Novel 64Cu-radiolabeled bile acid conjugates for targeted PET imaging

Article abstract of DOI:10.1016/j.bmcl.2015.01.008

A promising bifunctional chelate (N-NE3TA) was conjugated to bile acids, cholic acid (CA), deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA) as tumor targeting vectors. Bile acid conjugates of N-NE3TA (CA-N-NE3TA, DCA-N-NE3TA, and CDCA-N-NE3TA) were comparatively evaluated for complexation with ⁶⁴Cu, an imaging probe for positron emission tomography (PET). N-NE3TA-bile acid conjugates were evaluated for radiolabeling kinetics with ⁶⁴Cu, and the corresponding ⁶⁴Cu-radiolabeled conjugates were screened for complex stability in human serum and EDTA solution. The NE3TA-bile acid conjugates instantly bound to ⁶⁴Cu with excellent radiolabeling efficiency at room temperature. All NE3TA-bile acid conjugates radiolabeled with ⁶⁴Cu remained inert in human serum for 2 days without releasing a considerable amount of the radioactivity. The ⁶⁴Cu-radiolabeled complexes were further challenged by EDTA in a 100-fold molar excess. Bile acid-N-NE3TA conjugates radiolabeled with ⁶⁴Cu were quite stable with a minimal transfer of ⁶⁴Cu to EDTA at 4 h time point. The in vitro data indicate that the bile acid-N-NE3TA conjugates deserve further biological evaluation for ⁶⁴Cu-based targeted PET imaging applications.

Full text of DOI:10.1016/j.bmcl.2015.01.008

Bioorganic & Medicinal Chemistry Letters 25 (2015) 1082–1085
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel 64Cu-radiolabeled bile acid conjugates for targeted PET
imaging
⇑
Hyun-Soon Chong , Yunwei Chen, Chi Soo Kang, Xiang Sun, Ningjie Wu
Chemistry Division, Department of Biological and Chemical Sciences, Illinois Institute of Technology, 3101 S. Dearborn St., LS 182, Chicago, IL 60616, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A promising bifunctional chelate (N-NE3TA) was conjugated to bile acids, cholic acid (CA), deoxycholic
acid (DCA), and chenodeoxycholic acid (CDCA) as tumor targeting vectors. Bile acid conjugates of
N-NE3TA (CA–N-NE3TA, DCA–N-NE3TA, and CDCA–N-NE3TA) were comparatively evaluated for
complexation with 64Cu, an imaging probe for positron emission tomography (PET). N-NE3TA–bile acid
conjugates were evaluated for radiolabeling kinetics with 64Cu, and the corresponding 64Cu-radiolabeled
conjugates were screened for complex stability in human serum and EDTA solution. The NE3TA–bile acid
conjugates instantly bound to 64Cu with excellent radiolabeling efficiency at room temperature. All
NE3TA–bile acid conjugates radiolabeled with 64Cu remained inert in human serum for 2 days without
releasing a considerable amount of the radioactivity. The 64Cu-radiolabeled complexes were further
challenged by EDTA in a 100-fold molar excess. Bile acid–N-NE3TA conjugates radiolabeled with 64Cu
were quite stable with a minimal transfer of 64Cu to EDTA at 4 h time point. The in vitro data indicate
that the bile acid–N-NE3TA conjugates deserve further biological evaluation for 64Cu-based targeted
PET imaging applications.
Received 21 October 2014
Revised 1 January 2015
Accepted 5 January 2015
Available online 12 January 2015
Keywords:
Bifunctional chelator
Polyaminocarboxylate
PET imaging
Cu-64
Bile acid
Radiolabeling
Serum stability
Ó 2015 Elsevier Ltd. All rights reserved.
A sensitive diagnostic modality, positron emission tomography
(PET) has been demonstrated to give highly sensitive detection and
staging of cancers.1–3 Metallic radionuclides such as 64Cu, 68Ga, and
86Y have been explored for PET imaging. Among the radionuclides,
acid), DOTA (1,4,7,10-tetraazacyclododecane tetraacetic acid),
and TETA (2-[1,4,8,11-tetraazacyclotetradecane tetraacetic acid)
have been explored for PET imaging applications using 64Cu.1,4,5
We previously reported a bifunctional chelate N-NE3TA (Fig. 1)
containing both acyclic and macrocyclic binding moieties as a
promising chelate of 64Cu.8 N-NE3TA rapidly bound to 64Cu under
mild conditions, and in vitro and in vivo stability of 64Cu-N-NE3TA
was favorably compared to 64Cu-radiolabeled complex of C-DOTA,
one of the most frequently used chelate for PET imaging.8
Encouraged by the complexation kinetics and stability profile of
N-NE3TA with 64Cu, we were interested in utilizing the bifunc-
tional chelate for targeted PET imaging using a tumor targeting
vector. The primary bile acids (cholic acid and chenodeoxycholic
acid) and secondary bile acid (deoxycholic acid) are known to
target bile acid receptors or carriers overproduced in hepatic and
colorectal cancers.9–12 The amphifacial bile acids were shown to
form helical globular aggregates and enter into the cancer cells
due to their great cell permeability and have been explored as a
delivery shuttle of anti-cancer agents.10–12
b+
bÀ
c
64Cu (t1/2 = 12.7 h;
E
max
= 0.655 MeV;
E
max
= 0.573 MeV; Emax =
0.511 MeV) possesses half-life and decay property suitable for
PET imaging with extended imaging window.1,4,5 For development
of clinically viable 64Cu-based radiopharmaceuticals for targeted
PET imaging, it is essential to employ a bifunctional chelate that
can rapidly form a stable complex with Cu(II).5–7 Rapid radiolabel-
ing of 64Cu with a short half-life by a bifunctional chelate attached
to a sensitive biomolecule such as antibodies is required for
practical preparations of biologically active 64Cu-radiolabeled
complexes. 64Cu-radiolabeled complex must be stable in vivo with-
out undergoing transchelation with other metal-binding proteins
or biologically important metals. Cu(II) has a relatively small ionic
radius (73 ppm) and is known to display a high affinity for nitrogen
and oxygen donor atoms. Various acyclic and macrocyclic polyami-
nocarboxylate-based chelates including DTPA (diethylenetriamine
pentaacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic
We herein report synthesis of bile acid conjugates of N-NE3TA
and evaluation of the corresponding bile acid–NE3TA conjugates
for complexation with 64Cu for targeted PET imaging. The bifunc-
tional chelate N-NE3TA was conjugated to tumor-targeting bile
⇑
Corresponding author. Fax: +1 312 567 3494.
E-mail address: Chong@iit.edu (H.-S. Chong).
http://dx.doi.org/10.1016/j.bmcl.2015.01.008
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

H.-S. Chong et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1082–1085
1083
O
R1
NH
HO2C
HO2C
HO2C
N
N
N
N
CO2H
HO
R2
H
N
N
N
N
CO2H
CO2H
NO2
N-NE3TA
1
2
N-NE3TA-CA
(R = R = OH)
N-NE3TA-DCA (R1 = OH, R2 = H)
N-NE3TA-CDCA (R1 = H, R2 = OH)
Figure 1. Structure of N-NE3TA and N-NE3TA–bile acid conjugates.
CO2tBu
CO2t-Bu
O
OH
N
N
CO2tBu
(3)
CO2t-Bu
CO2t-Bu
N
N
(COCl)2, Et3N
DMSO, CH2Cl2
− 60 oC, 3 h
N
H
N
N
N
N
NaBH(OAc)3
DCE, RT, 18 h
CO2tBu
O2N
O2N
CO2tBu
4
1
2
NO2
O
R1
NH
CO2t-Bu
ButO2C
CH2Cl2, Et3N
5 d, reflux
H2 (15 psi)
10% Pd/c
CO2t-Bu
CO2t-Bu
N
N
N
N
N
N
N
R2
H
HO
N
EtOH
S
O
RT, 14 h
R1
CO2tBu
S
N
CO2tBu
7. R1 = R2 = OH
5
8. R1 = OH, R2 = H
9. R1 = H, R2 = OH
NH2
HO
R2
H
4M HCl (g)
1,4-dioxane
RT, 20 h
6a. R1 = R2 = OH
6b. R1 = OH, R2 = H
6c. R1 = H, R2 = OH
O
R1
NH
HO2C
N
N
R2
HO
N
N
H
CO2H
CO2H
10. R1 = R2 = OH (N-NE3TA-CA)
11. R1 = OH, R2 = H (N-NE3TA-DCA)
12. R1 = H, R2 = OH (N-NE3TA-CDCA)
Scheme 1. Synthesis of N-NE3TA analogues and N-NE3TA–bile acid conjugates.
acids, cholic acid (CA), deoxycholic acid (DCA), or chenodeoxycho-
lic acid (CDCA). The bile acid conjugates were evaluated for
radiolabeling kinetics with 64Cu for PET imaging application.
64Cu-radiolabeled bile acid conjugates were evaluated for complex
stability in human serum and a solution of EDTA.
Synthesis of bifunctional N-NE3TA analogue 5 and N-NE3TA–
bile acid conjugates 10–12 is shown in Scheme 1. Compound 2
was readily prepared from Swern oxidation of 1,13 and reductive
amination of 2 with 314 provided the key precursor macrocyclic
compound 4.13 The nitro group in 4 was converted to the amino
group in 5 which further was reacted with an activated bile acid
analogue 6a, 6b, or 6c which were prepared from reaction of bile
acid with 2-mercaptothiazoline as reported previously.15 tert-Butyl
N-NE3TA-NH2 (5) was reacted with the preactivated cholic acid
analogue (CA, 6a) in the presence of triethylamine under reflux
to provide N-NE3TA–CA conjugate 7. Similarly, N-NE3TA-DCA
and N-NE3TA-CDCA analogues 8 and 9 were prepared from reac-
tion of 5 with 6b and 6c, respectively. The removal of tert-butyl
protecting groups in 7–9 using 4 M HCl (g) in 1,4 dioxane provided
N-NE3TA–CA (10), N-NE3TA–DCA (11) and N-NE3TA–CDCA (12),
respectively.
The new N-NE3TA–bile acid conjugates were evaluated for
radiolabeling reaction kinetics with 64Cu at room temperature
(Table 1, Fig. 2, and Supporting information). Each conjugate
(20
(60
l
l
g) in 0.25 M NH4OAc (pH 5.5) was radiolabeled with 64Cu
Ci) at room temperature. During the reaction time (30 min),
the components were withdrawn at the designated time points
(1 min, 10 min, and 30 min), and the radiolabeling efficiency (%)
was determined using ITLC (20 mM EDTA in 0.15 M NH4OAc).
64Cu-EDTA migrated with the solvent front on TLC (Rf = 0.88), while

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H.-S. Chong et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1082–1085
Table 1
Table 2
Evaluation of bile acid conjugates for radiolabeling efficiency (%) with 64Cu (RT,
0.25 M NH4OAC, pH 5.5) using ITLC*
Evaluation of 64Cu-radiolabeled bile acid conjugates for in vitro stability in serum
(37 °C, pH 7) using radio-HPLC*
Time (min)
N-NE3TA–CA
N-NE3TA–DCA
N-NE3TA–CDCA
Day
N-NE3TA–CA
N-NE3TA–DCA
N-NE3TA–CDCA
1
10
30
99.7 0.3
99.9 0.1
99.9 0.1
89.6 3.8
97.0 0.4
98.9 0.5
97.6 0.3
98.2 0.4
99.2 0.1
0
1
2
98.0 0.3
97.7 0.3
99.0 0.4
97.3 0.8
95.5 1.3
97.0 1.0
97.7 0.2
98.7 0.1
99.7 0.3
*
*
Radiolabeling efficiency (mean standard deviation %) was measured in trip-
Bound complex (mean standard deviation %) was measured in duplicate using
licate using ITLC (eluent: 20 mM EDTA in 0.15 M NH4OAc).
radio-HPLC (solvent A: 0.1% TFA in H2O, solvent B: 0.1% TFA in CH3CN, 0–100% B/
15 min, flow rate: 1 mL/min).
64Cu-radiolabeled chelator complexes travel slower on the TLC
(Rf = 0.54). The 64Cu-radiolabeled complexes of the conjugates
and 64Cu-EDTA were well separated on the ITLC. All N-NE3TA bile
acid conjugates instantly bound to 64Cu with excellent radiolabel-
ing efficiency (>90%, 1 min time point, Table 1, Fig. 2a) at room
temperature. N-NE3TA–DCA was slightly slower in binding 64Cu
as compared to N-NE3TA–CA and N-NE3TA–CDCA, although
radiolabeling of the conjugate with 64Cu was nearly complete at
30 min time point. All 64Cu-radiolabeled complexes were shown
to be stable against EDTA present in the eluent of TLC (Supporting
information).
In vitro serum stability of the radiolabeled N-NE3TA–bile acid
conjugates was performed to determine if the 64Cu-radiolabeled
conjugates remained stable without loss of the radioactivity in
human serum. This was assessed by measuring the transfer of
64Cu from the complex to human serum proteins using radio-HPLC
(Table 2, Fig. 2d, and Supporting information). A fresh solution of
64Cu-radiolabeled conjugates were readily prepared from the
reactions of N-NE3TA–bile acid conjugates with 64Cu at room
temperature and directly used for serum stability studies (pH 7,
37 °C) using radio-HPLC by radio-HPLC analysis (solvent A: 0.1%
TFA in H2O, solvent B: 0.1% TFA in CH3CN, 0–100% B/15 min, flow
rate: 1 mL/min). The trace related to 64Cu bound to serum
(tR = 2.5 min) was clearly distinguished from the peaks of the
64Cu-N-NE3TA–bile acid conjugate (tR = 10–11 min for N-NE3TA–
CA and tR = 11–12 min for N-NE3TA–DCA and N-NE3TA–CDCA).
All 64Cu-radiolabeled conjugates remained quite stable in
human serum for 2 days as evidenced by radio-HPLC. While a tiny
amount of 64Cu (<1.0%) was detected from 64Cu-N-NE3TA–CA and
64Cu-N-NE3TA–CDCA over 2 days, 64Cu-N-NE3TA–DCA released
ꢀ3% of the radioactivity at 48 h time points (Fig. 2d) and appears
to be least stable in serum among the 64Cu-radiolabeled conjugates
tested.
64Cu-radiolabeled N-NE3TA–bile acid conjugates were further
challenged for complex stability in an excess amount of EDTA solu-
tion. 64Cu-radiolabeled complexes were treated with a solution of
EDTA at a 100-fold molar excess, and the resulting solution (pH
5.5) was incubated at 37 °C for 24 h. A sample was withdrawn at
Figure 2. (a) TLC chromatogram of 64Cu-radiolabeled complexes at 1 min time point of radiolabeling; (b) TLC chromatogram of 64Cu-radiolabeled complexes in a solution of
EDTA at 100-fold molar excess (1 h time point); (c) TLC chromatogram of 64Cu-radiolabeled complexes in a solution of EDTA at 100-fold molar excess at 24 h; (d) HPLC
chromatogram of 64Cu-radiolabeled complexes in human serum at 48 h.

H.-S. Chong et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1082–1085
1085
(>10%) was dissociated from 64Cu-radiolabeled N-NE3TA–bile acid
conjugates. The in vitro complexation kinetics and stability data
suggest that the N-NE3TA–bile acid conjugates can be further
evaluated for targeted PET imaging using animals.
Table 3
Stability of 64Cu-radiolabeled complexes against EDTA at a 100-fold molar excess
(37 °C)*
Time (h)
N-NE3TA–CA
N-NE3TA–DCA
N-NE3TA–CDCA
0
1
4
24
100.0 0.1
99.4 0.0
98.5 0.4
88.4 0.4
99.8 0.1
97.5 0.1
95.2 0.9
80.4 1.3
99.5 0.6
97.9 0.3
97.1 0.2
88.9 0.4
Supplementary data
*
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.01.
008.
Bound complex (mean standard deviation %) was measured in duplicate using
ITLC (eluent: 20 mM EDTA in 0.15 M NH4OAc).
different time points (0 h, 1 h, 4 h, and 24 h) and analyzed using
ITLC (Table 3, Fig. 2b and c, and Supporting information). All
64Cu-radiolabeled conjugates remained bound against EDTA chal-
lenge up to 4 h time point (Supporting information), although slow
release of the radioactivity was observed over 24 h. A small portion
of the activity (<5%) was transferred from the complexes to EDTA
(99.4% for N-NE3TA–CA, 97.9% for N-NE3TA–CDCA, 97.5% for
N-NE3TA–DCA) at 1 h time point (Fig. 2b). 64Cu-N-NE3TA–DCA
was shown to be less tolerant of EDTA treated. ꢀ20% of 64Cu was
dissociated from the complex at 24 h time point (Fig. 2c).
Approximately 10% of the radioactivity was leaked from 64Cu-N-
NE3TA–CA and 64Cu-N-NE3TA–CDCA at 24 h time point (Fig. 2c).
In summary, N-NE3TA–bile acid conjugates were evaluated for
complexation kinetics and stability with 64Cu for potential use in
targeted PET imaging. All N-NE3TA–bile acid conjugates rapidly
and almost completely bound to 64Cu. The corresponding 64Cu-
radiolabeled complexes remained quite stable in human serum,
and no considerable release of the radioactivity was observed with
the complexes. When rigorously challenged by excess amount of
EDTA at 37 °C for 24 h, a small amount of the radioactivity
References and notes
1. Wadas, T. J.; Wong, E. H.; Weisman, G. R.; Anderson, C. J. Curr. Pharm. Des. 2007,
13, 3.
2. Gambhir, S. S. Nat. Rev. Cancer 2002, 2, 683.
3. Chakravarty, R.; Hong, H.; Cai, W. Mol. Pharm. 2014, 11, 3777.
4. Wadas, T. J.; Wong, E. H.; Weisman, G. R.; Anderson, C. J. Chem. Rev. 2010, 110,
2858.
5. Smith, V. S. J. Inorg. Biochem. 1874, 2004, 98.
6. Cai, Z.; Anderson, C. J. J. Labelled Compd. Radiopharm. 2014, 57, 224.
7. Pandya, D. N.; Bhatt, N.; Dale, A. V.; Kim, J. Y.; Lee, H.; Ha, Y. S.; Lee, J.-E.; An, G.
I.; Yoo, J. Bioconjugate Chem. 2013, 24, 1356.
8. De Silva, R. A.; Jain, S.; Lears, K. A.; Chong, H. S.; Kang, C. S.; Sun, X.; Rogers, B. E.
Nucl. Med. Biol. 2012, 39, 1099.
9. Ho, N. F. Ann. N.Y. Acad. Sci. 1987, 507, 314.
10. Ballestero, M. R.; Monte, M. J.; Briz, O.; Jimenez, F.; Martin, F. G. S.; Marin, J. J. G.
Biochem. Pharmacol. 2006, 72, 729.
11. Torchia, E. C.; Agellon, L. B. Eur. J. Cell Biol. 1997, 74, 190.
12. Powell, A. A.; LaRue, J. M.; Batta, A. K.; Martinez, J. D. Biochem. J. 2001, 356, 481.
13. Chong, H-. S.; Ma, X.; Lee, H. S.; Bui, P.; Song, H. A.; Birch, N. J. Med. Chem. 2008,
51, 2208.
14. Chong, H-. S.; Song, H. A.; Birch, N.; Le, T.; Lim, S. Y.; Ma, X. Bioorg. Med. Chem.
Lett. 2008, 18, 3436.
15. Chong, H-. S.; Song, H. A.; Ma, X.; Lim, S.; Sun, X.; Mhaske, S. B. Chem. Commun.
2009, 3011