18F Labeled benzimidazole derivatives as potential radiotracer for positron emission tomography (PET) tumor imaging

Article abstract of DOI:10.1016/j.bmc.2010.02.060

This article reported the synthesis and bioevaluation of two [¹⁸F] labeled benzimidazole derivatives, 4-(5-(2-[¹⁸F] fluoro-4-nitrobenzamido)-1-methyl-1H-benzimidazol-2-yl) butanoic acid ([¹⁸F] FNBMBBA, [¹⁸F]a1) and 3-(2-fluoroethyl)-7-methyl-2-propyl-3H-benzimidazole-5-carboxylic acid ([¹⁸F] FEMPBBA, [¹⁸F]b1) for PET tumor imaging. The preparation [¹⁸F] FEMPBBA was completed in 1 h with overall radiochemical yield of 50-60% (without decay corrected). Biodistribution assay in S180 tumor bearing mice of both compounds were carried out, and the results are both meaningful. [¹⁸F] FEMPBBA which can be taken as a revision of [¹⁸F] FNBMBBA got an excellent result, and has significant advantages in some aspects compared with L-[¹⁸F] FET and [¹⁸F]-FDG in the same animal model, especially in tumor/brain uptake ratio. The tumor/brain uptake ratio of [¹⁸F] FEMPBBA gets to 4.81, 7.15, and 9.8 at 30 min, 60 min and 120 min, and is much higher than that of L-[¹⁸F] FET (2.54, 2.92 and 2.95) and [¹⁸F]-FDG (0.61, 1.02, 1.33) at the same time point. The tumor/muscle and tumor/blood uptake ratio of [¹⁸F] FEMPBBA is also higher than that of L-[¹⁸F] FET at 30 min and 60 min. This result indicates compound [¹⁸F] FEMPBBA is a promising radiotracer for PET tumor imaging.

Full text of DOI:10.1016/j.bmc.2010.02.060

Bioorganic & Medicinal Chemistry 18 (2010) 2394–2401
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
18F Labeled benzimidazole derivatives as potential radiotracer for positron
emission tomography (PET) tumor imaging
a
a
a
a
a
a
Shuting Zhang a, Xiao Wang a, , Yong He , Rui Ding , Hang Liu , Jingli Xu , Man Feng , Guixia Li ,
*
Ming Wang a, Cheng Peng b, Chuanmin Qi a,
*
a Key laboratory of Radiopharmaceuticals, College of Chemistry, Beijing Normal University, Beijing 100875, People’s Republic of China
b Center of PET, Xuan Wu Hospital, Beijing 100053, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
This article reported the synthesis and bioevaluation of two [18F] labeled benzimidazole derivatives, 4-(5-
Article history:
(2-[18F] fluoro-4-nitrobenzamido)-1-methyl-1H-benzimidazol-2-yl) butanoic acid ([18F] FNBMBBA,
Received 28 December 2009
Revised 25 February 2010
Accepted 26 February 2010
Available online 6 March 2010
[
[
18F]a1) and 3-(2-fluoroethyl)-7-methyl-2-propyl-3H-benzimidazole-5-carboxylic acid ([18F] FEMPBBA,
18F]b1) for PET tumor imaging. The preparation [18F] FEMPBBA was completed in 1 h with overall radio-
chemical yield of 50–60% (without decay corrected). Biodistribution assay in S180 tumor bearing mice of
both compounds were carried out, and the results are both meaningful. [18F] FEMPBBA which can be
taken as a revision of [18F] FNBMBBA got an excellent result, and has significant advantages in some
aspects compared with L-[18F] FET and [18F]-FDG in the same animal model, especially in tumor/brain
uptake ratio. The tumor/brain uptake ratio of [18F] FEMPBBA gets to 4.81, 7.15, and 9.8 at 30 min,
60 min and 120 min, and is much higher than that of L-[18F] FET (2.54, 2.92 and 2.95) and [18F]-FDG
(0.61, 1.02, 1.33) at the same time point. The tumor/muscle and tumor/blood uptake ratio of [18F] FEM-
PBBA is also higher than that of L-[18F] FET at 30 min and 60 min. This result indicates compound [18F]
FEMPBBA is a promising radiotracer for PET tumor imaging.
Keywords:
18F
Benzimidazole derivatives
Bendamustine
Positron emission tomography
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
methyl-1H-benzimidazol-2-yl) butanoic acid group plays in mak-
ing bendamustine behavior so unique.20,21
Benzimidazole and its derivatives are a kind of compound
which exerts a wide range of biological activities, such as anti-
inflammatory,1–3 histamine-H3 antagonist,4,5 anticancer6,7 and
antimetabolite.8 For their varied biological activities, benzimid-
azole derivatives have attracted continuing interest over the years
and were widely applied as an important pharmacophore or build-
ing block in drug discovery.9–11 Bendamustine is such a represen-
tative drug which contains the structure of benzimidazole and is
widely used as an antitumor agent in clinical.
Bendamustine is a DNA-alkylating agent shown clinical activity
against various human cancers including non-Hodgkin’s lym-
phoma,12,13 chronic lymphocytic leukemia,14,15 breast cancer16,17
and small-cell lung cancer.18,19 It contains three parts in its struc-
ture: a 2-chloroethylamine alkylating group, a benzimidazole ring,
and a butyric acid side chain. After the study of the action mecha-
nism and clinical activity of bedamustine, investigators found it
displays a distinct pattern of activity unrelated to other nitrogen
mustard DNA-alkylating agents such as cyclophosphamide, chlor-
ambucil and melphalan, which have the same alkylating group.
Moreover, there was no conclusion about the role of 4-(1-
Our group has been trying to develop some novel 18F labeled
radiotracers for positron emission tomography (PET) imaging espe-
cially for tumor imaging. PET is widely applied in clinical for tumor
imaging nowadays.22 Of all the most commonly used positron iso-
topes employed in PET, 18F is most favorable due to its optimal
physical half-life (t1/2 = 110 min).23 It is well established that
[
18F]-FDG is the most successful and widely used commercial PET
radiopharmaceutical in the clinical.24 However as there are also de-
fects of [18F]-FDG,25–28 some other radiotracers were developed by
labeling bioactive compounds with 18F such as [18F]-FET29 and
[
18F]-FLT30,31 as alternate radiotracers. As benzimidazole deriva-
tives also have a wide range of bioactivity and the structure is
special compared with the existed radiotracers, we are really inter-
ested about whether the 18F labeled benzimidazole derivatives can
get a good result at PET tumor imaging.
However, the labeling and application of compounds containing
benzimidazole nucleus in PET imaging were rarely reported. There-
fore, we decided to label benzimidazole derivatives based on the
structure of the building block of bendamustine with 18F, not only
because bendamustine is a representative drug which contains the
structure of benzimidazle, but also because it is the building block
of bendamustine which made this drug a unique DNA-alkylating
agent.
* Corresponding author. Tel.: +86 136 8138 0097; fax: +86 010 588 02075.
E-mail address: qicmin@sohu.com (C. Qi).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.060

S. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 2394–2401
2395
COOC 2H5
O2N
COOC2H5
H
N
H2N
O2N
NO2
Cl
Et3N, CH2Cl2
N
N
N
N
5steps
ref. 32
NO2
O
2,4-dinitrobenzoic acid
CH3
CH3
a2
a3
a1
O2N
O2N
COOH
COOC2H5
LiOH
Bu4N+F- 3H2O
DMF
H
N
H
N
N
N
N
CH3OH, H2O
F
O
F
O
N
CH3
CH3
a4
Scheme 1.
For this purpose we designed and synthesized [18F] 4-(5-(2-flu-
oro-4-nitrobenzamido)-1-methyl-1H-benzimidazol-2-yl)butanoic
acid ([18F] FNBMBBA). It is the labeling of 4-(1-methyl-1H-ben-
zimidazol-2-yl) butanoic acid (BBA, building block of bendamus-
tine) by the introducing of 2,4-dinitrobenzoyl group at 5- of
benzimidazole ring (Scheme 1). After that, we designed and
synthesized (Scheme 2) another compound [18F] 3-(2-fluoroethyl)-
7-methyl-2-propyl-3H-benzimidazole-5-carboxylic acid ([18F] FEM-
PBBA). It can be taken as a revision of the structure of [18F] FNBMBBA,
the carboxylic group was switched to a benzoic acid at the ring of
benzimidazole, and it was labeled by introducing an ethyltosylate
group at 3- of benzimidazole ring. A series of experiment was carried
out in order to evaluate the bioactivity of both compounds.
18FÀ using [18F] K2.2.2/KF complex by the following procedure
(Scheme 3).
[
18F] FNBMBBA was synthesized in approximately 100 min with
high radiochemical yields (35–45% without decay corrected), and
the radiochemical purity above 99%. The retention time of [18F]
FNBMBBA was 6.4 min (Altech C18 column 250 Â 4 mm, CH3CN/
H2O = 30:70 [vol/vol], 3 mL/min).
For the preparation [18F] FEMPBBA, the purification was used by
Sep-Pak Silica Plus cartridge instead of preparative HPLC method,
which resulted in a shorter time of about only 1 h with overall
radiochemical yield of 50–60% (without decay corrected), and the
radiochemical purity was above 99%. The retention time of [18F]
FEMPBBA was 6.8 min (Altech C18 column 250 Â 4 mm, CH3CN/
H2O = 30:70 [vol/vol], 1 mL/min). The HPLC chromatograms of
both compounds were shown in Figure 1.
2. Results and discussion
2.1. Radiochemistry
2.2. Stability of [18F] FNBMBBA and [18F] FEMPBBA in plasma
Both the radiosynthesis of compound [18F] FEMPBBA and [18F]
FNBMBBA was carried out by the nucleophilic substitution of
As the results of HPLC analysis for both compounds indicate,
they are all stable in the plasma after incubation for 3 h.
OTs
CH3
TsO
N
NaH/THF
N
H3COOC
CH3
CH3
6steps
NO2
N
b3
OTs
ref. 33
CH3
HOOC
CH3
N
H
H3COOC
N
N
N
N
F
Br
LiOH
b2
HOOC
H3COOC
CH3OH, H2O
NaH/THF
b1
F
F
b4
Scheme 2.
O2N
O2N
O2N
COOC2H5
COOC2H5
COOC2H5
H
N
H
N
H
[K+/K222]18F-
LiOH
N
N
N
N
N
N
18F
O
DMF
18F
O
NO2
O
CH3OH, H2O
N
CH3
CH3
CH3
18F a4
18F FNBMBBA (18F a1)
a3
CH3
CH3
N
CH3
[K+/K222]18F-
H3COOC
N
N
LiOH
CH3OH, H2O
N
N
N
HOOC
CH3CN
H3COOC
18F
18F
OTs
18F b4
18F FEMPBBA (18F b1)
b3
Scheme 3. Radiosynthesis procedures of [18F] FEMPBBA and [18F] FNBMBBA.

2396
S. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 2394–2401
2.3. Measurement of partition coefficient
For [18F] FEMPBBA (Table 2), the initial uptake in the tumor was
2.28 0.27%ID/g at 5 min, although it decreased as the time lapsed,
the rate of decrease was much slower than in other organs. That
makes the activity of [18F] FEMPBBA in the tumor remain at a certain
level in a long period with only a little decrease: 1.49 0.13%ID/g at
15 min, 1.41 0.26%ID/g at 30 min and 1.36 0.42%ID/g at 60 min.
On the other hand, the activity in other organs or tissue such as mus-
cle and blood had a more rapid clearance rate, which result in tumor/
blood and tumor/muscle uptake ratios get much higher as time
lapsed.
The partition coefficient of compound [18F] FNBMBBA and [18F]
FEMPBBA was determined to be log P = À1.79 and À0.50. It sug-
gests that the lipophilicity of compound [18F] FEMPBBA is much
higher than that of [18F] FNBMBBA.
2.4. Biodistribution of [18F] FNBMBBA and[18F] FEMPBBA in
S180 bearing mice
As the biodistribution result of [18F] FNBMBBA shown in Table
1, the accumulation of this compound in all of the organs or tissue
was highest in the 5 min post injection and then decreased. After
that the activity in all organs or tissue decreased rapidly and this
compound was quickly excreted out from the body through kidney
after 60 min, and there is nearly no accumulation in the brain of
this compound. We conclude this quickly clearance rate of [18F]
FNBMBBA is due to its high hydrophilicity.
The most interesting part of the biodistribution data of [18F] FEM-
PBBA is the uptake of this compound in the brain. As this compound
has a high lipophilicity (log P = À0.5), it was supposed to be hard to
penetrate the BBB, and the uptake in brain should be very low. How-
ever, in fact the initial [18F] FEMPBBA had a relatively high initial up-
take in the brain, with the activity of 1.03 0.18%ID/g in the brain at
5 min.
As the time lapsed, the activity in the brain rapidly decreased to
0.19 0.06%ID/g at 60 min and 0.05 0.02%ID/g at 120 min. This ra-
pid clearance rate in brain of [18F] FEMPBBA made the tumor/brain
uptake get a rather high ratio: 4.81 at 30 min, 7.15 at 60 min and
9.8 at 120 min.
Although the uptake in tumor of [18F] FNBMBBA is comparably
low, the clearance rate in the tumor is slower than in other organs
or tissue, as time lapses, the activity in the tumor exceeds than that
in most other organs or tissue. Consequently, [18F] FNBMBBA has a
high tumor/muscle uptake ratio which gets to 2.08 and 1.71 at
15 min and 60 min.
From the biodistribution data in S180 tumor bearing mice, we
can see this two compounds showed significant difference, and
Figure 1. HPLC chromatograms of [18F] FEMPBBA and [18F] FNBMBBA and their corresponding 19F standards.
Table 1
Biodistribution data of [18F] FNBMBBA ([18F] a1) in mice bearing S180 tumor (n = 4)
Organs
%ID/g SD
5 min
15 min
30 min
60 min
120 min
Heart
Liver
Pancreas
Lung
Kidney
Stomach
Muscle
Blood
Brain
Tumor
1.37 0.04
8.9 1.35
0.24 0.09
0.4 0.07
0.18 0.07
0.28 0.13
1.24 0.17
0.54 0.21
0.13 0.07
0.37 0.07
0.07 0.02
0.27 0.13
0.73
0.14 0.02
0.66 0.1
0.13 0.03
0.14 0.04
0.43 0.05
0.44 0.12
0.1 0.01
0.22 0.03
0.05 0.004
0.19 0.02
0.86
0.06 0.01
0.65 0.07
0.07 0.02
0.08 0.03
0.32 0.01
0.2 0.11
0.07 0.03
0.1 0.01
0.02 0.01
0.12 0.01
1.20
0.08 0.05
0.11 0.02
0.02 0.01
0.02 0.01
0.06 0.01
0.25 0.06
0.02 0.001
0.02 0.01
0.01 0.003
0.03 0.004
1.50
0.72 0.11
1.97 0.24
8.92 0.85
0.74 0.27
0.87 0.27
2.65 0.63
0.13 0.02
1
0.29
Tumor/blood
Tumor/muscle
0.38
1.15
2.08
1.90
1.71
1.50

S. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 2394–2401
2397
the reason may not simply be the difference of Partition Coefficient
of both compounds. Firstly, the uptake in tumor of two compounds
shows a different trend, [18F] FEMPBBA has maintained a compara-
bly high uptake in tumor for a long time which is not seen for [18F]
FNBMBBA. Secondly, the [18F] FEMPBBA has a much higher uptake
in brain than [18F] FNBMBBA while the hydrophilicities of these
two compounds are both too high to penetrate the BBB (blood–
brain barrier), and the initial uptake of [18F] FEMPBBA is compara-
bly high (Fig. 2). These results indicate the uptake of [18F] FEMPBBA
undergoes a special transport mechanism and apparently much
more suitable for PET tumor imaging.
Parallel biodistribution experiments of [18F] FDG and L-[18F] FET
using the same animal model was performed (Table 3 and 4) as a
comparison to demonstrate the potential of [18F] FNBMBBA as a
PET tumor imaging agents.
From the biodistribution data of [18F] FDG shown in Table 3, we
can discover that the clearance rate of [18F] FDG is really rapid and
consequently tumor/blood uptake ratio is extremely high. On the
other hand, the tumor/muscle uptake ratio of [18F] FDG is also opti-
mistic: 1.75, 2.23 and 2.20 at 30, 60 and 120 min. However, as the
high uptake in the brain of [18F] FDG, the tumor/brain uptake ratio
is comparably low, and kept being under 1 until 60 min after injec-
tion. For L-[18F] FET, the uptake in brain is much lower than [18F]
FDG, and it gets a quite high tumor/brain uptake ratio immediately
after inject (Table 4). The tumor/brain uptake ratio of L-[18F] FET
gets higher as time lapse (2.54, 2.92 and 2.95 at 30, 60 and
120 min) and is much higher than that of [18F] FDG.
3.5
3.0
18
[
F] FEMPBBA
18
L-[ F] FET
18
2.5
2.0
1.5
1.0
.5
[
F] FDG
This result is in accordance with the normal application of L-
[
18F] FET and [18F] FDG in clinical: although [18F] FDG is the most
widely applied PET radiopharmaceutical and have a high sensitiv-
ity for variety kinds of tumors, L-[18F] FET is always used in the
imaging of peripheral tumors as the contrast is by far superior to
that obtained with [18F] FDG because of the low uptake of L-[18F]
FET in normal brain tissue. As the result of biodistribution experi-
ment was shown to be capable of reflecting the specific character
of L-[18F] FET and [18F] FDG, we conclude the comparison of [18F]
FEMPBBA between them can also reflect the potential of it as a
radiotracer for PET tumor imaging.
0.0
0
20
40
60
80
100
120
140
Time (min)
Figure 2. Time course of activity located in the brain of [18F] FEMPBBA, L-[18F] FET
and [18F] FDG. All of the data in this figure except the uptake in the brain of [18F]
FEMPBBA at 120 min were considered statistically significant, P <0.01, two-tailed t-
test.
Table 2
Biodistribution data of [18F] FEMPBBA ([18F] b1) in mice bearing S180 tumor (n = 4)
Organs
%ID/g SD
5 min
15 min
30 min
60 min
120 min
Heart
Liver
Pancreas
Lung
Kidney
Stomach
Muscle
Blood
Brain
Tumor
4.48 0.73
4.13 0.25
2.33 0.38
8.33 1.65
35.9 1.39
5.52 0.51
1.78 0.27
3.17 0.39
1.03 0.18
2.28 0.27
0.72
2.66 0.48
4.02 1.03
1.76 0.48
2.53 0.28
1.03 0.20
3.49 0.35
7.4 0.65
3.87 1.32
1.17 0.05
1.36 0.24
0.31 0.06
1.49 0.13
1.09
1.08 0.35
1.55 0.23
0.67 0.31
2.35 0.85
4.2 1.95
3.23 1.46
0.62 0.05
0.95 0.19
0.19 0.06
1.36 0.42
1.43
0.26 0.08
0.38 0.12
0.2 0.16
0.66 0.18
1.1 0.17
1.14 0.52
0.19 0.07
0.3 0.09
0.05 0.02
0.49 0.09
1.63
1
0.40
5.44 1.43
13.79 8.40
4.33 1.48
1.22 0.26
2.25 0.77
0.44 0.06
1.41 0.26
0.63
Tumor/blood
Tumor/muscle
Tumor/brain
1.28
2.21
1.16
3.20
1.27
4.81
2.19
7.15
2.58
9.8
Table 3
Biodistribution data of [18F] FDG in mice bearing S180 tumor (n = 4)
Organs
%ID/g SD
30 min
5 min
15 min
60 min
120 min
Heart
Liver
Pancreas
Lung
Kidney
Muscle
Blood
Brain
Tumor
2.6 0.06
1.35 0.42
1.31 0.4
1.52 0.66
2.16 0.35
1.56 0.13
1.09 0.74
2.4 0.28
1.27 0.54
1.17
2.5 0.06
0.47 0.17
0.95 0.01
0.77 0.13
1.04 0.21
1.14 0.23
0.46 0.14
2.63 0.1
1.56 0.23
3.42
2.69 0.14
3.24 0.2
0.22 0.04
0.83 0.04
0.57 0.06
0.54 0.15
0.97 0.66
0.11 0.05
2.11 0.17
2.16 0.34
19.47
6.27 0.3
0.2 0.03
0.78 0.18
0.52 0.11
0.25 0.06
0.78 0.06
0.11 0.13
1.28 0.08
1.7 0.06
15.85
0.23
0
0.9 0.19
0.64 0.05
0.61 0.23
1.07 0.86
0.16 0.01
3.07 0.11
1.86 0.18
11.38
Tumor/blood
Tumor/muscle
Tumor/brain
0.81
0.53
1.37
0.59
1.75
0.61
2.23
1.02
2.2
1.33

2398
S. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 2394–2401
Table 4
Biodistribution data of [18F] FET in mice bearing S180 tumor (n = 4)
Organs
%ID/g SD
30 min
5 min
15 min
60 min
120 min
Heart
Liver
Pancreas
Lung
Kidney
Muscle
Blood
Brain
Tumor
5.89 1.23
7.67 1.34
0.93 0.35
16.8 2.98
6.58 0.84
2.88 1.33
6.81 0.95
0.99 0.24
2.08 0.49
0.31
3.41 0.31
5.60 0.30
0.22 0.02
9.82 0.45
6.79 0.23
3.34 0.41
3.30 0.25
1.03 0.12
2.50 0.23
0.76
3.27 0.37
4.23 0.28
0.11 0.07
8.57 0.63
2.38 0.43
2.69 0.96
3.23 0.33
1.29 0.09
3.28 0.69
1.02
3.14 0.26
3.56 0.29
0.03 0.01
4.70 0.41
3.75 0.49
2.37 0.38
2.51 0.36
0.94 0.23
2.75 0.36
1.10
0.94 0.36
1.22 0.51
0.09 0.05
1.55 0.73
1.29 0.21
2.10 1.33
1.12 0.37
0.73 0.21
2.15 1.36
1.92
Tumor/blood
Tumor/muscle
Tumor/brain
0.72
2.10
0.75
2.43
0.62
2.54
1.23
2.92
1.02
2.95
3.0
2.5
2.0
1.5
1.0
.5
10
8
18
F] FEMPBBA
[18F] FDG
L-[18F] FET
18
[
[
F] FNBMBBA
18
[
L- F] FET
18
[
F] FDG
6
4
2
0.0
0
0
5
15
30
60
120
5
15
30
60
120
Time (min)
Time (min)
Figure 3. Comparison of tumor/muscle uptake ratios of [18F] FEMPBBA with [18F]
Figure 4. Comparison of tumor/brain uptake ratios of [18F] FEMPBBA with [18F] FET
FET and [18F] FDG.
and [18F] FDG.
From the comparison of [18F] FEMPBBA between L-[18F] FET and
3. Conclusion
[
18F] FDG, we find [18F] FEMPBBA to be competitive in some aspect.
Our attempt of developing novel 18F labeled benzimidazole
derivatives has turned to be encouraging. Both compounds were
prepared by convenient procedures and got high radiochemical
preparation yields, and the biodistribution data of two compounds
in S180 tumor bearing mice has also revealed the promising pros-
pect of benzimidazole derivatives to be applied in PET tumor imag-
ing. The comparison of [18F] FEMPBBA with [18F] FDG and L-[18F]
FET showed it has an extremely high tumor/brain and optimistic
tumor/muscle and blood uptake ratios, which indicates more
investigation and experiments are needed to make further evalua-
tion of the potential of [18F] FEMPBBA and other 18F labeled benz-
imidazole derivatives to be suitable PET tumor imaging
radiotracers.
Firstly, compared with L-[18F] FET, [18F] FEMPBBA has a much
lower uptake in blood pool and a more rapid clearance rate from
blood. That make [18F] FEMPBBA get much higher tumor/blood up-
take ratio than L-[18F] FET at 30 min and 60 min after injection.
Secondly, [18F] FEMPBBA also has a much lower uptake in mus-
cle and a more rapid clearance rate from muscle than L-[18F] FET,
and get a much higher tumor/muscle uptake ratio. In contrast to
[
18F] FDG, although the initial uptake in muscle of [18F] FEMPBBA
a little higher, the clearance rate of [18F] FEMPBBA in muscle is
more rapid than that of [18F] FDG and it get a pretty much the same
tumor/muscle uptake ratio as [18F] FDG: 1.16, 2.19, 2.58 for [18F]
FEMPBBA and 1.37, 2.23, 2.2 for [18F] FDG at 15 min, 60 min and
120 min. It is interesting to find that the tumor/muscle uptake ra-
tio of [18F] FEMPBBA and [18F] FDG is nearly same at 60 min (2.19
for [18F] FEMPBBA and 2.23 for [18F] FDG), and the highest tumor/
muscle uptake ratio of [18F] FEMPBBA is higher than [18F] FDG
(Fig. 3).
4. Methods and experimental
4.1. General
The greatest advantage of [18F] FEMPBBA compared with L-[18F]
FET and [18F] FDG is the tumor/brain uptake ratio. Compared with
L-[18F] FET, the initial uptake of [18F] FEMPBBA in brain is slightly
higher, 1.03 for [18F] FEMPBBA and 0.99 for L-[18F] FET at 5 min.
However, the clearance rate of [18F] FEMPBBA from brain is really
rapid, and the activity of [18F] FEMPBBA get down immediately
after 5 min while the uptake of L-[18F] FET in brain increase at first
30 min, then decrease (Fig. 2). That make the [18F] FEMPBBA has
got a much higher tumor/brain uptake ratio than L-[18F] FET, and
certainly much higher than [18F] FDG (Fig. 4).
All commercial reagents and solvents were used without fur-
ther purification. No-carrier-added [18F] fluoride was produced
by the 18O(p, n)18F nuclear reaction at Beijing PET Center of Xuan-
wu Hospital. Semipreparative HPLC column (Altech C18 column
250 Â 4 mm) and analytical HPLC column (Altech C18 column
250 Â 4 mm) were purchased from Altech Inc. C18 Sep-Pak Car-
tridge was obtained from Waters Inc.
Melting points were determined in capillary tubes using a RY-1
apparatus and are uncorrected. Nuclear magnetic resonance spectra

S. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 2394–2401
2399
(1H NMR and 13C NMR) were performed on a Bruker spectrometer
(400 M and 100 M), and chemical shifts (d values) were reported
as parts per million (ppm) downfield from tetramethylsilane
(TMS). Mass spectra were recorded using a Brucker Apex IV FTM
instrument at Mass Spectroscopy Center at Beijing Normal Univer-
sity using high-resolution electrospray ionization (ESI), or electron
impact ionization (EI).
29.88, 26.71, 22.61, 14.21 ppm. 19F NMR (400 MHz, CDCl3) d
À109.21 ppm. MS (ESI+) m/z: 429.1 (M+H+). IR (KBr pellet, cmÀ1):
3337, 1732, 1651, 1598, 1561, 1530, 1486, 1431, 1423, 1377,
1350, 1318, 1269, 1263, 1189, 813, 736. Anal. Calcd for
C21H21FN4O5: C, 58.87; H, 4.94; N, 13.08. Found: C, 58.51; H,
5.08; N, 13.22.
4.2.3. Preparation of 4-(5-(2-fluoro-4-nitrobenzamido)-1-meth
yl-1H-benzimidazol-2-yl)butanoic acid (a1)
4.2. Synthesis
Compound a4 (60 mg, 0.14 mmol) was added into CH3CH2OH
(6 mL) and cooled to 0 °C in an ice bath, the solution of LiOH
(30 mg, 0.7 mmol) in H2O (1 mL) was then added. The reaction
mixture was stirred overnight and the pH was adjusted to 2–3 with
35% HCl. The product was precipitated and filtered, the crude prod-
uct was crystallized to give a light yellow solid (48 mg, 85%) mp
207–209 °C. 1H NMR (400 MHz, DMSO) d 11.22 (s, 1H, –COOH),
8.38 (d, J = 1.2 Hz, 1H, Ar-H), 8.33 (dd, J = 9.6 Hz, 2.0 Hz, 1H, Ar-
H), 8.22 (dd, J = 8.4 Hz, 2.0 Hz, 1H, Ar-H), 8.02 (m, 1H, Ar-H), 7.93
(d, J = 8.9 Hz, 1H, Ar-H), 7.76 (d, J = 9.0 Hz, 1H, Ar-H), 3.96 (s, 3H,
N–CH3), 3.20 (t, J = 7.6 Hz, 2H, –CH2CH2CH2–), 2.45 (t, J = 7.1 Hz,
2H, –CH2CH2CH2–), 2.06 (m, 2H, –CH2CH2CH2–) ppm. 13C NMR
(100 MHz, DMSO) d 173.64, 161.41, 158.39 (d, JCF = 253.0 Hz, 1C),
153.92, 149.35 (d, JCF = 8.6 Hz, 1C), 136.27, 131.17 (d, JCF = 3.4 Hz,
1C), 130.56, 130.40, 129.30, 119.70 (d, JCF = 3.5 Hz, 1C), 117.99,
112.80, 112.10 (d, JCF = 27.3 Hz, 1C), 104.54, 32.53, 30.99, 24.42,
21.30 ppm. 19F NMR (400 MHz, CDCl3) d À111.08 ppm. MS (ESI+)
m/z: 401.5 (M+H+). IR (KBr pellet, cmÀ1): 3432, 3043, 1724, 1682,
1624, 1562, 1529, 1500, 1446, 1419, 1352, 1287, 1191, 816, 739.
Anal. Calcd for C19H17FN4O5: C, 57.00; H, 4.28; N, 13.99. found: C,
56.76; H, 4.53; N, 13.87.
The preparation of compound a2 was carried out by following
the same procedure in Ref. 32, and the preparation of compound
b2 was carried out by the same procedure in Ref. 33. The prepara-
tion and the analysis data of other compounds were shown below.
4.2.1. Preparation of ethyl 4-(5-(2,4-dinitrobenzamido)-1-meth
yl-1H-benzimidazol-2-yl)butanoate (a3)
Compound a2 (0.261 g, 1 mmol) and TEA (0.14 mL, 1 mmol)
was added into CH2Cl2 (10 mL) and cooled to 0 °C in an ice bath.
Then the solution of 2,4-dinitrobenzoyl chloride (0.23 g, 1.2 mmol)
in CH2Cl2 (5 mL) was added dropwise. The reaction mixture was
stirred in 0 °C for 0.5 h then at room temperature for 6 h. The reac-
tion mixture was washed by NaHCO3 aqueous (10 mL Â 2), water
(10 mL Â 2), the organic layer was collected and dried over Na2SO4.
The solvent was removed by rotary evaporation under vacuum.
The crude product was recrystallized in acetic ether to give the
product as yellow–green solid. (391 mg, 83% yield) mp 176–
178 °C. 1H NMR (400 MHz, CDCl3) d 8.73 (s, 1H, –NHCO–), 8.69
(d, J = 2.0, 1H, Ar-H), 8.35 (dd, J = 8.3, 2.1, 1H, Ar-H), 7.81 (d,
J = 8.3, 1H, Ar-H), 7.59 (s, 1H, Ar-H), 7.48 (d, J = 8.6, 1H, Ar-H),
7.16 (d, J = 8.6, 1H, Ar-H), 4.04 (q, J = 7.1, 2H, –CH2CH3), 3.67 (s,
3H, N–CH3), 2.82 (t, 2H, –CH2CH2CH2–), 2.38 (t, J = 7.0, 2H,
–CH2CH2CH2–), 2.05 (m, 2H, –CH2CH2CH2–), 1.18 (t, J = 7.1, 3H,
–CH2CH3) ppm. 13C NMR (100 MHz, CDCl3) d 173.06, 162.72,
155.69, 147.97, 146.19, 142.15, 137.93, 133.66, 131.59, 130.46,
128.23, 119.93, 116.72, 111.63, 109.23, 60.50, 33.22, 29.87, 26.59,
22.52, 14.20 ppm. MS (ESI+) m/z: 456.8 (M + H+). IR (KBr pellet,
cmÀ1): 3210, 1729, 1641, 1612, 1597, 1546, 1527, 1483, 1341,
1305, 1222, 1050, 1017, 906, 808, 736. Anal. Calcd for
C21H21N5O7: C, 55.38; H, 4.65; N, 15.38. Found: C, 55.27; H, 4.73;
N, 15.52.
4.2.4. Preparation of methyl 7-methyl-2-propyl-3-(2-(tosyloxy)
ethyl)-3H-benzimidazole-5-carboxylate (b3)
To a 20 mL dried THF, NaH (0.18 g, 7.5 mmol) was dissolved and
the solution was stirred in an ice bath. Then compound b2 (0.58 g,
2.5 mmol) was added portionwise. After stirred in room tempera-
ture for another 1 h, ethyleneglycol-1,2-ditosylate (5 mmol) was
added and the reaction mixture was stirred in 40–50 °C overnight.
The solvent was removed and the residue was chromatographed
over a column of silica gel (A:P = 3:1) to give a white solid.
(0.45 g, 42% yield) mp 167–168 °C. 1H NMR (400 MHz, CDCl3) d
7.65 (s, 1H, Ar-H), 7.54 (s, 1H, Ar-H), 7.34 (d, J = 8.3 Hz, 2H,
Ar-H), 6.94 (d, J = 8.1 Hz, 2H, Ar-H), 4.33 (t, J = 5.1 Hz, 2H,
–CH2CH2–OTs), 4.26 (t, J = 5.1, 2H, –CH2CH2–OTs), 3.86 (s, 3H,
–OCH3), 2.79 (t, J = 8.0 Hz, 2H, –CH2CH2CH3), 2.58 (s, 3H, Ar-CH3),
2.23 (s, 3H, Ar-CH3), 1.79 (m, 2H, –CH2CH2CH3), 0.99 (t, J = 7.3 Hz,
3H, –CH2CH2CH3) ppm. 13C NMR (100 MHz, CDCl3) d 167.5,
157.5, 145.6, 145.0, 133.4, 131.4, 129.6, 129.0, 127.4, 123.9,
123.8, 108.4, 66.4, 52.0, 42.6, 29.4, 21.7, 21.5, 16.7, 14.0 ppm. MS
(ESI+) m/z: 431.1 (M+H+). IR (KBr pellet, cmÀ1): 1702, 1436, 1359,
1340, 1279, 1249, 1205, 1193, 1180, 1009, 899, 769, 664, 554. Anal.
Calcd for C22H26N2O5S: C, 61.38; H, 6.09; N, 6.51. Found: C, 61.53;
H, 6.34; N, 6.73.
4.2.2. Preparation of ethyl 4-(5-(2-fluoro-4-nitrobenzamido)-1-
methyl-1H-benzimidazol-2-yl)butanoate (a4)
Bu4NFÁ3H2O (0.73 g, 2 mmol) was added into dry CH3CN (1 mL),
The solvent was evaporated under a stream of nitrogen at 78 °C in
a flask, and repeated at least twice. Then the solution of compound
a3 (0.455 g, 1 mmol) in the DMF (10 mL) was added to the flask
and quickly heated to 120 °C. After stirred in this temperature for
0.5 h, the reaction was cooled down and added 20 mL H2O. The
mixture was extracted with acetic ether (20 mL Â 3), the organic
layer was collected and evaporated by rotary evaporation under
vacuum. The residue was chromatographed over a column of silica
gel (A:P = 3:1) to give a yellow solid (52 yield) mp 176–178 °C. 1H
NMR (400 MHz, CDCl3) d 8.40 (d, J = 13.6 Hz, 1H, Ar-H), 8.34 (t,
J = 8.2 Hz, 1H, Ar-H), 8.11 (dd, J = 8.6, 2.0 Hz, 1H, Ar-H), 8.02 (dd,
J = 11.2, 2.0 Hz, 1H, Ar-H), 7.86 (d, J = 1.8 Hz, 1H, –NH–CO–), 7.53
(dd, J = 8.6, 1.9 Hz, 1H, Ar-H), 7.24 (d, J = 8.6 Hz, 1H, Ar-H), 4.06
(q, J = 7.1 Hz, 2H, –OCH2CH3), 3.71 (s, 3H, N–CH3), 2.90 (t,
J = 7.5 Hz, 2H, –CH2CH2CH2–), 2.44 (t, J = 7.0 Hz, 2H,
–CH2CH2CH2–), 2.13 (m, J = 7.1 Hz, 2H, –CH2CH2CH2–), 1.19 (t,
J = 7.1 Hz, 3H, –OCH2CH3) ppm. 13C NMR (100 MHz, CDCl3) d
173.12, 160.78, 158.74 (d, JCF = 91.4 Hz, 1C), 155.60, 150.26 (d,
JCF = 10.1 Hz, 1C), 142.75, 133.69, 133.68 (d, JCF = 2.5 Hz, 1C),
131.59, 127.42 (d, JCF = 12.6 Hz, 1C), 119.82 (d, JCF = 3.5 Hz, 1C),
116.62, 112.21 (d, JCF = 30.7 Hz, 1C), 112.00, 109.13, 60.46, 33.27,
4.2.5. Preparation of methyl 3-(2-fluoroethyl)-7-methyl-2-prop
yl-3H-benzimidazole-5-carboxylate (b4)
To a 20 mL dried THF, NaH (0.29 g, 12 mmol) was dissolved and
the solution was stirred in an ice bath. Then compound b3 (0.93 g,
4 mmol) was added portionwise. After being stirred in room
temperature for another 1 h, 1-bromo-2-fluoroethane (1.4 mL,
13 mmol) was added dropwise and the reaction mixture was stir-
red in 40–50 °C overnight. The solvent was removed and the resi-
due was chromatographed over a column of silica gel (A:P = 2:1) to
give a white solid. (0.48 g, 83% yield). mp 103–105 °C. 1H NMR
(400 MHz, CDCl3) d 7.78 (s, 1H, Ar-H), 7.72 (s, 1H, Ar-H), 4.68 (dt,
J = 46.8, 4.7 Hz, 2H, –CH2CH2F), 4.39 (dt, J = 25.6 Hz, 4.6 Hz, 2H,

2400
S. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 2394–2401
–CH2CH2F), 3.87 (s, 3H, –OCH3), 2.61 (s, 3H, –CH3), 2.60 (s, 3H, –
CH3) ppm. 13C NMR (100 MHz, CDCl3) d 167.74, 154.29, 145.53,
134.12, 128.98, 124.07, 123.92, 108.61, 81.40 (d, JCF = 173.4 Hz,
1C), 52.14, 44.40 (d, JCF = 21.4 Hz, 1C), 16.67, 14.17 (d, JCF = 2.5 Hz,
1C) ppm. 19F NMR (400 MHz, CDCl3) d À221.32 ppm. MS (ESI+) m/z:
251.0 (M+H+). IR (KBr pellet, cmÀ1): 1712, 1518, 1435, 1408, 1346,
1292, 1274, 1232, 1215, 1203, 1034, 764. Anal. Calcd for
C13H15FN2O2: C, 62.39; H, 6.04; N, 11.19. Found: C, 62.43; H,
6.11; N, 11.08.
Cartridge, and the cartridge was washed with water (2 mL Â 2),
then eluted with 2 mL of CH3CN containing 0.1% TFA. The result
of HPLC analysis indicates the two compounds were both stable
in plasma.
4.3.2. Measurement of partition coefficient
The partition coefficient of [18F] FNBMBBA and [18F] FEMPBBA
was measured using 2.5 mL n-octanol as the organic phase and
2.5 mL 0.01 M PBS (pH 7.4) as the water phase. 0.01 mL radioactive
sample of [18F] FNBMBBA or [18F] FEMPBBA was added. After being
vigorously mixed for 5 min at room temperature, the radioactivity
of 0.01 mL of each phase was measured after centrifugation.
4.2.6. Preparation of 3-(2-fluoroethyl)-7-methyl-2-propyl-3H-
benzimidazole-5-carboxylic acid (b1)
Compound b4 (0.22 g, 0.79 mmol) was added into CH3OH
(7 mL) and cooled to 0 °C in an ice bath, the aqueous solution of
NaOH (10%, 3 mL) was then added. The reaction mixture was stir-
red overnight and the pH was adjusted to 2–3 with 35% HCl. The
product was precipitated and filtered, and the crude product was
crystallized to give a light white solid. (0.19 g, 90% yield) mp
237–239 °C. 1H NMR (400 MHz, DMSO) d 7.99 (s, 1H, Ar-H), 7.62
(s, 1H, Ar-H), 4.78 (s, 1H, –CH2CH2F), 4.66 (s, 2H, –CH2CH2F), 4.59
(s, 1H, –CH2CH2F), 2.87 (t, J = 7.6 Hz, 2H, –CH2CH2CH3), 2.54 (s,
3H, Ar-CH3), 1.83 (m, 2H, –CH2CH2CH3), 1.01 (t, J = 7.3 Hz, 3H,
–CH2CH2CH3) ppm.13C NMR (100 MHz, DMSO) d 167.96, 157.41,
144.95, 134.31, 127.44, 123.89, 122.87, 109.79, 82.56 (d,
JCF = 167.8 Hz, 1C), 43.52 (d, JCF = 19.6 Hz, 1C), 28.41, 20.41, 16.26,
13.81 ppm. 19F NMR (400 MHz, CDCl3) d À220.70 ppm. MS (ESI+)
m/z: 265.1 (M+H+). IR (KBr pellet, cmÀ1): 3448, 2960, 2929, 1687,
1613, 1595, 1459, 1413, 1348, 1248, 1227, 1215, 1189, 1098,
1041, 1020, 897, 777, 707. Anal. Calcd for C14H17FN2O2: C, 63.62;
H, 6.48; N, 10.60. Found: C, 63.45; H, 6.63; N, 10.45.
4.3.3. Biodistribution in S180 bearing mice
The biodistribution experiments of both compounds were car-
ried out with the same procedure.
Female Kunming mice with S180 were prepared for biodistribu-
tion studies. The S180 tumor model was generated by subcutaneous
injection of 5 Â 106 tumor cells into the right front flank of Female
Kunming mice without anesthesia. The experiment was performed
after the tumor cells were inoculated for 7–14 days, at which time
the mice weighed between 18–22 g. Each animal was injected with
saline solvent of [18F] FNBMBBA or [18F] FEMPBBA (10–20
100 L) via the tail vein, then sacrificed at 5, 15, 30, 60 and 120 min
lCi,
l
post injection. The interested organs and tissue samples were excised
immediately, blotted to remove adhering blood, weighed, and the
radioactivity was measured using a gamma counter. The uptake of
radiotracer in organs or tissue was expressed in counts per second
(cps) with decay correction normalized as mean SD (%ID/g, n = 4),
and the statistics were performed by two-tailed t-test.
4.2.7. Radiochemistry
Acknowledgments
[
18F] fluoride was generated from H2[18O]-enriched water via
proton bombardment. The [18F] fluoride was absorbed on an anion
exchange cartridge, and eluted with an aqueous mixture of
10 mg K2.2.2 and 3 mg K2CO3 in 0.5 mL CH3CN and 1 mL H2O to
get [18F] K2.2.2/KF complex. The solvent was evaporated under a
stream of nitrogen at 90 °C. Azeotropic drying was repeated more
than twice with 1 mL portions of CH3CN.
We acknowledged with thanks for financial supports from the
National Science foundation of China (No. 20371009 and
20671014) and Beijing Municipal Commission of Education. We
also would like to thank the cyclotron operator team of the PET
Centre of Xuan Wu hospital, for providing [18F] fluoride activity
and technical assistance.
However, as the leaving group of two compounds is different,
the condition of the substitution reaction is also different. After
the azeotropic drying was completed, the solution of approxi-
mately 4–5 mg a4 in 2 mL dry DMF for [18F] FNBMBBA (2 mL dry
CH3CN and 4–5 mg b3 for [18F] FEMPBBA) was added to the vial
containing [18F] K2.2.2/KF complex. The solution was heated to
115 °C (90 °C for [18F] FEMPBBA) for 20 min.
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