Syntheses of dendritic linkers containing chlorambucil residues for the preparation of antibody-multidrug immunoconjugates

Article abstract of DOI:10.1016/S0960-894X(02)00361-X

A novel dendritic molecule with nine chlorambucil (CBL) residues on the surface and a maleimide moiety at the core terminus was synthesized using a convergent synthetic methodology. This molecule is ready for attachment to single-chain Fv antibodies (scFv

Full text of DOI:10.1016/S0960-894X(02)00361-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2213–2215
Syntheses of Dendritic Linkers Containing Chlorambucil Residues
for the Preparation of Antibody–Multidrug Immunoconjugates
Chengzao Sun, Peter Wirsching* and Kim D. Janda*
Department of Chemistry, The Scripps Research Institute and the Skaggs Institute for Chemical Biology, 10550 N.Torrey Pines Road,
La Jolla, CA 92037, USA
Received 11 March 2002;accepted 19 April 2002
Abstract—A novel dendritic molecule with nine chlorambucil (CBL) residues on the surface and a maleimide moiety at the core
terminus was synthesized using a convergent synthetic methodology. This molecule is ready for attachment to single-chain Fv
antibodies (scFvs) to form antibody–multidrug immunoconjugates in an effort to study the relevance of drug/antibody molar ratio
and the potency of these drug–antibody immunoconjugates. A monomer and a trimer with a similar structural motif were also
prepared for comparative purposes. # 2002 Elsevier Science Ltd. All rights reserved.
Monoclonal antibodies (mAbs) directed to tumor-asso-
ciated antigens have been chemically conjugated with
anti-cancer drugs to form immunoconjugates.1 These
immunoconjugates are expected to both improve effi-
cacy and reduce systemic toxicity and therefore are
actively involved in chemotherapeutic research pro-
grams. Efforts to design and refine immunoconjugates
have focused on the selectivity of the mAbs as well as
drug-linking/drug-releasing strategies.1,2 To improve
tumor penetration and the pharmacokinetic properties
of these mAbs, smaller antibody fragments such as sin-
gle-chain Fv antibodies (scFvs) have been constructed.3
Recently, we have developed high-affinity human scFvs
to tumor-associated carbohydrate antigens sialyl Lewisx
and Lewisx using phage-display technology.4 To utilize
these antibodies to destroy cancer cells, we are moving
forward in the preparation of immunoconjugates. Since
the scFv unit is smaller than the whole IgG, there are
fewer sites available for linkage of the drug(s) to the
antibody. Consequently, we are exploring dendritic
molecules with two functionalized termini to be used as
linkers to connect multiple drug molecules to a scFv.
Our plan to prepare this kind of scFv-multidrug immuno-
conjugate is shown in Figure 1.
immunoconjugate.5 The increased molar ratio can be
controlled by varying the size or generation of the
dendritic molecule, which will allow investigations into
the relationship between the molar ratio of drug/antibody
and the potency of the immunoconjugate.
Figure 1. Cartoon depicting the preparation of an antibody–multidrug
immunoconjugate. Here a scFv is equipped with a cysteine residue and
a dendrimer carrying multiple drug residues is functionalized for con-
jugation at the core with a maleimide group.
Dendrimers are widely employed as macromolecular
vectors for drug delivery purposes and other biomedical
applications.6 Indeed many of them have been used as
encapsulating agents. In addition, drugs or other biolo-
gically active molecules can be attached to the surface of
the dendrimer. For example, antibodies and synthetic
porphyrins have been attached to the surface of a star-
burst dendrimer.7 However, there are very few reports
of using both termini, in another words, where the
starting core and the surface branches of the dendrimer
are functionalized in an orthogonal manner such that
each end serves a different purpose. We report herein
the design and synthesis of two dendritic compounds
A dendritic linker approach will increase the molar ratio
of drug/antibody, which is related to the potency of the
*Corresponding author. Tel.: +1-858-784-2516;fax: +1-858-784-
2595;e-mail: kdjanda@scripps.edu
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00361-X

2214
C.Sun et al./ Bioorg.Med.Chem.Lett.12 (2002) 2213–2215
Scheme 1. (a) HBTU, NMM, DMF;(b) 50% TFA, CH 2Cl2;(c)
NHSMP, NMM, CH2Cl2. HBTU=O-benzotriazol-1-yl-N,N,N’,N’-
tetramethyl-uronium hexafluoro-phosphate;NMM=4-methylmor-
pholine;NHSMP=3-maleimidopropionic acid N-hydroxysuccinimide
ester.
Figure 2. Drug molecules with one, three, nine CBL residues and a
maleimide functionality.
with up to nine drug molecules covalently attached on
the branches and the core functionalized for covalent
linkage to an antibody. This provides a novel approach
for using dendrimers for both multidrug carrying and
drug targeting purposes.
Scheme 2. (a) Acrylonitrile, KOH, 1,4-dioxane;(b) CbzCl, aq
.
NaHCO3, 1,4-dioxane;(c) NaBH 4, CoCl2 6H2O;(d) H 2SO4, EtOH,
reflux;(e) Boc 2O, TEA;(f) 3 N aq NaOH.
We designed and synthesized M-CBL1, M-G1-CBL3
and M-G2-CBL9, shown in Figure 2, as our model
compounds. M-CBL1 was used to explore the general
chemistry needed to access M-G1-CBL3 and M-G2-
CBL9 containing chlorambucil (CBL). The latter two
have dendritic linkers that carry three and nine CBL
molecules, respectively. CBL is a nitrogen mustard
alkylating agent used as an antineoplastic agent for
chronic lymphocytic leukemia and Hodgkin’s disease. It
has been conjugated to carrier proteins such as anti-
bodies8 and more recently transferrin.9 We chose this
drug for our study because it is readily available and
contains a carboxylic group for attachment to a den-
drimer. It has been demonstrated that amide bond
linkage to CBL does not impair its alkylating activity.10
However, ester conjugates of CBL may be not as active
as the free drug.9 Consequently, the backbone of the
dendrimer is designed as a polyamido-polyether linkage
with amino functionality at the terminus to attach the
CBL molecule. At the core of the dendron is an amino
group, which allows introduction of a maleimide moiety
for facile conjugation to a cysteine residue in an scFv.
with 10% Pd/C did not affect the chloroethyl moiety on
CBL and yielded 5 almost quantitatively. The amine 5
was then treated with NHSMP to afford the desired
trimeric M-G1-CBL3 (Scheme 3).
Our initial plan to synthesize M-G2-CBL9 was based on
a seemingly straightforward and convenient divergent
construction.13 Thus, generation-2, a nona-acid dendrimer
611b would be coupled with the hydrazide derivative of
CBL as shown in Scheme 4.
However, even with a large excess of H2NNHCBL we
failed to obtain pure nonamer. In all attempts, the LC/MS
results showed a mixture of hexamer through nonamer
products. Therefore, we decided to change our
approach to a convergent synthesis,14 which hopefully
would guarantee pure nonamer.
Thus, 3.3 mol equiv of 5 was coupled to the carboxylic
terminus of 2 to furnish the generation-2 dendrimer-CBL
compound 7 in 64% yield. Following a standard 50%
TFA/CH2Cl2 deprotection of the Boc group to unmask
the free amine 8, attempts to add the maleimide group
M-CBL1 was synthesized in three steps using 3-mal-
eimidopropionic acid N-hydroxysuccinimide ester
(NHSMP), CBL and N-Boc-1,4-diaminobutane in 70%
overall yield (Scheme 1).
Syntheses of M-G1-CBL3 and M-G2-CBL9 proceeded
with the construction of building blocks 1 and 2
(Scheme 2). Tri-acid 2 was synthesized following litera-
ture precedent11 while tri-amine 1 was synthesized using
the same intermediate 3. Thus, protection of the amino
group in 3 with a Cbz group followed by reduction of
the nitrile functionality to a primary amine using cobalt
(II) ion-catalyzed NaBH4 reduction in methanol gave
1.12 Coupling of 3.3 mol equivalents of CBL to 1 with
HBTU gave 4 in 72% yield. Hydrogenation of trimer 4
Scheme 3. (a) HBTU, NMM, DMF;(b) 10% Pd/C, MeOH;(c)
NHSMP, NMM, CH2Cl2.

C.Sun et al./ Bioorg.Med.Chem.Lett.12 (2002) 2213–2215
2215
Acknowledgements
This work was supported in part by grants from the
NIH NIAID-AI47127, California Cancer Research
Program (00–00757V-20012) and the Skaggs Institute
for Chemical Biology.
References and Notes
Scheme 4. (a) Isobutylchloroformate, TEA, THF;(b) 50% TFA,
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at one terminus and a maleimide moiety on the
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the potency of antibody–drug immunoconjugates and
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