Synthesis and evaluation of [123I] labeled iodovinyl amino acids syn-, anti-1-amino-3-[2-iodoethenyl]-cyclobutane-1-carboxylic acid, and 1-amino-3-iodomethylene-cyclobutane-1-carboxylic acid as potential SPECT brain tumor imaging agents

Article abstract of DOI:10.1016/j.bmcl.2008.01.035

syn- and anti-1-amino-3-[2-iodoethenyl]-cyclobutane-1-carboxylic acid (syn-, anti-IVACBC 16, 17) and their analogue 1-amino-3-iodomethylene-cyclobutane-1-carboxylic acid (gem-IVACBC 18) were synthesized and radioiodoinated with [¹²³I] in 34-43%

Full text of DOI:10.1016/j.bmcl.2008.01.035

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1264–1268
Synthesis and evaluation of [123I] labeled iodovinyl amino acids syn-,
anti-1-amino-3-[2-iodoethenyl]-cyclobutane-1-carboxylic acid,
and 1-amino-3-iodomethylene-cyclobutane-1-carboxylic acid
as potential SPECT brain tumor imaging agents
Weiping Yu,a Larry Williams,a Eugene Malveaux,a Vernon M. Camp,a
Jeffrey J. Olsonb and Mark M. Goodmana,*
aDepartment of Radiology, Emory University, School of Medicine, Atlanta, GA 30322, USA
bDepartment of Neurosurgery, Emory University, School of Medicine, Atlanta, GA 30322, USA
Received 3 December 2007; revised 8 January 2008; accepted 10 January 2008
Available online 13 January 2008
Abstract—syn- and anti-1-amino-3-[2-iodoethenyl]-cyclobutane-1-carboxylic acid (syn-, anti-IVACBC 16, 17) and their analogue 1-
amino-3-iodomethylene-cyclobutane-1-carboxylic acid (gem-IVACBC 18) were synthesized and radioiodoinated with [123I] in 34–
43% delay-corrected yield. All these amino acids entered 9L gliosarcoma cells primarily via L-type transport in vitro with high
uptake of 8–10% ID/1 · 106 cells. Biodistribution studies of [123I]16, 17 and 18 in rats with 9L gliosarcoma brain tumors demon-
strated high tumor to brain ratios (4.7–7.3:1 at 60 min post-injection). In this model, syn-, anti-, and gem-[123I]IVACBC are prom-
ising radiotracers for SPECT brain tumor imaging.
ꢀ 2008 Elsevier Ltd. All rights reserved.
The development of radiotracers for detecting cancer in
patients is a major focus of radiopharmaceutical re-
search. A number of classes of compounds that accumu-
late preferentially in neoplastic tissues have been
investigated for this purpose, including metabolically
based radiotracers such as amino acids. Amino acids
are important biological substrates in virtually all bio-
logical processes and are required nutrients for cell
growth. A variety of radiolabeled amino acids have been
developed as potential tumor imaging agents for posi-
tron emission tomography (PET) and single photon
emission computerized tomography (SPECT). The ami-
no acids developed for oncologic imaging can be divided
into two major categories, naturally occurring amino
acids and their structurally similar analogues, and
non-natural amino acids. One potential advantage of
non-natural amino acids over naturally occurring amino
acids is their increased metabolic stability. Additionally,
non-natural amino acids may not participate effectively
in protein synthesis. These properties can potentially
simplify the analysis of tracer kinetics with non-natural
amino acids.
Non-natural amino acid PET tracers anti-1-amino-3-
[18F]fluorocyclobutane-1-carboxylic
acid
(anti-
[18F]FACBC)1,2 and syn- and anti-1-amino-3-[18F]fluo-
romethyl-cyclobutane-1-carboxylic acid (syn- and anti-
[18F]FMACBC)3 have been prepared in this lab and
have shown great potential in imaging brain and pros-
tate cancer.4–6 The radionuclides used for PET and
SPECT have complementary properties. While PET
has higher temporal and spatial resolution, SPECT
radionuclides possess certain advantages. Most com-
monly used SPECT radionuclides, including iodine-123
and technecium-99m, are available without an onsite
cyclotron. SPECT radionuclides have longer half-lives
(e.g., 13.2 h for iodine-123 and 6 h for technecium-
99m) than most widely used PET radionuclides (e.g.,
110 min for fluorine-18 and 20 min for carbon-11).
The longer half-lives of most SPECT radionuclides facil-
itate chemical synthesis, allow for the study of processes
that occur over longer time courses, and are suitable for
remote distribution of the radiolabeled product to sites
that do not have radiosynthetic capabilities. There are
Keywords: SPECT; Iodine-123; Rats; 9L gliosarcoma; Amino acids;
LAT; Brain tumor.
*
Corresponding author. Tel.: +1 404 727 9366; fax: +1 404 727
3488; e-mail: mgoodma@emory.edu
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.035

W. Yu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1264–1268
1265
currently fewer radiolabeled amino acids suitable for
SPECT than for PET, with [123I]IMT7 representing the
most widely used radiolabeled amino acid for SPECT.
bonyl)amino]-3-[2-trimethylstannylethenyl]-cyclobutane-
1-carboxylic acid tert-butyl esters 8 and 9 were prepared
from vinyl iodides 6 and 7, respectively, using hexameth-
ylditin and catalytic tetrakis(triphenylphosphine)
palladium(0).12,13
In an effort to develop the radiotracers for SPECT tu-
mor imaging, we report here the syntheses of syn-,
anti-1-amino-3-[2-iodoethenyl]-cyclobutane-1-carboxylic
acid (syn-, anti-IVACBC 16, 17), 1-amino-3-iodomethyl-
ene-cyclobutane-1-carboxylic acid (gem-IVACBC 18),
which are analogues of FACBC and FMACBC, their
The protected gem-IVACBC stannylated precursor for
radiolabeling was prepared starting from epibromohy-
drin (10). The key intermediate, a 5:1 mixture of syn/
anti-5-(3-benzyloxycyclobutane)hydantoins (11), was
prepared in 4 steps as described previously,1 and hydro-
lyzed at 120 ꢁC with 3 N NaOH to the syn/anti-1-amino-
3-benzyloxycyclobutane-1-carboxylic acids. Subsequent
N-protection with di-tert-butyl dicarbonate and tert-bu-
tyl esterification with tert-butyl 2,2,2-trichloroacetimi-
date followed by Pd0 catalyzed debenzylation3 gave
the syn/anti-1-[N-(tert-butoxycarbonyl)amino]-3-hydro-
xy-cyclobutane-1-carboxylic acid tert-butyl esters (12).
The alcohols were transformed to the corresponding ke-
tone (13) by Swern oxidation followed by reaction with
CHI3/CrCl2 to form protected gem-IVACBC (14) which
was treated with (SnMe3)2/Pd0(PPh3)4 to afford 1-[N-
(tert-butoxycarbonyl)amino]-3-trimethylstannylmethyl-
ene-cyclobutane-1-carboxylic acid tert-butyl ester (15) as
the radiolabeling precursor12 (Scheme 2).
[
123I] radiolabeling and biological evaluation with a rat
9L gliosarcoma cell line and in Fischer rats with 9L tu-
mors implanted intracranially (Fig. 1).
The syntheses of syn- and anti-IVACBC stannylated
precursors for radiolabeling were prepared in a series
of synthetic steps starting from allylbenzylether (1),
which is shown in Scheme 1. The intermediates, the
syn-
and
anti-1-[N-(tert-butoxycarbonyl)amino]-3-
hydroxymethyl-cyclobutane-1-carboxylic acid tert-butyl
esters (2,3), were prepared in 7 steps according to
Martarello’s method.3 The alcohol isomers were trans-
formed to the corresponding aldehydes (4,5) by Swern
oxidation.8 In the key synthetic step, reaction of 4 or 5
with iodoform in tetrahydrofuran using chromium(II)
chloride as a catalyst gave exclusively trans (E) isomers
1
of 6 and 79–12 as determined by H NMR. The stannyl
The 127I references of syn-, anti-, and gem-IVACBC 16,
17, and 18 were prepared from the N-Boc amino acid
labeling precursors syn- and anti-1-[N-(tert-butoxycar-
F
F
F
CO2H
NH2
CO2H
NH2
NH2
CO2H
H
H
I
H
I
anti-FMACBC
anti-FACBC
syn-FMACBC
NH2
CO2H
NH2
I
CO2H
NH2
CO2H
H
H
H
gem-IVACBC 18
anti-IVACBC 17
syn-IVACBC 16
Figure 1. Structures of FACBC and its fluoro and iodovinyl analogues.
HO
O
CH
NHBoc
CO2t-Bu
NHBoc
CO2
7 steps
a
Ph
O
t
-Bu
H
H
2
3
(syn)
(anti)
4
5
(syn)
(anti)
1
Sn(CH3)3
I
NHBoc
CO2t-Bu
NHBoc
b
c
CO2t-Bu
H
H
8
9
(syn)
(anti)
6
7
(syn)
(anti)
Scheme 1. Syntheses of syn- and anti-IVACBC stannylated precursors 8 and 9. Reagents and conditions: (a) (COCl)2, DMSO, DCM, À50 ꢁC then
Et3N, 79% (4); 64% (5); (b) CHI3, CrCl2, THF, 0 ꢁC—rt, 59% (6); 60% (7); (c) (CH3)3Sn–Sn(CH3)3, Pd0(PPh3)4, THF, 50–60 ꢁC, 26% (8); 18% (9).

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W. Yu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1264–1268
O
HN
BocHN CO2-t-Bu
O
NH
4 steps
4 steps
O
Br
OH
O
Ph
10
12
11
BocHN CO2-t-Bu
BocHN CO2-t-Bu
BocHN CO2-t-Bu
a
b
c
O
Sn(CH3)3
I
13
14
15
Scheme 2. Synthesis of gem-IVACBC stannylated precursor 15. Reagents and conditions: (a) (COCl)2, DMSO, DCM, À50 ꢁC then Et3N, 92% (13);
(b) CHI3, CrCl2, THF, 0 ꢁC—rt, 27% (14); (c) (CH3)3Sn–Sn(CH3)3, Pd0(PPh3)4, THF, 50–60 ꢁC, 76% (15).
tert-butyl esters 6, 7 and 14, respectively, by treatment of
trifluoroacetic acid (TFA) and purified by ion-retarda-
tion resin chromatography12 (Scheme 3).
dation resin chromatography12. The procedure required
approximately 100 min with decay-corrected yields
(d.c.y.) of 41.9 18.3% (n = 17, syn-[123I]IVACBC 16),
43.4 23.0% (n = 12, anti-[123I]IVACBC 17) and
33.7 18.2% (n = 7, gem-[123I]IVACBC 18) in over
99% radiochemical purity as measured by radiometric
TLC. Based on 200 nmol of tin precursor utilized for
labeling, and approximate yields of 1 mCi of product,
the minimum specific activities for syn-, anti-, and
Radioiodinated syn-, anti-[123I]IVACBC 16, 17, and
gem-[123I]IVACBC 18 were prepared using no-carrier-
added (NCA) sodium[123I]iodide under oxidizing condi-
tions followed by deprotection with TFA (Scheme 4).
The radioiodinated products were purified by ion-retar-
I
I
NH2
NHBoc
CO2H
H
CO2t-Bu
H
a
6
7
(syn)
(anti)
16 (syn)
17 (anti)
NHBoc
CO2t-Bu
NH2
I
I
a
CO2H
18 (gem)
14 (gem
)
Scheme 3. Syntheses of syn-, anti-, and gem-IVACBC 16, 17, and 18. Reagents and conditions: (a) TFA, DCM, rt, 78% (16); 71% (17); 68% (18).
123I
123I
Sn(CH3)3
NHBoc
NH2
CO2H
NHBoc
CO2t-Bu
a
b
CO2t-Bu
123I]16 (syn)
123I]17 (anti)
[
[
123I]6 (syn)
123I]7 (anti)
[
[
8
9
(syn)
(anti)
NHBoc
CO2t-Bu
123I
123I
NHBoc
NH2
CO2H
(H3C)3Sn
a
b
CO2t-Bu
123I]14(gem)
[
123I]18 (gem)
15 (gem)
[
Scheme 4. Radiosyntheses of syn-, anti-, and gem-IVACBC 16, 17, and 18. Reagents and conditions: (a) [123I]NaI, H2O2, H+, rt; (b) TFA, DCM,
85 ꢁC, 42% (16); 43% (17); 34% (18).

W. Yu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1264–1268
1267
gem-[123I] 16, 17, and 18 are estimated at 5 mCi/lmol.
The final product was not assayed for the presence of
tin.
16, 17, and 18 are selective substrates for L-type amino
acid transport in 9L gliosarcoma cells in vitro.
The in vivo biodistribution studies were performed in
Fischer rats with 9L tumors implanted intracranially.
The radioactivity in tumors and in normal tissues of tu-
mor-bearing rats (n = 5 each time point) was calculated
at 30, 60, 120 min post-injection (p.i.) and normalized as
percent injected dose per gram tissue (% ID/g). The up-
take of radioactivity of [123I]IVACBC 16, 17, and 18 in
tumor and brain is presented in Figure 3. The experi-
ments showed that these amino acids had rapid and pro-
longed accumulation in tumors, ranged from 0.83 to
1.85% ID/g and was significantly higher than in normal
brain tissue (p < 0.001 at all time points, one-way ANO-
VA). The uptake in normal brain tissue was less than
0.4% ID/g at all time points for all these compounds
thus the tumor to normal brain uptake ratios were
7.3:1, 6.5:1, and 4.7:1 at 60 min p.i. for [123I]IVACBC
16, 17, and 18, respectively. Low uptake was found in
heart, liver, lung, bone, and thyroid. The low thyroid
uptake with all radiotracers indicates that free iodide
was not generated during the time course of the study.
The in vitro studies were performed in 9L rat gliosar-
coma cells in Hank’s Balanced Salt Solution (HBSS)
incubated for 30 min at 37 ꢁC with or without inhibitors
to evaluate the compounds tumor cell uptake and
transport mechanism. 2-Amino-bicyclo[2.2.1]-heptane-
2-carboxylic acid (BCH) (10 mM) and N-methyl-a-
aminoisobutyric acid (MeAIB) (10 mM) were used as
L- and A-type amino acid transport inhibitors, respec-
tively. The results of these amino acid uptake assays
are depicted in Figure 2. In the absence of inhibitors,
[
123I]IVACBC 16, 17, and 18 showed high levels of intra-
cellular accumulation, 8.4–9.9% of the initial dose per
million cells (% ID/1 · 106 cells) in 9L gliosarcoma cells.
In the presence of BCH, 92.3–97.5% of inhibition was
observed in all these compounds compared to controls
(p < 0.001 in all cases, one-way ANOVA). In contrast,
no significant uptake inhibition occurred with MeAIB
for 16 and 18 and 14.3% inhibition for 17 relative to
controls. These results demonstrate that compounds
Figure 2. 9L cell uptake and inhibition assays with [123I]16, [123I]17, and [123I]18.
Figure 3. Radioactive uptake in tumor and brain of 9L tumor-bearing Fisher rats with [123I]IVACBC 16, 17, and 18.

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W. Yu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1264–1268
The uptake of radioactivity of [18F] labeled anti-FAC-
BC,2 syn-FMACBC3, and anti-FMACBC3 in tumors
at 60 min p.i. were 1.72, 1.59, and 2.50% ID/g in the
same animal model, respectively, which resulted in tu-
mor to brain ratios of 6.6:1, 6.9:1 and 8.9:1, respec-
tively.2,3 Thus, the characteristics of candidate
compounds 16, 17, and 18 are comparable with anti-
FACBC, syn-FMACBC, and anti-FMACBC, by enter-
ing 9L cells via L-type transport system in vitro and
showing high levels of uptake in 9L brain tumors
in vivo in comparison to normal brain.
studies. Research supported by Nihon Medi-Physics
Co., Ltd.
References and notes
1. McConathy, J.; Voll, R. J.; Yu, W.; Crowe, R. J.;
Goodman, M. M. Appl. Radiat. Isot. 2003, 58, 657.
2. Shoup, T. M.; Olson, J.; Hoffman, J. M.; Votaw, J.;
Eshima, D.; Eshima, L.; Camp, V. M.; Stabin, M.; Votaw,
D.; Goodman, M. M. J. Nucl. Med. 1999, 40, 331.
3. Martarello, L.; McConathy, J.; Camp, V. M.; Malveaux,
E. J.; Simpson, N. E.; Simpson, C. P.; Olson, J. J.; Bowers,
G. D.; Goodman, M. M. J. Med. Chem. 2002, 45, 2250.
4. Nye, J. A.; Schuster, D. M.; Yu, W.; Camp, V. M.;
Goodman, M. M.; Votaw, J. R. J. Nucl. Med. 2007, 48,
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5. Oka, S.; Hattori, R.; Kurosaki, F.; Toyama, M.; Williams,
L. A.; Yu, W.; Votaw, J. R.; Yoshida, Y.; Goodman, M.
M.; Ito, O. J. Nucl. Med. 2007, 48, 46.
6. Schuster, D. M.; Votaw, J. R.; Nieh, P. T.; Yu, W.; Nye, J.
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In summary, three new SPECT tumor imaging agents,
syn-, anti-, and gem-IVACBC 16, 17, and 18 have been
synthesized, [123I] labeled and biologically evaluated.
All these compounds demonstrated high levels of tumor
uptake in vitro and in vivo in a 9L rat gliosarcoma brain
tumor model and they are L-type amino acid trans-
porter substrates. These results are comparable with
those of anti-FACBC, syn-FMACBC, and anti-
FMACBC in the same animal model, which support
the candidacy of syn-, anti-, and gem-IVACBC 16, 17,
and 18 as promising SPECT brain tumor imaging
agents.
7. Jager, P. L.; Vaalburg, W.; Pruim, J.; de Vries, E. G.;
Langen, K. J.; Piers, D. A. J. Nucl. Med. 2001, 42, 432.
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108, 7408.
10. De Lera, A. R.; Torrado, A.; Iglesias, B.; Lopez, S.
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Acknowledgments
11. Wulff, W. D.; Powers, T. S. J. Org. Chem. 1993, 58, 2381.
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13. Panekand, J. S.; Jain, N. F. J. Org. Chem. 2001, 66, 2747.
We are grateful to Dr. Bing Wang of the NMR Center
of Emory University for his assistance with NMR stud-
ies and Zhaobin Zhang for his assistance with the in vivo