Synthesis and evaluation of a bifunctional chelate for development of Bi(III)-labeled radioimmunoconjugates

Article abstract of DOI:10.1016/j.bmcl.2011.06.107

A new bifunctional ligand C-DEPA was designed and synthesized as a component for antibody-targeted radiation therapy (radioimmunotherapy, RIT) of cancer. C-DEPA was conjugated to a tumor targeting antibody, trastuzumab, and the corresponding C-DEPA-trastu

Full text of DOI:10.1016/j.bmcl.2011.06.107

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7513–7515
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of a bifunctional chelate for development
of Bi(III)-labeled radioimmunoconjugates
Mamta Dadwal a, Chi Soo Kang a, Hyun A. Song a, Xiang Sun a, Anzhi Dai a, Kwamena E. Baidoo b,
Martin W. Brechbiel b, Hyun-Soon Chong a,
⇑
a Chemistry Division, Biological, Chemical, and Physical Sciences Department, Illinois Institute of Technology, 3101 S. Dearborn St., LS 182, Chicago, IL 60616, USA
b Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A new bifunctional ligand C-DEPA was designed and synthesized as a component for antibody-targeted
radiation therapy (radioimmunotherapy, RIT) of cancer. C-DEPA was conjugated to a tumor targeting
antibody, trastuzumab, and the corresponding C-DEPA-trastuzumab conjugate was evaluated for
radiolabeling kinetics with 205/6Bi. C-DEPA-trastuzumab conjugate rapidly bound 205/6Bi, and 205/6Bi-C-
DEPA-trastuzumab conjugate was stable in human serum for 72 h. The in vitro radiolabeling kinetics
and serum stability data suggest that C-DEPA is a potential chelate for preclinical RIT applications using
212Bi and 213Bi.
Received 29 May 2011
Revised 23 June 2011
Accepted 24 June 2011
Available online 7 July 2011
Keywords:
Radioimmunotherapy
Bifunctional ligand
212Bi
Ó 2011 Published by Elsevier Ltd.
213Bi
Trastuzumab
Radioimmunotherapy (RIT) using
a-particle emitting radionuc-
a superior bifunctional ligand that can tightly and rapidly bind the
lides has been proven to be effective in treatment of leukemia, lym-
short-lived radioisotopes is required for safe and potent
application.
a-RIT
phoma, and micrometastatic cancers.1–3 Among those radionuclides
for
ergy (5–8 MeV) and a short emission range (50–80
posed to selectively and effectively kill tumor cells with
minimum damage to normal cells.3 Preclinical and clinical studies
a
-RIT, 212Bi (t1/2 = 60.6 m) and 213Bi (t1/2 = 45 m) with high
a
en-
We previously reported that decadentate DEPA (7-[2-(bis-
carboxymethyl-amino)-ethyl]-4,10-bis-carboxymethyl-1,4,7,10-
tetraazacyclododec-1-yl-acetic acid) is a promising chelator for the
radioisotopes of Bi(III).12 DEPA was designed to possess a donor
system integrating both macrocyclic DOTA (1,4,7,10-tetraaza-
cyclododecane-1,4,7,10-tetracarboxylic acid) and acyclic DTPA
(diethylenetriamine pentaacetic acid) and expected to rapidly form
a stable complex with a metal having relatively large ionic radii
such as Bi(III), and Ac(III) based on cooperative binding of both
the macrocyclic and acyclic moieties. 205/6Bi-DEPA was stable in
human serum without any loss of the radioactivity for two weeks
and displayed excellent in vivo stability in mice.12 We herein re-
port synthesis and evaluation of the bifunctional analog, C-DEPA,
as a potential chelator of the Bi(III) radionuclides.
The synthetic route to the new bifunctional ligand C-DEPA is
outlined in Schemes 1 and 2. We wanted to prepare the macrocy-
clic backbone compound 5 via nucleophilic ring opening reaction
of N-BOC protected aziridine 2 by cyclen (1,4,7,10-tetraazacyclod-
odecane) 1 followed by removal of the BOC group in 4 (Scheme 1).
Alkylation of 5 was expected to provide the key precursor molecule
10 for the synthesis of C-DEPA (Scheme 2). As expected, selective
mono-substitution of 1 with aziridine derivative 2 produced 4 by
the opening of the aziridine ring in 2 at the less hindered methy-
lene carbon. However, the isolated yield of 4 was low as the
lm) were pro-
a
have demonstrated therapeutic efficacy of
a-particle emitting
radionuclides in treatment of the blood-born cancers.4–7 In particu-
lar, RIT using monoclonal antibody (mAb) labeled with 213Bi was
found to be effective in the treatment of chemo- and external radia-
tion-resistant leukemia cells.8 An effective bifunctional chelator of
212Bi or 213Bi possessing a functional group for conjugation to a
tumor targeting antibody is an essential component for a
-RIT.1 Sev-
eral less optimal bifunctional ligands including C-DOTA(2-(4-nitro-
benzyl)-1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra-acetic
acid) and C-DTPA ((2-(4-nitrobenzyl)-diethylenetriamine pentaace-
tic acid) have been explored for RIT of 213Bi and 212Bi.1,9–11 C-DTPA
displays rapid and high yield radiolabeling with Bi(III), while the
chelator produces an unstable complex both in vitro and in vivo.1
The excellent in vitro or in vivo stability of Bi(III) complex of DOTA
is reported.1 However, slow kinetics of DOTA in binding Bi(III) under
mild conditions limits RIT application of the ligand.1 Development of
⇑
Corresponding author. Tel.: +1 312 567 3235; fax: +1 312 567 3494.
E-mail address: Chong@iit.edu (H.-S. Chong).
0960-894X/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2011.06.107

7514
M. Dadwal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7513–7515
O2N
NO2
H
H
i) 4M HCl/
1,4-dioxane
rt, 14 h
H
H
NO2
H
H
H
H
N
N
N
N
N
2
NHBOC
N
N
N
N
N
N
N
N
BOC
NH2
ii) NaOH (aq)
CHCl3
MeOH
reflux, 21 h
H
4
H
1
5
+
O2N
i) 4M HCl/1,4-dioxane
rt, 14 h
ii) NaOH (aq)/ CHCl3
3
NHBOC
BOC
H
BOC
N
N
N
N
NO2
BOC
BOC
(COCl)2
DMSO, Et3N
O2N
N
N
N
O2N
BOC
NHBOC
BOC
8
HN
OH
O
N
HN
BOC
CH2Cl2
NaBH(OAc)3
DCE
-60 oC, 2.5 h
BOC
rt, 18 h
6
9
7
Scheme 1. Synthesis of key intermediate compound 5.
CO2H
NO2
CO2t-Bu
NO2
NO2
H
N
N
N
N
N
N
N
4M HCl
N
N
BrCH2CO2t-Bu
N
CO2H
CO2H
1.4-dioxane
NH2
HO2C
N
N
K2CO3, CH3CN
reflux, 8 h
N
N
CO2t-Bu
CO2t-Bu
t-BuO2C
rt, 14 h
HO2C
H
H
5
t-BuO2C
11
10
H2 (20 psi)
10% Pd/C
H2O
rt , 14 h
CO2H
CO2H
NH2
NCS
CO2H
CO2H
CSCl2
CHCl3/H2O
N
N
N
N
N
N
N
N
N
N
CO2H
CO2H
HO2C
HO2C
rt, 4 h
HO2C
HO2C
12
13
Scheme 2. Synthesis of C-DEPA (11) and C-DEPA-NCS (13).
reaction also formed polysubstitution byproducts and elimination
product 3, and purification of the polar intermediate compound 4
was challenging. Different reaction conditions at varying tempera-
ture, time, or solvent were examined to optimize the isolated yield
of 4. The reaction of 1 (1 equiv), 2 (1 equiv), and DIPEA (3 equiv) in
DMF (45 °C, 25 h) provided elimination product 3 as the exclusive
product. The reaction of 1 with 2 in CH3CN for 10–24 h provided 4
in poor yield (12–21%). When the reaction was refluxed in CH3OH
for 48 h, a mixture of 3 and 4 in a 1:1 mole ratio was obtained as
evidenced by analytical HPLC. The best isolated yield of 4 (56%)
was achieved from the reaction of 1 (1 equiv) and 2 (2 equiv) in
MeOH under reflux for 21 h. The BOC group in 4 was removed by
the treatment of 4 with 4 M HCl in 1,4-dioxane to provide 5. Reg-
iochemistry in the ring opening of aziridine analog 2 was con-
firmed by independently preparing compound 5 starting from 6
(Scheme 1). Swern oxidation of 6 provided 7 in good isolated yield
(78%), and BOC-protected macrocyclic compound 9 was obtained
from reductive amination of 7 with 813 using sodium triacetoxy-
borohydride provided in excellent isolated yield (85%). Removal
of the BOC groups in 9 using 4 M HCl in 1,4-dioxane provided 5.
Preparation of 5 via this alternative route was found to be more
reproducible and practical as compared to the original synthetic
route via selective substitution of 1 which we initially accom-
plished. The 1H and 13C NMR spectral data of 5 obtained from
two different synthetic routes were essentially identical and
confirms the regiochemistry observed in the ring opening of the
aziridine compound 2. Synthesis of the desired bifunctional ligands
C-DEPA and C-DEPA-NCS is shown in Scheme 2. Reaction of 5 with
tert-butyl bromoacetate in acetonitrile produced compound 10.
This substitution reaction turned out to be very challenging, and
10 was isolated in poor yield (10%) using semi-prep HPLC purifica-
tion, and the isolated yield of the reaction is yet to be improved.
Subsequent removal of the tert-butyl groups in 10 using HCl(g)
in 1,4-dioxane provided C-DEPA (11). The nitro group in 11 was
transformed into the amino group to provide 12. Reaction of 12
with thiophosgene provided the bifunctional ligand C-DEPA-NCS
(13) containing the isothiocyanate group for conjugation to an
antibody.
C-DEPA-NCS was conjugated to a tumor targeting antibody,
trastuzumab which was approved by the FDA for the treatment

M. Dadwal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7513–7515
7515
and Supplementary data). 205/6Bi-C-DTPA-trastuzumab conjugate
was evaluated for comparison. The 205/6Bi-C-DEPA-trastuzumab
conjugate was prepared at 37 °C and pH 5.5, purified on a PD-10
column, and incubated in human serum (37 °C). At each time point
(0, 24, 48, and 72 h), aliquots of the reaction mixture were ana-
lyzed using SE-HPLC (PBS, pH 7.4). 205/6Bi-C-DEPA-trastuzumab
was stable without release of the radioactivity into serum, and
no transchelation of 205/6Bi from the complex to the serum protein
was observed over the course of 3 days. However, ꢀ23% of the
radioactivity was released from 205/6Bi-C-DTPA-trastuzumab con-
jugate in 72 h.
Table 1
Radiolabeling efficiency (%) of C-DEPA-trastuzumab, C-DTPA-trastuzumab, and
C-DOTA-trastuzumab conjugates with 205/6Bi (pH 5.5, RT)a
Time
(min)
C-DEPA-
trastuzumab
C-DTPA-
trastuzumab
C-DOTA-
trastuzumabb
1
5
10
20
30
60
88.7 1.4
90.4 1.0
91.2 1.2
91.1 1.4
92.6 0.8
92.7 0.8
93.6 0.5
95.1 0.3
95.3 0.6
96.0 0.3
95.9 0.3
95.8 0.1
8.4 1.9
17.2 4.3
28.7 4.5
38.0 4.9
49.7 9.0
60.2 8.0
a
SE-HPLC (mobile phase: PBS, pH 7.4). Radiolabeling efficiency (mean standard
In summary, the new bifunctional ligand C-DEPA was prepared,
characterized, and conjugated to a tumor-targeting antibody, trast-
uzumab. The corresponding C-DEPA-trastuzumab conjugate was
evaluated for complexation kinetics with 205/6Bi. C-DEPA-trast-
uzumab conjugate rapidly formed a complex with 205/6Bi with an
excellent radiolabeling efficiency, and 205/6Bi-C-DEPA-trastuzumab
conjugate was stable in human serum for 3 days. Based on the
promising result of the in vitro evaluations, C-DEPA will be further
evaluated for RIT with 212Bi and 213Bi using tumor bearing mice.
deviation %) was measured in triplicate.
b
The data was cited for comparison (Ref. 16)
Table 2
In vitro serum stability (SE-HPLC) of 205/6Bi-radiolabeled trastuzumab-ligand conju-
gates (pH 7 and 37 °C)
Radioimmunoconjugate
Time (h)
205/6Bi-bound conjugate
complex (%)
205/6Bi-C-DEPA-trastuzumab
0
24
48
72
100.0
100.0
100.0
100.0
Acknowledgments
We appreciate the financial support from the National Insti-
tutes of Health (K22CA102637 and R01CA112503). This research
was supported in part by the Intramural Research Program of the
NIH, National Cancer Institute, Center for Cancer Research.
205/6Bi-C-DTPA-trastuzumab
0
24
48
72
100.0
73.4
76.7
77.0
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.06.107.
of metastatic breast cancer and is known to selectively target the
HER2 protein over-expressed in various tumors including colon
and breast cancers.14,15 Protein concentration in the C-DEPA-trast-
uzumab conjugate was quantified using the UV spectroscopic
method as described in the experimental.16 The Pb(II)-AAIII based
UV–Vis spectrophotometric assay as previously reported16,17 was
used for the determination of the number of C-DEPA ligand linked
to trastuzumab (L/P ratio). The spectroscopic assay data indicate
that C-DEPA was conjugated to trastuzumab with a L/P ratio of
0.84:1 (Supplementary data).
The purified C-DEPA-trastuzumab conjugate was further evalu-
ated for radiolabeling reaction kinetics with 205/6Bi (t1/2 = 15.3 d for
205Bi; t1/2 = 6.24 d for 206Bi), a surrogate of 212Bi and 213Bi at room
temperature for 1 h. For comparison, C-DTPA-trastuzumab conju-
gate was prepared and radiolabeled with 205/6Bi. During the incu-
bation time (1 h), the radiolabeling reaction kinetics was
determined by taking aliquots of the reaction solutions at six dif-
ferent time points (1, 5, 10, 20, 30, and 60 min). The components
were analyzed using SE-HPLC after challenging the reaction mix-
ture with 10 mM DTPA, and the radiolabeling efficiency (%) at each
time point was determined (Table 1 and Supplementary data). The
data in Table 1 indicate that C-DEPA-trastuzumab conjugate was
rapid in binding 205/6Bi (1 min, 89%; 60 min, 93%) at room temper-
ature which was comparable to labeling of C-DTPA-trastuzumab
with 205/6Bi (1 min, 94%; 60 min, 96%). However, C-DOTA-trast-
uzumab conjugate displayed slow complex formation kinetics with
205/6Bi (60 min, 60%) as previously reported.16 The result confirms
that C-DOTA is not qualified for use in RIT applications using the
short-lived radioisotopes 212Bi and 213Bi.
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In vitro serum stability of the radiolabeled complexes was
performed to determine if 205/6Bi-C-DEPA-trastuzumab conjugate
remained stable without loss of 205/6Bi in human serum (Table 2