Synthesis and biological evaluation of a novel decadentate ligand DEPA

Article abstract of DOI:10.1016/j.bmcl.2008.09.063

An efficient and short synthetic route to a novel decadentate ligand 7-[2-(bis-carboxymethyl-amino)-ethyl]-4,10-bis-carboxymethyl-1,4,7,10-te traaza-cyclododec-1-yl-acetic acid (DEPA) with both macrocyclic and acyclic binding moieties is reported. A reproducible and scalable synthetic method to a precursor molecule of DEPA, 1,4,7-tris(tert-butoxycarbonylmethyl)tetraazacyclododecane was developed. DEPA was evaluated as a chelator of ¹⁷⁷Lu, ²¹²Bi, and ²¹³Bi for potential use in an antibody-targeted cancer therapy, radioimmunotherapy (RIT) using Arsenazo III based spectroscopic complexation kinetics, in vitro serum stability, and in vivo biodistribution studies.

Full text of DOI:10.1016/j.bmcl.2008.09.063

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5792–5795
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of a novel decadentate ligand DEPA
a
b
b
a
c
Hyun-Soon Chong a, , Sooyoun Lim , Kwamena E. Baidoo , Diane E. Milenic , Xiang Ma , Fang Jia ,
*
Hyun A. Song a, Martin W. Brechbiel b, Michael R. Lewis c
a Chemistry Division, Biological, Chemical, and Physical Sciences Department, Illinois Institute of Technology, 3101 S. Dearborn Street, LS 182, Chicago, IL 60616, USA
b Radioimmune & Inorganic Chemistry Section, Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA
c Department of Veterinary Medicine and Surgery, Department of Radiology, Nuclear Science and Engineering Institute, University of Missouri-Columbia, Columbia, MO, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 June 2008
Revised 15 September 2008
Accepted 16 September 2008
Available online 19 September 2008
An efficient and short synthetic route to a novel decadentate ligand 7-[2-(bis-carboxymethyl-amino)-
ethyl]-4,10-bis-carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl-acetic acid (DEPA) with both macrocy-
clic and acyclic binding moieties is reported. A reproducible and scalable synthetic method to a precursor
molecule of DEPA, 1,4,7-tris(tert-butoxycarbonylmethyl)tetraazacyclododecane was developed. DEPA
was evaluated as a chelator of 177Lu, 212Bi, and 213Bi for potential use in an antibody-targeted cancer ther-
apy, radioimmunotherapy (RIT) using Arsenazo III based spectroscopic complexation kinetics, in vitro
serum stability, and in vivo biodistribution studies.
Keywords:
Radioimmunotherapy
DEPA
Ó 2008 Published by Elsevier Ltd.
177Lu
212Bi
213Bi
Bimodal ligand
Macrocyclic and acyclic ligands that possess amino and carbox-
ylate groups as metal binding moieties have been employed for
biomedical and radiopharmaceutical applications such as magnetic
resonance (MR)1 and positron emission tomography (PET)2 imag-
ing, iron depletion therapy (IDT),3 and radioimmunotherapy
(RIT).4 Recently, we have developed novel bimodal polyaminocarb-
oxylate ligands in the NETA ({4-[2-(bis-carboxymethyl-amino)-
ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl}-acetic acid) and
cyclic cavity (12-membered ring) than NETA. The most frequently
explored polyaminocarboxylates in RIT are 1,4,7,10-tetraazacy-
clododecane-1,4,7,10-tetracarboxylic acid (DOTA) and diethylene
triamine pentaacetic acid (DTPA) derivatives. The novel bimodal li-
gand DEPA is proposed to rapidly form a stable complex with a me-
tal having relatively large ionic radii such as Lu(III), Bi(III), and
Ac(III) using the donor system integrating both macrocyclic DOTA
and acyclic DTPA.
NE3TA
({4-carboxymethyl-7-[2-(carboxymethyl-amino)-ethyl]-
Herein, we report an efficient and short synthetic route to the
novel bimodal ligand DEPA ({7-[2-(bis-carboxymethyl-amino)-
ethyl]-4,10-bis-carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-
yl}-acetic acid, Fig. 1). The precursor molecule to DEPA, 1,4,7-tris
(tert-butoxycarbonylmethyl)tetraazacyclododecane was prepared
from cyclen using a convenient synthetic method involving a sim-
ple pH-controlled work-up. The structurally new ligand DEPA was
evaluated as a potential chelator of 177Lu, 212Bi, and 213Bi for RIT
applications.
Synthesis and isolation of polar macrocyclic polyaminocarboxy-
lates remain challenging. Chromatographic purification of the po-
lar macrocycles is often complicated due to the formation of
polar polyalkylated by-products which are quite indistinguishable
from the desired product by TLC. An efficient and short method to
prepare DEPA (Scheme 1) is based on a coupling reaction of pre-
alkylated precursor molecules 2 and 3. Reaction of trisubstituted
cyclen derivative 213 and N,N-dialkylated bromide 314 was ex-
pected to provide the desired macrocycle 4 while minimizing the
formation of polyalkylated by-products. A short and reproducible
[1,4,7]triazonan-1-yl}-acetic acid) series (Fig. 1) possessing both
macrocyclic and acyclic moieties.5–8 The bimodal ligands NETA
and NE3TA were evaluated as chelators of various metals such as
90Y, 205/6Bi, 203Pb, 177Lu, and 64Cu and showed their potential for
use in cancer therapeutic and diagnostic applications.5–7 In partic-
ular, NETA was proven to be an effective chelator for RIT, an anti-
body-targeted radiation therapy4 that employs a tumor-targeting
mAb for selective delivery of a cytotoxic radioisotope.5 Previous
studies have suggested that 177Lu (t1/2 = 6.7 d), 212Bi (t1/2 = 60.6 m),
and 213Bi (t1/2 = 45.7 m) are promising radioisotopes for RIT.9,10
For a safe and potent RIT, a ligand that can form a stable complex
with the radioisotope with clinically acceptable complexation
kinetics is required.
As an ongoing effort to develop an effective chelator for use in
RIT, we designed decadentate DEPA (Fig. 1) having a larger macro-
* 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 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2008.09.063

H.-S. Chong et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5792–5795
5793
CO2H
HO2C
N
N
N
N
N
N
N
CO2H
CO2H
HO2C
HO2C
CO2H
HO2C
CO2H
DOTA
DTPA
N
HO2C
N
CO2H
HO2C
CO2H
N
N
N
N
N
N
HO2C
N
N
CO2H
N
CO2H
CO2H
CO2H
CO2H
CO2H
HO2C
N
N
DEPA
H
NE3TA
NETA
Figure 1. Ligands in preclinical evaluation for RIT.
O
O
O
O
C
C
C
N
N
N
Ot-Bu
N
Br
C
N
N
N
N
t
-BuO
H
H
Ot-Bu
t
-BuO
H
H
OBn
C
O
N
CO2Bn
N
N
N
N
BnO2C
BrCH2CO2t-Bu
CHCl3
rt, 17 h
(3)
H
C
O
N
C
O
O
CH3CN, DIPEA
72 h, reflux
Ot-Bu
O
C
t
-Bu
2
1
4
BnO
O
O
C
C
N
N
N
N
OH
HO
OH
C
O
C
O
N
OH
O
C
DEPA
HO
Scheme 1. Synthesis of DEPA.
synthetic route to trisubstituted cyclen 2 which appears as a non-
UV responsive tailing spot on TLC was developed since the known
syntheses required either a lengthy procedure or complicated col-
umn chromatographic purification of the polar macrocycle.12,13
The efficient synthetic procedure reported herein provides for iso-
lation of 2 by a simple pH-controlled work-up without complicated
column chromatography in highly reproducible isolated yield.
When no base and an equimolar molar quantity of tert-butyl bro-
moacetate (3.0 equiv) were employed, 2 was actually produced
in a higher yield (47%). The base-promoted coupling reaction of 2
and N,N-dialkylated bromide 3 in CH3CN successfully provided 4
in moderate yield (56%). Isolation of 4 having benzyl groups which
could be monitored by HPLC and TLC analysis was achieved by
flash column chromatography. Both benzyl and tert-butyl groups
in 4 were removed by treatment with 6 M HCl(aq) to afford the de-
sired chelator DEPA.
little at this wavelength. The absorbance (A652) for the AAIII–Lu(III)
or AAIII–Bi(III) complex was measured in the absence and in the
presence of the ligands over 1 h at room temperature. The com-
plexation kinetics of Lu(III) and Bi(III) with DEPA was determined
0.06
0.05
0.04
0.03
0.02
0.01
0.00
The complexation kinetics of the ligand DEPA with Bi(III) and
Lu(III) was determined using a well-known spectroscopic compet-
ing reaction with Arsenazo III (AAIII) according to a modification of
a previously reported procedure.5 AAIII is known to form a weak
complex with many different metals producing a UV–vis absor-
bance maximum at ꢀ652 nm,15 while uncomplexed AAIII absorbs
0
600
1200 1800 2400 3000 3600
Reaction Time (s)
Figure 2. Plot of absorbance (652 nm) versus time of Lu(III)–AAIII (d), DOTA (-),
and DEPA (4) at pH 4.5 (0.15 M NH4OAc) and 25 °C.

5794
H.-S. Chong et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5792–5795
at pH 4.5 or 4.0, respectively, as hydrolysis occurs at a higher pH
0.06
0.05
0.04
0.03
0.02
0.01
0.00
and radiolabeling reaction are routinely performed at this pH.7,16
The complexation of the new ligands studied herein was compared
to that of DOTA, which is known to form an inert complex with
metallic radionuclides, but with extremely slow kinetics.5 A plot
of absorbance at 652 nm versus time is shown in Figures 2 and 3.
The data in Figure 2 indicate that decadentate DEPA is quite slow
and inefficient in binding Lu(III) resulting in slight decrease in
the absorbance (ꢀ0.02) for AAIII–Lu(III) (Fig. 2). At this point, it is
not clear why the more flexible DEPA with 10 donor groups dis-
played slow complexation kinetics with Lu(III) versus the octaden-
tate DOTA ligand. The spectroscopic kinetics data indicate that
DOTA displayed sluggish complexation with both Lu(III) and Bi(III),
while DEPA showed enhanced complexation kinetics with Bi(III) as
compared to DOTA. The complexation of DEPA with Bi(III) was al-
most complete at the starting point (To), although the ligand pro-
0
600
1200 1800 2400 3000 3600
Reaction Time (s)
duces
a
reaction equilibrium curve at the absorbance
(A652 = ꢀ0.01).
Figure 3. Plot of absorbance (652 nm) versus time of Bi(III)–AAIII (d), DOTA (-), and
DEPA (4) at pH 4.0 (0.15 M NH4OAc) and 25 °C.
The new ligand DEPA was radiolabeled with 177Lu and 205/6Bi (a
surrogate of 212Bi and 213Bi) and the corresponding radiolabeled
complexes were evaluated for in vitro serum stability as described
previously.6,17,18 For comparison, DOTA was also radiolabeled with
177Lu. DEPA and DOTA (0.25 M NH4OAc buffer, pH 4.0) was radio-
labeled with 177Lu at 45 °C for 0.5 h to afford 177Lu–DEPA (Rf = 0.6)
and 177Lu–DOTA (Rf = 0.4) in respective radiochemical yields of 90%
and 95% as determined by radio-TLC. DEPA (0.25 M NH4OAc buffer,
pH 5.0) was successfully radiolabeled with 205/6Bi at room temper-
ature for 1 h to afford 205/6Bi–DEPA in 96% yield (radio-TLC).6,18
177Lu–DOTA, 177Lu–DEPA, and 205/6Bi–DEPA were purified from un-
bound 177Lu or 205/6Bi by ion-exchange chromatography using a
Chelex-100 column (1 mL volume bed, 100–200 mesh, Na+ form,
Bio-Rad, Richmond, CA) eluted with PBS (pH 7.4). In vitro serum
stability of the purified radiolabeled complexes was performed to
determine if DEPA or DOTA radiolabeled with 177Lu or 205/6Bi re-
mained stable without loss of the radionuclide in human serum.
This was assessed by measuring the transfer of radionuclide from
the complex to serum proteins. The data in Table 1 indicate that
205/6Bi–DEPA was extremely stable in serum, and no radioactivity
was released over 14 days (Table 1). 177Lu–DEPA remained intact
without being dissociated in serum. However, 10% of the radio-
activity was released from 177Lu–DOTA in 4 days.
Table 1
In vitro serum stability of 177Lu–DOTA, 177Lu–DEPA, and 205/6Bi–DEPA at pH 7 and
37 °C
Time (h)
Radiolabeled complex
177Lu–DEPA
177Lu–DOTA
205/6Bi–DEPA
0
100
100.5
99.0
100.1
99.3
97.0
97.5
98.2
91.2
89.6
—
100
100
100
100
100
100
100
100
100
100
—
100
100
100
100
100
100
100
100
100
100
100
100
100
100
0.25
0.5
1
2
4
6
24
48
96
120
192
288
336
—
—
—
—
—
—
5.0
4.0
3.0
2.0
1.0
0.0
0.5 hr
1 hr
4 hr
8 hr
24 hr
Blood
Liver
Spleen
Kidney
Lung
Heart
Femur
Figure 4. Biodistribution of 205/6Bi–DEPA (iv injection) in non-tumor bearing athymic mice.

H.-S. Chong et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5792–5795
5795
Based on the promising data obtained from the AAIII based
Acknowledgments
spectroscopic complexation kinetics and serum stability experi-
ments, the stability of 205/6Bi–DEPA was further evaluated by per-
This research is supported by the National Institutes of Health
(R01CA112503-01A2 to C.H.S.). This material is in part based upon
work supported by the Intramural Research Program of the NIH,
National Cancer Institute, Center for Cancer Research (M.W.B.)
and by the Office of Research and Development (Medical Research
Service), Department of Veterans Affairs, through a Merit Review
Type I Award (M.R.L.).
forming
a biodistribution study in normal athymic mice as
described previously.8 Blood levels and organ uptake of the radio-
labeled complexes in mice were measured at five time points, 0.5,
1, 4, 8, and 24 h post-injection of 205/6Bi–DEPA. The data in Figure 4
illustrate that DEPA radiolabeled with 205/6Bi was essentially inert
in vivo and rapidly cleared from the body. Radioactivity that was
detected in the blood and the organs was less than 2.44 %ID/g at
all points. At 24 h post-injection, the %ID/g in the kidneys and
spleen was 0.36 0.03% and 0.39 0.07%, respectively, which was
slightly higher than that observed in other organs. The bone accu-
mulation of the radioactivity was 2.02 0.96 %ID/g at 0.5 h which
rapidly decreased to 0.38 0.09 %ID/g at 1 h. Previously, we re-
ported that 177Lu–NETA displayed very low organ uptake and rapid
blood clearance, while 205/6Bi–NETA exhibited very high retention
in liver at the longer time intervals (5.93 0.78 %ID/g at 0.5 h
and 7.31 1.521 %ID/g at 24 h) due to possible dissociation of the
complex in vivo. Although NETA formed a stable complex with
Lu(III) (89 pm), the ligand seems to be inadequate for larger metal
Bi(III) (117 pm) due to its smaller cavity size compared to the mac-
rocyclic ring in DEPA. The in vivo biodistribution result suggest
that the enhanced in vivo stability of 205/6Bi–DEPA compared to
205/6Bi–NETA may result from size-match between Bi(III) and mac-
rocyclic DOTA backbone.19
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.09.063.
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In summary, the novel decadentate ligand DEPA having both
macrocyclic and acyclic metal binding moieties was efficiently
prepared. The complexation stability and kinetics data suggest
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