CA2239183C - Branched hydrazone linkers - Google Patents
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- CA2239183C CA2239183C CA002239183A CA2239183A CA2239183C CA 2239183 C CA2239183 C CA 2239183C CA 002239183 A CA002239183 A CA 002239183A CA 2239183 A CA2239183 A CA 2239183A CA 2239183 C CA2239183 C CA 2239183C
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Abstract
Branched hydrazone linkers for linking a targeting ligand such as an antibody to a therapeutically active drug. The point of branching is at a polyvalent atom and the number of drugs increases by a factor of two for each generation of branching. A preferred drug is doxorubicin.
Description
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 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, ,
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 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-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
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
-3-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 WO 97!23243 PCT/US96120513 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.
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 WO 97!23243 PCT/US96120513 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.
-5-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
or a moiety of the formula 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
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
or a moiety of the formula 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
-6-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
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
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
'(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
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
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
-7--NH-NH2 or O
li -NHNHC-NHNH2 .
In another preferred embodiment the novel branched linker of the invention has the formula H
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 Preferred branched linkers of formula II are where d is 2, f is 0, g is l, and/or b is 0.
_g_ 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~
O N[(CH2)mC0-X~2 _g_ WO 97!23243 PCT/US96/20513 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;
WO 97/23243 PCTlUS96/Z05I3 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, 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 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 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 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
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 , 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.
-1fi-W097IZ3243 CA 02239183 2005-02-22 pCT~S96/205I3 The most preferred spacer has the formula H
--N
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.
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
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
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 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
Rs Rs (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 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.
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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 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 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 "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 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_ 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.
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.
Goldman et al., Pediatrics, 105:252, 1984.
Glioma BF7, GE2, CG12 N. de Tribolet stet al., in loc. cit. p. 81.
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 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, 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 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 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.
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 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$
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 .
O O
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
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
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
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 $
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
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
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$
WO 97/23243 PCT/US96/ZOSi3 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
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
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
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
SCHEME VI1. SYNTHESIS OF 4~C
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 ~,~.~/~
~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
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.
~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.
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 WO 97fZ3243 PCTlUS96l205i3 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 $x~ rile 3 Glutamyldi(Boc)hydrazide (Compouad ao. 5) Z-Glutamyldi(Boc)hydrazide (4_) (19.59 g, 38.44 rmnole) was hydrogenated along with 2g 10~ Pd-C in 200 ml MeOH at 50 psi for 3 hr. The reaction was filtered through Celite*and rotary evaporated. The resulting foam was dried under high vacuum to yield 5_ (14.448, 100$).
* Trade-mark 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 _48_ * Trade-mark 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 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~) .
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)+
WO 97!23243 PCT/US96/20513 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)+
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.
~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~) .
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 (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.
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 _57_ * Trade-mark 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 * Trade-mark WO 97!23243 PCT/US96/20513 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.
WO 97/23243 PCT/US96l20513 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.
WO 97/23243 PCT/LTS96/ZOSi3 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}+.
WO 97/23243 PCT/US96/2fl513 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 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.
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 W~ 9~~3~3 CA 02239183 2005-02-22 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.
-fi5-* Trade-mark 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 WO 97/23243 PCTlUS96/20513 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 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.
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).
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_ WO 97/23243 PCT/US9b/205I3 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.
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+).
* Trade-mark WO 97/23243 PCTlUS96/20513 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~).
WO 97123243 PCTlUS96/20513 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.
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
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 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~).
* Trade-mark 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).
_79_ WO 9?/23243 PCTJUS96/20513 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_ 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).
* Trade-mark WO 97/23243 PCTlUS96/20513 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 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, WO 97/23243 PCTlLTS96/205I3 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.
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.
WO 97!23243 PCT/LTS96/20513 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 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 _87_ WO 97/23243 PCT/US96/205i3 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
li -NHNHC-NHNH2 .
In another preferred embodiment the novel branched linker of the invention has the formula H
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 Preferred branched linkers of formula II are where d is 2, f is 0, g is l, and/or b is 0.
_g_ 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~
O N[(CH2)mC0-X~2 _g_ WO 97!23243 PCT/US96/20513 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;
WO 97/23243 PCTlUS96/Z05I3 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, 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 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 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 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
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 , 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.
-1fi-W097IZ3243 CA 02239183 2005-02-22 pCT~S96/205I3 The most preferred spacer has the formula H
--N
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.
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
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
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 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
Rs Rs (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 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.
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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 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 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 "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 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_ 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.
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.
Goldman et al., Pediatrics, 105:252, 1984.
Glioma BF7, GE2, CG12 N. de Tribolet stet al., in loc. cit. p. 81.
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 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, 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 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 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.
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 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$
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 .
O O
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
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
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
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 $
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
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
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$
WO 97/23243 PCT/US96/ZOSi3 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
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
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
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
SCHEME VI1. SYNTHESIS OF 4~C
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 ~,~.~/~
~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
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.
~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.
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 WO 97fZ3243 PCTlUS96l205i3 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 $x~ rile 3 Glutamyldi(Boc)hydrazide (Compouad ao. 5) Z-Glutamyldi(Boc)hydrazide (4_) (19.59 g, 38.44 rmnole) was hydrogenated along with 2g 10~ Pd-C in 200 ml MeOH at 50 psi for 3 hr. The reaction was filtered through Celite*and rotary evaporated. The resulting foam was dried under high vacuum to yield 5_ (14.448, 100$).
* Trade-mark 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 _48_ * Trade-mark 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 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~) .
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)+
WO 97!23243 PCT/US96/20513 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)+
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.
~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~) .
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 (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.
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 _57_ * Trade-mark 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 * Trade-mark WO 97!23243 PCT/US96/20513 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.
WO 97/23243 PCT/US96l20513 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.
WO 97/23243 PCT/LTS96/ZOSi3 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}+.
WO 97/23243 PCT/US96/2fl513 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 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.
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 W~ 9~~3~3 CA 02239183 2005-02-22 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.
-fi5-* Trade-mark 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 WO 97/23243 PCTlUS96/20513 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 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.
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).
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_ WO 97/23243 PCT/US9b/205I3 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.
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+).
* Trade-mark WO 97/23243 PCTlUS96/20513 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~).
WO 97123243 PCTlUS96/20513 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.
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
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 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~).
* Trade-mark 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).
_79_ WO 9?/23243 PCTJUS96/20513 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_ 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).
* Trade-mark WO 97/23243 PCTlUS96/20513 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 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, WO 97/23243 PCTlLTS96/205I3 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.
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.
WO 97!23243 PCT/LTS96/20513 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 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 _87_ WO 97/23243 PCT/US96/205i3 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
-8$-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 _89_ 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.
-90_ WO 97123243 PG"T/US96I205I3 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 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 MC-G1u-(DOX)2 16 ~ 33878-05811.8 0.7 33878-06414.6 0.5 33878-14115.1 0.2 32178-17416.1 0 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 MP-Glu-(f3-Ala-DOX)227 ,~ 33878-17311.7 0.5 _9j_ 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 _92_ 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 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)).
WO 97!23243 PCT/US96l205I3 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.
_g5_ Table 3. Antitumor ac tivityof BMS-187852; MB-Glu-(DOX)2 conjugates against established 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 -96_ WO 97/23243 PCT/fJS96JZO5I3 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 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 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.
_g7_ 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 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 -s$-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 _99_ 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 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-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 MP-Glu-(i3-Ala-DOX)211.82 2.2558 >4 52.0 48.0 $ Means
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 _89_ 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.
-90_ WO 97123243 PG"T/US96I205I3 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 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 MC-G1u-(DOX)2 16 ~ 33878-05811.8 0.7 33878-06414.6 0.5 33878-14115.1 0.2 32178-17416.1 0 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 MP-Glu-(f3-Ala-DOX)227 ,~ 33878-17311.7 0.5 _9j_ 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 _92_ 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 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)).
WO 97!23243 PCT/US96l205I3 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.
_g5_ Table 3. Antitumor ac tivityof BMS-187852; MB-Glu-(DOX)2 conjugates against established 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 -96_ WO 97/23243 PCT/fJS96JZO5I3 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 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 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.
_g7_ 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 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 -s$-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 _99_ 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 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-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 MP-Glu-(i3-Ala-DOX)211.82 2.2558 >4 52.0 48.0 $ Means
Claims (77)
1. 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 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 acylhydrazone bonds with aldehyde or keto groups derived from a drug moiety, wherein the polyvalent atom is carbon or nitrogen.
2. The branched linker of Claim 1 wherein n is 1, 2, 3, or 4.
3. The branched linker of Claim 1 wherein n is 1, 2, or 3.
4. The branched linker of Claim 1 wherein n is 1 or 2.
5. The branched linker of Claim 1 linked to a targeting ligand which is an antibody or fragment thereof.
6. The branched linker of Claim 1 linked to drug moieties which are anthracyclines.
7. The branched linker of Claim 1 wherein n is 1 or 2, said polyvalent atom is carbon or nitrogen, said targeting ligand is an antibody or fragment thereof, and said drug moieties are anthracyclines.
8. The branched linker of Claim 1 having the formula 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 or a moiety of the formula wherein W, a, b and m are as defined hereinbefore, and X1 is a moiety -of the formula -NH-NH2 or or a moiety of the formula wherein W, a, b, and m are defined hereinbefore, and X2 is a moiety of the formula NH-NH2 or or a moiety of the formula wherein W, a, b, and m are as defined hereinbefore, and X3 is a moiety of the formula -NH-NH2 or or a moiety of the formula wherein W, a, b and m are as defined hereinbefore, and X4 is a moiety of the formula -NH-NH2 or
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 or a moiety of the formula wherein W, a, b and m are as defined hereinbefore, and X1 is a moiety -of the formula -NH-NH2 or or a moiety of the formula wherein W, a, b, and m are defined hereinbefore, and X2 is a moiety of the formula NH-NH2 or or a moiety of the formula wherein W, a, b, and m are as defined hereinbefore, and X3 is a moiety of the formula -NH-NH2 or or a moiety of the formula wherein W, a, b and m are as defined hereinbefore, and X4 is a moiety of the formula -NH-NH2 or
9. The branched linker of Claim 8 wherein X is -NH-NH2 or
10. The branched linker of Claim 8 wherein X1 is -NH-NH2 or
11. The branched linker of Claim a wherein A is a Micheal Addition acceptor.
12. The branched linker of Claim 8 wherein W is of the formula wherein g is an integer of 1 to 6
13. The branched linker of Claim 8 wherein W is of the formula
14. The branched linker of Claim 8 wherein b is 0 and a is 2.
15. The branched linker of Claim 13 wherein b is 0 and a is 2.
16. The branched linker of Claim 8 wherein Q is of the formula -(CH2)f-(Z)g-(CH2)h-wherein f is an integer of 1 to 10, h is an integer of 1 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 S, O, NH, SO2, 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 O, N, or S.
17. The branched linker of Claim 16 wherein f is 1 or 2, h is 1 or 2, g is 1, and Z is phenyl, pyridyl, or a cycloaliphatic hydrocarbon ring containing 3 to 6 carbon atoms.
18. The branched linker of Claim 17 wherein Z is phenyl or cyclohexyl.
19. The branched linker of Claim 16 wherein g is 0 and f + h is an integer of 1 to 4.
20. The branched linker of Claim 16 wherein g is 0 and f + h is an integer of 2.
21. A linker/drug having the formula of Claim 8 wherein X is -NH-N=Drug, or
22. The linker/drug of Claim 21 wherein said Drug is an anthracycline antibiotic.
23. The linker/drug of Claim 22 wherein said anthracycline antibiotic is of the formula in which R1 is -CH3. -CH2OH, -CH2OCO(CH2)3CH3 or -CH2OCOCH(OC2H5)2;
R3 is -OCH3, -OH or hydrogen;
R4 is -NH2, -NHCOCF3, 4-morpholinyl, 3-cyano-4-morpholinyl, 1-piperidinyl, 4-methoxy-1-piperidinyl, benzylamine, dibenzylamine, cyanomethyl amine or 1-cyano-2-methoxyethyl amine;
R5 is -OH, -OTHP or hydrogen; and R6 is -OH or hydrogen, provided that R6 is not -OH
when R5 is -OH or -OTHP.
R3 is -OCH3, -OH or hydrogen;
R4 is -NH2, -NHCOCF3, 4-morpholinyl, 3-cyano-4-morpholinyl, 1-piperidinyl, 4-methoxy-1-piperidinyl, benzylamine, dibenzylamine, cyanomethyl amine or 1-cyano-2-methoxyethyl amine;
R5 is -OH, -OTHP or hydrogen; and R6 is -OH or hydrogen, provided that R6 is not -OH
when R5 is -OH or -OTHP.
24. A linker/drug of the formula 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.
25. The linker/drug of Claim 24 wherein X5 is of the formula wherein R3 is -OCH3, -OH, or hydrogen.
26. The linker/drug of Claim 25 wherein m is 0 and n is 2 or 3.
27. A linker/drug of the formula wherei n n is an integer of 1 to 6;
a is an integer of 0, 2, 2, or 3, m is an integer of 0 or 1, and X5 is an anthracycline antibiotic.
a is an integer of 0, 2, 2, or 3, m is an integer of 0 or 1, and X5 is an anthracycline antibiotic.
28. The linker/drug of Claim 27 wherein X5 is wherein R3 is -OCH3, -OH, or hydrogen.
29. The linker/drug of Claim 28 wherein m is 0 and n is 2 or 3.
30. The branched linker of Claim 1 having the formula.
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;
A is a thiol acceptor;
T is of the formula wherein d is an integer of 2 to 6, m is an integer of 1 or 2, b is an integer of 0 or 1, f 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
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;
A is a thiol acceptor;
T is of the formula wherein d is an integer of 2 to 6, m is an integer of 1 or 2, b is an integer of 0 or 1, f 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
31. The branched linker of Claim 30 wherein A is a Michael Addition acceptor.
32. The branched linker of Claim 30 wherein T is of the formula
33. The branched linker of Claim 30 wherein T is of the formula
34. The branched linker of Claim 30 wherein d is 2, f is 0, and g is 1.
35. The branched linker of Claim 30 having the formula
36. The branched linker of Claim 30 having the formula
37. The branched linker of Claim 30 having the formula
38. The branched linker of Claim 30 having the formula
39. The branched linker of Claim 30 having the formula
40. A conjugate having the formula wherein A is a thiol adduct, W is a spacer moiety, Q is a bridging group, m is an integer of 0 or 1, 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, Y is O or NH2+Cl-, z is an integer of 0 or 1, 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 or a moiety of the formula wherein W, a, b and m are as defined hereinbefore, and X1 is a moiety of the formula -NH-N=Drug, or or a moiety of the formula wherein W, a, b and m are as defined hereinbefore, and X2 is a moiety of the formula -NH-N=Drug, or or a moiety of the formula wherein W, a, and m are defined hereinbefore, and X3 is a moiety of the formula -NH-N=Drug, or or a moiety of the formula wherein W a, b, and m are defined hereinbefore, and X4 is a moiety of the formula -NH-N=Drug or
41. The conjugate of Claim 40 wherein X is -NH-N=Drug or
42. The conjugate of Claim 40 wherein X1 is -NH-N=Drug or
43. The conjugate of Claim 40 wherein A is a Micheal Addition adduct.
44. The conjugate of Claim 40 wherein W is of the formula wherein g is an integer of 1 to 6.
45. The conjugate of Claim 40 wherein W is of the formula
46. The conjugate of Claim 40 wherein b is 0 and a is 2.
47. The conjugate of Claim 40 wherein Q is of the formula - (CH2)f-(Z)g - (CH2)h-wherein f is an integer of 1 to 10, h is an integer of 1 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 S, O, NH, SO2, 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 O, N, or S.
48. The conjugate of Claim 47 wherein f is 1 or 2, h is 1 or 2, g is 1, and Z is phenyl, pyridyl, or a cycloaliphatic hydrocarbon ring containing 3 to 6 carbon atoms.
49. The conjugate of Claim 48 wherein Z is phenyl or cyclohexyl.
50. The conjugate of Claim 47 wherein g is 0 and f + h is an integer of 1 to 4.
51. The conjugate of Claim 47 wherein g is 0 and f + h is an integer of 2.
52. The conjugate of Claim 40 wherein said Drug is an anthracycline antibiotic.
53. The conjugate of Claim 52 wherein said anthracycline antibiotic is of the formula in which R1 is -CH3, -CH2OH, -CH2OCO(CH2)3CH3 or -CH2OCOCH(OC2H5)2:
R3 is -OCH3, -OH or hydrogen;
R4 is -NH2, -NHCOCF3, 4-morpholinyl, 3-cyano-4-morpholinyl, 1-piperidinyl, 4-methoxy-1-piperidinyl, benzylamine, dibenzylamine, cyanomethyl amine or 1-cyano-2-methoxyethyl amine;
R5 is -OH, -OTHP or hydrogen; and R6 is -OH or hydrogen, provided that R6 is not -OH
when R5 is -OH or -OTHP.
R3 is -OCH3, -OH or hydrogen;
R4 is -NH2, -NHCOCF3, 4-morpholinyl, 3-cyano-4-morpholinyl, 1-piperidinyl, 4-methoxy-1-piperidinyl, benzylamine, dibenzylamine, cyanomethyl amine or 1-cyano-2-methoxyethyl amine;
R5 is -OH, -OTHP or hydrogen; and R6 is -OH or hydrogen, provided that R6 is not -OH
when R5 is -OH or -OTHP.
54. The conjugate of Claim 40 wherein G is an immunoglobulin or a fragment thereof.
55. The conjugate of Claim 54 in which G is an immunoglobulin selected from the group consisting of BR96, BR64, L6, a relaxed BR96, a relaxed BR64, a relaxed L6, a chimeric BR96, a chimeric BR64, a chimeric L6, a relaxed chimeric BR96, a relaxed chimeric BR64, a relaxed chimeric L6; and, fragments thereof.
56. The conjugate of Claim 55 in which G is a chimeric BR96, a relaxed chimeric BR96; or a fragment thereof.
57. The conjugate of Claim 40 in which G is a ligand selected from the group consisting of bombesin, EGF, transferrin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, TGF-.alpha., TGF-.beta., VGF, insulin and insulin-like growth factors I and II.
58. The conjugate of Claim 57 in which G is bombesin.
59. The conjugate of Claim 40 in which G is a ligand selected from the group consisting of carbohydrates, steroids and lectins.
60. A conjugate having the formula 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 6, c is an integer of 0 or 1, p is an integer of 1 to 6, Y is O or NH2+Cl-, 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 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
61. The conjugate of Claim 60 wherein A is a Michael Addition adduct.
62. The conjugate of Claim 60 wherein T is of the formula
63. The conjugate of Claim 60 wherein T is of the formula
64. The conjugate of Claim 60 wherein d is 2, f is 0, and g is 1.
65. The conjugate of Claim 60 wherein said Drug is an anthracycline antibiotic.
66. The conjugate of Claim 65 wherein said anthracycline antibiotic is of the formula in which R1 is -CH3, -CH2OH, -CH2OCO(CH2)3CH3 or -CH2OCOCH(OC2H5)2;
R3 is -OCH3, -OH or hydrogen;
R4 is -NH2, -NHCOCF3, 4-morpholinyl, 3-cyano-4-morpholinyl, 1-piperidinyl, 4-methoxy-1-piperidinyl, benzylamine, dibenzylamine, cyanomethyl amine or 1-cyano-2-methoxyethyl amine;
R5 is -OH, -OTHP or hydrogen; and R6 is -OH or hydrogen, provided that R6 is not -OH
when R5 is -OH ar -OTHP.
R3 is -OCH3, -OH or hydrogen;
R4 is -NH2, -NHCOCF3, 4-morpholinyl, 3-cyano-4-morpholinyl, 1-piperidinyl, 4-methoxy-1-piperidinyl, benzylamine, dibenzylamine, cyanomethyl amine or 1-cyano-2-methoxyethyl amine;
R5 is -OH, -OTHP or hydrogen; and R6 is -OH or hydrogen, provided that R6 is not -OH
when R5 is -OH ar -OTHP.
67. The conjugate of Claim 66 wherein said anthracycline antibiotic is of the formula wherein R3 is -OCH3, -OH, or hydrogen.
68. The conjugate of Claim 60 wherein G is an immunoglobulin or a fragment thereof.
69. The conjugate of Claim 68 in which G is an immunoglobulin selected from the group consisting of BR96, BR64, L6, a relaxed BR96, a relaxed BR64, a relaxed L6, a chimeric BR96, a chimeric BR64, a chimeric L6, a relaxed chimeric BR96, a relaxed chimeric BR64, a relaxed chimeric L6; and, fragments thereof.
70. The conjugate of Claim 69 in which G is a chimeric BR96, a relaxed chimeric BR96; or a fragment thereof.
71. The conjugate of Claim 60 in which G is a ligand selected from the group consisting of bombesin, EGF, transferrin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, TGF-.alpha., TGF-.beta., VGF, insulin and insulin-like growth factors I and II.
72. The conjugate of Claim 71 in which G is bombesin.
73. The compound of Claim 60 in which G is a ligand selected from the group consisting of carbohydrates, steroids and lectins.
74. Use of a pharmaceutically effective amount of one or more conjugates according to Claim 40 or Claim 60 to kill a selected cell population wherein the targeting ligand is reactive With said selected cell population.
75. A pharmaceutically acceptable composition useful in the treatment of disease which comprises a pharmaceutically effective amount of at least one conjugate according to Claim 40 or Claim 60 and a pharmaceutically acceptable carrier.
76. Use of a pharmaceutically effective amount of at least one conjugate according to Claim 40 or Claim 60 for treating mammalian diseases selected from the group consisting of cancers, non-malignant tumors, non-cytocidal viral or pathogenic infections and autoimmune disorders.
77. Use of a pharmaceutically effective amount of at least one conjugate according to Claim 40 or Claim 60 for treating mammalian tumors.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US910095P | 1995-12-22 | 1995-12-22 | |
| US60/009,100 | 1995-12-22 | ||
| PCT/US1996/020513 WO1997023243A1 (en) | 1995-12-22 | 1996-12-17 | Branched hydrazone linkers |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2239183A1 CA2239183A1 (en) | 1997-07-03 |
| CA2239183C true CA2239183C (en) | 2006-08-29 |
Family
ID=29405601
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002239183A Expired - Lifetime CA2239183C (en) | 1995-12-22 | 1996-12-17 | Branched hydrazone linkers |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2239183C (en) |
-
1996
- 1996-12-17 CA CA002239183A patent/CA2239183C/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| CA2239183A1 (en) | 1997-07-03 |
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