Synthesis and biological evaluation of radioiodinated 2NUBTA as a cerebral ischemia marker

Article abstract of DOI:10.1016/j.bmcl.2008.12.052

N-[4-(Benzothiazol-2-yl)phenyl]-11-(2-nitroimidazole-1-yl)undecanamide (2NUBTA) was synthesized and radiolabeled with iodine-131. In vitro evaluation of the [¹³¹I]2NUBTA using murine sarcoma S180 cells showed increase in radioactivity in hypoxic cells up to 4 h, while it was not in aerobic cells. Its potential as a cerebral ischemia marker was evaluated using gerbil stroke models that had been subjected to right common carotid artery ligation to produce cerebral ischemia. The uptake in the right cerebral hemisphere decreased slowly than that of the left and the right/left hemisphere uptake ratios increased with time going on. It indicated that [¹³¹I]2NUBTA might be a possible cerebral ischemia marker.

Full text of DOI:10.1016/j.bmcl.2008.12.052

Bioorganic & Medicinal Chemistry Letters 19 (2009) 658–661
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of radioiodinated 2NUBTA as a cerebral
ischemia marker
*
Taiwei Chu , Zejun Li, Xiangyun Wang
Bejing National Laboratory for Molecular Sciences (BNLMS), Department of Applied Chemistry, College of Chemistry and Molecular Engineering, Peking University,
Beijing 100871, China
a r t i c l e i n f o
a b s t r a c t
Article history:
N-[4-(Benzothiazol-2-yl)phenyl]-11-(2-nitroimidazole-1-yl)undecanamide (2NUBTA) was synthesized
and radiolabeled with iodine-131. In vitro evaluation of the [131I]2NUBTA using murine sarcoma S180
cells showed increase in radioactivity in hypoxic cells up to 4 h, while it was not in aerobic cells. Its
potential as a cerebral ischemia marker was evaluated using gerbil stroke models that had been subjected
to right common carotid artery ligation to produce cerebral ischemia. The uptake in the right cerebral
hemisphere decreased slowly than that of the left and the right/left hemisphere uptake ratios increased
with time going on. It indicated that [131I]2NUBTA might be a possible cerebral ischemia marker.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 7 August 2008
Revised 8 December 2008
Accepted 11 December 2008
Available online 24 December 2008
Keywords:
Cerebral ischemia marker
Radiolabeling
Hypoxia
Lipophilicity
Intact blood–brain barrier
2-Nitroimidazole
Thioflaven-T
Stroke is the third cause of mortality and the first cause of dis-
ability in adults and until recently there is no efficacious treatment
for acute cerebral ischemic stroke. To help the stroke sufferers,
optimal management now requires delineation of infarction prior
to intervention.1 Nuclear medicine which can offer a non-invasive
method for demonstrating cerebral ischemia will receive more and
more attention.2 However, the marker of stroke ischemia is scarce
up to now because of the difficulty for it to permeate across the in-
tact blood–brain barrier (BBB) into the brain and for it to retain in
the cerebral ischemia.3
It is well known that the 2-nitroimidazole derivatives have the
property of selective accumulation in hypoxic/ischemic tissue.3,4 In
order to design the molecule easy to permeate across the blood–
brain barrier (BBB), 2-nitroimidazole was conjugated to BTA
(derivative of Thioflaven-T, developed by Mathis et al. for imaging
amyloid in Alzheimer’s disease),5,6 then it could be possibly used as
cerebral ischemia marker. N-[4-(benzothiazol-2-yl)phenyl]-3-(2-
nitroimidazole-1-yl) propanamide (2NPBTA, see Fig. 1) has been
synthesized.7 Biodistribution results showed that [131I]2NPBTA
could locate in the cerebral ischemia, but it cleaned fast.8 In order
to improve the biological properties of the cerebral ischemia mar-
ker, a new derivative with higher partition coefficient (P) N-[4-
(benzothiazol-2-yl)phenyl]-11-(2-nitroimidazole-1-yl)undecana-
mide (2NUBTA) was synthesized and radiolabeled with iodine-131
in this study. Its biological evaluation in hypoxic cells and gerbil
cerebral ischemia models was also explored.
Synthesis, radiolabeling, determination of P value. 11-Bromoun-
decanoic acid (0.70 g, 2.64 mmol) and thionyl chloride (2.0 mL,
27 mmol) were mixed and heated slightly in the fume hood. When
there was no bubble, 11-bromoundecanoyl chloride (1) was
obtained (Fig. 2). The excess thionyl chloride was removed under
vacuum. BTA (2, 0.60 g, 2.65 mmol) was dissolved in a 3 mL solu-
tion of DMF and triethylamine (50/50, v/v), then poured into the
vial of 11-bromoundecanoyl chloride. The mixture was stirred at
0 °C for 1 h. After adding diluted HCl solution, the resulting precip-
itate was filtered and washed with deionized water and ethyl ace-
tate. The residue was recrystallized from methanol to give 0.50 g of
N-[4-(benzothiazol-2-yl)phenyl]-11-bromoundecanamide
(3),
yield 40%, mp 159–160 °C. 1H NMR d (DMSO-d6): 10.20 (s, 1H),
O
S
NH
CH2 CH2
N
N
C
N
NO2
2NPBTA
* Corresponding author. Tel./fax: +86 10 62754319.
E-mail address: twchu@pku.edu.cn (T. Chu).
Figure 1. Structure of N-[4-(benzothiazol-2-yl)phenyl]-3-(2-nitroimidazole-1-yl)
propanamide.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.052

T. Chu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 658–661
659
SOCl2
Br(CH2)10COCl
1
Br(CH2)10COOH
S
N
SH
+
NH2
PPA
NH2
HOOC
NH2
180° C, 4h, 40%
2
S
N
3
DMF
NHCO(CH2)10Br
1 + 2
0° C, 1h, 40%
K2CO3 / DMF
110° C ,3h, 76%
N
NH +
NO2
3
S
NH
O
N
(CH2)10
N
NO2
N
2NUBTA
Figure 2. Synthesis of N-[4-(benzothiazol-2-yl)phenyl]-11-(2-nitroimidazole-1-yl)undecanamide.
8.13 (d, 1H), 8.04 (d, 2H), 8.01 (d, 1H), 7.80 (d, 2H), 7.53 (t, 1H), 7.43
1.0
0.8
0.6
0.4
0.2
0.0
hypoxic
aerobic
(t, 1H), 3.51 (t, 2H), 2.35 (t, 2H), 1.78 (m, 2H), 1.61 (m, 2H), 1.32 (m,
12H). EI-MS calculated for C24H29BrN2OS (M+): m/z 472. Found:
472. Analytical data calculated for C24H29BrN2OS: C, 60.88; H,
6.17; N, 5.92. Found: C, 60.81; H, 6.21; N, 5.81.
The mixture of 2-nitroimidazole (0.051 g, 0.45 mmol), N-[4-
(benzothiazol-2-yl)phenyl]-11-bromoundecanamide (3, 0.281 g,
0.59 mmol), and K2CO3 (0.082 g, 0.59 mmol) in 2 mL of DMF was
stirred at 110 °C for 3 h. After cooling and adding the deionized
water, the resulting precipitate was filtered and washed with
deionized water several times. The residue was recrystallized from
methanol, and 0.173 g (0.34 mmol) of N-[4-(benzothiazol-2-yl)-
phenyl] -11-(2-nitroimidazole-1-yl)undecanamide (2NUBTA), as
pale yellow crystals, was obtained. mp 155–156 °C. Yield: 76%.
1H NMR d (DMSO-d6): 10.20 (s, 1H), 8.12 (d, 1H), 8.05 (d, 2H),
8.02 (d, 1H), 7.79 (d, 2H), 7.69 (d, 1H, imi-H), 7.52 (t, 1H), 7.43 (t,
1H), 7.17 (d, 1H, imi-H), 4.35 (t, 2H), 2.34 (t, 2H), 1.75 (t, 2H),
1.60 (t, 2H), 1.26 (s, 12H). EI-MS calculated for C27H31N5O3S
(M+): m/z 505. Found: 505. Analytical data calculated for
C27H31N5O3S: C, 64.14; H, 6.18; N, 13.85. Found: C, 64.27; H,
6.25; N, 13.22.
0
50
100
150
Time (min)
200
250
Figure 3. Accumulation (Cin/Cout) of [131I]2NUBTA in hypoxic (—d—) and aerobic
(—j—) S180 cells as function of time (means standard deviation of five
independent parallel experiments).
a
The carrier-free Na131I (10
chloramines-T were added to 100
nol/pH 7.4 PBS (50/50, v/v) solution. Standing at room temperature
for 10 min, 20 L of 5 mg/mL Na2S2O5 was added in to terminate
l
L, 7.4 MBq) and 20
lL of 5 mg/mL
lL of 1 mg/mL 2NUBTA in etha-
strip was used as the fixed phase and ethanol as the developer.
The radioiodinated product migrated to the top half of the strip
(Rf = 0.8 ꢀ 0.9) while the insoluble radiochemical impurities and
131IÀ remained near the origin (Rf = 0 ꢀ 0.2). The radiochemical
purity was >98%. The ethanol eluent was concentrated and diluted
with an isotonic saline solution to a 10% ethanol (v/v) solution for
l
the reaction. A polyamide column chromatography (ethanol as elu-
ent) was used to separate [131I]2NUBTA from 131IÀ. The radiochem-
ical yield was 40% and the specific activity was 1.5 Â 1010 Bq/
mmol. The product was determined by TLC in which a polyamide

660
T. Chu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 658–661
Table 1
Uptake of [131I]2NPBTA8 and [131I]2NUBTA in the gerbil ischemic and normal brain hemisphere
Brain
[
131I]2NPBTA8
[
131I]2NUBTA
4 h
8 h
12 h
4 h
8 h
12 h
Right
Left
Right/left
0.042 0.005
0.036 0.004
1.18 0.13
0.034 0.006
0.025 0.003
1.39 0.10
0.025 0.004
0.014 0.002
1.76 0.10
0.233 0.021
0.151 0.029
1.57 0.29
0.174 0.015
0.097 0.017
1.82 0.28
0.127 0.008
0.049 0.012
2.76 0.96
Each value is mean standard deviation.
further partition coefficient determination and in vitro and in vivo
study. Being kept at room temperature for one week, the radio-
chemical purity of the product was still >95%. Five milliliters of
1-octanol, 5 mL of water, and 0.1 mL of radioiodinated product
were mixed by vigorously vortexing at room temperature for
5 min. Then it was centrifuged at 4000 rpm for 5 min, and the
phases were separated. Sample (0.1 mL) was taken from each
phase and counted. The octanol/water partition coefficient (P)
was the ratio of the radioactivity of octanol phase to that of aque-
ous phase. Then the water layer was removed and the same vol-
ume of fresh water was added until consistent P values were
obtained. This measurement was repeated for three times. The
logP of [131I]2NUBTA was 1.99, higher than that of previously re-
ported cerebral ischemia marker [131I]2NPBTA (logP = 1.56).7,8
Obviously, by adding the length of the linker between 2-nitroimi-
dazole and BTA, the lipophilicity of new cerebral ischemia marker
was incomplete. Therefore, the blood supply of each hemisphere
was isolated from the contralateral carotid and basilar arteries.
Ligation of one carotid artery caused ischemia in the ipsilateral
hemisphere, while the other side was unaffected, providing neigh-
boring normal tissue as an internal control. The unique anatomical
feature of the gerbil made them widely used as a model in global
ischemia.
Adult mongolian gerbils (male, 80 g) were used for stroke mod-
els. They had been subjected to right common carotid artery liga-
tion to produce cerebral hypoxia–ischemia (HI) as initially
described by Levine and Payan9 and the stroke index described
by Ohno et al.10 was calculated. Animals with total stroke indices
of >10 were used for injection.
[
131I]2NUBTA (0.5 mL,
1.0 Â 105 Bq) was injected intraperitoneally (ip) into the gerbils.
At the time of sacrifice, animals with total stroke indices of >10
were killed (no anesthesia) by cervical dislocation in groups of
three at 4, 8, and 12 h after injection. The whole brain was re-
moved, placed on dry ice for 2 min, and then cut in half along
the cerebral longitudinal fissure. The right and left halves were
weighed and radioactivity counted. The percent injected dose per
gram of tissue, that is, % ID/g, was determined for the right and left
hemispheres. The right/left hemispheral uptake ratios were calcu-
lated. All experiments were carried out following the principles of
laboratory animal care and the China law on the protection of
animals.
[
131I]2NUBTA increased.
Cellular accumulation. S180 cells were suspended in 20 mL Dul-
becco’s modified Eagle’s medium (DMEM) containing 10% (v/v) of
fetal bovine serum at a concentration of (1–2) Â 106 cells/mL and
incubated at 37 °C. The hypoxic and aerobic conditions were gen-
erated by stirring the cell suspension under an atmosphere of
nitrogen or air, respectively (both containing 5% CO2). In the hyp-
oxic conditions, after pre-equilibrated for 40 min, the oxygen sen-
sor of the Dissolved Oxygen Meter read 0.00–0.01 mg/L. In the
aerobic conditions, no pre-equilibration was performed, the oxy-
The final results were expressed as means standard deviation
(SD) as listed in Table 1. The uptake of [131I]2NUBTA in the right
hemisphere was higher than that in the left at 4, 8, and 12 h
post-injection. The results indicated that the clearance from ische-
mic brain tissue was slower than that from normal brain tissue.
The right/left uptake ratios, that is, the uptake ratios of ischemic
to normal brain tissues were gradually increasing for 2NUBTA,
from 1.57 at 2 h to 2.76 at 12 h. The difference between the uptake
of the right hemisphere and the left hemisphere was very signifi-
cant (P value <0.01) at 8 and 12 h.
gen meter read ꢀ6.00 mg/L.
[
131I]2NUBTA (0.2 mL, 148 kBq,
L, in
13.5 nmol) was added to the suspension and aliquots (200
l
pentaplicate) were pipetted using a syringe through rubber plug
to keep the oxygen out of the cell suspension at 5, 30, 60, 90,
120, 150 min, and centrifuged at 1500 rpm for 5 min. A 90
ple of each supernatant was removed for counting (A), each tube
containing cells and 110 L medium with resuspending before
counting was also counted (B). The accumulation ratio, Cin/Cout
lL sam-
l
,
was defined as concentration of radioactivity within the cells di-
vided by that in the external medium, and calculated as [(B À A)/
And the results of the uptake of [131I]2NPBTA, with lower lipo-
A], because the volume of cells in 200 lL of cell suspension is about
philicity (logP = 1.56) reported previously8 were also listed in
20
l
L. The final results were expressed as means standard devia-
Table 1 for comparison. It was found that the uptakes of
tion (SD) of five independent parallel experiments. During the
whole procedure, the cells maintained >90% viability.
[
131I]2NUBTA (logP = 1.99) in both the right hemisphere and the
left hemisphere were higher than those of [131I]2NPBTA, and espe-
cially the right/left uptake ratio of [131I]2NUBTA was also higher
than that of [131I]2NPBTA, such as 2.76 for [131I]2NUBTA, 1.76 for
The Cin/Cout ratios under hypoxic and aerobic conditions were
plotted as a function of time as showed in Figure 3. The accumula-
tion of [131I]2NUBTA steadily increased with time in hypoxic cells,
but fluctuated with time and had no fixed trend in aerobic cells.
The Cin/Cout values at 5 min for hypoxic and aerobic cells were
0.52 and 0.31, respectively, for [131I]2NUBTA, while at 4 h the Cin/
Cout was 0.74 for hypoxic cells and 0.29 for aerobic cells, represent-
ing a 2.6-fold hypoxic/aerobic difference. At 4 h, the difference be-
tween hypoxic cells and aerobic cells was very significant and the P
values (two-tailed) <0.01 (P value was determined using Student’s
t-test). The results indicated [131I]2NUBTA could retain in hypoxic/
ischemic tissue but flow out from normal tissue with time going,
when it was injected intraperitoneally (ip) into animals.
[
131I]2NPBTA at 12 h post-injection, respectively.
Therefore, after adding the length of the linker between 2-nitro-
imidazole and BTA to increase the lipophilicity, [131I]2NUBTA may
be a more suitable cerebral ischemia marker than [131I]2NPBTA.
Acknowledgments
We thank the National Natural Science Foundation of China
(Grant No. 20771011) and the Ministry of Science and Technology
of China (Grant No. 2006CB705700) for financial support. The
assistance of Dr. Weihong Cong and Prof. Jianxun Liu, Xiyuan Hos-
pital, China Academy of Traditional Chinese Medicine in the
in vitro and in vivo studies is gratefully acknowledged.
Evaluation in gerbil cerebral ischemia models. The gerbils did not
have a posterior communicating artery, that is, the circle of Willis

T. Chu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 658–661
661
5. Mathis, C. A.; Bacskai, B. J.; Kajdasz, S. T.; McLellan, M. E.; Frosch, M. P.; Hyman,
B. T.; Holt, D. P.; Wang, Y. M.; Huang, G. F.; Debnath, M. L.; Klunk, W. E. Bioorg.
Med. Chem. Lett. 2002, 12, 295.
6. Mathis, C. A.; Wang, Y. M.; Holt, D. P.; Huang, G. F.; Debnath, M. L.; Klunk, W. E.
J. Med. Chem. 2003, 46, 2740.
7. Li, Z. J.; Chu, T. W.; Liu, X. Q.; Wang, X. Y. Nucl. Med. Biol. 2005, 32, 225.
8. Chu, T. W.;Li, Z. J.;Liu, X. Q.; Wang, X. Y. Int. J. Biomed. Imag. 2007. ArticleID49791.
9. Levine, S.; Payan, H. Exp. Neurol. 1966, 16, 255.
References and notes
1. Beauchamp, N. J.; Barker, P. B.; Wang, P. Y.; van Zijl, P. C. M. Radiology 1999, 212,
307.
2. Guadagno, J. V.; Donnan, G. A.; Markus, R.; Gillard, J. H.; Baron, J. C. Curr. Opin.
Neurol. 2004, 17, 61.
3. Takasawa, M.; Moustafa, R. R.; Baron, J. C. Stroke 2008, 39, 1629.
4. Chu, T. W.; Li, R. J.; Hu, S. W.; Liu, X. Q.; Wang, X. Y. Nucl. Med. Biol. 2004, 31,
199.
10. Ohno, K.; Ito, U.; Inaba, Y. Brain Res. 1984, 297, 151.