known in the
art. ee, e-a., Morrison, S. L., et al., Proc. ~lat'1
Aced. Sci, 81 6851 (1984).
Encompassed by the term "chimeric antibody" is the
concept of "humanized antibody", that is those antibodies
in which the framework or "complementarity determining
regions ("CDR") have been modified to comprise the CDR of
an immunoglobulin of different specificitry as compared
to that of the parent immunoglobulin. In a preferred
embodiment, a murine CDR is grafted into the framework
region of a human antibody to prepare the "humanized
antibody". She, ea., L. Riechmann et ., Nature 332,
323 (1988); M. S. Neuberger et al., Nature 3~, 268
(1985). Particularly preferred CDR's correspond to those
representing sequences recognizing the antigens noted
above for the chimeric and bifunctional antibodies. The
reader is referred to the teaching of EPA 0 239 400
_2~_
WO 97123243 CA 02239183 2005-02-22 p~~~s96/205I3
(published September 30, 1987)
for its teaching of CDR modified antibodies.
One skilled in the art will recognize that a
bifunctional-chimeric antibody can be prepared which
would have the benefits of lower immunogenicity of the
chimeric or humanized antibody, as well as the
flexibility, especially for therapeutic treatment, of the
bifunctional antibodies described above. Such
bifunctional-chimeric antibodies can be synthesized, for
instance, by chemical synthesis using cross-linking
agents andlor recombinant methods of the type described
above. In any event, the present invention should not be
construed as limited in scope by any particular method of
production of an antibody whether bifunctional, chimeric,
bifunctional-chimeric, humanized, or an antigen
recognizing fragment or derivative thereof.
In addition, the invention encompasses within its
scope immunoglobulins (as defined above) or
immunoglobulin fragments to which are fused active
proteins, for example, an enzyme of the type disclosed in
Neuberger, etet al., PCT appiicaation, W086I01533,
published March 13, 1986.
As noted, "bifunctional", "fused". "chimeric"
(including humanized), and "bifunctional-chimeric"
(including humanized) antibody constructions also
include, within their individual contexts constructions
comprising antigen recognizing fragments. As one skilled
in the art will recognize, such fragments could be
prepared by traditional enzymatic cleavage of intact
bifunctional, chimeric, humanized, or chimeric-
bifunctional antibodies. If, however, intact antibodies
are not susceptible to such cleavage, because of the
nature of the construction involved, the noted
constructions can be prepared with immunoglobulin
fragments used as the starting materials; or, if
-27-
W097ts3243 CA 02239183 2005-02-22 p~~Sg~O$13
recombinant techniques are used, the DNA sequences,
themselves, can be tailored to encode the desired
"fragment" which, when expressed, can be combined in vivo
or 3n vitro, by chemical or biological means, to prepare
the final desired intact i~arnunoglobulin "fragment". It
is in this context, therefore, that the term "fragment"
is used.
Furthermore, as noted above, the immunoglobulin
(antibody), or fragment thereof, used in the present
invention may be polyclonal or monoclonal in nature.
Monoclonal antibodies are the preferred immunoglobulins,
however. The preparation of such polyclonal or
monoclonal antibodies now is well known to those skilled
in the art who, of course, are fully capable of producing
useful immunoglobulins which can be used in the
invention. ee, e~c~., G. Kohler and C. Milstein, Nature
256, 495 (1975). In addition, hybridomas andlor
monoclonal antibodies which are produced by such
hybridomas and which are useful in the practice of the
present invention are publicly available from sources
such as the American Type Culture Collection ("AT~C"?
12301 Parklawn Drive, Rockville, Maryland 20852 or,
comanercially, for example, from Boehringer-Mannheim
Biochemicals, P.O. Box 50816, Indianapolis, Indiana
X6250.
Particularly preferred monoclonal antibodies for use
in the present invention are those which recognize tumor
associated antigens. Such monoclonal antibodies, a=e not
to be so limited, however, and may include; for example,
the following
..2g_
CA 02239183 1998-06-O1
WO 97/Z3243 PCT/US96/205I3
Antigen Site Monoclonal
$~ 2~ RP Fir np~p
Lung Tumors KS1/4 N. M. Varki etetan~Ar
al., f
Res. 44:681,
1984.
534,F8;604A9 F. Cuttitta stet
al., in: G.
L.
Wright (ed) ~Sonoclonal
An 'bp ; a n , NTarCel
-n r
Dekker, Inc., 161,
NY., p.
1984.
Squamous Lung Gl, LuCa2, LuCa3,Kyoizumi et al-,r Re
n .,
LuCa4 45:3274, 1985.
Small Cell Lung TFS-2 Okabe , Ca_n_cerRes.,
Cancer 45:1930, 1985.
Colon Cancer 11.285.14 G. Rowland stet n~ar
al., C'a
14.95.55 Imnninol. I mo 19:1
h r,
,
1985.
NS-3a-22,NS-10
NS-19-9,NS-33a Z. Steplewski Carar
et al.,
NS-52a,17-1A .~, 41:2723,
1982.
Carcinoembryonic MoAb 35 or ZCE025Acolla, R. S. Proc.
stet al.,
Nat. Acad. Sci.,
(USA),
77:563, 2980.
Melanoma 9.2.27 T. F_ Bumol and Reiseld,
R. A.
Proc. Natl. Acad., (USA)
S ~
,
79:1245, 1982.
-29-
CA 02239183 1998-06-O1
WO 97/23243 PCT/C1S96J20513
p97 96.5 K. E. Hellstrom etet al.,
Monoclonal 1-~ntibod-es and
C'anrPr~ ~pC. Cit. p. 31.
Antigen T65 T101 Boehringer-Maiuzheim
P.O. Box 50816
Indianapolis, IN 46250
Ferritin Antiferrin Boehringer-Mannheim
P.O. Box 50816
Indianapolis, IN 46250
R24 W. G. Dippold etet al., Proc.
Natl. Acad. Sci. (USAy,
77:6114, 1980.
Neuroblastoma P1 153/3 R. H. Kennet and F. Gilbert,
SCi_anr_rg, 203:1120, 1979.
M~ 1 J. T. Kemshead in Monoclonal
tib
A
di
d C
l
n
o
es an
ancer,
oc.
Clt. p. 49.
UJ13A
Goldman et al., Pediatrics,
105:252, 1984.
Glioma BF7, GE2, CG12 N. de Tribolet stet al., in
loc. cit. p. 81.
-30-
CA 02239183 1998-06-O1
WO 97/23243 PCT/US96/20513
Ganglioside L6 I. Hellstrom stet al., Proc.
Natl. Acad. Sci. (USA),
83:7059 (1986); U.S. Patent
Nos. 4.906,562, issued
March 6,.1990 and 4,935,495,
issued June 19, 1990.
Chimeric L6
U.S. Serial No. 07/923,244,
filed October 27, 1986,
equivalent to PCT Patent
Publication, WO 88/03145,
published May 5, 1988.
Lewis Y BR64 U.S. Serial No. 07/2$9,635,
filed December 22, 1988,
and
U.S. Serial No. 07/443,696,
filed November 29, 1989,
equivalent to European
Patent
Publication, EP A 0
375 562,
published June 27, 1990.
fucosylated BR96, Chimeric U_S. Serial No. 07/374,947,
Lewis Y BR96 filed June 30, 2989,
and U.S.
Serial No. 07/544,246,
filed
June 26, 1990, equivalent
to
PCT Patent Publication,
WO
91/00295, published
January 10, 1991.
Breast CancerB6.2, B72.3 D. Colcher stet al.,
in
M
l
l A
ib
di
onoc
ona
nt
o
es and
cer, Ioc. cit. p. 121.
Osteogenic 791T/48, M. J. Embleton, , p.
181.
Sarcoma 791T/36
-31 -
W097J23243 CA 02239183 2005-02-22 p~~gg~pgl3
Leukemia CALL 2 C. T. Teng et aJ.., Lancet,
1:01, 1982.
anti-idiotype
R.A. Miller et al., N. Engl.
J. Med., 306:517, 1982.
ovarian Cancer OC I25 R. C. Bast , J. C1
Imrest. ,, 68:1331.. 1981.
Prostrate Cancer D83.21, P6.2, Turp- J. J. Starling stet al., in
27 lN~oclon~al Antil~~s and
Cancer, loc. cit, p. 253.
Renal Cancer A6H, D5D P. H. Large stet al., Suraerv,
98:143, 1985.
In a preferred embodiment, the ligand containing
conjugate is derived from chimeric antibody BR96,
"ChiBR96", disclosed in U.S. Serial No. 07/544,246, filed
June 26, 1990, and 'which is equivalent to PCT Published
Application. WO 91100295, published January 10, 1991.
ChiBR96 is an internalizing murine/human
chimeric antibody and is reactive, ad noted, with the
fucosylated Lewis Y antigen expressed by human carcinoma
cells such as those derived from breast, lung, colon, and
ovarian carcinomas. Modified and/or humanized BR96
antibody can also be used in the present invention;
exa~les of such anitbodies are disclosed in U.S. Serial
No. 08!285,936, filed August 4, 1994, and U.S. Serial No.
08/487,860, filed June 7, 2995; the disclosures of which
are incorporated herein by reference. The hybridoma
expressing chimeric BR96 and identified as ChiBR96 was
deposited on May 23, 1990, under the terms of the
Budapest Treaty, with the American Type Culture
Collection ("ATCC"), 12301 Parklawn Drive, Rockville,
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CA 02239183 1998-06-O1
WO 97/23243 fCT/US96/20513
Maryland 20852. Samples of this hybridoma are available
under the accession number ATCC 10460. ChiBR96 is
derived, in part, from its source parent, BR96. The
hybridoma expressing BR96 was deposited, on February 21,
- 5 1989, at the ATCC, under the terms of the Budapest Treaty
arid is available under the accession number HB 10036.
The desired hybridoma is cultured and the resulting
antibodies are isolated from the cell culture supernatant
using standard techniques now well known in the art.
See, ea., "Monoclonal Hybridoma Antibodies: Techniques
and Applications", Hurell (ed.) (CRC Press, 1982).
Thus, as used "immunoglobulin" or "antibody"
encompasses within its meaning all of the
immunoglobulin/antibody forms or constructions noted
above.
The conjugates of the invention demonstrate improved
activity relative to linear conjugates. The present
invention also encompasses pharmaceutical compositions,
combinations and methods for treating diseases such as
cancers and other tumors, non-cytocidal viral or other
pathogenic infections, and auto-immune diseases. More
particularly, the invention includes methods for treating
disease in mammals wherein a pharmaceutically effective
amount of at least one conjugate of the invention is
administered in a pharmaceutically acceptable manner to
the host mammal, preferably humans.
Alternative embodiments of the methods of the
invention include the administration, either
simultaneously or sequentially, of a number of different
conjugates, i.e., bearing different drugs or different
targeting ligands, for use in methods of combination
chemotherapy. For example, an embodiment of this
invention may involve the use of a number of conjugates
wherein the specificity of the antibody component of the
conjugate varies, i.e., a number of conjugates are used,
each one having an antibody that binds specifically to a
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CA 02239183 1998-06-O1
WO 97/23243 PCT/US96/20513
different antigen or to different sites or epitopes on
the same antigen or to different sites or epitopes on the
same antigen present on the cell population of interest.
The drug component of these conjugates may be the same or
may vary. For example, this embodiment may be especially
useful in the treatment of certain tumors where the
amounts of the various antigens on the surface of a tumor
is unknown or the tumor cell population is heterogeneous
in antigen expression and one wants to be certain that a
sufficient amount of drug is targeted to all of the tumor
cells at the tumor site. The use of a number of
conjugates bearing different antigenic or epitope
specificities for the tumor increases the likelihood of
obtaining sufficient drug at the tumor site.
Additionally, this embodiment is important for achieving
a high degree of specificity for the tumor because the
likelihood that normal tissue will possess all of the
same tumor-associated antigens is small (see,
Immunol., 127(1), pp. 157-60 (1981)).
Alternatively, a number of different conjugates can
be used, wherein only to drug component of the conjugate
varies. For example, a particular antibody can be
linked to two or more doxorubicins to form one conjugate
arid can be linked to two or more daunomycins to form a
second conjugate. Both conjugates can then be
administered to a host to be treated and will localize,
due to the antibody specificity, at the site of the
selected cell population sought to be eliminated. Both
drugs will then be released at that site. This
embodiment may be important where there is some
uncertainty as to the drug resistance of a particular
cell population such as a tumor because this method
allows the release of a number of different drugs at the
site of or within the target cells. An additional
embodiment includes the conjugation of more than one drug
to a particular antibody to form a conjugate bearing a
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CA 02239183 1998-06-O1
WO 97/23243 PCT/CTS96/20513
variety of different drugs along its surface - all linked
to the antibody via acylhydrazone bonds. Administration
of the conjugate of this embodiment results in the
release of a number of different drugs at the site of or
- 5 within the target cells. Furthermore, a combination of
drug-targeting ligand conjugates can be used wherein the
drug can be targeted to a cell population carrying a
specific antigen as well as a receptor for a specific
ligand on its surface. Again, one type of drug or number
of different drugs can be used in this combination
therapy.
The conjugates of the invention can be administered
in the form of pharmaceutical compositions using
conventional modes of administration including, but not
limited to, intravenous, intraperitoneal, oral,
intralymphatic, or administration directly into the site
of a selected cell population such as a tumor.
Intravenous administration is preferred. In the case of
the conjugates, for in vivo treatment, it may be useful
to use conjugates comprising antibody fragments such as
Fab or Flab")2 or chimeric or humanized antibodies.
The pharmaceutical compositions of the invention-
comprising the conjugates - may be in a variety of dosage
forms which include, but are not limited to, solid, semi-
solid and liquid dosage forms such as tablets, pills,
powders, liquid solutions or suspensions, suppositories,
polymeric microcapsules or microvesicles, liposomes, and
injectable or infusible solutions. The preferred form
depends upon the mode of administration and the
therapeutic application.
The pharmaceutical compositions may also include
conventional pharmaceutically carriers known in the art
such as serum proteins such as human serum albumin,
buffersubstances such as phosphates, water or salts or
electrolytes.
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CA 02239183 1998-06-O1
WO 97/23243 PCT/LJ596/20513
The most effective mode of administration and dosage
regimen for the conjugates of this invention depends upon
the severity and course of the disease, the patient's
health and response to treatment and the judgment of the
treating physician. Accordingly, the dosages of the
conjugates and any accompanying compounds should be
titrated to the individual patient. Nevertheless, an
effective dose of the conjugates may be in the range of
from about 1 to about 100 mg/m2 drug or from about 500-
5000 mg/m2 antibody. An effective dose of the conjugates
containing ligands other than antibodies may be in the
range of from about 1 to about 100 mg/m2 drug or from
about 1 to about 100 mg/m2 ligand.
Preoarat3.on of the Molecules of the Invention
The carbon-branched linker is derived from a bis-
carboxylic acid, which also contains a protected amine
functionality. Through a multi-step process, the
carboxylic acid groups are converted to terminal
hydrazide groups, whereby the amino group is elaborated
to yield a terminal thiol acceptor. Condensation of the
multiple hydrazide with a drug containing an aldehyde or
ketone groups yields a multiple acylhydrazone of the
drug.
The nitrogen-branched linker is derived from an
oligoamine, differentially protected in such a way that
all but one amino group are elaborated to yield terminal
N, N-dialkanoylhydrazide groups. The remaining amino
group is elaborated to yield a terminal thiol acceptor.
Condensation of the multiple hydrazides with-an drug
containing an aldehyde or ketone group yields a multiple
acylhydrazone of the drug. -
Conjugation of the linker to the targeting ligand is
accomplished by the reaction of free thiol groups of the
ligand, generated under controlled atmospheric
-36-
CA 02239183 1998-06-O1
WO 97/23243 PCT/US96/205I3
conditions, with the terminal thiol acceptor of the
linker.
Exemplary reaction schemes for preparation of the
compounds of the invention are illustrated below. The
compound numbers are cross referenced in the Example
section hereof.
EXEMPLARY BIS- AND TETRA-DOX 1~9YDRAZONES
O NHN=DOX n Configuration
O ~ 2 L
NHN=DOX ~ 3 L and D
O H O c_ 5 L
H
NHN=DOX n Configuration
p ~ 2 L
O
NHN=DOX ~ 3 L
O n H O O g 5 L
O NHN=DOX
O NHN=DOX n Configuration
O H O $ 2 all L
O
H O ~ 3 all L
N 5 all L
O H O NHN=DOX
O NHN=DOX
j$
-37-
CA 02239183 1998-06-O1
WO 97/23243 PCT/US96/205I3
SCHEME i. SYNTHESIS OF ~
O NHNH-BOC O NHNH-BOC
1. DCC/NHS H2
Z-C,.lu --' --
2. BOC-NHNH2 NHNH-BOC 10°!° Pd-C NHNH-BOC .
Z-NH H2
O O
4
p ~ O NHNH-BOC
N
O ~ x / O O TFA
0 o N~ NHNH-BOC
X= o~o~ or o~N~ O l~Jn H O
0
O NHNH2 O NHN=DOX
/ DOX O
R / O
N~ NHNH2 N NHN=DOX
O ~l''''JJ H O O ~ H
~ 2 TFA O
Z
-38-
CA 02239183 1998-06-O1
WO 97/23243 PCT/US96/20513
SCHEME Il. SYNTHESES OF ~
1. DCC/NHS - Hz O
Z-(i-AIa ~- - ~~ 'I
2. BOGNHNHp Z H NHNH-BOC ~Q°~ Pd-C HzN _ NHNH-BOC
$ $
H H
N~NHNH-BOC O N~NHNH-BOC
7. DCCMHS '' ~O HH
Z-GIU H H
N NHNH-BOC 1a°~ Pd-C N NHNH-BOC
Z-NH ~ HzN
O O O O
O H
O O N' ~ 'NHNH-BOC
~(N
~(~ x O O TFA
O
o / N~ N' ~ 'NHNH-BOC
~ ~N
X= o o ~ or o- N' , O H
O O
0
12
H H
N~ NHNHz N~ NHN=DOX
H 'OI DOX ~ O H flO
N N~ NHNHz ~ N N' ~ ' NHN=DOX
O O H O '' jO( O n H O ~' ~O
~ 2'i'FA
13 $
-39-
CA 02239183 1998-06-O1
WO 97/23243 PCT/US96I20513
SCHEME III. SYNTHESIS OF 18
O NHNH-BOC O NHNH-BOC
O N NHNH-BOC N NHNH-BOC
1. DCC/NHS H p Hz _ H O
Z-Glu
2. ~ Z-NH N O 1 D°/ Pd-C NH N O
O ~ NHNH-BOC 2 O ~ NHNH-BOC
L O NHNH-BOC ~ O NHNH-BOC
O NHNH-BOC
00
O N NHNH-BOC
O n X O H O
p O .L~
o N H
X= o o~ or o- H N NHNH-BOC
O
0
O NHNH-BOC
O NNNHz O NHN=DOX
N NHNHZ O N NHN=DOX
O O H O O O H O
TFA ~ H O DCX ~ H O
O N n H O N NHNHz O N n H O N NHN=DOX
O NHNH2 O NHN=DOX
1$
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SCHEME tV. SYNTHESIS OF ~6
CUNHNH-BUC
NH-BOC NHa NvCONHNH~BOC
Z-NH~ ~ Z-NH~ /---~ - X TFA ---~- Z-NH~ ~---
~NH-BOC ~NH2 ~N~ CONHNH-BOC
CONHNH-BOC
~1
~ONHNH-SOC
CONHNH~BOC
N~CONHNH-BOC N~CONHNH-BOC
HpN~ N N
COOH
N~CONHNH-BOC ~~ CONHNH-BOC
~CONHNH-BOC ~ CONHNH-BOC
/CONHNH-BOC /CONHNH2
I N ~IN~CONHNH-BOC ~ ~IN~CONHNH2
~N
O O
N'~CONHNH~eOC ~~ CONHNH2
'CONHNH-BOC ~ CONHNHz
CONHN-DOX
N CONHN=DOX
N~N
O
N CONHN=DOX
CONHN~OX
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SCHEME V. SYN'~HESIS OF 3 2
Z-NH Z-NH~ ~CONHNH-BOC HaN~ ~CONHNH-BOC
~NH ' HC( -----
~CONHNH-BOC L CONHNH-BOC
2Z ?~
O
~NH~ rCONHNH-BOC ~ N~ ~--CONHNH-BOC
COOH ~ ~ N
'-CONHNH-BOC p ~ CONHNH-BOC
~N~ ~--CONHNH2 ~N~ ~-CONHN=DOX
O ~ CONHNHZ \\O ~ CONHN=DOX
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iOC
H= NHCONNHCONHNH-HOC
triphosgene ~ Z NH-~ N
BOC~NHNHZ
ELsN EtaN NHCONNHCONHNH-BOC
I
BOC
BOC O
NHZ NHCONNHCONHNH-80C NHCONNHCONHNH~BOC
H
O N
NHCO iNHCONHNH~BOC ~NHCONNHCONHNH-BOC
BOC
ate.
BOC O
O I ((//
NHCONNHCONHNN~BOC ~ JJHCONHNNCONHNH=
~N ~J~
N ~'' H\\~~~~
O ~ O \,1
NHCONNHCONHNH~BOC ~HCONHNHCONNNH,
IBOC
O
NHCONHNHCONHN=OOX
~~N
O
NHCONHNHCONHN~DOX
SCHEME Vl. SYNTHESIS OF 38
Hi
Z-NH
N~ ' x TFA
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SCHEME VI1. SYNTHESIS OF 4~C
Z-NH NH-80C
BOGNH ~ N ~ NH-BOC ~ B~ Z-NH~ ~--J '~-FA
H
NH-BOC
/CONHNH-BOC
Hz B~ NHNH-BOC N ~CONHNH-BOC
Z-NH~ Z-NH ~,~.~/~
N KHC03 N
~NHz t~ CONHNH-BOC
4t? - x TF ~A
CONHNH-BOC
O
~CONHNH-BOC II .O O ~CONHNH-BOC
H2 ~~CONHNH-BOC ~O N~CONHNH-BOC
H~~ ~ ~ NH~
Pd-C ~ COOH N
NI ~ CONHNH-BOC ~' ~ CONHNH-BOC
'CONHNH-BOC ~ 'CONHNH-BOC
~CONHNH-BOC O /CONHNHz
1. EDCI I N ,NvCONHNH-BOC P-TsOH ~ ~IN~CONHNHz
2. HOBt ~ N~---~
O O
DMF ~N'~CONHNH-BOC ~NI~CONHNHz
'CONHNH-BOC ~ 'CONHNHz
~CONHN=DOX
DOX~HCI ~ ~CONMN=DOX
N
N
O
ff ~CONHN=DOX
'CONHN=DOX
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The abbreviations in the above reaction schemes have
the following definitions: Z is carbobenzoxy, DCC is
dicyclohexylcarbodiimide, BOC is t-butoxy carbonyl, TFA
is trifluoroacetic acid, and DOx is doxorubicin_
The following examples are to illustrate the
invention but should not be interpreted as a limitation
_ thereon.
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~xamule 1
Z-Glutamyldi(Boa)hydrazide (Compound no. 4)
Z-Glutamic acid (42.20 g, 150 mmole) and N-hydroxy
succinimide (34.53 g, 300 mmole} were dissolved in 150 ml
DMF at 0°C under dry N2. A 0.5M solution of
dicyclohexylcarbodiimide in methylene chloride (600 ml,
300 mmole) was added dropwise over a 1 hour period with
stirring. The reaction was stored at 4°C in the
refrigerator for 18 hr. Dicyclohexylurea precipitate
(65.48 g, 98~} was filtered, and the filltrate was added
directly to solid t-butylcarbazate (39.65 g, 300 mmole).
After stirring at room temperature for 48 hr., the
reaction was rotary evaporated to an oil, which was
redissolved in 300 ml ethyl acetate/200 ml ether. The
organic layer was extracted three times with 200 ml 10~
citric acid, 3 times with 200 ml saturated aqueous sodium
bicarbonate, and once with 100 ml brine. The organic
layer was dried over sodium sulfate and rotary evaporated
to a foam. Flash chromatography was carried out on silica
gel (4 in. X 19 in.) with ethyl acetate-hexane 2:1, 12L.
Pure fractions containing product (4} were pooled and
concentrated to a foam by rotary evaporation to yield,
after drying under high vacuum, 55.24 g (72~).
1H-NMR (CDC13}: b 1.44 and 1.47 (2s, 18H}, 1.9-2.4 (bm,
4H), 4.32 (bm, 1H), 5.06 (dd, 2H), 5.55 (d, 1H), 6.5 (bd,
2H), 7.31 (bm, 5H), 9.6 (s, 1H), and 9.9 (s, 1H).
TLC: Rf 0.64, CH2C12/MeOH (9:1).
Mass Spec.: FAB 510 (M+H+} 532 (M+Na+), 548.1 (M+K+)
Elemental Analysis for C23H35N508: Theoretical C, 54.21;
H, 6.92; N, 13.74. Found C, 53.96; H, 6.91; N, 13.41.
2
Z-(D)-Glutamyldi(8oc)hydrazide
(Compound no. D-4)
Z-(D)-Glutamic acid (42.20 g, 150 mmole) and N-
hydroxy succinimide (34.53 g, 300 mmole) were dissolved
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in 150 ml DMF at 0°C under dry N2. A 0.5M_ solution of
dicyclohexylcarbodiimide in methylene chloride (600 ml,
300 mmole) was added dropwise over a 1 hour period with
stirring. The reaction was stored at 4°C in the
refrigerator for 18 hr~. Dicyclohexylurea precipitate
(64.97 g, 97~) was filtered, and the filltrate was added
directly to solid t-butylcarbazate (39.65 g, 300 mmole).
After stirring at room temperature for 48 hr., the
reaction was rotary evaporated to an oil, which was
redissolved in 300 ml ethyl acetateJ200 ml ether. The
organic layer was extracted three times with 200 ml 10~
citric acid, 3 times with 200 ml saturated aqueous sodium
bicarbonate, and once with 100 ml brine. The organic
layer was dried over sodium sulfate and rotary evaporated
to a foam. Flash chromatography was carried out on silica
gel (4 in. X 18 in.) with the following gradient: (1)
CH2C12, 2L, (2) CH2C12-methanol 25:1, 4L, and (3) CH2C12-
methanol 9:1, 6L. Pure fractions containing product (,~),
which eluted in CH2C12-methanol 9:1, were pooled and
concentrated to a foam by rotary evaporation to yield,
after drying under high vacuum, 59.11 g (77~).
1H-131~t (CDC13) : 8 1.44 and 1.47 (2s, 18H) , 1.9-2:4 (bin,
4H) , 4.32 (lxn, 1H) , 5 . 06 (dd, 2H) , 5. 5? (d, 1H) , 6 .6 (m,
2H), 7.31 (bm, 5H), 9.60 (s, 1H), and 9.87 (s, 1H).
TLC: Rf 0.64, CH2C12JMe0H (9:1).
Mass Spec.: FAB 532 (M+Na+), 549 (M+K+)
Elemental Analysis for C23H35N50g: Theoretical C, 54.21;
H, 6.92; N, 13.74. Found C, 53.99;H, 6.92; N, 13.50.
3 0 $x~ rile 3
Glutamyldi(Boc)hydrazide (Compouad ao. 5)
Z-Glutamyldi(Boc)hydrazide (4_) (19.59 g, 38.44
rmnole) was hydrogenated along with 2g 10~ Pd-C in 200 ml
MeOH at 50 psi for 3 hr. The reaction was filtered
through Celite*and rotary evaporated. The resulting foam
was dried under high vacuum to yield 5_ (14.448, 100$).
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WO 97l232d3 CA 02239183 2005-02-22 PCT/US96IZOSI3
1H-NMR (d4-Methanol): b 1.42 and 1.45 (2s, 18H), 1.9 (bm,
2H), 2.35 (t, 2H), 3.34 (t, 1H).
TLC: Rf 0.34, CH2C12/MeOH (9:1).
Mass Spec.: DCI 376 (M+H)+.
Elemental Analysis for C15H2gN506 ~ 0.5 H20: Theoretical
C, 46.87; H, ?.87; N, 18.22. Found C, 46.96; H, 7.74; N,
18.02.
Example 4
(D)-Glutamyldi(Boc)hydrazide
(Compound no. D-5_)
Z-(D)-Glutamyldi(Boc)hydrazide (D-4_) (23.05 g, 45.2
mmole) was hydrogenated along with 2g 10% Pd-C in 200 ml
MeOH at 50 psi.for 4 hr. After filtration through Celite
and rotary evaporation, a foam was obtained. Flash
chromatography on silica gel (2 in. X 20 in.) was carried
out with the following gradient: (1) CH2C12-methanol
25:1, 600 ml, (2) CH2C12-methanol 9:1, 6L, and (3)
CH2C12-methanol 8:2, 4L. Pure fractions were pooled and
rotary evaporated. Drying under high vacuum,yielded D-5
(13.51 g, 80%).
1H-NMR (d4-Methanol): b 1.46 and 1.47 (2s, 18H), 1.94
(bm, 2H), 2.33 (t, 2H), 3.34 (t,lH).
TLC: Rf 0.34, CH2C12JMe0H (9:1).
Mass Spec.: FAB 376 (M+H)+, 398 (M+Na)+, 414 (M+K)+.
Elemental Analysis for C15H29N506 ~ 0.5 H20: Theoretical
C, 46.87; H, 7~87: N, 18.22. Found C, 46.85; H, 7.63; N,
17.9$.
Examale 5
Malei~nidopropionylglutamylditBoc)hydrazide
(Compound no. 6a)
Malei.mi.dopropionic acid (636 mg, 3.76 mmole) and N-
hydroxysuccinimide (476 mg, 4.14 mmole) were dissolved in
10 ml DMF at 0°C. A 0.5~I solution of DCC in CH2C12 (7.6
ml, 3.8 mmoie) was added, and the reaction allowed to
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stand for 20 hr. at 4°C. After filtration of the DCU
precipitate, the filtrate was added to 5 (1.27g, 3.38
mmole) and stirred at room temperature for 2.5 days.
Solvents were partially removed by rotary evaporation.
. 5 The oil was dissolved in 100 ml ethyl acetate, then
extracted three times with 100 m1 10~ citric acid, three
times with 100 ml saturated aqueous sodium bicarbonate,
and three times with 100 ml H20. The organic layer was
dried over sodium sulfate and rotary evaporated to a
foam. This was purified by flash chromatography on silica
gel (2 in. X 12 in.) with CH2C12-acetic acid-methanol
93:2:5. Pure fractions were pooled, rotary evaporated,
and dried under high vacuum to yield &a as a foam (1.22
g, 69~) .
1H-NMR (d4-Methanol): 8 1.46 (s, 18H), 2.01 (m), 2H},
2.33 (t, 2H), 2.51 (t, 2H), 3.76 (t, 2H), 4.34 (t, 1H),
6.80 (s, 2H).
TLC: Rf 0.54, CH2C12-acetic acid-methanol 90:2:8.
Mass Spec.: FAB 549.4 (M+Na}+, 565.3 (M+K}+
Elemental Analysis for C22H34N609 ~ 2HOAc: Theoretical C,
48.29; H, 6.55; N, 13.00. Found C, 48.15; H, 6.48; N,
13.28.
Examflle 6
Maleimidobutyrylglutamyldi(8oc)hydrazide
(Compouad no. 6b)
Maleimidobutyric acid (1.9 g, 20.3 mmole) and N-
hydroxy succinimide (2.7 g, 23.5 mmole) were dissolved in
25 ml DMF at 0°C. A 0.5~! solution of DCC in CH2C12 (45
ml, 22.5 mmole) was added, and the reaction allowed to
stand for 16 hr. at 4°C_ After filtration of the DCU
precipitate, the filtrate was added to 5 (7.7 g, 20.5
mmole) and the reaction stored at 4°C for four days.
Solvents were removed by rotary evaporation. The oil was
dissolved in 100 ml ethyl acetate, then extracted three
times with 100 ml 10~ citric acid, three times with 100
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ml saturated aqueous sodium bicarbonate, and three times
with 100 ml H20. The organic layer was dried over sodium
sulfate and rotary evaporated to a foam. This was
purified through a plug of silica gel with CH2C12-acetic
acid-methanol 93:2:5, rotary evaporated, and dried under
high vacuum to yield 6~h as a foam (3.50 g, 63~).
1H-NMR (d4-Methanol): b 1.36 and 1.37 (2s, 18H), 1.77 (p,
2H), 2.00 (bm, 2H), 2.14 (t, 2H), 2.26 (t, 2H), 3.43 (t,
2H) , 4.26 (t, 1H) , 6.71 (s, 2H) .
TLC: Rf 0.58, CH2Cl2-acetic acid-methanol 90:2:8.
Mass Spec.: 541 (M+H)+, 563 (M+Na)+, 579 (M+K)+
Elemental Analysis for C23H36N6Og ~0.75 H20: Theoretical
C, 49.86; H, 6.82; N, 15.17. Found C, 50.21; H, 6.72; N,
14.79.
Examr~le 7
Maleimidobutyryl-(D)-glutamyld3.(8oc)hydrazide
( Compound ao . D-~~)
Maleimidobutyric acid (1.832 g, 10.0 mmole) was
dissolved with N-Methylmorpholine (1.21 m1, 11.0 mmole}
in 60 ml dry THF under N2 at O°C. Isobutylchloroformate
(1.30 ml, 10.0 mmole) was added dropwise, followed 10
minutes later by the addition of (D)-
Glutamyldi(Boc)hydrazide (D-~) (3.754 g, 10.0 mmole).
Stirring was continued for 1 hour at O°C. The reaction
was rotary evaporated to a foam, which was then dissolved
in 150 ml EtOAc. The organic-layer was washed two times
with 100 ml 10~ citric acid and two times with 100 ml
saturated NaHC03. The organic layer was concentrated to a
foam, which was purified by flash chromatography on
silica gel (2 in. X 11 in.) with CH2C12-acetic acid-
methanol 95:2:3, 2L followed by CH2C12-acetic acid-
methanol 93:2:5, 1L. Pure fractions were pooled and
rotary evaporated to a foam. Drying under high vacuum
yielded 3 (3.25 g, 60~) .
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1H-NMR (d4-Methanol): 8 1_45 and 1.46 (2s, 18H), 1.86 (m,
2H), 2.09 (bm, 2H), 2.24 (t, 2H), 2.35 (t, 2H), 3.52 (t,
2H), 4.35 (t, 1H), 6.81 (s, 2H).
TLC: Rf 0.51, CH2C12-acetic acid-methanol 90:5:5.
Mass Spec.: 563 (M+Na)+, 579 (M+K)+
Elemental Analysis for C23H36N6~9 ~ 0.75 H20: Theoretical
C, 49.86; H, 6.82; N, 15.17. Found C, 50.25; H, 6.65; N,
14.80.
~amx~le 8
Maleimidocaproylglutamyldi(8oc)hydrazide
(Compound ao. 6c)
Maleimidocaproic acid (4.22 g, 20 mmole) and N-
hydroxysuccinimide (2.53 g, 22 mmole) were dissolved in
25 ml DMF at 0°C. A 0.5M solution of DCC in CH2C12 (40
ml, 20 mmole) was added, and the reaction allowed to
stand for 20 hr. at 4°C. After filtration of the DCU
precipitate, the filtrate was added to 5 (7.88 g, 21
mmole) and the reaction stirred at room temperature for 6
hr. Solvents were removed by rotary evaporation. The oil
was dissolved in 100 ml ethyl acetate, then extracted
three times with 100 ml 10~ citric acid, three times with
100 m1 saturated aqueous sodium bicarbonate, and three
times with 100 ml H20_ The organic layer was dried over
sodium sulfate and rotary evaporated to a foam. This was
purified by flash chromatography on silica gel (2 in. X
10 in.) with 4L CH2C12-acetic acid-methanol 97:1:2. Pure
fractions were pooled, rotary evaporated, and dried under
high vacuum to yield 6~ as a foam (6.40 g, 56~).
1H-NMR (d4-Methanol): 8 1.2 (p, 2H), 1.40 (s, 18H), 1.5
(m, 4H), 2.0 (bm, 2H), 2.14 (t, 2H), 2.28 (t, 2H), 3.41
(t, 2H), 4.29 (t, 1H), 6.72 (s, 2H).
TLC: Rf 0.30, CH2C12-acetic acid-methanol 93:2:5.
Mass Spec.: FAB 569 (M+H)+, 591 (M+Na)+, 607 (M+K)+
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Elemental Analysis for C25H4pN60g ~ 0.5 H20: Theoretical
C, 51.98; H, 7.15; N, 14.55. Found C, 51.79; H, 6.96; N,
14.39.
Example 9
lMaleimidopropionylglutamyld3.hydrazide
ditrifluoroacetate (Compound ao. 7~)
Maleimidopropionylglutamyldi(Boc)hydrazide (6~,)
(1.50 g, 2.85 mmole} was stirred in 15 ml
CH2C12/trifluoroacetic acid (1:1) under N2 for 1.5 hr.
Solvents were removed by rotary evaporation. Ether was
added and co-evaporated three times, then the resulting
solid was triturated with ether. The solid was filtered
and dried under high vacuum to yield 7a (1.6 g, 1000
~-H-NMR (d4-Meth nol): 8 1.99 and 2.16 (2m, 2H), 2.41 (t,
2H}, 2.53 (t, 2H), 3.80 (t, 2H}, 4.38 (dd, 1H), 6.81 (s,
2H) .
Mass Spec.: FAB 349.2 (M+Na}+, 365.1 (M+K)+
Elemental Analysis for C12H18N605 ~ 2.8 TFA: $
Theoretical C, 32.75; H, 3.25; N, 13.02. Found C, 33.04;
H, 3.37; N, 12.72.
Examule 10
Maleim3.dobutyrylglutamyld3.hydrazide
ditrifluoroacetate (Compound ao. 7 b)
Maleimidobutyrylglutamyldi(Boc)hydrazide (6b) (3.50
g, 6.47 mmole) was stirred in 40 ml
CH2C12/trifluoroacetic acid (1:1) under N2 for 2 hr.
Solvents were removed by rotary evaporation. Ether was
added and co-evaporated three times, then the resulting
solid was triturated with ether. The solid was filtered
and dried under high vacuum to yield 7b (3.8 g, 1000
1H-NMR (d4-Methanol): 8 1.87 (p, 2H), 2.0 and 2.2 (2m,
2H), 2.27 (t, 2H), 2.44 (m, 2H), 3.53 (t, 2H), 4.42 (dd,
1H}, 6.82 (s, 2H).
Mass Spec.: FAB 341 (M+H)+, 363 (M+Na)+, 379 (M+K)+
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Elemental Analysis for C13H20N605~ 3.15 TFA: Theoretical
C, 33.14; H, 3.34; N, 12.01. Found C, 33.49; H, 3.52; N,
11.64.
Exams~le 11
Maleimidobutyryl-(D)-glutamyldihydrazide
ditrifluoroacetate (Compound no. D-7 b)
Maleimidobutyryl-(D)-glutamyldi{Boc)hydrazide {D-~)
(2.06 g, 3.81 mmole) was stirred in 40 ml CH2C12 with 40
ml trifluoroacetic acid under N2 for lhr. Solvents were
removed by rotary evaporation. Ether was added and co-
evaporated three times, then the resulting solid was
triturated with ether. The solid was filtered and dried
under high vacuum to yield D-7b (2.2 g, 100$)
1H-NMR (d4-Methanol): 8 1.79 (p, 2H), 1.9 and 2.1 (2m,
2H), 2.18 (t, 2H), 2.37 (m, 2H), 3.45 (t, 2H), 4.35 (dd,
1H), 6.73 (s, 2H).
Examine ~
Maleimidocaproylglutamyldihydrazide
ditrifluoroacetate (Compound no.
Maleimidocaproylglutamyldi(Boc)hydrazide (6c) {5.96
g, 10.5 mmole) was stirred in 100 ml
CH2C12/trifluoroacetic acid (1:1) under N2 for 1 hr.
Solvents were removed by rotary evaporation. Ether was
added and co-evaporated three times, then the resulting
solid was triturated with ether. The solid was filtered
and dried under high vacuum to yield 7c (6.3 g, 1000
1H-NMR (d4-Methanol): b 1.22 (p, 2H), 1.52 (s, 4H), 1.92
and 2.09 (2m, 2H}, 2.18 (t, 2H), 2.35 {m, 2H}, 3.41 (t,
2H), 4.35 (dd, 1H), 6.72 (s, 2H).
Mass Spec.: FAB 369 (M+H)+, 391 (M+Na}+, 407 (M+K}+
Elemental Analysis for C15H24N605~ 2.5TFA: Theoretical C,
36.76; H, 4.09; N, 12.86. Found C, 36.66; H, 4.22; N,
12.72.
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~x~.~tnle 13
Dlaleimidoprop3.onylglutamyldihydrazone of
Doxorubicin
(Compound no. 2a "MP-Glu(DOX)2")
Maleimidopropionylglutamyldihydrazide
ditrifluoroacetate (7a) (600 mg, 1.07 mmole} and DOX~HC1
(1.24 g, 2.14 mmole) were dissolved in 600 ml methanol
over a period of 3 hours. The reaction was concentrated
to 100 ml by rotary evaporation, then stirred for 3 days.
The reaction was further concentrated to 12 ml and eluted
on an LH-20 column (2" X 10") with methanol.
Chromatography was repeated in the same system on mixed
fractions. The purified product was rotary evaporated to
a red film and dried under high vacuum to yield ?~ (776
mg, 50~).
1H-NMR (d4-Methanol): (selected peaks) 8 1.34 (2d, 6H),
4.07 (2s, 6H), 6.79 (s, 2H), 7.5-8.0 (m, 6H).
Mass Spec.: FAB 1375.4 (M-H)'~; Ionspray 1377.2 MH+.
Elemental Analysis for C66H72N8025 ~ 2HC1 ~ 3.0H20:
Theoretical C, 52.70; H, 5.36; N, 7.45. Found C, 52.57;
H, 5.25; N, 7.33.
ample 14
Maleimidobutyrylglutamyldihydrazone of Doxorubicin
(Compound no. 2b "MBGlu(DOX)2")
Maleimidobutyrylglutamyldihydrazide
ditrifluoroacetate (~) (1.00 g, 1.76 mmole) and DOX~HC1
(2.05 g, 3.53 mmole) were dissolved in 800 ml methanol
over a period of 3 hours. The reaction was concentrated
to 150 ml by rotary evaporation, then stirred for 1.5
days. The reaction was further concentrated to 20 ml and
eluted on an LH-20 column (2" X 12") with methanol.
Chromatography was repeated in the same system on mixed
fractions. The purified product was rotary evaporated to
a red film and dried under high vacuum to yield 2~ (2.32
g, 51~) .
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1H-NMR (d4-Methanol): {selected peaks) 8 1.33 (2d, 6H),
4.06 (2s, 6H), 6.80 (s, 2H), 7.5-8.0 (m, 6H).
Mass Spec.: FAB 1392 MH+, 1413.4 (M+Na)+, 1429 (M+K)+.
Ionspray 1392.5 (M+H)+, 1414.4 (M+Na)+
' S Elemental Analysis for C67H74N8025 ~ 2HC1 ~ 4.OH20:
Theoretical C, 52.38; H, 5.51; N, 7.29. Found C, 52.38;
' H, 5.58; N, 7.50.
Examule 1S
Maleimidobutyryl-(D)-glutamyldihydrazoae of
Doxorubicin
(Compound no. D-2 b '°M8-D-Glu(DOX)2°°)
Maleimidobutyryl-(D)-glutamyldihydrazide
ditrifluoroacetate (D-7b) (570 mg, 1.00 mmole) and
DOX~HC1 (1.34 g, 2.30 mmole) were dissolved in &00 ml
methanol over a period of 3 hours. The reaction was
concentrated to 100 ml by rotary evaporation, then
stirred for 2.5 days. The reaction was further
concentrated to 50 ml and eluted on an LH-20 column (2" X
10") with methanol. Chromatography was repeated in the
same system on mixed fractions. The purified product was
rotary evaporated to a red film and dried under high
vacuum to yield D-?h {420 mg, 30~).
1H-NMR (d4-Methanol): (selected peaks) 8 1.30 (2d, 6H),
4.07 (2s, 6H), 6.80 (s, 2H), 7.5-8.0 (m, 6H).
Mass Spec.: FAB 1392.0 MH+, 1414.9 (M+Na)+, 1429.7
( M+K ) + .
Elemental Analysis for Cg7H74N8025 ~ 2HC1 ~ 3.5H~0:
Theoretical C, 52.69; H, 5.48; N, 7.34; C1, 4.64. Found
C, 52.74; H, 5.57; N, 7.47; Cl, 5.28.
Example 16
Maleimidocaproylglutamyldihydrazone of Doxorubicin
(Compound no. 2c "MCGlu(DOX)2°)
Maleimidocaproylglutamyldihydrazide
ditrifluoroacetate {7c) (298 mg, 0.50 mmole) and DOX~HCl
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(580 mg, 1.00 mmole) were dissolved in 350 ml methanol
over a period of 3 hours. The reaction was concentrated
to 50 ml by rotary evaporation, then stirred for 3 days.
The reaction was further concentrated to 5 m1 and eluted
on an LH-20 column (2" X 20") with methanol. The purified
product was rotary evaporated to a red film and dried
under high vacuum to yield 2c {510 mg, 68~).
1H-NMR (d4-Methanol): (selected peaks) 8 1.34 (2d, 6H),
4.08 (2s, 6H), 6.76 (s, 2H), 7.5-8.0 (m, 6H).
Mass Spec.: FAB 1420 MH+, 1442.3 (M+Na)+. Ionspray 1419.6
(M+H)+.
HRMS: calculated 1419.5156; observed 1419.5191.
Elemental Analysis for C69H78N8025 ~ 2HC1 ~ 4H20:
Theoretical C, 52.98; H, 5.67; N, 7.16. Found C, 52.96;
H, 5.39; N, 7.45.
~x~~ule 17
Z-~3-Alaayl(BOC)hydr~.zide (Compound ao. 8)
Z-~3-Alanine {8.93 g, 40 mmole), t-butylcarbazate
(5.29 g, 40 mmole), and EDCI (8.00 g, 42 mmole} were
stirred in 200 ml CH2C12 for 1.5 hr. at room temperature.
The reaction was extracted three times with 200 m1 0.1 ~I
acetic acid, twice with 200 ml saturated aqueous sodium
bicarbonate, and once with 200 ml water. The organic
layer was dried over sodium sulfate, rotary evaporated,
and dried under high vacuum to yield $ as a foam, 12.42 g
(92~) .
1H-NMR (d6-DMSO): b 1.38 (s, 9H), 2.25 (t, 2H), 3.19 (q,
2H}, 4.99 (s, 2H), 7.3 (m, 6H), 8.21 (s, 1H), 9.56 {s,
1H) .
TLC: Rf 0.58, CH2Cl2/MeOH (9:1).
Mass Spec.: FAB 338 (M+H)+.
Elemental Analysis for C16H23N305: Theoretical C, 56.96;
H, 6.87; N, 12.45. Found C, 57.19; H, 7.05; N, 12.57.
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CA 02239183 2005-02-22 p~/pg9~0513
Examale l8
~i-Alanyl(BOC~hydrazide (Compouad ao. 9)
_8 (15.25 g, 45.2 mmole) was hydrogenated at 50 psi
in 200 ml methanol with 3 g 10~ Pd-C for 4 hours. The
reaction was filtered through Celite, rotary evaporated,
and dried under high vacuum to yield 9_ as a hygroscopic
foam, 9.2g (1000.
1H-NNgt (d4-Methanol): b 1.40 (s, 9H), 2.32 (t, 2H), 2.88
(t, 2H).
Mass Spec.: FAB 204.2 (M+H)+.
Elemental Analysis for C8H17N303 ~ 0.5H20: Theoretical C,
45.27; H, 8.55; N, 19.80. Found C, 45.51; H, 8.17; N,
19.49.
Exaamle 19
Z-Glutamyldi[~3-Alanyl(Eoc~hydrazidel (Compouad no.
Z-Glutamic acid (3.86 g, 13.7 mmole) and N-hydroxy
succinimide (3.17 g, 27.5 mmole) were dissolved in 80 ml
DMF at 0°C under dry N2. A 0.5~ solution of
dicyclohexylcarbodiimide in methylene chloride (55 m1,
27.5 mmole) was added and the reaction was stored at 4°C
for 24 hr. Dicyclohexylurea precipitate was filtered, and
the filltrate was added to ~ (6.00 g, 29.5 mmole). After
stirring at room temperature for 15 hr., the reaction was
rotary evaporated to an oil, which was redissolved in 150
ml ethyl acetate. The organic layer was extracted three
times with 100 ml 10~ citric acid, 3 times with 100 ml
saturated aqueous sodium bicarbonate, and three times
with 100 ml brine. The organic layer was dried over
sodium sulfate and rotary evaporated to a foam. Flash
chromatography was carried out on silica gel (2 in. X 11
in.) with 1L CHaCl2/methanol 25:1 followed by 3L
CH2C12lmethanol 9:1. Pure fractions containing product
(~Q) were pooled and concentrated to a foam by rotary
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WO 97f13243 CA 02239183 2005-02-22 p~/pS96/10513
evaporation to yield, after drying under high vacuum,
6.70 g (75~).
1H-NMR (CDC13): 8 1.42 (s, 18H), 2.03 and 2.32 (2m, 8H),
3.5 (m, 4H), 4.35 (t, 1H), 5.05 (dd, 2H), 6.22 (d,
1H),
6.49 (d, 2H), 7.30 (s, 5H), 7.42 (m,~lH), ?.58 (m,
1H).
TLC: Rf 0.40,CH2C12/MeOH 9:1.
Mass Spec.: DCI 652 (M+H)+, 674 ~(M+Na)+, 690 (M+K)+.
Elemental Analysis for C2gH45N7010: Theoretical C, 53.45;
H, 6.96; N, 15.04. Found C, 53.10; H, 6.90; N, 14.91.
Examule 20
Glutamyldi(~-Alanyl(Soc)hydrazide]
(Compound no. 1~)
Z-Glutamyldi[~i-Alanyl(Boc)hydrazide] (10) (3.52 g,
5.40 mmole) was hydrogenated along with 1g 10~ Pd-C in 75
ml MeOH at 50 psi for 2 hr. The reaction was filtered
through Celite and rotary evaporated. The resulting foam
was dried under high vacuum to yield 11 (2.778, 99~).
1H-NMR (d4-Methanol): b 1.46 (s, 18H), 1.91 (m, 2H), 2.25
(t, 2H), 2.42 (q, 4H), 3.35 (t, 1H), 3.44 (m, 4H).
Mass Spec.: FAB 518 (M+H)+, 540 (M+Na)+, 556 (M+R)+.
Elemental Analysis for C21H39N708 ~ 1.5H20: Theoretical
C, 46.31; H, 7.77; N, 18.OO..Found C, 46.34; H, ?.42; N,
17.90.
Example 21
MaleimidopropionylglutamyldiL~-
Alanyl(Hoc)hydrazide] (Compound no. 12a)
Maleimidopropionic acid (0.399 mg, 2.36 mmole) and
N-hydroxy succinimide (272 mg, 2.36 mmole) were dissolved
in 30 ml CH2C12/3 ml DMF at 0°C. A 0.5M_ solution of DCC
in CH2C12 (4.7 ml, 2.36 mmole) was added, and the
reaction stirred for 3 hr. at room temperature. After
filtration of the DCU precipitate, the filtrate was
added to 11 (1.10 g, 2.13 mmole) and the reaction stirred
at room temperature for one day. Solvents were rezaoved by
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rotary evaporation. The oil was purified by flash
chromatography on silica gel (2 in. X 10 in.) with 500 m1
CH2C12, 2L CH2C12/methanol 95:5, and 2L CH2C12/methanol
9:1. Pure fractions were pooled, rotary evaporated, and
dried under high vacuum to yield 12a as a foam (850 mg,
60~).
1H-NMR (d4-Methanol): S 1.46 (s, 18H), 1.82 and 2.04 (2m,
2H), 2.23 (t, 2H), 2.40 (m, 4H), 2.52 (t, 2H), 3.45 {m,
4H), 3.78 (t, 2H), 4.20 (dd, 1H), 6.81 (s, 2H).
TLC: Rf 0.22, CH2C12/MeOH 9:1.
Mass Spec.: FAB 669 (M+H)+, 691 (M+Na)+, 707 (M+K)+.
Elemental Analysis for C28H44N8011 ~ 2H20: Theoretical C,
47.72; H, 6.87; N, 15.90. Found C, 47.70; H, 6.57; N,
15.83.
Bxamx~ I. a 2 2
Malezm3.dobutyrylglutamyldi[(3-Alaayl(Boc)hydrazide]
(Compound ao. 12b)
Maleimidobutyric acid (432 mg, 2.36 mmole) and N-
hydroxy succinimide (272 mg, 2.36 mmole) were dissolved
in 30 ml CH2C12/3 ml DMF at 0°C. A 0.5M solution of DCC
in CH2C12 (4.7 ml, 2.36 mmole) was added, and the
reaction stirred for 3 hr. at room temperature. After
filtration of the DCU precipitate, the filtrate was
added to ,~1 (1.10 g, 2.13 mmole) and the reaction stirred
at room temperature for one day. Solvents were removed by
rotary evaporation. The oil was purified by flash
chromatography on silica gel (2 in. X 10 in.) with 500 ml
CH2C12, 2L CH2C12/methanol 95:5, and 2L CH2C12/methanol
9:1. Pure fractions were pooled, rotary evaporated, and
dried under high vacuum to yield 12b as a foam {800 mg,
55~).
1H-NMR (d4-Methanol): 8 1.46 (s, 18H), 1.87 {m, 3H), 2.08
(m, 1H), 2.24 (m, 4H), 2.41 (m, 4H), 3.45 (m, 6H), 4.23
(dd, 1H), 6.82 (s, 2H).
TLC: Rf 0.20, CH2C12/MeOH 9:1.
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Mass Spec.: FAB 683 (M+H)+, 705 (M+Na)+, 721 (M+K)+.
Elemental Analysis for C2gH46N8011 ~ 1.5H20: Theoretical
C, 49.08; H, 6.96; N, 15.79.-Found C, 48.85; H, 6.65; N,
15.73.
Exams 1 a 2 3
Maleimidocaproylglutamyldi[(3-Alaayl(Boc)hydrazide7 -
(Compound ao.l2c)
Maleimidocaproic acid (453 mg, 2.14 mmole) and N-
methylmorpholine (239 mg, 2.36 mmole) were dissolved in
25 ml dry THF under Ar at -5°C. Isobutylchloroformate
(263 mg, 1.93 mmole) was added. After 5 min., 11 (1.0 g,
1.93 mmole) was added as a THF solution, and the reaction
stirred for 3 hr. with warming to room temperature. Ethyl
acetate (150 ml) was added, and then the solution was
extracted three times with 75 ml 10~ citric acid, three
times with 75 ml saturated aqueous sodium bicarbonate,
and three times with 75 ml water. The organic layer was
dried over sodium sulfate, then passed through a plug of
silica gel with CH2C12/methanol 9:1. The purified product
was rotary evaporated, and dried under high vacuum to
give 12c, 800 mg (58~).
1H-NMR (d4-Methanol): 8 1.30 {m, 2H), 1.46 (s, 18H),
1.60 (m, 4H), 1.88 and 2.06 (2m, 2H), 2.22 (t, 4H), 2.41
{t, 4H) , 3 .44 (m, 6H) , 4.24 {dd, 1H) , 6.80 (s, 2H) .
TLC: Rf 0.24, CH2C12lMeOH 9:1.
Mass Spec.: FAB 711.4 (M+H)+, 733.2 (M+Na)+, 749.3
(M+K)+.
Elemental Analysis for C31H50N8011 ~ 1.OH20: Theoretical
C, 51.09; H, 7.19; N, 15.38. Found C, 51.43; H, 7.00; N,
15.08.
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E~camnle 24
Maleimidopropionylglutamyldi((3-Alanylhydrazide3
( Compound no . 13 a )
Maleimidopropionylglutamyldi[~3-Alanyl(Boc)-
hydrazide] (12a) (850 mg, 1.27 mmole) was stirred in 15
ml CH2C12/trifluoroacetic acid (1:1) under N2 for 1.5 hr.
Solvents were removed by rotary evaporation. Ether was
added and co-evaporated three times, then the resulting
solid was triturated with ether. The solid was filtered
and dried under high vacuum to yield 13a (890 mg, 1000
1H-NMR (d4-Methanol}: S 1.83 and 2.02 (2m, 2H}, 2.23 (t,
2H), 2.52 (q, 6H), 3.47 (m, 4H}, 3.78 (m, 2H), 4_13 (dd,
1H), 6.82 (s, 2H).
Mass Spec.: FAB 469.0 (M+H)+, 491.1 (M+Na)+, 507.1
(M+K) +.
Elemental Analysis for C18H2gN807 ~ 3.75TFA ~ 0.25Et20:
Theoretical C, 34.80; H, 3.77; N, 12.25. Found C, 34.63;
H, 4.04; N, 12.20.
~xamt~le 25
Maleimidobutyrylglutamyldi((3-Alanylhydrazide~
(Compound no. 13b)
Maleimidobutyrylglutamyldi[(3-Alanyl(Boc)-hydrazide]
(12b) (800 mg, 1.17 mmole) was stirred in 15 ml
CH2C12/trifluoroacetic acid (1:1) under N2 for 1.5 hr.
Solvents were removed by rotary evaporation. Ether was
added and co-evaporated three times, then the resulting
solid was triturated with ether. The solid was filtered
and dried under high vacuum to yield 13b (840 mg, 1000
1H-NMR (d4-Methanol): 8 1.88 (m, 3H), 2.06 (m, 1H), 2.26
(t, 4H), 2.51 (t, 4H}~, 3.50 (m, 6H), 4.18 (dd, 1H), 6.82
(s, 2H) .
Mass Spec.: FAB 483.2 (M+H)+, 505.1 {M+Na)+, 521.1
(M+K}+.
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Elemental Analysis for C1gH30N807 ~ 3.5TFA ~ 0.25Et20:
Theoretical C, 36.03; H, 4.03; N, 12.45. Found C, 36.00;
H, 4.29; N, 12.26.
~xam~le 26
Maleimidocaproylglutamyldi((3-Alanylhydrazide~
(Compound no. 13c)
Ma.leimidocaproylglutamyldi[(3-Alanyl(Boc)-hydrazide]
(1~c) (800 mg, 1.13 mmole) was stirred in 15 ml
CH2C12/trifluoroacetic acid (1:1) under N2 for 1.5 hr.
Solvents were removed by rotary evaporation. Ether was
added and co-evaporated three times, then the resulting
solid was triturated with ether. The solid was filtered
and dried under high vacuum to yield 13c (870 mg, 100 0
1H-NMR (d4-Methanol): 8 1.31 (p, 2H), 1.61 (s, 4H), 1.85
and 2.03 (2m, 2H), 2.24 (t, 4H), 2.50 (t, 4H), 3.47 (m,
6H), 4.19 (dd, 1H), 6.80 (s, 2H).
Mass Spec.: 2onspray 511.2 (M+H)+, 533.0 (M+Na}'~'.
Elemental Analysis for C21H34N807 ~ 2.75TFA ~ 0.25Et20:
Theoretical C, 39.20; H, 4.70; N, 13.30. Found C, 39.32;
H, 4.58; N, 13.06.
Exams~le 27
Malei=nidopropionylglutamyldi[(3-Alanyl-hydrazone~
of Doxorubicin (Compound no. 3~a '°MP-Glu- (8-Ala-
DOX)2")
Maleimidopropionylglutamyldi[(3-Alanyl-hydrazide]
ditrifluoroacetate (13a) (1.0 g, 1.44 mmole) and DOX~HC1
(1.68 g, 2.88 mmole) were dissolved in 600 ml methanol
over a period of 3 hours. The reaction was concentrated
to 100 ml by rotary evaporation, then stirred for 1 day.
After further concentration to 10 ml, elution on an LH-20
column (2" X 10"} with methanol/DMF (1:1) was carried
out. The purified product was concentrated by rotary
evaporation and precipitated by the addition of
acetonitrile. The red solid was isolated by
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centrifugation and dried under high vacuum to yield 3a
(450 mg, 20~) .
1H-NMR (d4-Methanol): $ 1.29 (2bd, 6H), 4.04 (s, 6H),
&.80 (s, 2H), 7.5-8.0 (m, 6H).
Mass Spec.: Ionspray 1519.6 (M+H)t, 1541.2 (M+Na)+.
Elemental Analysis for C72Hg2N10027 ~ 2HC1 ~ 7H20:
Theoretical C, 50.32; H, 5.75; N, 8.15. Found C, 50.20;
H, 5.49; N, 8.44.
Examule 28
Maleimidobutyrylglutamyldi ( ~i-Alaxiylhydrazaael of
Doxorubicia
(Compound xio. 3b ~~MB-Glu- ($-Ala-DOX) 2 ~~ )
Maleimidobutyrylglutamyldi[(3-Alanylhydrazide]
ditrifluoroacetate (13b) (280 mg, 0.395 mmole) and
DOX~HCl (458 mg, 0.790 mmole) were dissolved in 250 ml
methanol over a period of 3 hours. The reaction was
concentrated to 50 ml by rotary evaporation, then stirred
for 2 days. After further concentration to 5 ml, elution
on an LH-20 column (1" X 15") with methanol/DMF (1:1) was
carried out. The purified product was concentrated by
rotary evaporation and precipitated by the addition of
acetonitrile. The red solid was isolated by
centrifugation and dried under high vacuum to yield 3b
(325 mg, 51~).
1H-NMR (d4-Methanol): 8 1.30 (m, 6H), 4.04 (s, 6H), 6.78
(s, 2H) , 7.4-8.0 (m, 6H) .
Mass Spec.: FAB 1533.7 (M+H)'", 1555.5 (M+Na)+, 1572.4
( M-t-K ) + _
Elemental Analysis for C73Hg4N10027 ~ 2HC1 ~ 7H20:
Theoretical C, 50.61; H, 5.82; N, 8.08. Found C, 50.83;
H, 5.so; N, 7.41.
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Bxamnle 29
Maleimidocaproylglutamyldi[~i-Alaaylhydrazone] of
Doxoruh3.c3.a
(Compound ao. 3 c "MC-Glu- (i3-A1a-DOX) 2" )
Maleimidocaproylglutamyldi[(3-Alanylhydrazide]
ditrifluoroacetate (13c) (148 mg, 0.20 mmole) and DOX~HCl
(232 mg, 0.40 mmole) were dissolved in 150 ml methanol
over a period of 3 hours. The reaction was concentrated
to 10 ml by rotary evaporation, then stirred for 2 days.
After further concentration to 2 ml, elution on an LH-20
column (1" X 10") with methanol/DMF (1:1) was carried
out. The purified product was concentrated by rotary
evaporation and precipitated by the addition of
acetonitrile. The red solid was isolated by
centrifugation and dried under high vacuum to yield ~c
(162 mg, 50~).
1H-NMR (d6-DMSO): 8 1.20 (m, 6H), 4.0 (ppm) 6H, 6.95 (s,
2H), 7.5-8.1 (m, 6H).
Mass Spec.: FAB 1561 (M+H)+, 1583.4 (M+Na)+, 1599.9
(M+K)+.
Elemental Analysis for C~5HggN100~ ~ 2HC1 ~ 7H20:
Theoretical C, 51.17; H, 5.95; N, 7.96. Found C, 51.04;
H, 5 . 41; N, 10 . 23 .
example 30
Z-Glutamyldi[glutamyldi(Boc)hydrazide]
Compound no . 1~.)
Z-Glutamic acid (844 mg, 3.0 mmole) and N-hydroxy
succinimide (691 mg, 6.0 mmole) were dissolved in 6 ml
DMF at 0°C under dry N2. A 0.5~ solution of
dicyclohexylcarbodiimide in methylene chloride (12.0 ml,
6.0 mmole) was added. The reaction was stirred for 4 hr.
Dicyclohexylurea precipitate was filtered, and the
filtrate was added to 5 (2.253 g, 6.0 mmole). After
stirring at room temperature for 60 hr., the reaction was
rotary evaporated to an oil, which was redissolved in 200
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ml ethyl acetate. The organic layer was extracted three
times with 125 ml 10~ citric acid, 3 times with 125 ml
saturated aqueous sodium bicarbonate, and once with 125
ml brine. The organic layer was dried over sodium sulfate
and rotary evaporated to a foam. Flash chromatography was
carried out on silica gel (2 in. X 12 in.) with
CH2C12/methanol/acetic acid 93:5:2. Pure fractions
containing product (14_) were pooled and concentrated to a
foam by rotary evaporation to yield, after drying under
high vacuum, 2.30 g (77~).
1H-NMR (d4-Methanol): 8 1.35 (s, 36H), 1.7-2.4 (m, 12H),
3.90 (bt, 1H), 4.35 (m, 2H), 4.98 (q[AB], 2H), 7.25 (m,
5H) .
TLC: Rf 0.61, CH2C12/MeOH 9:1.
Mass Spec.: FAB 1018.5 (M+Na)+, 1034.4 (M+K)+.
Elemental Analysis~for C43H6gN11016.~ 2H20: Theoretical
C, 50.04; H, 7.13; N, 14.93. Found C, 50.20; H, 6.85; N,
14.90.
Exaamule 31
Glutamylditglutamyldi(Boc)hydrazide~
{Compound no. 15)
Z-Glutamyldi[glutamyldi{Boc)hydrazide] (1~) (1.86 g,
1.87 mmole) was hydrogenated along with 1g 10~ Pd-C in 75
ml MeOH at 50 psi for 3 hr. The reaction was filtered
through Celite and rotary evaporated. The resulting foam
was dried under high vacuum to yield ,1~ (1.59 g, 99~).
1H-NMR (d4-Methanol): 8 1.46 {s, 36H), 1.6-2.4 {m, 12H),
3.23 (m, 1H), 4,.4D {2t, 2H).
Mass Spec.: FAB 862 {M+H)+, 884 (M+Na)+, 900 (M+K)+.
Elemental Analysis for C35H63N11014 ~ 1H20: Theoretical
C, 47.77; H, 7.45; N, 17.51. Found C, 47.67; H, 7.28; N,
17.33.
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le 32
Maleimidopropioaylglutamyldi(glutamyldi(Boc)
hydrazide~ (Compound x~,o. 16a)
The N-hydroxysuccinimide ester of maleimidopropionic
acid (300 mg, 1.13 mmole) was prepared as in the -
synthesis of ~, then stirred with
glutamyldi[glutamyldi(Boc)hydrazide] (~5) (883 mg, 1.02
mmole) and triethylamine (143 ul, 1.02 mmole) in 25 ml
DMF at room temperature for 16 hr. Solvent was removed by
rotary evaporation, and the residue was purified by flash
chromatography on silica gel (1 in. X 10 in.) with
CH2C12-acetic acid-methanol 93:2:5. Pure fractions were
pooled, rotary evaporated, and dried under high vacuum to
give 16a (400 mg, 39~).
1H-NMR (d4-Methanol): 8 1.46 (s, 36H}, 1.8-2.4 (m, 12H),
2.50 (t, 2H), 3.76 (t, 2H}, 4-.11 (m, 1H), 4.39 (2t, 2H),
6.81 (s, 2H).
Mass Spec.: FAB 1035.6 (M+Na}+, 1051 (M+K)+.
Exa~nnole 33
Maleimidobutyrylglutamyldi[glutamyldi(Boa)
hydrazide~ (Compound no. ~.6b)
Maleimidobutyric acid (227 mg, 1.24 mmole} was
dissolved with N-methylmorpholine (178 ul, 1.61 mmole) in
10 ml dry THF under N2 at O°C. Isobutylchloroformate (144
ul, 1.11 mmole) was added, followed 5 minutes later by
the addition of glutamyldi[glutamyldi(Boc)hydrazide]
(960 mg, 1.11 mmole) as a solution in 15 ml DMF. The
reaction was stored at 4°C for 16 hours. The reaction was
concentrated by rotary evaporation, then dissolved in 200
ml EtOAc_ The organic layer was washed three times with
50 ml 10~ citric acid, three times with 50 ml saturated
NaHC03, and three times with 50 ml H20_ The organic layer
was concentrated to a foam, which was purified by flash
.chromatography on silica gel (1 in. X 12 in.) with
CH2C12-acetic acid-methanol 93:2:5. Pure fractions were
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pooled and rotary evaporated to a foam. Drying under high
vacuum yielded 16b (900 mg, 79~).
1H-NMR (d4-Methanol): 8 1.46 (s, 36H), overlapping
signals 1.86 (t), 2.22 (t), and 1.9-2.4 (m) 16H total,
3.50 (t, 2H), 4.11 (m, 1H), 4.40 (2t, 2H), 6.82 (s, 2H).
Mass Spec.: FAB 1049.5 (M+Na)+, 1065.4 (M+K)+.
Elemental Analysis for C43H7pN12017 ~ 3-$H20 ~ 3HOAc:
Theoretical C, 46.33; H, 7.06; N, 13.23. Found C, 46.24;
H, 6.52; N, 13.37.
Examr~le 34
Maleimidocaproylglutamyldi[glutamyld3.(Boc)
hydrazzdeI (Compound ao. 16c)
This compound was synthesized following the
procedure used for 16b. Yield of 16c was 330 mg, 54~.
1H-NMR (d4-Methanol): 8 1.28 (m, 2H}, 1.46 (s, 36H), 1.56
(m, 4H), overlapping signals 1.9-2.5 (m) and 2.20 (t) 14H
total, 3.48 (t, 2H), 4.10 (m, 1H), 4.40 (m, 2H}, 6.80 (s,
2H) .
Mass Spec.: FAB 2078.8 (M+Na)+, 1093.5 (M+K)+.
Elemental Analysis for C45H74N12017 ~ 3H20 ~ 3HOAc:
Theoretical C, 47.51; H, 7.19; N, 13.04. Found C, 47.44;
H, 6.48; N, 13.24.
Examx~le 35
Ma.le3.m3.dopropion.ylglutamyldi [glutamyld3.-hydrazide~
( Compound ao . 17 a )
Maleimidopropionylglutamyldi[glutamyldi(Boc}hydrazid
e] (1 a) (400 mg, 0.395 mmole) was stirred in 15 ml
CH2C12/trifluoroacetic acid (1:1) under N2 for 1.5 hr.
Solvents were removed by rotary evaporation. Ether was
added and co-evaporated three times, then the resulting
solid was triturated with ether. The solid was filtered
and dried under high vacuum to yield 17a (250 mg, 59~).
1H-NMR (d4-Methanol) of the crude material verified
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complete removal of the BOC groups. This was used in the
synthesis of 18a without further purification.
Exa.mgl a 3 6
Maleimidobutyrylglutamyldi[glutamyl-dihydrazide~
( Compound no . 17 ~,)
Maleimidobutyrylglutamyldi[glutamyldi(Boc}-
hydrazide] (26b) (900 mg, 0.877 mmole) was stirred in 15
ml CH2C12/trifluoroacetic acid (1:1) under N2 for 1.5 hr.
Solvents were removed by rotary evaporation. Ether was
added and co-evaporated three times, then the resulting
solid was triturated with ether. The solid was filtered
and dried under high vacuum to yield 17b (817 mg, 86~)
1H-NMR (d4-Methanol): 8 overlapping signals 1.7-2.5 (m),
1.80 (t), and 2.17 (t} total 16H, 3.45 (t, 2H), 4.04 (t,
1H) , 4.36 (m, 2H) , 6.75 (s, 2H) .
Elemental Analysis for C23H38N12~9 ~ 6.5 TFA: Theoretical
C, 31.61; H, 3.28; N, 12.29. Found C, 31.76; H, 3.49; N,
12.06.
Exa~n.~le 37
Maleimidocaproylglutamyldi[glutamyl-dihydrazidel
(Compound no. 17c)
Maleimidocaproylglutamyldi[glutamyldi(Boc}-
hydrazide] (16c) (330 mg, 0.313 mmole) was stirred in 15
ml CH2C12/trifluoroacetic acid (1:1} under N2 for 1.5 hr.
Solvents were removed by rotary evaporation. Ether was
added and co-evaporated three times, then the resulting
solid was triturated with ether. The solid was filtered
and dried under high vacuum to yield 17c (350 mg, 1000
1H-NMR (d4-Methanol): 8 1.30 (m, 2H), 1.60 (2t, 4H),
overlapping signals 1.9-2.5 (m) and 2.22 (t) total 14H,
3.47 (t, 2H), 4.09 (t, 1H), 4.43 (2t, 2H), 6.80 (s, 2H).
Elemental Analysis for C25H42N120g ~ 6.2 TFA: Theoretical
C, 32.99; H, 3.57; N, 12.34. Found C, 32.76; H, 3.73; N,
12.72.
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l~xamnle 38
Maleimidopropionylglutamyldilglutamyl-dihydrazone]
of Doxorubicin
. 5 . ( Compound no . 18 a )
Maleimidopropionylglutamyldi[glutamyl-dihydrazide]
. (17a) (250 mg, 0.230 mmole) and DOX~HCl (588 mg, 1.01
mmole) were dissolved in 100 ml methanol then
concentrated to 25 ml by rotary evaporation and stirred
for 2 days. The reaction was further concentrated to 15
ml and eluted on an LH-20 column (1" X 10") with
methanol. The purified product was rotary evaporated to a
red film and dried under high vacuum to yield 18a (180
mg, 27~).
1H-NMR (d4-Methanol): (selected peaks) 8 1.33 (m, 12H),
4.04 and 4.06 (2d, 12H), 6.72 (s, 2H), 7.4-8.0 (m, 12H).
Mass Spec.: FAB Ianspray 2713.5 (M+H)+.
Elemental Analysis for C130H144N26049 ~ 4HC1 ~ 4H20
4TFA: Theoretical C, 48.91; H, 4.76; N, 6.61. Found C,
48.49; H, 5.28; N, 7.06.
Example 39
Maleimidobutyrylglutamyldi(glutamyl-dihydrazone~
of Doxorubicin
(Compound no. 18b)
Maleimidobutyrylglutamyldi[glutamyl-dihydrazide]
(17b) (300 mg, 0.273 mmole) and DOX~HC1 (697 mg, 1.20
mmole) were dissolved in 100 ml methanol then
concentrated to 25 ml by rotary evaporation and stirred
for 2 days. The reaction was further concentrated to 15
ml and eluted on an LH-20 column (1" X 10") with
methanol. The purified product was rotary evaporated to a
red film and dried under high vacuum to yield 18b (500
mg, 64~).
1H-NMR (d4-Methanol): (selected peaks) S 1.36 (m, 12H),
4.04 and 4.10 (2d, 12H), 6.69 (s, 2H), 7.5-8.0 (m, 12H).
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Mass Spec.: FA8 Ionspray 2728 (M+H)+.
Elemental Analysis for C131H146N16049 ~ 4HC1 ~ 2TFA
4H20: Theoretical C, 51.08; H, 5.08; N, 7.06. Found C,
51.02; H, 5.05; N, 7.16.
~xamt~le 40
Maleimidocaproylglutamylditglutamy3-dihydrazox~.e~
of Doxorubicin
(Compound no. 18c °MC-Glu(DOX)4~~)
Maleimidocaproylglutamyldi[glutamyl-dihydrazide]
(17~) (233 mg, 0.210 mmole) and DOX~HCl (489 mg, 0.843
mmole) were dissolved in 100 ml methanol then
concentrated to 25 ml by rotary evaporation and stirred
for 2 days. The reaction was further concentrated to 15
ml and eluted on an LH-20 column (1" X 10") with
methanol. The purified product was rotary evaporated to a
red film and dried under high vacuum to yield 18c (430
mg, 71~).
1H-NMR (d4-Methanol): (selected peaks) S 1.36 (m, 12H),
4.04 and 4.10 (2d, 12H), 6.69 (s, 2H), 7.5-8_0 (m, 12H).
Mass Spec.: FAB Ionspray 1379 (M+H)2+.
Elemental Analysis for C133H150N16049 ~ 4HC1 ~ 4TFA
4H20: Theoretical C, 49.36; H, 4.88; N, 6.53. Found C,
49.34; H, 4.79; N, 6.66.
Examt~le 41
Compound no. l9
Z-NHCH2CH2-Br (3.168, 12.3 mmole) and (BOC-NHCH2CH2)2-NH
(3.728, 12.3 mmole) were stirred in 60 ml ACN/40 ml
3 0 phosphate buf f er ( 0 .1M, pH 9 ) at 5 5°C f or 2 days . Af ter
cooling, the reaction was diluted with 200 ml H20 and
extracted twice with 200 ml Et20. The organic layers were
combined, dried over Na2S04, and evaporated under vacuum.
The oily residue was chromatographed on Merck silica gel
60 (2" x 11") with (1) CH2C12, 2L, (2) CH2C12/MeOH
97.5:2.5, 1.5L, and (3) CH2C12/MeOH 95:5, 2L. The desired
_7p_
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product 29, which elutes in (2)-(3), was pooled,
evaporated under vacuum, and dried under high vacuum to
yield 1.938 (33~).
1H-NMR (CDC13): 8 1.37 {s, 18H), 2.47 (m, 6H), 3.15 (m,
~ 5 6H), 5.07 (s, 2H), 7.28 (m, 5H).
13C_~ (CDC13): 8 28.38, 38.55, 39.01, 53.90, 54.27,
65.18, 66.60, 79.29, 126.94, 127.50, 127.96, 128.14,
128.41, 128.47, 136.66, 156.38, 156.78.
Mass Spec . : FAB 481. 2 (MHO' )
Elemental Analysis for C24H40N4~6: Theoretical C, 59.98;
H, 8.39; N, 11.66. Found C, 60.26;H, 8.43; N, 11.60.
FT2R: 3336, 2976, 1694, 1524, 1366, 1252, 1170, 736, 69E
cm 1.
~xamnle 42
Compound ao.20
(1.928, 3.99 mmole) was stirred in50~ TFA/CH2C12 (60
ml) for 3 hr. Solvents were removed by rotary
evaporation, then repeated co-evaporations with Et20. The
oily product was triturated with 50 ml Et20 three times,
then dried under high vacuum to yield 20 as a foam
(2.298, 100}.
1H-NMR (d4-MeOH): S 2.64 (t, 2H), 2.78 (t, 4H), 3.01 (t,
4H), 3.21 {t, 2H), 5.08 (s, 2H), 7.34 (m, 5H).
13C-NMR (d4-MeOH): 8 38.36, 39.53, 52.61, 54.95, 67.69,
128.91, 229.12, 129.51, 138.2, 159.2.
Mass Spec.: FAB 281.1 (MH+)
High Res. Mass Spec.: Theoretical, 281.1977;
Experimental, 281.1984-(MH+).
Elemental Analysis for C14H24N402 ~2.6TFA: Theoretical C,
39.98; H, 4.65; N, 9.71; F, 25.69.
Found C, 39.85;H, 4.60; N, 9.68; F, 25.38.
FTIR: 3036, 1680, 1532, 1260, 1204, 1136, 838, 800, 722
czri 1.
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W097IZ3243 CA 02239183 2005-02-22
rcrws~nosl3
Examflle 43
Compouad ao.2_~
BrCH2CONHNH-BOC (10.128, 40.0 m~aole) was added in several
portions over a 5 minute period to a stirring suspension
of ~0 (6.228, 10.0 mQnole) and KHC03 ( 8.018, 80 mmole) in
100 ml ~' at 0°C. The reaction was then stirred at room
temperature for 60 hours. Solvents were removed by rotary
evaporation to an oily residue. This was dissolved in 500
ml of Et20/EtOAc 1:1 and extracted 5 times with 150 ml
saturated NaHC03 followed by two times with water. The
organic layer was dried over Na2S04 and rotary evaporated
to an oil. Further drying under high vacuum yielded 22
{9.678, 1000.
1H-NMR (d4-MeOH): b 1.45 (s, 36H), 2.69 (m, 10H), 3.23
(t, 2H) . 3.37 (s, 8H) , 5.06 (s, 2H) , 7.33 (iri, 5H) .
13C_~ (d4_M~H): 8 28.64, 53.28, 53.88, 54.56, 58.59,
66.92, 67.54, 81.94, 129.07, 129.51, 138.35, 157.63,
158.89, 173.20.
Mass Spec.: Ionspray 969.6 (MH+)
Elemental Analysis for C42H72N12014 ~0.5H20: Theoretical
C, 51.57; H, 7.52; N, 17.18. Found C, 51.73;H, 7.52; N,
16.84.
FTIR: 3296, 2980, 1728, 1696, 1518, 1394, 1368, 1248,
1162, 1048, 1016, 874, 756, 698 cm 1.
~xamale 44
Compound ao.2~
?~1 (2.11 g, 2.18 mmole) was hydrogenated at 35 psi in 50
ml MeOH for 2 hours. The reaction was filtered through
Celite* rotary evaporated, and dried under high vacuum to
yield ~ as a foam ( 1.65 g,' 91~) .
1H-NMR (d4-MeOH): 8 1.46 (s, 36H), 2.71 (m, 12H), 3.34
(s, 8H) .
13C_M~ (d4_MeOH): b 28.64, 34.77. 53.11, 53.91, 58.12,
81.90, 157.64, 172.88.
Mass Spec.: Ionspray 835.5 (MH+).
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Elemental Analysis for C34H66N12012 ~1.0H20 ~l.OMeOH:
Theoretical C, 47.50; H, 8.20; N, 18.99. Found C,
47.41; H, 7.88; N, 18.74.
FT2R: 3292, 2980, 1720, 1690, 1484, 1368, 1248, 1162,
' S 1048, 1016, 880, 773, 574 cm 1.
Exaxnule 4B
Compound no.23
A solution of 22 {1.03 g, 1.23 mmole) and malefic
anhydride (121 mg, 1.23 mmole) was stirred in 25 ml
CH2C12 for 2.5 hours. Solvents were removed by rotary
evaporation to yield 23 (1.16 g, 1000 .
1H-NMR (d4-MeOH): b 1.45 (s, 36H), 3.13 (m, 4H), 3.45 {m
and s, 16H), 3.68 (m, 2H), 6.17 (dd, 2H).
Mass Spec.: Ionspray 933.6 (MH'~'), 955.5 (M+Na+).
Examule 46
Compound xio . 2 4
(603 mg, 0.646 mmole) and EDCI (149 mg, 0.775 mmole)
were stirred in 25 ml dry CH2C12 under N2 for 2.5 hr. at
room temperature. The reaction was then extracted three
times with 25 ml saturated aqueous NaHC03 solution, then
once with 25 ml water. The organic layer was dried over
Na2S04, rotary evaporated, and dried under high vacuum to
yield the isomaleimide intermediate (494 mg, 84~).
1H-NMR (CDC13): ~ 1.45 (s, 36H), 2.8 (m, 10H), 3.31 (s,
8H), 3.7 (m, 2H), 6.57 and 7.40 (dd, 2H).
This product was stirred with HOBt (35 mg, 0.259 mmole)
in 8 ml DMF for 7 hours at room temperature. Solvent was
removed by rotary evaporation. The oily residue was
dissolved in 60 ml Et20/EtOAc 1:1 and extracted five
times with 25 ml saturated aqueous NaHC03 solution, then
once with 25 ml water. The organic layer was dried over
Na2S04, rotary evaporated, and dried under high vacuum to
yield the maleimide product ~4 (463 mg, 94~).
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1H-NMR (CDC13): S 1.45 {s, 36H), 2.7 (m, 10H), 3.32 (s,
8H), 3.57 (m, 2H), 6.68 (s, 2H).
13C-~ (CDC13): 8 28.16, 81.73, 134.25, 155.5, 170.79.
Mass Spec.: Electrospray 915.5 (MHO'), 937.5 (M+Na+).
FTIR: 3300, 2982, 1738, 1708, 1680 (sh), 1498, 1394,
1368, 1248, 1162, 1048, 1016, 72, 696 cm-1.
~xamnle 47
Compound r~.o . 2 ~,
?~, (214 mg, 0.234 mmole) was stirred with p-
toluenesulfonic acid {450 mg, 2.37 mmole) in 25 ml dry
CH2C12 under N2 for 3 hours. Solvent was removed by
rotary evaporation. The residue was triturated four times
with 125 ml Et20, then dried under high vacuum to yield
~ {378 mg, 94~).
1H-NMR (d4-MeOH): b 2.36 (s, 21H), 3.22 (t, 2H), 3.52 (m,
8H), 3.71 (s, 8H), 3.94 (t, 2H), 6.85 (s, 2H), 7.23 (d,
14H), 7.70 (d, 14H).
Mass Spec.: F.AB 515.1 (MH+).
Example 48
Compound no.2~
(100 mg, 58 umole) and Doxorubicin HCl (277 mg, 305
umole) were stirred in 25 ml dry methanol for 24 hour.
The reaction was concentrated by rotary evaporation to 4
ml, then purified on Sephadex LH-20 (1" x 18") with
methanol. Fractions containing pure product were pooled,
rotary evaporated, and dried under high vacuum to yield
{123 mg, 59~) .
1H-NMR (d4-MeOH): 8 1.2 (m, 12H), 3.9 (s, 12H), 6.8 (s,
2H), 7.2-8.0 (m) superimposed with 7.2 (d), and 7.7 (d)
total 24 H.
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W097123243 CA 02239183 2005-02-22 pC,j,~sg6~~~3
Example 49
Compound ao.~
Mono-Z-ethylene diamine HC1 (3.46 g, 15 mmole),
BrCH2CONHNH-BOC (7.59 g, 30 mmola), and KHC03 (5.26 g,
52.5 mmole) were stirred in 60 ml DMF under N2 at room
temperature for 24 hours. The reaction was partitioned
between 25 ml Et20 and 150 ml saturated aqueous NaHC03.
The Et20 layer was washed with 100 ml saturated aqueous
NaHC03. All aqueous layers were extracted with 100 ml
Et20. The combined Et20 layers were washed with brine,
dried over Na2S04, and rotary evaporated to yield 6.5 g
crude product. This material was flash chromatographed on
2" x 20" silica gel 60 (Merck) column with (1)
CH2C12/MeOH 95:5, 2L, (2) CH2C12/MeOH 92.5:7.5, 1L, and
(3) CH2C12/MeOH 90:10. 2L. Fractions containing the
desired product were pooled, rotary evaporated, and dried
under high vacuum to yield ~7 as a foam (4.64 g, 57$).
1H-NMR (CDC13): b 1.36 (s, 18H), 2.70 (m, 2H), 3.22 (s,
4H), 3.28 {m, 2H}, 5.01 (s, 2H), 7.25 (m, 5H).
13C-NMR (CDC13): 8 28.08, 38.75, 55.67, 57.19, 66.77,
81.85, 128.02, 128.41, 136.47, 155.95, 158.10, 170.79.
Mass Spec.: Tonspray 539.3 {l~i'~), 561.2 (M+Na+), 577.1
(M+K+).
Elemental Analysis for C24H38N60g ~0.SH20: Theoretical C,
52.64; H, 7.18; N, 15.35. FOUnd C, 52.53;H, 7.05, N,
15.30.
FTIR: 3300, 2980, 1724, 1694, 1528, 1368, 1250, 1160,
1016, 880, 754, 698 cm-1.
Lxam~le 50
Compound ao.~
27 was hydrogenated in 100 ml EtOH along with 2 g 10~ Pd-
C at 45 psi for 4.5 hours. After filtration of the
catalyst through Celite, the solvent was rotary
evaporated and dried under high vacuum to yield ~_8 as a
foam {3.06 g, 92~).
* Trade-mark T
CA 02239183 1998-06-O1
WO 97/23243 PCT/US96/20513
1H-NMR (CDC13): 8 1.43 and 1.44 (2s, 18H), 2.80 (t, 2H),
3.23 (d, 4H), 3.39 (m, 2H}. (d4-MeOH): 2.24 and 1.26
(2s, 18H), 2.59 (t, 2H), 3.02 (d, 4H), 3.15 (t, 2H).
Mass Spec.: Ionspray 405.3 (MHO').
Elemental Analysis for C16H32N606 ~0.5H20: Theoretical C, '
46.48; H, 8.04; N, 20.33. Found C, 46.57;H, 8.04; N,
20.37.
FTIR: 3328, 2980, 1698, 1672, 1500, 1368, 1300, 1252,
1162, 778, 692 crri 1.
Example 51
Compound no.2~
Malefic anhydride (98 mg, 1.0 mmole) and 28 (405 mg, 1.0
mmole) were stirred in 15 ml CH2C12 for 2 hours at room
temperature. The reaction was rotary evaporated, and the
crude product triturated with Et20. The residue was dried
under high vacuum, yielding ~ (400 mg, 80~).
1H-NMR (CDC13): 8 1.47 and 1.48 (2s, 18H}, 2_89 (t, 2H),
3.32 (d, 4H), 3.46 (m, 2H}, 6.42 (dd, 2H).
~x~le 52
Compoutzd no . 3 0
(503 mg, 2.0 mmole} and EDCI (230 mg, 1.2 mmole) are
stirred in 25 ml dry CH2C12 under N2 for 2.5 hr. at room
temperature. The reaction is then extracted three times
with 25 ml saturated aqueous NaHC03 solution, then once
with 25 ml water. The organic layer is dried over Na2S04,
rotary evaporated, and dried under high vacuum to yield
the isomaleimide intermediate.
This product is stirred with HOBt (54 mg, 0.40
mmole) in 8 ml DMF for 7 hours at room temperature.
Solvent is removed by rotary evaporation. The oily
residue is dissolved in 60 ml Et20/EtOAc 1:1 and
extracted five times with 25 ml saturated aqueous NaHC03
solution, then once with 25 ml water. The organic layer ,
is dried over Na2S04, rotary evaporated, and dxied under
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W097123243 CA 02239183 2005-02-22
PCT/US96I205I3
high vacuum to yield the maleimide product 30 (455 mg,
94%) .
Example. 53
Compound no.3~
(485 mg, 1.0 mmole) is stirred with p-toluenesulfonic
acid (1.90 g, 10 nunole) in 50 ml dry CH2C12 under N2 for
3 hours. Solvent is removed by rotary evaporation. The
residue is triturated four times with 125 ml Et20, then
dried under high vacuum to yield ~1_ (800 mg, 94%).
Exaa~nle 54
Compound no.3 22
31 (200 mg, 0.25 mmole) and Doxorubicin HCl (377 mg, 0.65
mrnole) are stirred in 25 ml dry methanol for 24 hour. The
reaction is concentrated by rotary evaporation t,o 4 ml,
then purified in two equal portions on Sephadex LH-20 (1"
x 18") with methanol. Fractions containing pure product
are pooled, rotary evaporated, and dried under high
vacuum to yield ~2 (200 mg, 50%).
Examflle 55
Compound no.33
t-Butyl carbazate (396 mg, 3 mmole) is stirred in 10 ml
dry CH2C12 under N2,~ then triethylamine (0.6 g, 6 m~nole)
is added followed by triphosgene (296 mg, 1 mmole) in a
single portion. When the initial reaction subsides, ~0_
(934 mg, 1.5 nnnole) is added in 20 ml CH2C12 along with
additional triethylamine (0.45 g, 4.5 mmole). The mixture
is stirred at room temperature'for 1.5 hr., diluted with
CH2C12, then partitioned with water (100 ml). The organic
layer is dried over Na2S04, and rotary evaporated. Flash
chromatography on silica gei 60 yields pure product ~3
(684 mg, 50%).
* Trade-mark
WO 97/Z3243 CA 02239183 2005-02-22 p~~gg~O5I3
Exa~tv 1 a 5 6
Compound no.34
33 (650 mg, 0.71 mmole) is hydrogenated in 50 ml EtOH
along with 1 g 10~ Pd-C at 45 psi for 4.5 hours. After
filtration of the catalyst through Celite, the solvent is
rotary evaporated and dried under high vacuum to yield
as a foam (550 mg, 100$).
Example 57
Compound no.35
Malefic anhydride (63 mg, 0.64 mnnole) and 34 (500 mg, 0.64
mmole) are stirred in 15 ml CH2C12 for 2 hours at room
temperature. The reaction is rotary evaporated, and the
crude product triturated with Et20. The residue is dried
under high vacuum, yielding ~5 (448 mg, 80~).
Examine 58
Compouad no.36
35 (438 mg, 0.5 mmole) and EDCI (115 mg, 0.6 mmole) are
stirred in 25 ml dry CH2C12 under N2 for 2.5 hr. at room
temperature. The reaction is then extracted three times
with 25 ml saturated aqueous NaHC03 solution, then once
with 25 ml water. The organic layer is dried over Na2S04,
rotary evaporated, and dried under high vacuum to yield
the isomaleimide intermediate.
This product is stirred with HOBt (27 mg, 0.20
mmole) in 8 ml D~ for 7 hours at room temperature.
Solvent is removed by rotary evaporation. The oily
residue is dissolved in 60 ml Et20lEt0Ac 1:1 and
extracted five times with 25 ml saturated aqueous NaHC03
solution, then once with 25 ml water. The organic layer
is dried over Na2S04, rotary evaporated, and dried under
high vacuum to yield the maleimide product 3~, (400 mg,
94~).
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CA 02239183 1998-06-O1
WO 97/23243 PCT/US96/20513
Exams~le S9
Compound no.37
36 (400 mg, 0.47 mmole) is stirred with p-toluenesulfonic
acid (894 mg, 4.7 mmole) in 50 ml dry CH2C12 under N2 for
~ 5 3 hours. Solvent is removed by rotary evaporation. The
residue is triturated four times with 125 ml Et20, then
dried under high vacuum to yield 37 (455 mg, 94~).
Example 60
Compoux~d ao . 3 8
,~7 (257 mg, 0.25 mmole) and Doxorubicin HCl (377 mg, 0.65
mmole) are stirred 'in 25 ml dry methanol for 24 hour. The
reaction is concentrated by rotary evaporation to 4 ml,
then purified in two equal portions on Sephadex LH-20 (1"
x 18") with methanol. Fractions containing pure product
are pooled, rotary evaporated, and dried under high
vacuum to yield 3$ (222 mg, 50~).
Examule 61
Compound ao. ~9
Z-NHCH2CH2-Br (3.16g, 12.3 mmole) and (BOC-NHCH2CH2)2-~
(3.72g, 12.3 mmole) were stirred in 60 ml ACN/40 ml
phosphate buffer (0.1M, pH 9) at 55°C for 2 days. After
cooling, the reaction was diluted with 200 ml H20 and
extracted twice with 200 ml Et20. The organic layers were
combined, dried over Na2S04, and evaporated under vacuum.
The oily residue was chromatographed on Merck silica gel
60 (2" x 11") with (1) CH2C12, 2L, (2) CH2C12/MeOH
97.5:2.5, 1.5L, and (3) CH2C12/MeOH 95:5, 2L. The desired
product 1~, which elutes in (2)-(3), was pooled,
evaporated under vacuum, and dried under high vacuum to
yield 1.938 (33~}.
1H-NMR (CDC13): S 1.37 (s, 18H), 2.47 (m, 6H), 3.15 (m,
6H}, 5.07 (s, 2H), 7.28 (m, 5H).
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13C_~ (CDC13): 8 28.38, 38.55, 39.01, 53.90, 54.27,
65.18, 66.60, 79.29, 126.94, 127.50, 127.96, 128.14,
128.41, 128.47, 136.66, 156.38, 156.78.
Mass Spec.: FAB 481.2 (MHO')
Elemental Analysis for C24H4pN4O6: Theoretical C, 59.98;
H, 8.39; N, 21.66. Found C, 60.26;H, 8.43; N, 11.60.
FTIR: 3336, 2976, 1694, 1524, 1366, 1252, 1170, 736, 698
cm 1.
Exmle 62
Compound r~.o .
102 (1.92g, 3.99 mmole) was stirred in 50~ TFA/CH2C12 (60
ml) for 3 hr. Solvents were removed by rotary
evaporation, then repeated co-evaporations with Et20. The
oily product was triturated with 50 ml Et20 three times,
then dried under high vacuum to yield X03 as a foam
(2.298, 1000 .
1H-NMR (d4-MeOH): 8 2.64 (t, 2H), 2.78 (t, 4H), 3.01 (t,
4H), 3.21 (t, 2H), 5.08 (s, 2H), 7.34 (m, 5H).
13C-NMR (d4-MeOH): b 38.36, 39.53, 52.61, 54.95, 67.69,
128.91, 129.12, 129.51, 138.2, 159.2.
Mass Spec.: FAB 281.1 (MF3+)
High Res. Mass Spec.: Theoretical, 281.2977;
Experimental, 281.1984 (MH+).
Elemental Analysis for C14H24N402 ~2.6TFA: Theoretical C,
39.98; H, 4.65; N, 9.71; F, 25.69. Found C, 39.85;H,
4.60; N, 9.68; F, 25.38.
FTIR: 3036, 1680, 1532, 1260, 1204, 1136, 838, 800, 722
1_
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WO 97123243 CA 02239183 2005-02-22 p~~g9~pg13
Examgl_e 63
Compouad ao . ~1,
BrCH2CONHNH-BOC (10.128, 40.0 mmole) was added in several
portions over a 5 minute period to a stirring suspension
of 103 (6.228, 10.0 rnmole) and RHC03 ( 8.018, 80 mmole)
in 100 ml DMF at 0°C. The reaction was then stirred at
room temperature for 60 hours. Solvents were removed by
rotary evaporation to an oily residue. This was dissolved
in 500 ml of Et20/EtOAc 1:1 and extracted 5 times with
150 ml saturated NaHC03 followed by two times with water.
The organic layer was dried over Na2S04 and rotary
evaporated to an oil. Further drying under high vacuum
yielded 3Q~. (9.678, 100%).
1H-NMR (d4-MeOH): 8 1.45 (s, 36H), 2.69 (m, lOH), 3.23
(t, 2H), 3.37 (s, 8H), 5.06 (s, 2H), 7.33 (m, 5H).
13C-NMR (d4-MeOH): 8 28.64, 53.28, 53.88, 54.56, 58.59,
66.92, 67.54, 81.94, 129.07, 129.51, 138.35, 157.63,
158.89, 173.20.
Mass Spec.: Ionspray 969.6 (MH+)
Elemental Analysis for C42H72N12414 ~0-5H20: Theoretical
C, 51.57; H, 7.52: N, 17.18. Found C, 51.73;H, 7.52; N,
16.84.
FTIR: 3296, 2980, 1728, 1696, 1518, 1394, 1368, 1248,
1162, 1048, 1016, 874, 756, 698 cm 1.
Example 64
Compouad no.
(2.11 g, 2.18 manole) was hydrogenated at 35 psi in 50
ml MeOH for 2 hours. The reaction was filtered through
celite, rotary evaporated, and dried under high vacuum to
yield 105 as a foam ( 1.65 g, 92%).
1H-Nl~t (d4-MeOH): S 1.46 (s, 36H), 2.71 (m, 12H), 3.34
(s, 8H).
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13C_~ (d4_MeOH): 8 28.64, 34.77, 53.11, 53.91, 58.12,
81.90, 157.64, 172.88.
Mass Spec.: 2onspray 835.5 (MH+).
Elemental Analysis for C34H66N12012 ~1.OH20 ~l.OMeOH:
Theoretical C, 47.50; H, 8.20; N, 18.99. Found C,
47.41;H, 7.88; N, 18.74.
FTIR: 3292, 2980, 1720, 1690, 1484, 2368, 1248, 1162,
1048, 1016, 880, 773, 574 crri 1.
Exa~nx~ 1 a 6 5
Compound xio . ~3
A solution of 105 (1.03 g, 1.23 mmole) and malefic
anhydride (122 mg, 1.23 mmole) was stirred in 25 ml
CH2C12 for 2.5 hours. Solvents were removed by rotary
evaporation to yield 106 (1..16 g, 1000 .
1H-NMR (d4-MeOH): 8 1.45 (s, 36H), 3.13 (m, 4H), 3.45 (m
and s, 16H}, 3.68 (m, 2H), 6.17 (dd, 2H}.
Mass Spec.: Ionspray 933.6 (MH+), 955.5 (M+Na+).
~xa~ nle 66
Compound a.o . 4 4
106 (603 mg, 0.646 mmole) and EDCI (149 mg, 0.775 mmole)
were stirred in 25 ml dry CH2C12 under N2 for 2.5 hr. at
room temperature. The reaction was then extracted three
times with 25 ml saturated aqueous NaHC03 solution, then
once with 25 ml water_ The organic layer was dried over
Na2S04, rotary evaporated, and dried under high vacuum to
yield the isomaleimide intermediate (494 mg, 84~).
1H-NMR (CDC13): b 1.45 (s, 36H), 2.8 (m, 10H), 3.31 (s,
8H), 3.7 (m, 2H), 6.57 and 7.40 (dd, 2H).
This product was stirred with HOBt (35 mg, 0.259
mmole) in 8 ml DMF for 7 hours at room temperature.
Solvent was removed by rotary evaporation. The oily
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residue was dissolved in 60 ml Et20/EtOAc 1:1 and
extracted five times with 25 ml saturated aqueous NaHC03
solution, then once with 25 ml water. The organic layer
was dried over Na2S04, rotary evaporated, and dried under
high vacuum to yield the maleimide product ,~07 (463 mg,
94~ ) .
1H-NMR (CDCI3): 8 1.45 (s, 36H), 2.7 (m, 10H), 3.32 (s,
8H), 3.57 (m, 2H), 6.68 (s, 2H).
13C_~ (CDC13): 8 28.16, 81.73, 134.25, 155.5, 270.79.
Mass Spec.: Electrospray 915.5 (MH+), 937.5 (M+Na+).
FTIR: 3300, 2982, 1738, 1708, 1680 (sh), 1498, 1394,
1368, 1248, 1162, 1048, 1016, 72, 696 cm-1.
~xamgle 67
Compound no. 4 5
107 (214 mg, 0.234 mmole) was stirred With p-
toluenesulfonic acid (450 mg, 2.37 mmole) in 25 ml dry
CH2C12 under N2 for 3 hours. Solvent was removed by
rotary evaporation. The residue was triturated four times
with 125 ml Et20, then dried under high vacuum to yield
X08 (378 mg, 94~).
1H-NMR (d4-MeOH): 8 2.36 (s, 21H), 3.22 (t, 2H), 3.52 (m,
8H), 3.71 (s, 8H), 3.94 (t, 2H), 6.85 (s, 2H), 7.23 (d,
14H), 7.70 (d, 14H).
Mass Spec.: FAB 515.1 (MH+).
E~a~ns~le 68
Compound ao. 4 6
208 (100 mg, 58 umole) and Doxorubicin HC1 (177 mg, 305
umole) were stirred in 25 ml dry methanol for 24 hour.
The reaction was concentrated by rotary evaporation to 4
ml, then purified on Sephadex LH-20 (1" x 18") with
methanol. Fractions containing pure product were pooled,
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rotary evaporated, and dried under high vacuum to yield
1~f 9 (113 mg, 59~) .
1H-NMR (d4-MeOH): 8 1.2 (m, 12H), 3.9 (s, 12H), 6.8 (s,
2H), 7.2-8.0 (m) superimposed with 7.2 (d), and 7.7 (d)
total 24 H.
Example 69
Conjugate Synthesis
Thiolati.on:
Method A. On a scale __<3 g, (see Willner, D., Trail,
P.A., Hofstead, S.J., King, H.D., Lasch, Braslawsky,
G.R., Greenfield, R.S., Kaneko, T., Firestone, R.A.
(1993) (6-Maleimidocaproyl)-hydrazone of Doxorubicin:A
new derivative for the preparation of inununoconjugates of
Doxorubicin. I~iocon-iuaate Chem., 4, 521.) 2n typical
example, 1.54 g BR96 (180 ml at 53.4 uM, 9.5 umole) was
de-oxygenated by several cycles of alternating vacuum and
Ar atmosphere. This was then treated with 34 mM DTT (2.0
ml, 68.0 umole in Ar-bubbled PBS, pH 7.0) and stirred at
37°C under Ar for 3 hr. Removal of low molecular weight
compounds was accomplished by ultrafiltration against
PBS, pH 7.0 in an Amicon stirred cell at 4°C. A 400 ml
Amicon cell was fitted with an Amicon YM30 filter
(molecular weight cut-off 30,000), and charged to 40 psi
with Ar. Cell eluant was monitored for thiol content
with Ellman's reagent until a baseline reading at 412 nm
was obtained. Concentration of protein and thiol groups
were determined according to the previously reported
method. In this example, 1.47 g reduced BR96 (190 ml at
48.57 uM MAb, 412.7 uM thiol) was obtained, for a yield
of 95~ and a thiol titer of 8.5 mole thiol groups/mole
BR96.
Method B. On a scale >3 g, the same procedure was
utilized for the DTT reaction, with the exception that
the MAb solutions were de-oxygenated by bubbling with Ar.
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Purification after DTT reduction was accomplished by
ultrafiltration in a Filtron Minisette unit. The
Minisette was fitted with two Filtron 30K cassettes and
was connected to a Watson Marlow 604S pump with Bioprene
tubing. The MAb solution was ultrafiltered at 0°C under
Ar against Ar-bubbled PBS, pH 7.0 (eluant flow rate 100-
150 ml/min., 25 psi backpressure), while continually
monitoring eluant for thiol content as above. In a
typical example, a 6.6 g batch of BR96 (550 ml at 75.3
uM) yielded 6.1 g reduced BR96 (800 ml at 47.6 uM MAb,
398 uM thiol) for a yield of 92~ and thiol titer of 8.4
mole thiol groups/mole BR96.
Coxsjuyatios:
The following procedure, for the conjugation of BR96
and ~, is typical of that used for all linkers cited
herein. (See Riddles, P.W., Blakeley, R.L., Zerner, B.,
(1979) Ellman's reagent: 5,5'-Dithiobis(2-nitrobenzoic
acid)-A reexamination. Anal. Biochem., 94, 75.) To
reduced BR96 from Method A (125 ml, 6.07 umole MAb, 51.5
umole thiol) was added dropwise at 0°C under Ar a
solution of 2b (93 mg, 67.2 umole) in 5 m1 Ar-bubbled
H20. After stirring for 30 min., the reaction was
filtered through a 0.22u sterile filter. Conjugate was
purified at 4°C by percolation (approximately 2 ml/min.)
through a 1"x36" Bio-Beads column (initially prepared by
swelling and packing in methanol, then equilibrated in
H20, and finally PBS, pH 7.0}. The purified conjugate
was filtered again through a 0.22u sterile filter to
yield 155 ml of BR96-2b (BR96, 39.13 uM; DOX, 589.0 uM;
MR, 15.1 mole DOX/mole BR96; yield, 1000 . Conjugate was
frozen in liquid n2 and stored at -80°C.
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Exambla 70
Biolog3.cal Studses
Mater3.als and Methods
Monoclonal Antibodies and Immunoconjugates.
MAb BR64 {murine IgG1) and MAb BR96 (mouse/human
chimeric; human IgG1) identify Ley related tumor
associated antigens expressed on carcinomas of the lung,
colon, breast, and ovary. The MAbs are rapidly
internalized following antigen-specific binding
(Hellstrbm, I., Garrigues, H. J., Garrigues, U. and
Hellstrom, K. E. (1990). Highly tumor-reactive,
internalizing, mouse monoclonal antibodies to Lei'-related
cell surface antigen, Cancer Research 50, 2183-2190.
Trail et al., 1992; Trail et al., 1993; Willner, D.,
Trail, P.A., Hofstead, S.J., King, H.D., Lasch, S.J.,
Braslawsky, G.R., Greerifield, R.S., Kaneko, T. and
Firestone, R.A. (1993). (6-Maleimidocaproyl)-hydrazone of
doxorubicin - a new derivative for the preparation of
immunoconjugates of doxorubicin. Biocon~uaate Chem 4,
521-527). Doxorubicin immunoconjugates of various
DOX/MAb molar ratios were prepared with both chimeric
BR96 and control human IgG.
Tumor Cell Lines. L2987 is a human lung line which
expresses the BR64 and BR96 antigens. L2987 was obtained
from I. Hellstbm (Bristol-Myers Squibb, Seattle, WA).
In vitro cytotoxi.city assays. In vitro cytotoxicity
assays were performed as described previously (Trail et
al., 1992). Briefly, monolayer cultures of L2987 human
carcinoma cells were harvested using trypsin-EDTA {GIBCO,
Grand Island, NY), and the cells counted and resuspended
to 1 x 105/ml in RPMI-1640 containing 10~ heat
inactivated fetal calf serum (RPMI-10~FCS). Cells {0.1
ml/well) were added to each well of 96 well microtiter
plates and incubated overnight at 37°C in a humidified
atmosphere of 5~ C02. Media was removed from the plates
and serial dilutions of DOX or MAb-DOX conjugates added
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to the wells. All dilutions were performed in
quadruplicate. Cells were exposed to DOX or MAb-DOX
conjugates for various times (2h-48h as denoted in
results) at 37°C in a humidified atmosphere of 5$ C02.
Plates were then centrifuged (200 x g,5 min), the drug or
conjugate removed, and the cells washed 3x with RPMI-
10~FCS. The cells were cultured in RPMI-10~FCS (37°C, 5~
C02) for an additional 48 h. At this time the cells were
pulsed for 2 h with 1.0 uCi/well of [3H]thymidine New
England Nuclear, Boston, MA). The cells were harvested
onto glass fiber mats (Skatron Instruments, Inc.,
Sterling, VA), dried, and filter bound [3H]thymidine
radioactivity determined (f~-Plate scintillation counter,
Pharmacia LKB Biotechnology, Piscataway, NJ). Inhibition
of [3H]thymidine uptake was determined by comparing the
mean CPM for treated samples with that of the mean CPM of
the untreated control. In studies designed to evaluate
the stability of various linkers, cells were exposed to
BR96 or control IgG conjugates for varying periods of
time (2-48h) and the specificity ratio (IC50 IgG-DOX/IC50
BR96-DOX) calculated for the various exposure times.
Experimental An3.mals. Congenitally athymic female
mice of Balb/c background (Balb/c nu/nu; Harlan Sprague-
Dawley, Indianapolis, IN) were used in thse studies.
Mice were housed in Thoren caging units on sterile
bedding with controlled temperature and humidity.
Animals received sterile food and water ad libiturn.
Human Tumor Xeaograft Models . The L2987 human tumor
line was established as tumor xenografts in athymic mice
and maintained by serial passage as described previously
(Trail et al., 1992). L2987 tumors were measured in 2
perpendicular directions at weekly or biweekly intervals
using calipers. Tumor volume was calculated according to
the equation: V=2xw2/2 where: V=volume (mm3),
1=measurement of longest axis (mm), and w=measurement of
axis perpendicular to 1. In general, there were 8-10
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mice per control or treatment group. Data are presented
as median tumor size for control or treated groups.
Antitumor activity is expressed in'terms of median log
cell kill (LCK): where LCK = T-C/TVDT x 3.3. T-C is
defined as the median time -(days) for treated tumors to
reach 500mm3 size minus the median time for control
tumors to reach 500mm3 in size and TVDT is the time
(days) for control tumors to double in volune (250-
500mm3). Partial tumor regression reflects a decrease in
tumor volume to <_ 50~ of the initial tumor volume:
complete tumor regression refers to a tumor which for a
period of time is not palpable; and cure is defined as an
established tumor which is not palpable for a period of
time >_10 TVDT's.
Therapy. Treatments were administered by the ip or iv
route on various schedules as denoted. DOX was diluted
in normal saline and MAb and MAb-DOX conjugates were
diluted in PBS. All therapy was administered on a mg/kg
basis calculated for each animal and doses are presented
as mg/kg/injection. Control animals were not treated.
Doses of immunoconjugate are reported based on the drug
(equivalent DOX) and antibody content. The maximum
tolerated dose (MTD) for a treatment regimen is defined
as the highest dose on a given schedule which resulted in
< 20~ lethality.
Results:
Relationship between drug/MAb molar ratio and is
vitro potency of Linear and branched DOX hydrazone
conjugates
The relationship between conjugate molar ratio and
the in vitro potency of DOXHZN conjugates was reported
previously (Trail et al., 1992). In these studies BR64-
DOXHZN (disulfide linked) conjugates were prepared with
conjugate ratios ranging from 1-8. The in vitro potency
of the immunoconjugates varied over a 33 fold range (TC50
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values of 1-33 uM DOX) and potency was correlated with
conjugate molar ratio; conjugates of higher mole ratio
were significantly (p<0.05) more potentin vitro on both a
DOX and MAb basis than those conjugates prepared at lower
mole ratios. However, the number of DOX molecules which
can be directly linked to a given MAb without a
~ subsequent reduction in MAb binding affinity is limited.
For example, Shih et al., demonstrated a reduction in MAb
avidity and antigen-specific potency was as molar ratios
of directly linked DOX conjugates exceeded 10 (Shih,
L.B., Sharkey, R_M., Primus, F.J. and Goldenber, D.M.
(1988). Site-specific linkage of methotrexate to
monoclonal antibodies using an intermediate carrier.
International Journal of Cancer 41, 8320839; Shih et al.,
25 1991). Therefore, the use of branched linkers which
increase the drug/MAb molar ratio by a factor of 2n
(wherein n is a positive integer) without increasing the
number of conjugation sites on the MAb molecule was
employed.
As shown in Table 1, the conjugate molar ratios of the
various singly branched conjugates (i.e., 2n wherein n=1)
ranged from 11-16 and that of the doubly branched
conjugates (i.e., 2n wherein n=2) was 24. On an
individual lot basis (Table 1), the singly branched
DOXHZN conjugates were 2-20 fold (IC50 values of 0.1-1.0
uM equivalent DOX), and the doubly branched conjugates
(IC50 of 0.2uM) were 10 fold, more potent than the
straight chain DOXHZN conjugate BMS-182248 (2 uM DOX).
As used herein "BMS-182248" refers to the straight chain
conjugate as disclosed by
Trail,P.A.,Willner,D.,Lasch,S.J.,
Henderson,A.J.,Hofstead,S.J.,Casazza,A.M.,Firestone
R.A.,Hellstrom,K.E.(1993), Cure of xenografted human
carcinomas by BR96-Doxorubicin Immuno-conjugates, Science
261,212-215. Thus, increasing the concentration of DOX
delivered per BR96 MAb, by increasing the conjugate molar
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ratio (M.R.) resulted in a significant increase in the in
vitro potency of the conjugates. As shown in Table 2,
the mean in vitro potency of various single and double
branches conjugates was similar (0.2-0.5 uM DOX) and each
offered an in vitro potency advantage over that of HMS-
182248 on both a DOX and MAb basis.
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Table 1. Cytotoxicity of individual lots branched DOX
of
hydrazone conjugates to BMS-182248.
relative
F~campleCompound
Conjugate No. No. Lot M.R.
No. IC50(uM
DOX)
SMS-182248 pooled 2.0
date
8
MC-Glu-(13-Ala-DOX)229 ,3~ 33878-02013.9 0.2
' 1 0 MC-GLU-(DOX)4 40 ~ 33878-03124.0 0.2
33878-03424.4 0.2
MB-Glu-(DOX)2 14 ~ 33119-166a11.3 0.9
33878-13211.7 1.0
1 5 33878-13312.3 0.7
33878-13412.4 0.4
32178-18013.7 0_4
33119-164a14.1 0.5
33878-05216.2 0.1
2 ~ 33878-6015.0 0.6
MB-G1u-(f~-Ala-DOX)228 ~ 33878-06611.6 0_5
34616-5311.9 0.4
33878-05012.1 0.3
25
MC-G1u-(DOX)2 16 ~ 33878-05811.8 0.7
33878-06414.6 0.5
33878-14115.1 0.2
32178-17416.1 0
1
32252-19313.8 .
0.2
MP-Glu(DOX)2 13 ,~ 33878-12714.5 0.3
32178-18215.4 0_2
33878-12015.5 0.2
3 5 33878-11315.6 0.1
MB-D-Glu(DOX)2 15 D-~ 33119-19115.3 0.2
33119-19711.2 0.2
40
MP-Glu-(f3-Ala-DOX)227 ,~ 33878-17311.7 0.5
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97/23243
Table 2. In vitro otency branched
p and specificity
of
chain conjugates.
Co~ound MR IC5 0(~ IC50(uM Specificity
~) MAb)
Conjugate No. (range) (Mean) (Mean) ratios
BMS-182248 8 2.0 0.25 >5
MC-Glu-(13-Ala-DOX)2 14 0.2 0.01 NDb
3~
MC-GLu-(DOX)4 ~ 24 0.2 0.008 ND
MB-Glu-(DOX)2 ,2~ 11.3-16.20.5 0.04 >16
1 MB-Glu-(fS-Ala-DOX)2 11.6-12.10.4 0.03 >25
5 ~
MC-Glu-(DOX)2 ~ 11.8-16.10.3 0.02 31
MP-Glu-(DOX)2 ?~ 14.5-15.60.2 0.01 >40
MB-D-Glu-(DOX)2 L~~ 11.2-15.30.2 0.02 35
MP-Glu-(i~-Ala-DOX)2 11.7 0.5 0.04 >20
~
2 5 ~ Specificity Ratio defined as: IC50 IgG-DOX/IC50 BR96-DOX
b Not determined
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In vitro stability of Singly branched DOX
Conjugates
Among the characteristics desirable for efficacous
MAb-drug conjugates are linker chemistries which are
- 5 extremely stable in the extracellular environment yet
liberate drug efficiently upon internalization into
. antigen-expressing cells. One method for assessing
extracellular stability, and in part, intracellular
hydrolysis rates is to evaluate antigen-specific
cytotoxicity of binding relative to non-binding
conjugates over various exposure times. In these types
of experiments, extracellular stability will be reflected
by the lack of potency of non-binding immunoconjugates_
Rapid intracellular hydrolysis following antigen-specific
internalization will result in a high level of potency
which does not change significantly with increased
exposure time. Several experiments have been performed
with BR96-DOX conjugates prepared with linear or branched
linkers. In the following experiments, L2987 cells were
exposed to the various drug conjugates for 2, 8, 24 or 48
h and the IC50 values of both BR9& (binding) and IgG
(non-binding) conjugates determined. The results are
presented in Figures 1 and 2. As shown in Figure 1, the
MCDOXHZN (BMS-182248) conjugate was less potent than the
branched hydrazone, MB-Glu-(DOX)2; BMS-187852, conjugate
during the first 24 h of exposure. The potency of the
MCDOXHZN conjugate was increased over time whereas that
of the branched DOXHZN remained essentially unchanged
over 48h of exposure. These data suggest that the
intracellular rates of hydrolysis for the branched DOXHZN
conjugate was more rapid than that of the DOXHZN
conjugate.
The characteristic of extracellular stability was
evaluated by examining the kinetics of cell killing of
non-binding IgG conjugates prepared with. the different
linker chemistries. As shown in Figure 2, the potency of
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both the IgG conjugates prepared as straight chain
MCDOXHZN and branched chain MB-Glu-(DOX)2, hydrazone
conjugates increased with longer exposure times. The
increase in potency of non-binding conjugates likely
reflects cytotoxicity of DOX itself following release of
DOX from the conjugate over time. The potency of both
the linear and branched hydrazone conjugates increased in
parallel, suggesting that the extracellular stability of
these conjugates was quite similar. In summary, the BR96
branched hydrazone conjugates were more potent in vitro
at short exposure times than were the MCDOXHZN (BMS-
182248) conjugates. However, the extracellular stability
of the branched conjugates was not different from that of
the straight chain MCDOXHZN conjugate. Taken together,
these data suggest that the branched hydrazone offers a
potential advantage in the rate of intracellular release
of DOX, but does not offer an increase in extracellular
stability.
In vi.vo Biology of branched chain DOX hydrazoae
conjugates
To evaluate the effect on antitumor activity of
increasing the conjugate MR approximately 2 fold, BR96
and IgG conjugates were produced using six different
branched linkers and the conjugates evaluated for
antigen-specific activity in vivo against L2987 human
tumor xenografts.
The structure and substantial purity (in particular
lack of unconjugated drug) was established for each
conjugate, however, unidentified impurities were present.
In particular, a high MW aggregate, which is most likely
a dimeric form of the conjugate was present. Therefore,
antitumor activities of these branched chain conjugates
were compared with that of research. grade BMS-
182248;{BMS-182248{RG)).
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In the tables describing antitumor activity, the
optimal dose of BR96-DOX conjugates is defined as the
lowest dose administered which produced > 4 log cell kill
and >_70~ tumor regression. The antitumor activity of
~ 5 IgG-DOX conjugates at the maximum dose tested is included
for demonstration of antigen-specific activity.
1. BMS-187852; MB-Glu-(DOX)2
The molar ratio of the BMS-187852 conjugates varied
from 13.7-15. As shown in Table 3, 3 lots
of BMS-187852 were tested. The optimal dose for both
BMS-187852 and BMS-282248 was 2.5 mg/lcg DOX. However,
because of the doubling of the molar ratio of BMS-187852,
the branched conjugate was approximately 2 fold more
potent than BMS-182248(RG) on a MAb basis. The antitumor
activity of BMS-187852 was antigen-specific.
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Table 3. Antitumor ac tivityof BMS-187852; MB-Glu-(DOX)2
conjugates against established
L2987
tumors.
Molar Optimal Log ~TmmnrRPffYPRf7'IhTC
DOSe Cell
Antibody Lot# DQX Complete
Ratio Antibody Partial
Kill
BMS-182248 8 2.5 88 5 64 21
Research Gr.
1 0 BR96 33878-06015 2.5 45 >8 78 22
IgG 33878-05618 >10 >260 2.6 0 0
BR96 32178-18013.72.5 50 >6 100 0
IgG 32178-17815.2>5 >451.7 0 p
BR96 34616-16915_12.5 48 5.8 90 10
IgG 34616-17814.5>5 >950 0 0
2. BMS-187853; MB-Glu-(fS-Ala-DOX)2
Two lots of BMS-187853 conjugate (molar ratios
approximately 11.5) were evaluated against established
L2987 lung tumor xenografts. The antitumor activity of
the 2 lots was similar; both produced optimal antigen-
specific antitumor activity at doses of approximately 2.0
mg/kg DOX, 45 mg/kg BR96. Overall, these conjugates were
similar to BMS-182248(RG) on a DOX and 2 fold more-potent
on a MAb basis.
Table 4. Antitumor activity of BMS-187853;
MB-Glu-(fS-
Ala-DOX)2 conjugates against established L2987tumors.
Molar Optimal Dose ar ~s~o
Log Cell ~ Timor
R
Antibody Lot# Ratio DQX An tibody Kill CompletePartial
~7S-182248 8 2.5 88 5 64 21
Research Gr.
BR96 33878-066 11.6 2.5 44 >6_7 55 22
4 ~ IgG 33878-078 16.2 >10 >178 1.1 0 0
BR96 32878-158 11.5 2.0 46 5.2 50 20
IgG 32878-162 13.7 >5 >100 0 0 0
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3. BMS-188077; MC-GLU(DOX)2
The DOX/BR96 molar ratio of BMS-188077 conjugates
was in the range of 14.6-16.1. As shown in Table 5,
antigen-specific antitumor activity was observed for BMS-
- 5 188077. BMS-188077 was of similar potency as BMS-
182248(RG) on a DOX equivalent basis but due to the
increase in the molar ratio, approximately 2 fold more
potent on a MAb basis.
Table 5. Antitumor activity of BMS-188077;MC-Glu-(DOX)2
conjugates against established L2987 tumors.
Molar Optimal Dose Log Cell ~ 'm_mor Reare~R~ons
Antibody Lot# Ratio DOX Antibody Kill Complete Partial
BMS-182248 8 2.5 88 5 64 21
Research
Gr_
BR96 33878-06414.6 2.5 48 4.6 30 70
2 IgG 33878-05416.2 >10 >163 1.7 0 0
0
SR96 32178-17416.1 2_5 42 >4 87.5 12.5
IgG 32178-17612.2 >5 >114 0.7 0 0
2 BR96 33878-14115.1 2.5 45 >6 75 25
5
IgG 33878-14615.5 >5.0 84 0.8 0 0
30 4. BMS-189099; MP-Glu-(DOX)2
Three lots of BMS-189099 conjugates were evaluated
in parallel with non-binding IgG conjugates (BMS-188078)
produced with the same linker chemistry. The mole ratios
of the BR96 conjugates were in the range of 14.5-15.5.
35 The antitumor activity of BMS-189099 and non-binding
conjugates is presented in Table 6. Antigen-specific
antitumor activity was observed in vivo. The BMS-189099
conjugates were of similar potency as BMS-182248(RG) on a
DOX basis but approximately 2 fold more potent on a MAb
40 basis.
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Table 6. Antitumor activity of BMS-189099
(MP-Glu-
(DOX)2) conjugates against established L2987
tumors_
Molar Optimal Log ~ '1'i~mnrC(r aaiOria
Dose Cell
Antibody Lot# Ra tio DoX Antibody CompletePartial
Kill
BMS-182248 8 2.5 88 5 64 21
Research .
Gr
1 0 BR96 33878-120 15.5 2.5 44 >7.6 90 10
IgG 33878-118 15_9 >5 >79 0.3 0 0
BR96 32178-182 15.35 1.25 >6.3 50 25
23
IgG 32178-184 15.91 >5 >86 1.9 0 0
BR96 33878-127 14.5 2.5 48 >4 80 20
IgG 33878-125 14.7 >5 >95 0.9 0 0
5. BMS-189812; MB-[D]-GLU(DOX)2
The molar ratios of the BMS-189812 conjugates were
in the range of 11-15 moles DOX/moles BR96_ Data for the
antitumor activity of BMS-189812 is summarized in Table
7. The optimal dose of BMS-189812 was approximately 2
mg/kg DOX, 50 mg/kg BR96. The potency on a DOX basis was
similar to BMS-182248 (RG) and the conjugate was two fold
more potent on a MAb basis.
Table 7. Antitumor activity of BMS-189812; MB-[D]-Glu-
(DOX)2 conjugates against established L2987 tumors.
Molar optimal Dose Log Cell ~ m"rnor uparPCs;o s
Antibody Lot# Ratio DoX Antibody Kill Complete Partial
BMS-182248 8 2.5 88 5 64 21
Research Gr.
BR96 33119-191 15.3 2.5 45 >5 75 25
4 a IgG 33119-189 18 >5 >89 0.8 0 0
BR96 32119-197 11.2 1.0 27 5.2 20 50
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6. BMS-190385; MB-Glu-(f~-Ala-DOX)2 conjugates
The BMS-190385 conjugates demonstrated antigen-
specific activity in vivo. The antitumor activity of BMS-
190385 conjugates is presented in Table 8. As shown two
' 5 lots of BR96-DOX conjugate are currently being evaluated
against established L2987 lung xenografts. Antigen-
specific antitumor activity was observed. Although the
data is~still developing, it appears that the optimal
dose of thse conjugates is 2 mg/kg DOX, 60 mg/kg BR96.
This is similar to that of BMS-182248 on a DOX basis and
slightly more potent on a MAb basis.
Table 8. Antitumor activity of BMS-190385; MB-Glu-(~~-
Ala-(DOX)2 conjugates against established L2987 tumors.
~ 5 Molar Optimal Dose Log Cell ~ tumor R m- s gone
Antibody Lot# Ratio DOX Antibody Kill Complete Partial
BMS-182248 8 2.5 88 5 64 21
2 0 Research Gr.
BR96 34616-24 11 5 2.0 60 >4 60 40
IgG 34616-29 12.6 >5.0 >108 2.7 0 0
2 5 BR96 35255-2 12_14 2.5 56 5.5 44 56
IgG 33119-199 14.7 >5.0 >92 0.7 0 0
30 Summary of branched chain DOXHZN conjugates
The branched chain DOXHZN conjugates evaluated
herein typically had molar ratios in the range of 11-15.
This is 1.5-1.8 fold higher than the molar ratio
typically observed for BMS-182248. all of the conjugates
35 evaluated demonstrated antigen-specific activity both in
vitro and in vivo. Among the various branched chain
conjugates, there were no significant differences in
either in vitro (Table 2) or in vivo (Table 9) potency.
When evaluated in vitro, the branched conjugates offered
40 an increase in potency on both a DOX and a MAb basis.
This likely reflects the fact that conjugates were
assayed using a 2h exposure and as shown in Figure 1, the
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branched conjugates appear to release DOX more rapidly
than the straight chain MCDOXHZN conjugate following
antigen-specific internalization. The dose of equivalent
DOX which produced >_4 log cell kill and >_70~ tumor
regressions was the same for both the branched chain
DOXHZN and single chain DOXHZN (BMS-182248) conjugates
(Summarized in Table 9). However, because the molar
ratio of the branched chain conjugates was increased by
1.5-1.8 fold over that of BMS-182248, these conjugates
were approximately 2 fold more potent than BMS-182248 on
a MAb basis.
Table 9. Antitumor activity of optimal doses of branched
chain DOXHZN conjugates against lung
established
L2987
tumor xenografts.
Compound MolarsOptimal Log ~ Wmo-r rees;onsa
Doses Cell$ Re~
no. Conjugate Ratio DQXAntibodyKill Complete
Partial
~NtS-1822488 2.588 5 64 21
MB-Glu-(DQX)214.4 2.547_5 >6 89_0 11.0
2 ~ r~-cLU-
5 ( t~-
Ala-DOX)2 11.55 3.7579.5 >5 52.5 21.0
MC-Glu-(DOX)215.27 2.545.0 >4 64.2 35.8
3 ~ MP-
0
Glu-(DOX)2 15.1 2.138.3 >4 73.3 18.3
-
D-~ MiS- [D
] -
Glu-(DOX)2 13.25 2.2550 >5 47.5 37.5
35
MP-Glu-
(i3-Ala-DOX)211.82 2.2558 >4 52.0 48.0
$ Means
40
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