Doxorubicin immunoconjugates containing bivalent, lysosomally-Cleavable dipeptide linkages

Article abstract of DOI:10.1016/S0960-894X(02)00194-4

Bivalent doxorubicin (DOX)-dipeptides (16a-c) were prepared and conjugated to the monoclonal antibody BR96. The dipeptides are cleaved by lysosomal proteases following internalization of the resulting immunoconjugates. Conjugate 18b demonstrated antigen-s

Full text of DOI:10.1016/S0960-894X(02)00194-4

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1529–1532
Doxorubicin Immunoconjugates Containing Bivalent,
Lysosomally-Cleavable Dipeptide Linkages
Gene M. Dubowchik,a,* Shilpa Radia,a Harold Mastalerz,a Michael A. Walker,a
Raymond A. Firestone,a,y H. Dalton King,a Sandra J. Hofstead,a David Willner,a
Shirley J. Laschb and Pamela A. Trailb,{
aBristol-Myers Squibb Pharmaceutical Research Institute, PO Box 5100, Wallingford, CT 06492-7660, USA
bBristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543-4000, USA
Received 13 November 2001; accepted 15 February 2002
Abstract—Bivalent doxorubicin (DOX)-dipeptides (16a–c) were prepared and conjugated to the monoclonal antibody BR96. The
dipeptides are cleaved by lysosomal proteases following internalization of the resulting immunoconjugates. Conjugate 18b demon-
strated antigen-specific in vitro tumor cell killing activity (IC50=0.2 mM) that was equipotent to DOX with a near doubling of drug
molecules/MAb. Size exclusion chromatography showed 18b to be a noncovalent dimer that was formed immediately upon con-
jugation. # 2002 Elsevier Science Ltd. All rights reserved.
The success of antigen-dependent drug delivery to target
tumor cell populations can be limited by the cell surface
density of expressed antigen by saturation of the recep-
tors with immunoconjugate that is endocytosed at a
given rate.1 In this regard, the more drug molecules an
immunoconjugate is able to carry without compromis-
ing tumor localization, drug release, and aqueous solu-
bility the better. One way of increasing drug loading
without coating the surface of a monoclonal antibody
(MAb) with (usually lipophilic) drug moieties is to use
polyvalent linkers in conjunction with carefully con-
trolled conjugation methods. Herein we report the
synthesis of branched drug-dipeptide linker compounds
that can be conjugated to free thiol groups generated on
MAbs by treatment with limiting amounts of dithio-
threitol (DTT).2 The dipeptides are cleaved by lysoso-
mal proteases following internalization of the resulting
immunoconjugates.3 The liberation of active drug by
these dipetide conjugates requires the presence of a self-
immolative p-aminobenzyloxycarbonyl (PABC) spacer,4
presumably because of steric constraints in the enzyme
active site.3
Design and Synthesis
To make the linkers as hydrophilic as possible, we
wanted to minimize their size while ensuring that the
site of desired proteolytic cleavage was unencumbered
so that liberation of free drug could occur as rapidly as
with single-chain dipeptide linkers. Previous hydrazone-
containing branched linkers used glutamic acid as the
branching device.5 However, to generate symmetrical
linkers and to avoid the issue of chirality we chose to
use iminodiacetyl (IDA) and iminodipropionyl (IDP)
units in order to vary the distance between the branch
point and the dipeptide. The maleimidoethyl-containing
intermediates 7a and 7b were prepared as shown in
Scheme 1. Mono-protected ethylenediamine 3 was pre-
pared in 55% overall yield by treatment with 2 equiv of
benzyloxycarbonyl chloride followed by partial acid-
olysis in concd HCl/acetic acid.6 Alkylation using either
t-butyl bromoacetate or t-butyl acrylate, followed by
hydrogenolysis in the presence of acetic acid gave the
sensitive amino diesters 5a and 5b. These were immedi-
ately treated with maleic anhydride followed by
TMSCl-mediated cyclization to give the mal-
eimidoesters 6a and 6b in ca. 60% yield. Significant
decomposition occurred when deprotection was
attempted with TFA or HCl in dioxane. However,
overnight stirring with 3 equiv of p-toluenesulfonic acid
(TsOH) in CH2Cl2 provided the diacid ammonium salts
7a and 7b. In each case, a solid precipitate was formed
*Corresponding author. Fax: +1-203-677-7702;
e-mail: gene.dubowchik@bms.com
yPresent address: Boehringer Ingelheim Pharmaceuticals Inc., 900
Ridgebury Rd., PO Box 368, Ridgefield, CT 06877-0368, USA.
{Present address: Bayer Research Centre, 400 Morgan Lane, West
Haven, CT 06516-4175, USA.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00194-4

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G. M. Dubowchik et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1529–1532
containing 1.2–1.4 equiv of TsOH that did not interfere
in the next reaction.
molecular weights identical to 16a. This decomposition
could be inhibited to a varying extent by the presence of
2–10% acetic acid and by ‘capping’ the maleimide with
mercaptoethanol, giving the corresponding thioether
18a. In addition, 16b and 16c were completely stable
under the same conditions. To us, this evidence suggests
the formation of the diastereoisomeric 18-membered
cyclic amides 17 perhaps initiated by transient proton
transfer from Lys to the weakly-basic IDA nitrogen
(Scheme 4). 1H NMR of the newly-formed products was
consistent with the structural assignment 17 in that
there appeared additional complexity in an already
complex spectrum–as might be expected upon cycli-
zation–and the usually prominent maleimide CH signal
(d=7.0 ppm) was no longer visible.
N-Maleimidopropionyl-iminodiacetic acid 11 was pre-
pared as shown in Scheme 2 in a straightforward man-
ner from commercially available maleimido-propionic
acid 8 and di-t-butyliminodiacetate.
Coupling of the diacids (7a, 7b, or 11) with two equiv of
the dipeptide-benzyl alcohol, Phe-Lys(MMT)-PABOH
12,7 was accomplished under standard conditions by in
situ formation of the NHS active esters using DCC
(Scheme 3), giving the branched benzylic alcohols 13 in
very good yields. Formation of the activated p-nitro-
phenyl carbonates 14 and coupling with DOX were
carried out essentially as described for the single-chain
peptide linkers.7 The two p-monomethoxytrityl (MMT)
protecting groups were cleanly removed by treatment of
15 with excess dichloroacetic acid (20 equiv)/anisole
(200 equiv) in CH2Cl2. Interestingly, in the case of the
IDP compound 16b the branching nitrogen atom was
able to remain protonated giving, after precipitation
with ethyl acetate, the tris–Cl2CHCO2H salt. On the
Cathepsin B-mediated Drug Release and Plasma Stability
Thioether 18a, a model compound representing the
most sterically-demanding bivalent linker, was tested
for cathepsin B-mediated liberation of DOX under
conditions used for single-chain linkers.3 In this case,
complete drug release occurred nearly as rapidly (t1/2
=20 min) as measured for the model compound Z-Phe-
Lys-PABC-DOX (t1/2=8 min) suggesting that the pre-
sence of the second drug-peptide arm should present no
liability. In addition, 18a showed no detectable
instability in freshly-drawn human plasma at 37 ꢀC over
6 h.
1
other hand, H NMR of 16a showed the less basic IDA
amine to be unprotonated following isolation.8
This clear pKa difference between the two amines led to
unusual side products. When a sample of 16a was left in
DMFat room temperature, HPLC showed its gradual
disappearance over several hours coinciding with the
appearance of two new components in a 1:1 ratio having
Scheme 1. (a) CBZ-Cl, KOH, quant; (b) concd HCl, AcOH, Á, 55%;
(c) KHCO3, DMF, 73%; (d) Et3N, CH3OH/CH2Cl2, 96%; (e) H2, Pd-
C, AcOH, quant; (f) (1) maleic anhydride, CH2Cl2; (2) TMSCl, Et3N,
MeCN, Á, 55–63%; (g) TsOH (3 equiv), CH2Cl2, quant.
Scheme 3. (a) DCC, HOSu, DME, 76–90%; (b) PNP2CO, DIEA,
CH2Cl2, 68–94%; (c) DOX-HCl, DIEA, NMP, 60–93%; (d)
Cl2CHCO2H, anisole, CH2Cl2, 90–100%.
Scheme 2. (a) oxalyl chloride, cat. DMF, CH2Cl2; (b) HN(CH2CO2t-
Bu)2, DIEA, quant; (c) TFA, CH2Cl2, 90%.

G. M. Dubowchik et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1529–1532
1531
Scheme 4.
potency that one would expect for an ‘unaggregated’
conjugate. Whether this means that the ‘site of aggre-
gation’ is far from the site of drug linkage or whether
disaggregation occurs prior to approach of the protease-
perhaps as a result of the lower pH of the lysosome-is
unknown. In subsequent reports, we will show how this
problem was successfully circumvented.
Conjugation and In Vitro Cytotoxicity
Bivalent maleimide 16a was conjugated to chimeric
BR96 and a control IgG that had been thiolated as
reported previously,9 using a protocol modified for less
soluble dipeptide-drug-linker compounds.10 Immuno-
conjugate 18b had a drug/MAb mole ratio (MR) of
14.1, close to the theoretical limit of 16 that would result
from functionalization of all eight free thiol residues
generated by controlled DTT reduction (protein yield
64%), while the MR for the nonbinding IgG conjugate
18c was 11.4.
References and Notes
1. Dubowchik, G. M.; Walker, M. A. Pharmacol. Ther. 1999,
83, 67.
2. Trail, P. A.; Willner, D.; Lasch, S. J.; Henderson, A. J.;
Hofstead, S.; Casazza, A. M.; Firestone, R. A.; Hellstrom, I.;
Hellstrom, K. E. Science 1993, 261, 212.
3. Dubowchik, G. M.; Firestone, R. A. Bioorg. Med. Chem.
Lett. 1998, 8, 3341.
4. Carl, P. L.; Chakravarty, P. K.; Katzenellenbogen, J. A. J.
Med. Chem. 1981, 24, 479.
5. King, H. D.; Yurgaitis, D.; Willner, D.; Firestone, R. A.;
Yang, M. B.; Lasch, S. J.; Hellstrom, K. E.; Trail, P. A. Bio-
conj. Chem. 1999, 10, 279.
Against the antigen-positive L2987 lung carcinoma cell
line (2 h exposure),5 18b had an IC50 of 0.2 mM (equiva-
lent DOX), while 18c was much less active (IC50=
5.0 mM). Thus, 18b demonstrated antigen-specific cell
killing activity that was equipotent to DOX with a near
doubling of drug molecules/MAb. However, size exclu-
sion chromatography (SEC) showed 18b and 18c to
exist almost exclusively as non-covalent dimers.11
Manipulation of conjugation conditions failed to pre-
vent this aggregation which was found to occur almost
immediately upon addition of 16a to the thiolated
MAb. Although the dimeric nature of 18b does not
appear to impair its in vitro activity, protein aggregates,
even noncovalent ones, are unlikely to be effective ther-
apeutics in vivo as they are liable to be recognized as
foreign bodies and cleared by the reticuloendothelial
system.12 Without exception, we find that non-cova-
lently aggregated immunoconjugates are susceptible to
cathepsin B-mediated drug release and show in vitro
6. Lawson, W. B.; Leafer, M. D.; Tewes, A.; Rao, G. J. S.
Hoppe-Seyler’s Z. Physiol. Chem. 1968, 349, 251.
7. Dubowchik, G. M.; Radia, S. Tetrahedron Lett. 1997, 38, 5257.
8. All new compounds gave satisfactory analytical data. For
1
16a: H NMR (CDCl3/CH3OD) d 1.19 (d, 6H), 1.33 (m, 4H),
1.52 (m, 6H), 1.74 (m, 6H), 2.14 (ABq, 4H), 2.49 (brt, 2H),
2.93 (m, 12H), 3.25 (brs, 4H), 3.51 (m, 2H), 3.77 (m, 2H), 3.90
(s, 6H), 4.04 (m, 2H), 4.32 (m, 2H), 4.65 (s, 4H), 4.82 (m, 4H),
5.05 (brs, 2H), 5.33 (brs, 2H), 5.82 (brs, 2H), 6.63 (s, 2H), 7.01
(m, 14H), 7.32 (m, 6H), 7.63 (t, 2H), 7.79 (d, 2H). MS (ESI)
1078.8 (MH2)2+, 2156.8 (MH)+.

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9. Willner, D.; Trail, P. A.; Hofstead, S. J.; King, H. D.;
Lasch, S. J.; Braslawsky, G. R.; Greenfield, R. S.; Kaneko, T.;
Firestone, R. A. Bioconj. Chem. 1993, 4, 521.
10. Dubowchik, G. M.; Firestone, R. A.; Padilla, L.; Willner,
D.; Hofstead, S. J.; Mosure, K.; Knipe, J. O.; Lasch, S. J.;
Trail, P. A. Manuscript submitted for publication.
tides, only DOX-substituted heavy and light chains. Under
normal conditions, no monomeric species were detected for
18b and 18c. In comparison with BR96, the control IgG
is much more heterogeneous, making it difficult to
directly compare the degree of aggregation of IgG- and
BR96-conjugates.
11. When subjected to gel electrophoresis under denaturing
conditions, 18b showed no covalently-crosslinked polypep-
12. Finbloom, D. S.; Abeles, D.; Rifai, A.; Plotz, P. H.
J. Immunol. 1980, 125, 1060.