Synthesis of [18F] 4-amino-N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide (IDO5L): A novel potential PET probe for imaging of IDO1 expression

Article abstract of DOI:10.1002/jlcr.3263

To synthesize ¹⁸F-labeled positron emission tomography (PET) ligands, reliable labeling techniques inserting ¹⁸F into a target molecule are necessary. The ¹⁸F-fluorobenzene moiety has been widely utilized in the synthesis of ¹⁸F-labeled compounds. The present study utilized [¹⁸F]-labeled aniline as intermediate in [¹⁸F]-radiolabeling chemistry for the facile radiosynthesis of 4-amino-N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide ([¹⁸F]IDO5L) as indoleamine 2,3-dioxygenase 1 (IDO1) targeted tracer. IDO5L is a highly potent inhibitor of IDO1 with low nanomolar IC₅₀. [¹⁸F]IDO5L was synthesized via coupling [¹⁸F]3-chloro-4-fluoroaniline with carboximidamidoyl chloride as a potential PET probe for imaging IDO1 expression. Under the optimized labeling conditions, chemically and radiochemically pure (>98%) [¹⁸F]IDO5L was obtained with specific radioactivity ranging from 11 to 15GBq/μmol at the end of synthesis within ～90min, and the decay-corrected radiochemical yield was 18.2±2.1% (n=4).

Full text of DOI:10.1002/jlcr.3263

Research Article
Received 03 October 2014,
Revised 25 November 2014,
Accepted 24 December 2014
Published online 18 February 2015 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3263
1
8
Synthesis of [ F] 4-amino-N-(3-chloro-4-
fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-
carboximidamide (IDO5L): a novel potential
PET probe for imaging of IDO1 expression
Xuan Huang, Robert J. Gillies, and Haibin Tian*
1
8
18
To synthesize F-labeled positron emission tomography (PET) ligands, reliable labeling techniques inserting F into a
1
8
18
target molecule are necessary. The F-fluorobenzene moiety has been widely utilized in the synthesis of F-labeled
compounds. The present study utilized [ F]-labeled aniline as intermediate in [ F]-radiolabeling chemistry for the facile
radiosynthesis of 4-amino-N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide ([ F]IDO5L) as
1
8
18
1
8
indoleamine 2,3-dioxygenase 1 (IDO1) targeted tracer. IDO5L is a highly potent inhibitor of IDO1 with low nanomolar
1
8
18
IC50. [ F]IDO5L was synthesized via coupling [ F]3-chloro-4-fluoroaniline with carboximidamidoyl chloride as a potential
PET probe for imaging IDO1 expression. Under the optimized labeling conditions, chemically and radiochemically pure
1
8
(
~
>98%) [ F]IDO5L was obtained with specific radioactivity ranging from 11 to 15 GBq/μmol at the end of synthesis within
90 min, and the decay-corrected radiochemical yield was 18.2 ± 2.1% (n = 4).
Keywords: indoleamine 2; 3-dioxygenase; IDO1; PET imaging; fluorine-18; radiolabeling
1
1
Introduction
increased α- C-AMT uptake by cells is a complicated issue
because of many other enzymes involved in tryptophan
transportation and metabolism pathway. Therefore, there is
urgent need to develop a specific PET imaging agent targeted
to IDO1 for cancer imaging.
Indoleamine 2,3-dioxygenase-1 (IDO1) catabolizes the
L-tryptophan (Trp) to yield L-N-formylkynurenine (Kyn), which is
the initial and rate-limiting step in Trp degradation pathway.
IDO1 is expressed in a variety of tissues and particularly high
level expression observed in placenta of pregnant females,
various human tumor cells, and dendritic cells that localize to
Among all potent inhibitors reported in literature, the most
potent inhibitor of IDO1 is INCB024360 (IC50 = 7.1 nM, HeLa cell
assay). However, the structure of INCB024320 has not yet been
6
1
–3
the tumor-draining lymph nodes.
Increasing in vivo and in
disclosed. The carboximidamide compound IDO5L is one of
the highest potent inhibitors of the IDO1 (IC50 = 19 nM, HeLa
vitro pieces of evidence implicated that IDO1 is involved in
immune escape of tumor cells, and blockage of its activity can
directly increase the ability of tumor-bearing mice to reject
14
cell assay). Moreover, the reported rapid clearance rate
(
t1/2 <0.5 h, via oral administration) of IDO5L indicated the faster
clearance rate after i.v administration during PET imaging. This
fast clearance may be preferred for F-PET imaging agent for
2
,4–7
tumors.
Recent studies demonstrated that the expression
level of IDO increased in various tumors such as in lungs,
prostate, and pancreas, lymphoma, and breast cancers. It is
reported that patients with high level of IDO1 expression were
correlated with later clinical phases and larger tumors and
indicated a worse prognosis in various cancers such as diffuse
large B-cell lymphoma and chronic lymphocytic leukemia.
Currently, two IDO1 inhibitors 1-methyl-D-tryptophan and
INCB024360 are used in phase II clinical trials for the treatment
18
less imaging background. We believe that fluorine-18-labeled
carboximidamide compound IDO5L can serve as a novel probe
for the PET imaging to generate IDO1 activity profile in vivo that
is useful to predict IDO1-related cancer diagnostic and monitor
therapeutic efficacy of IDO1 inhibitors. For the first time, we
8–10
18
synthesized F-carboximidamide compound IDO5L as a novel
18
potential PET agent by utilizing [ F]3-chloro-4-fluoroaniline as
intermediate.
11
of breast cancer and melanoma, respectively.
Molecular imaging methods such as positron emission
tomography (PET) have potential to generate IDO1 expression
profile in vivo and provide valuable information on how the
IDO pathway responds to the immune-modulating therapies. Department of Cancer Imaging and Metabolism, H. Lee Moffitt Cancer Center &
11
11
Research Institute, Tampa, FL, USA
α-[ C] methyl-L-tryptophan (α- C-AMT), an IDO1 substrate, has
been reported to be associated with the IDO1 expression levels
*
Correspondence to: Haibin Tian, H. Lee, Department of Cancer Imaging and
12,13
in the brain and lung tumors.
However, IDO1 only was
Metabolism, Moffitt Cancer Center & Research Institute, Tampa, FL, USA.
involved in the first step of the kynurenine pathway, while E-mail: Haibin.Tian@moffitt.org
J. Label Compd. Radiopharm 2015, 58 156–162
Copyright © 2015 John Wiley & Sons, Ltd.

X. Huang et al.
Results and discussion
Chemistry
The reference compound IDO5L (4-amino-N-(3-chloro-
4
-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-3-carboximidamide)
was synthesized using the reported three-step method with
14
minor modification, which is illustrated in Scheme 1. Firstly,
malononitrile was treated with hydroxylamine, sodium nitrite,
and hydrochloric acid to give hydroxyamidine 2 in 91% yield.
The hydroxyamidine 2 was then diazotized under acidic
conditions to provide the hydroximoyl chloride 2 in 57% yield.
Coupling of compound 2 with 3-chloro-4-fluoroaniline was
carried in the basic conditions to produce IDO5L as brown
powder in the yield of 88%.
The synthesis of the two triflate precursors 6 and 10 started
from commercially available 2-chloro-4-nitroaniline 4 (Scheme 2).
Aniline 4 was deprotonated by sodium hydride followed by
methylation to afford 2-chloro-N,N-dimethyl-4-nitroaniline 5 in
7
9% yield. After further methylation of compound 5, the triflate
Scheme 2. Synthesis of two triflate precursors 6 and 10. Conditions: (a) NaH, THF,
°C to RT., 30 min and then MeI 17 h, 79%; (b) methyl trifluoromethanesulfonate,
dichloromethane, RT., 24 h, 76%; (c) NaBH , Pd/C, MeOH, RT. 1 h, 99%; (d)
ethanol, O, 60°C, 30 min, 96%; (e) 1,1′-
carbonyldiimidazole, THF, 70°C, 1 h, 92%; and (f) methyl trifluoromethanesulfonate,
dichloromethane, RT., 12 h, 82%.
precursor 6 was obtained in 76% yield. To produce another triflate
precursor 10, the nitro group of compound 5 was firstly reduced
to aniline 7 in 99% yield by sodium borohydrate under the compound 3, NaHCO
catalysis of palladium on activated charcoal. Compound 8 was
then achieved by coupling aniline 7 and compound 3 in 96%
yield under the basic conditions. The N-hydroxyamidine group
of compound 8 was then cyclized with 1,1′-carbonyldiimidazole
to give compound 9 in 92% yield. Finally, compound 9 was
methylated by methyl trifluoromethanesulfonate to produce the
triflate 10 in the yield of 82%.
0
4
3
,
H
2
15
decomposition of the carboximidamide protection group and
trimethylammonium triflate salts 10.
Although attractive as a one-step radiolabeling procedure, the
18
triflate precursor failed to give the target [ F]IDO5L.
Consequently, a three-step radiochemical route has been
Radiochemistry
18
established by using [ F]-labeled aniline as intermediate
18
Because it is attractive as a one-step radiolabeling procedure, (Scheme 3). The 3-chloro-4-[ F]fluoroaniline intermediate was
aryltrimethylammonium was often used by many as a leaving synthesized using the reported two-step method with minor
1
8
15,16
group in the nucleophilic substitution of no-carrier-added [ F] modification.
The first step involved the nucleophilic
18
fluoride, the triflate precursor, can be used for F-labeling strategy aromatic substitution of triflate precursor 6 by Kryptofix
to replace the conventional complex and long process of multiple- 222/K
18
2 3
CO -activated [ F] in acetonitrile. After optimization, the
step radiolabeling procedure, which shortens reaction time and labeling reaction was completed in 5 min with 98% labeling yield
1
8
labor significantly. The synthesis of the target tracer F-IDO5L (determined by radio-thin-layer chromatography (TLC)) at 70°C
Scheme 3) was performed initially by the conventional Kryptofix- (Figure 1a). Compared with the reported labeling conditions
(
18
15
mediated nucleophilic F-substitution of triflate precursor 10 (room temperature in 25 min), our method saved 20-min
followed by NaOH hydrolysis. However, not F-IDO5L but reaction time while still achieved good labeling yield. The
unlabeled F anion was observed in the radio-HPLC profiles of solvent and reagents used in nucleophilic aromatic substitution
18
1
8
the reaction mixture under various reaction conditions such as were removed by solid-phase extraction on C18 Sep-Pak.
1
8
different solvents (dimethyl sulfoxide (DMSO)/MeCN), temperature
90°C/120°C), and reaction time (5–30min). Analysis of reaction borohydrate under the catalysis of palladium on activated
samples before NaOH hydrolysis found that the precursor 10 charcoal and constant stirring at room temperature for 5 min to
Then, the nitro group of F-11 was reduced by sodium
(
1
8
decomposed to compound 8 and compound 9 as well as other afford F-12 in 89–96% labeling yield determined by radio-
unknown compounds. TLC (Figure 1b). It is worth to mention that relatively lower
Considering that the target product IDO5L is stable under the labeling yield (49–60%) was observed without using stirrer
labeling conditions, we believed that the failure of labeling is because of poor mixing of reactant and catalyst (Table 1). The
because of the difficulty of nucleophilic aromatic fluorination excess NaBH was quenched by concentrated hydrochloric acid,
4
on the weak activated aromatic ring and the ease of and the reaction mixture was passed through 0.2-μm filter to
Scheme 1. Synthesis of standard IDO5L. Conditions: (a) NaNO
2
, HCl (aq.) 2 N; then, 50% NH
2
OH reflux, 2 h; HCl 10 N to pH = 7.0, 91%; (b) NaNO
2 2
, HCl, H O, 0°C, 1.5 h, 57%;
and (c) 3-chloro-4-fluoroaniline, NaHCO , EtOH, room temperature (RT.) to 60°C, 1 h, 88%.
3
J. Label Compd. Radiopharm 2015, 58 156–162
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org

X. Huang et al.
1
8
18
18
4
Scheme 3. Radiosynthesis of F-IDO5L. Conditions: (a) [ F]KF/K222, MeCN, RT., 120°C, 30 min, then NaOH (aq.) 1 N, 15 min; (b) [ F]KF/K222, MeCN, 70°C., 5 min; (c) NaBH ,
Pd/C, MeOH, 70°C, 5 min; and (d) compound 3, NaHCO
3
(aq.), methanol, 60°C, 15 min.
Table 1. Labeling yields of each reaction step
Time
Product
(min)
Conditions
Labeling yield*
1
8
F-11
2
5
94
98
1
0
5
5
98
1
1
8
8
F-12
No stirrer
With stirrer
49–60 (n > 3)
89–98 (n > 3)
0 (n = 3)
F-IDO5L
15
Et N (0.7 mmol,
3
†
3
5 eq.)
a)
1
5
5
NaHCO (aq.;
51–64 (n > 3)
20–40 (n > 3)
3
†
(0.3 mmol, 15 eq.)
1
Na CO (aq.;
2
3
†
0.15 mmol, 7.5 eq.)
*
By radio-TLC, n = 1 unless otherwise stated
†
By radio-TLC, n = 1 unless otherwise stated. B. eq. equivalent
of compound 3
overall radiochemical yields (decay corrected, based on starting
1
8
[
(
F]fluoride) are 47–64%, which is less than the reported yield
15
58–72%), possibly because ~25% activity was volatilized
b)
during the solvent removal process.
The final step is the coupling of compound 3 with F-12 to
18
18
give [ F]IDO5L under base conditions. To neutralize remaining
HCl and basicify the reaction mixture, excess amount of three
different bases (100 μL Et N, 300 μL 1 M NaHCO , and 150 μL
3
3
1
M Na CO ) was tested. Among them, NaHCO gave the best
2
3
3
18
labeling yield (51–64%) of [ F]IDO5L determined by radio-TLC
Figure 1c), while Et N produced no labeled product, and Na CO
(
3
2
3
gave low radiochemistry yield (<40%). Considering the absence
1
8
of compound 3 and the presence of unreacted F-12 in HPLC
profiles of the crude products, we believed that decomposition
of compound 3 under base condition is a major side reaction.
Therefore, lower labeling yield achieved under Na CO can be
c)
2
3
1
8
explained as a stronger base caused faster decomposition of
Figure 1. Representative radio-TLC profiles of (a) crude sample of F-11 (EtOAc/
18
1
8
compound 3. After filtration, [ F]IDO5L was purified by
preparative HPLC and eluted at 10.9–11.7 min (Figure 2).
Formulation of the labeled product for i.v. injection was prepared
hexanes = 1:3,
R
R
f
= 0.80), (b) crude sample of
F-12 (EtOAc/hexanes = 1:3,
= 0.45).
1
8
f
= 0.34), and (c) crude sample of F-IDO5L (EtOAc/hexanes = 1:3, R
f
remove Pd/C. After being dried at 100°C under a stream of as follows: The HPLC solvents were first diluted with water and
1
8
nitrogen, the residue F-12 was redissolved in methanol. The then passed through C18 Sep-Pak column. After being washed
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J. Label Compd. Radiopharm 2015, 58 156–162

X. Huang et al.
1
8
Figure 2. Representative chromatogram from the preparative HPLC separation of the F-IDO5L product.
1
8
with water, the [ F]IDO5L was eluted by methanol. The elution 4-amino-N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-oxadiazole-
18
solvent was removed by evaporation, and the residue was 3-carboximidamide [ F]IDO5L as a novel IDO1-targeted tracer.
redisolved in physiological saline. The identity of the new tracer The tracer has been synthesized and optimized by a three-step
18
[
F]IDO5L was confirmed in HPLC analysis by coinjection with radiolabeling method with good radiochemical yield. PET
IDO5L (Figure 3). Typically, starting from 0.34 to 0.74 GBq imaging studies are currently being carried out in various cancer
1
8
(
9.2–19.9 mCi) [ F]fluoride, 35 to 77 MBq (0.94–2.09 mCi) of animal models in order to evaluate the in vivo potential of this
1
8
purified [ F]IDO5L could be obtained in ~90 min. The overall IDO1 inhibitor.
three-step decay-corrected radiochemical yield was 18.2± 2.1%
(
n = 4) with the radiochemical purity exceeding 98%. Specific
Experimental
activity, determined by using online measurements of radioactivity
and UV absorption, was 11–15 GBq/μmol at end of synthesis.
General
All reagents and solvents were purchased from Sigma-Aldrich or Fluka
and used as received without further purification. Solid-phase extraction
cartridges (Sep-Pak QMA and Sep-Pak C18) were purchased from Water
Conclusion
18
In conclusion, we explored [ F]-labeled aniline as intermediate in Corp., Milford, MA, USA. Column chromatography was performed on
F]-radiolabeling chemistry for the facile radiosynthesis of silica gel (60 Å, 230–400 mesh, for flash chromatography). TLC was
18
[
1
8
Figure 3. Representative chromatogram from the HPLC analysis of the purified F-IDO5L, coinjection with reference IDO5L.
J. Label Compd. Radiopharm 2015, 58 156–162
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org

X. Huang et al.
performed on aluminum plates precoated with silica (200 μm, 60 F254), was added. The reaction was stirred and heated at 60°C for 1 h. The
which were visualized either by quenching of ultraviolet fluorescence reaction mixture was concentrated by a rotary evaporator and dissolved
1
13
(
λ
max = 254 nm) or by iodine stain. H and C spectra were obtained in ethyl acetate (100 mL), after being washed by brine (50 mL) twice and
on a Varian Mercury 400-MHz spectrometer with CDCl , MeCN-d , and dried over Na SO After solvent evaporation, the residue was
DMSO-d as the solvent. All coupling constants were measured in hertz
Hz), and the chemical shifts (δ and δ ) were quoted in parts per
million relative to the internal standard tetramethylsilane (δ 0).
chemical shifts were measured with respect to CFCl . High-resolution
mass spectroscopy (HRMS) was carried out on an Agilent 6210 LC–MS
3
3
2
4
.
6
recrystallized in ethyl acetate/hexanes to afford IDO5L (2.38 g, 88%) as
(
H
C
a brown solid.
1
9
F
R (ethyl acetate/hexanes: 1/1 (v/v)): 0.45.
f
1
3
6
H-NMR (400 MHz, DMSO-d ): δ 11.41 (s, br, 1 H), 8.91(s, br, 1 H), 7.21
(dd, J = 9.2 and 9.2 Hz, 1 H), 6.98 (dd, J = 6.4 and 2.8 Hz, 1 H), 6.72–6.76
(
ESI–time of flight).
HPLC analysis and purification were performed on Agilent 1260 using
(m, 1 H), and 6.27 (s, 2 H).
1
3
1
C-NMR (100 MHz, DMSO-d
6
): δ 155.3, 152.2 (d, JC-F = 239.3 Hz),
3
3
an in-line UV detector (254 nm) and a NaI crystal flow-count radioactivity
detector (Lablogic Flow-RAM detector). The analytical HPLC was
performed on an Agilent Eclipse XDB C18 column (5 μm, 4.6 × 250 mm)
140.4, 139.3, 137.9 (d, JC-F = 2.9 Hz), 122.0, 120.8 (d, JC-F = 6.8 Hz), 118.7
2
2
(d, JC-F = 18.5 Hz), and 116.2 (d, JC-F = 21.7 Hz).
HRMS calculated for C ClFN [M + H]+: m/z = 272.0345, found
272.0340.
9
H
8
5 2
O
with the flow rate 1.0 mL/min using MeCN/0.1% acetic acid in H
0/50, 12/88, or 40/60 as an eluent. Semipreparative HPLC purification
system was performed on an Agilent Eclipse XDB C18 column (5 μm, 2-Chloro-N,N-dimethyl-4-nitroaniline (5)
.6 × 250 mm) with the flow rate 5.0 mL/min using MeCN/0.1% acetic
acid in H O 40/60 for 20 min. Radio-TLC Imaging Scanner (AR-2000,
2
O
5
9
2
-Chloro-4-nitroaniline 4 (5.18 g, 30.0 mmol) in 60 mL anhydrous THF was
2
added to 60% NaH (60%, 2.64 g, 66.0 mmol) under argon protection at 0°
C. The resulting solution was stirred in an ice/water bath for 10 min and
then stirred for 30 min at room temperature. Methyl iodide (10.65 g,
BioScan USA) was used for the radiochemical purity measurements. A
dose calibrator (ATOMLAB 500, Biodex) was used for all radioactivity
measurements.
7
5.0 mmol) was added to the reaction mixture and stirred for 17 h at
room temperature. Then, ice (~10.0 g) and water (40 mL) were added
to the reaction mixture to quench the reaction. After 5 min, the organic
solvent was removed by vacuum. The aqueous layer was extracted by
ethyl acetate (3 × 100 mL), and the combined organic layer was washed
Chemistry
4
-Amino-N-hydroxy-1,2,5-oxadiazole-3-carboximidamide (2)
by brine (100 mL) and dried over anhydrous Na
evaporation, the resulting residue was purified by flash chromatography
2 4
SO . After solvent
Malononitrile (13.2 g, 200 mmol) was added to preheated water (280 mL,
5°C) and stirred for 5 min. The resulting solution was cooled in an
4
(
(
SiO
4.73 g, 79%) as a yellow solid.
(ethyl acetate/hexanes: 1/19 (v/v)): 0.20.
2
) and eluted with ethyl acetate/hexanes (1/19, v/v) to afford 5
ice/water bath, and sodium nitrite (15.18 g, 220 mmol) was added and
stirred for 5 min. Then, HCl (10 N, 13.2 mL) was added to start the mild
exothermic reaction, while bubbles were observed. After 3 min, the
ice/water bath was removed, and the reaction mixture was stirred 1.5 h
at room temperature. The hydroxylamine (39.6 g, 600 mmol) was added
to the light yellow reaction mixture in an ice/water bath. After being
stirred at room temperature for 1 h, the reaction mixture was refluxed
for 2 h and then cooled in an ice/water bath. HCl (10 N, 32.0 mL) was
added in portions to the reaction mixture till neutral (pH = 7.0 via a pH
meter). The precipitate was collected by filtration, washed well with
water, and dried in a vacuum (oil pump) to afford the desired product 2
R
f
1
H-NMR (400 MHz, CDCl
dd, J = 9.2 and 2.8 Hz, 1H, Ar–H), 6.96 (d, J = 8.8 Hz, 1H, Ar–H), and
.01 (s, 6H, N(CH ).
C-NMR (100 MHz, CDCl
and 43.0.
HRMS calculated for C
01.0433.
3
): δ 8.18 (d, J = 2.8 Hz, 1H, Ar–H), 8.03
(
3
3 2
)
1
3
3
): δ 155.4, 140.6, 127.1, 124.7, 123.3, 117.8,
+
8
H
9
ClN
2
O
2
[M + H] : m/z = 201.0425, found
2
2
-chloro-N,N,N-trimethyl-4-nitrobenzenaminium
(26.0 g, 91%).
trifluoromethanesulfonate (6)
R
f
(ethyl acetate/hexanes: 1/1 (v/v)): 0.22.
1
H-NMR (400 MHz, DMSO-d
.19 (s, 2H, NH ).
C-NMR (100 MHz, CD
3 6 5 2
HRMS Calcd for C H N O Na [M + Na] , 166.0336, found, 166.0337.
6 2
): δ 10.46 (s, 1 H, OH), 6.27(s, 2H, NH ), and
To a solution of 2-chloro-N,N-dimethyl-4-nitroaniline (0.60 g, 3.0 mmol) in
anhydrous dichloromethane (20 mL), methyl trifluoromethanesulfonate
6
2
1
3
3
OD): δ 155.86, 145.73, and 141.13.
+
(
0.99 g, 6.0 mmol) was added under argon protection at room
temperature. The resulting red solution was stirred for 24 h at room
temperature, and precipitation was observed. The off-white precipitate
was collected by filtration and washed well with dichloromethane
4
-Amino-N-hydroxy-1,2,5-oxadiazole-3-carboximidamidoyl
chloride (3)
(
3 × 20 mL) and ethyl ether (20 mL). The precipitate was then evaporated
4
-Amino-N-hydroxy-1,2,5-oxadiazole-3-carboximidamide (2; 14.31 g, 100 mmol) on a rotary evaporator and dried by vacuum to offer off the product 6
and NaCl (17.0 g, 290 mmol) were added to a mixture of water (190 mL),
acetic acid (100 mL), and HCl (10 M, 50 mL). The suspension was stirred
at room temperature till complete solution was achieved and then cooled
by an ice/water bath. A solution of sodium nitrite (6.81 g, 98 mmol) in water
(1.09 g, 76%) as a white solid.
1
6
H-NMR (400 MHz, DMSO-d ): δ 8.60 (d, J = 2.4 Hz, 1 H, Ar–H), 8.41
(dd, J = 9.2 and 2.4 Hz, 1 H, Ar–H), 8.34 (d, J = 9.2 Hz, 1 H, Ar–H), and
3.85 (s, 9 H, N(CH ).
C-NMR (100 MHz, DMSO-d
3 3
)
1
3
(24.0 mL) was added, and the reaction mixture was stirred from 0°C to room
6
): δ 148.7, 146.3, 129.3, 127.7, 126.5, 124.1,
), and 56.2.
2 2
12ClN O [M] : m/z = 215.0587, found
1
temperature overnight. The white precipitate was collected by filtration,
washed well with water, taken in ethyl acetate, and dried over anhydrous
121.1 (q, JC-F = 320.2 Hz, CF
HRMS calculated for
215.0587.
3
+
C
9
H
2 4
Na SO . The suspension was filtered, and the filtrate was evaporated on a
rotary evaporator to offer the product 3 (9.24 g, 57%).
1
1
1
2
-chloro-N ,N -dimethylbenzene-1,4-diamine (7)
6
H-NMR (400 MHz, DMSO-d ): δ 13.40 (s, 1 H, OH) and 6.29(s, br, 2H,
NH1
2
).
2
(
-Chloro-N,N-dimethyl-4-nitroaniline 5 (3.00 g, 15.0 mmol) in methanol
50 mL) was added with palladium on activated charcoal (42 mg). Then,
NaBH (1.14 g, 30.0 mmol) was added to the solution dropwise under
stirring at room temperature. Determine reaction completeness by TLC
silica, ethyl acetate/hexanes = 1:2, UV light and stain). When
completed (~1 h), cold HCl solution (2 N, 20 mL) was added dropwise to
0.0 mmol) and 3-chloro-4-fluoroaniline (1.60 g, 11.0 mmol) were added the mixture to quench the reaction (caution: hydrogen gas bubble
3
3
C-NMR (100 MHz, CD OD): δ 155.21, 142.91, and 129.20.
4
4
-Amino-N-(3-chloro-4-fluorophenyl)-N′-hydroxy-1,2,5-
oxadiazole-3-carboximidamide (IDO5L)
(
I
2
4
1
-Amino-N-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride (3; 1.63 g,
in ethanol (40 mL). Then, NaHCO
3
(2.10 g, 25.0 mmol) in water (20 mL)
generated). The reaction solvent was removed by rotary evaporation.
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Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 156–162

X. Huang et al.
1
3
Then, saturated NaHCO
aqueous solution was extracted by ethyl acetate (3 × 50 mL). The
combined organic layer was washed by brine (50 mL) and dried over
3
solution (100 mL) was added, and the resultant
6
C-NMR (100 MHz, DMSO-d ): δ 156.8, 155.7, 148.9, 142.8, 134.7,
1
3
134.4, 133.7, 129.1, 126.4, 125.9, 121.1 (q, JC-F = 320.3 Hz, CF ),
and 56.2.
HRMS calculated for
337.0796.
+
Na
2
SO
4
. After solvent evaporation, the resulting residue was purified by
) and eluted with ethyl acetate/hexanes
1/2, v/v) to afford the product 7 (2.54 g, 99%) as a black solid.
(ethyl acetate/hexanes: 1/2 (v/v)): 0.40.
C
13
H14ClN
6
O
3
[M] : m/z = 337.0810, found
flash chromatography (SiO
(
2
R
f
Radiochemistry
1
H-NMR (400 MHz, CDCl
d, J = 2.8 Hz, 1H, Ar–H), 6.53(dd, J = 8.4 and 2.8 Hz, 1H, Ar–H), 3.54 Synthesis of [ F]11
s, 2H, NH ), and 2.70 (s, 6 H, NCH ).
3
): δ 6.92 (d, J = 8.4 Hz, 1H, Ar–H), 6.71
18
(
(
2
3
18
1
3
Aqueous [ F]fluoride (1.0–2.0 mL, 5–20 mCi, from Cardinal Health) was
trapped on a preconditioning QMA cartridge and eluted with a mixture
of Kryptofix [2.2.2] (475 μL of a 23-mg/mL stock solution in MeCN) and
C-NMR (100 MHz, CDCl
and 44.5.
HRMS calculated for C
71.0678.
3
): δ 142.8, 142.0, 129.5, 121.1, 117.0, 114.2,
+
8
H11ClN
2
[M + H] : m/z = 171.0684, found
K
[
2
CO
3
stock solution (25 μL of a 400-mg/mL stock solution in water).
F]Fluoride was dried at 120°C under a stream of nitrogen by
azeotropic distillation with anhydrous acetonitrile (3 × 0.3 mL) to give
1
18
4
-Amino-N-(3-chloro-4-(dimethylamino)phenyl)-N′-hydroxy-1,2,5-
+
18
À
the no-carrier-added [K/K222
After cooling to room temperature, a solution of triflate precursor 6
]
F complex as a white semisolid residue.
oxadiazole-3-carboximidamide (8)
1
1
2
4
1
-Chloro-N ,N -dimethylbenzene-1,4-diamine 7 (1.79 g, 10.5 mmol) and (3.6 mg, 10 μmol) in anhydrous acetonitrile (0.5 mL) was added into
-amino-N-hydroxy-1,2,5-oxadiazole-3-carboximidoyl chloride 3 (1.63 g, the reaction vial. After being heated at 70°C for 5 min, the mixture was
0 mmol) in ethanol (50 mL) were added in NaHCO
in 25 mL H O). The reaction mixture was stirred at 60°C for
0 min, and the reaction completeness was determined by TLC. After
being extracted by ethyl acetate (2 × 50 mL), the combined organic layer
was washed by brine (50 mL) and dried over anhydrous Na SO
solvent evaporation, the resulting residue was purified by flash
chromatography (SiO ) and eluted with ethyl acetate/ hexanes (3/7, v/v)
to afford the product 8 (2.85 g, 96%) as a gray solid.
(ethyl acetate/hexanes: 3/7 (v/v)): 0.37.
3
solution ( 2.01 g mixed with water (4.0 mL), and the aqueous solution was passed
NaHCO
3
2
through an activated C18 Sep-Pak (pretreated by passing through
5 mL of methanol, 10 mL of water and 40 mL of air). The Sep-Pak was
rinsed with 10% acetonitrile in water (5.0 mL), and the residue solvent
3
18
2
4
. After was partly removed by air (20 mL). The product 2-chloro-4-[ F]fluoro-
1
8
nitrobenzene ([ F]11) was slowly eluted through the column with
methanol (1.5 mL). Radio-TLC analysis of the crude reaction mixture
showed 98% radiochemical yield (EtOAc/hexanes: 1/3 (v/v), R = 0.80)
with unreacted [ F]fluoride as the main impurity.
2
f
1
8
R
f
1
H-NMR (400 MHz, DMSO-d
6
): δ 11.23 (s, 1H, OH), 8.65 (s, 1H, NH), 6.92
18
Synthesis of [ F]12
(d, J = 8.4 Hz, 1H, Ar–H), 6.83 (d, J = 2.4 Hz, 1H, Ar–H), 6.59 (dd, J = 8.4 and
2
1
2
2 3 2
.4 Hz, 1H, Ar–H), 6.20 (s, 2H, NH ), and 2.57 (s, 6 H, N(CH ) ).
13
The elution from the previous step was collected in a reaction vial
containing Pd/C (1 mg) and NaBH (5 mg) under nitrogen protection. The
4
reaction mixture was stirred at room temperature for 5 min before being
quenched by concentrated HCl (25 μL). After filtering off the catalyst, the
solution was concentrated at 100°C under a stream of nitrogen in
C-NMR (100 MHz, DMSO-d
22.8, 120.4, 120.1, and 43.6.
6
): δ 155.4, 144.8, 140.5, 139.6, 136.1, 126.9,
+
HRMS calculated for C11
97.0872.
H13ClN
6
O
2
[M + H] : m/z =297.0861, found
1
8
5 min. Radio-TLC analysis of the crude reaction mixture showed
9–96% conversion to product (EtOAc/hexanes: 1/3 (v/v), R = 0.34).
3
-(4-Amino-1,2,5-oxadiazol-3-yl)-4-(3-chloro-4-(dimethylamino)
f
phenyl)-1,2,4-oxadiazol-5(4H)-one (9)
18
Synthesis of [ F]IDO5L
4
3
-Amino-N-(3-chloro-4-(dimethylamino)phenyl)-N′-hydroxy-1,2,5-oxadiazole-
-carboximidamide 8 (0.864 g, 2.91 mmol) and 1,1′-carbonyldiimidazole The residue of [ F]12 was redissolved in 200 μL of methanol and then
mixed with NaHCO solution (300 μL, 1 M). The acidity of the resulting
mixture was examined by a pH paper to confirm pH ~ 9. A solution of
1
8
(0.519 g, 3.20 mmol) in THF (10 mL) were stirred at 70°C for 1 h. The
3
reaction completeness was determined by TLC. After solvent evaporation,
the resulting residue was purified by chromatography (SiO
product 9 (0.866 g, 92%) as a gray solid.
2
) to afford the 4-amino-N-hydroxy-1,2,5-oxadiazole-3-carboximidamidoyl chloride
3
(3.2 mg, 20 μmol) in methanol (200 μL) was added, and the reaction
R
f
(ethyl acetate/hexanes: 3/7 (v/v)): 0.26.
mixture was stirred at 60°C for 15 min. Radio-TLC analysis of the crude
H-NMR (400 MHz, DMSO-d
): δ 7.68 (d, J = 2.4 Hz, 1H, Ar–H), 7.47 reaction mixture showed 51–64% conversion to [ F]IDO5L (EtOAc/
f
dd, J = 8.8 and 2.4 Hz, 1H, Ar–H), 7.23 (d, J = 8.8 Hz, 1H, Ar–H), 6.61 hexanes: 1/1 (v/v), R = 0.45). The solution was subsequently filtered,
1
18
6
(
(
s, 2H, NH
2
), and 2.79 (s, 6 H, N(CH3)2).
C-NMR (100 MHz, DMSO-d ): δ 157.1, 155.3, 151.4, 149.1, 134.3, 129.9,
27.6, 125.9, 125.2, 120.3, and 43.0.
and the filter was washed with methanol (200 μL). The methanol solution
was purified by HPLC with retention time of 11 min. The collected HPLC
fraction (~4 mL) was diluted with water and then passed through the
1
3
6
1
3
+
HRMS calculated for C12
23.0662.
H11ClN
6
O
3
[M + H] : m/z = 323.0654, found activated C18 Sep-Pak column. After being washed by 10 mL of water,
the labeled product was eluted by 1.5 mL methanol and dried under a
stream of nitrogen in 100°C. Finally, the residue was dissolved in 0.3 mL
4
-(3-(4-Amino-1,2,5-oxadiazol-3-yl)-5-oxo-1,2,4-oxadiazol-4(5H)-yl)-2-
chloro-N,N,N-trimethylbenzenaminium trifluoromethanesulfonate
10)
physiological saline for animal study. Radiochemical purity and specific
1
8
activity of the product [ F]IDO5L were determined by analytical HPLC
1
8
(
with the retention time of 13.5 min. The identity of [ F]IDO5L was
confirmed by a coinjection with a nonradioactive standard IDO5L.
To
dimethylamino)phenyl)-1,2,4-oxadiazol-5(4H)-one 9 (0.806 g, 2.50 mmol)
in dichloromethane (30 mL), methyl trifluoromethanesulfonate (548 μL,
.0 mmol) was added under the protection of nitrogen gas. After being
a
solution of the 3-(4-amino-1,2,5-oxadiazol-3-yl)-4-(3-chloro-4-
18
Typically, starting from 0.34 to 0.74 GBq (9.2–19.9 mCi) [ F]fluoride, 35
(
18
to 77 MBq (0.94–2.09 mCi) of purified [ F]IDO5L could be obtained in
~90 min.
5
stirred at room temperature overnight (~12 h), the reaction mixture
was filtered, and the resulting solid was washed by dichloromethane.
The powder was then dissolved in methanol and precipitated by ethyl
Acknowledgements
ether to afford the product 10 (1.00 g, 82%) as a white solid.
This project was supported by the American Cancer Society’s
1
6
H-NMR (400 MHz, DMSO-d ): δ 8.32 (d, J = 8.8 Hz, 1H, Ar–H), 8.17 Institutional Research Grant project number 60-16318-99-19.
(d, J = 2.4 Hz, 1H, Ar–H), 7.92(dd, J = 8.8 and 2.4 Hz, 1H, Ar–H), 6.67 We thank Adam Weaver and Robert Sanders for the technical
(
s, 2H, NH
2
), and 3.85 (s, 9 H, NCH
3
).
assistance.
J. Label Compd. Radiopharm 2015, 58 156–162
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org

X. Huang et al.
[
8] T. Yoshikawa, T. Hara, H. Tsurumi, N. Goto, M. Hoshi, J. Kitagawa, N.
Kanemura, S. Kasahara, H. Ito, M. Takemura, K. Saito, M. Seishima,
T. Takami, H. Moriwaki, Eur. J. Haematol. 2010, 84, 304.
9] V. Lindström, J. Aittoniemi, J. Jylhävä, C. Eklund, M. Hurme, T.
Paavonen, S. S. Oja, M. Itälä-Remes, M. Sinisalo, Clin Lymphoma
Myeloma Leuk 2012, 12, 363.
Conflict of Interest
The authors did not report any conflict of interest.
[
References
[
10] X.-Q. Liu, K. Lu, L.-L. Feng, M. Ding, J.-M. Gao, X.-L. Ge, X. Wang, Leuk.
Lymphoma 2013, 55, 405.
[11] The clinical trials website. https://www.clinicaltrials.gov/ct2/
results?term=NCT01604889, https://www.clinicaltrials.gov/ct2/
results?term=NCT01961115, https://www.clinicaltrials.gov/ct2/
results?term=NCT01042535.
[1] X. Liu, R. C. Newton, S. M. Friedman, P. A. Scherle, Curr. Cancer Drug
Targets 2009, 9, 938.
[2] C. X. Uyttenhove, L. Pilotte, I. Theate, V. Stroobant, D. Colau, N.
Parmentier, T. Boon, B. J. Van den Eynde, Nat. Med. 2003, 9,
1269.
[
[
3] D. H. Munn, E. Shafizadeh, J. T. Attwood, I. Bondarev, A. Pashine, A. L.
Mellor, J. Expt. Med. 1999, 189, 1363.
4] M. Friberg, R. Jennings, M. Alsarraj, S. Dessureault, A. Cantor, M.
[12] C. Batista, C. Juhász, O. Muzik, W. Kupsky, G. Barger, H. Chugani, S.
Mittal, S. Sood, P. Chakraborty, D. Chugani, Mol. Imaging Biol. 2009,
11, 460.
Extermann, A. L. Mellor, D. H. Munn, S. J. Antonia, Int. J. Cancer [13] C. Juhász, O. Muzik, X. Lu, M. S. Jahania, A. O. Soubani, M. Khalaf, F.
2
002, 101, 151.
Peng, T. J. Mangner, P. K. Chakraborty, D. C. Chugani, J. Nucl. Med.
2009, 50, 356.
[14] E. W. Yue, B. Douty, B. Wayland, M. Bower, X. Liu, L. Leffet, Q. Wang, K.
J. Bowman, M. J. Hansbury, C. Liu, M. Wei, Y. Li, R. Wynn, T. C. Burn, H.
K. Koblish, J. S. Fridman, B. Metcalf, P. A. Scherle, A. P. Combs, J. Med.
Chem. 2009, 52, 7364.
[15] Y. Seimbille, M. E. Phelps, J. Czernin, D. H. S. Silverman, J. Label
Compds. Radiopharm. 2005, 48, 829.
[16] H. F. Vanbrocklin, J. P. O’Neil, D. L. Hom, A. R. Gibbs, J. Label Compds.
Radiopharm. 2001, 44, S880.
[
5] A. J. Muller, J. B. DuHadaway, P. S. Donover, E. Sutanto-Ward, G. C.
Prendergast, Nat. Med. 2005, 11, 312.
[
6] X. Liu, N. Shin, H. K. Koblish, G. Yang, Q. Wang, K. Wang, L. Leffet, M. J.
Hansbury, B. Thomas, M. Rupar, P. Waeltz, K. J. Bowman, P. Polam, R.
B. Sparks, E. W. Yue, Y. Li, R. Wynn, J. S. Fridman, T. C. Burn, A. P.
Combs, R. C. Newton, P. A. Scherle, Blood 2010, 115, 3520.
7] H. K. Koblish, M. J. Hansbury, K. J. Bowman, G. Yang, C. L. Neilan, P. J.
Haley, T. C. Burn, P. Waeltz, R. B. Sparks, E. W. Yue, A. P. Combs, P. A.
Scherle, K. Vaddi, J. S. Fridman, Mol. Cancer Ther. 2010, 9, 489.
[
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J. Label Compd. Radiopharm 2015, 58 156–162