Synthesis and biological evaluation of novel F-18 labeled pyrazolo[1,5-a]pyrimidine derivatives: Potential PET imaging agents for tumor detection

Article abstract of DOI:10.1016/j.bmcl.2011.06.072

Two novel pyrazolo[1,5-a]pyrimidine derivatives, 7-(2-[¹⁸F] fluoroethylamino)-5-methylpyrazolo[1,5-a]pyrimidine-3-carbonitrile ([ ¹⁸F]FEMPPC, [¹⁸F]1) and N-(2-(3-cyano-5-methylpyrazolo[1, 5-a]pyrimidin-7-ylamino)ethyl)-2-[¹⁸F]fluoro-4-nitrobenzamide ([ ¹⁸F]FCMPPN, [¹⁸F]2), have been designed and successively labeled with ¹⁸F by the nucleophilic substitution employing tosylate and nitryl as leaving groups, respectively. The radiochemical synthesis of both compounds was completed within 60 min with final high-performance liquid chromatography purification included. The corresponding radiochemical yields (without decay correction) were approximately 35% and 30%, respectively. Meanwhile, we compared the uptake characteristics of [¹⁸F]1 and [¹⁸F]2 with those of [¹⁸F]FDG and L-[¹⁸F]FET in S180 tumor cells. Furthermore, the tumor uptake of [¹⁸F]1 and [ ¹⁸F]2 was assessed in mice bearing S180 tumor and compared with [¹⁸F]FDG and L-[¹⁸F]FET in the same animal model. In vitro cell uptake studies showed [¹⁸F]1 had higher uptake than [ ¹⁸F]FDG, [¹⁸F]2 and L-[¹⁸F]FET over the 2 h period. In ex vivo biodistribution showed tumor/brain uptake ratios of [ ¹⁸F]2 were 12.35, 10.44, 8.69 and 5.13 at 15 min, 30 min, 60 min and 120 min post-injection, much higher than those of L-[¹⁸F]FET (2.43, 2.54, 2.93 and 2.95) and [¹⁸F]FDG (0.59, 0.61, 1.02 and 1.33) at the same time point. What's more, the uptake of [¹⁸F]1 in tumor was 1.88, 4.37, 5.51, 2.95 and 2.88 at 5 min, 15 min, 30 min, 60 min and 120 min post-injection, respectively. There was a remarkable increasing trend before 30 min. The same trend was present for L-[¹⁸F]FET before 30 min and [¹⁸F]FDG before 60 min. Additionally, the tumor/brain uptake ratios of [¹⁸F]1 were superior to those of [¹⁸F]FDG at all the selected time points, the tumor/muscle and tumor/blood uptake ratios of [ ¹⁸F]1 at 30 min were higher than those of L-[¹⁸F]FET at the same time point. MicroPET image of [¹⁸F]1 administered into S180 tumor-bearing mouse acquired at 30 min post-injection illustrated that the uptake in S180 tumor was obvious. These results suggest that compound [ ¹⁸F]1 could be a new probe for PET tumor imaging.

Full text of DOI:10.1016/j.bmcl.2011.06.072

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4736–4741
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel F-18 labeled pyrazolo[1,5-a]
pyrimidine derivatives: Potential PET imaging agents for tumor detection
Jingli Xu a, Hang Liu a, Guixia Li a, Yong He a, Rui Ding a, Xiao Wang a, Man Feng a, Shuting Zhang a,
b
Yurong Chen a, Shilei Li a, Mingxia Zhao a, Chuanmin Qi a, , Yonghong Dang
⇑
a Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, China
b Department of Nuclear Medicine, PUMC Hospital, Beijing 100730, China
a r t i c l e i n f o
a b s t r a c t
Two novel pyrazolo[1,5-a]pyrimidine derivatives, 7-(2-[18F]fluoroethylamino)-5-methylpyrazolo[1,5-
a]pyrimidine-3-carbonitrile ([18F]FEMPPC, [18F]1) and N-(2-(3-cyano-5-methylpyrazolo[1,5-a]pyrimi-
din-7-ylamino)ethyl)-2-[18F]fluoro-4-nitrobenzamide ([18F]FCMPPN, [18F]2), have been designed and
successively labeled with 18F by the nucleophilic substitution employing tosylate and nitryl as leaving
groups, respectively. The radiochemical synthesis of both compounds was completed within 60 min with
final high-performance liquid chromatography purification included. The corresponding radiochemical
yields (without decay correction) were approximately 35% and 30%, respectively. Meanwhile, we com-
pared the uptake characteristics of [18F]1 and [18F]2 with those of [18F]FDG and L-[18F]FET in S180 tumor
cells. Furthermore, the tumor uptake of [18F]1 and [18F]2 was assessed in mice bearing S180 tumor and
compared with [18F]FDG and L-[18F]FET in the same animal model. In vitro cell uptake studies showed
Article history:
Received 12 February 2011
Revised 3 June 2011
Accepted 16 June 2011
Available online 23 June 2011
Keywords:
F-18 labeled
Pyrazolo[1,5-a]pyrimidine derivatives
PET imaging agents
Tumor detection
[
18F]1 had higher uptake than [18F]FDG, [18F]2 and L-[18F]FET over the 2 h period. In ex vivo biodistribu-
tion showed tumor/brain uptake ratios of [18F]2 were 12.35, 10.44, 8.69 and 5.13 at 15 min, 30 min,
60 min and 120 min post-injection, much higher than those of L-[18F]FET (2.43, 2.54, 2.93 and 2.95)
and [18F]FDG (0.59, 0.61, 1.02 and 1.33) at the same time point. What’s more, the uptake of [18F]1 in
tumor was 1.88, 4.37, 5.51, 2.95 and 2.88 at 5 min, 15 min, 30 min, 60 min and 120 min post-injection,
respectively. There was a remarkable increasing trend before 30 min. The same trend was present for
L-[18F]FET before 30 min and [18F]FDG before 60 min. Additionally, the tumor/brain uptake ratios of
[
18F]1 were superior to those of [18F]FDG at all the selected time points, the tumor/muscle and tumor/
blood uptake ratios of [18F]1 at 30 min were higher than those of L-[18F]FET at the same time point.
MicroPET image of [18F]1 administered into S180 tumor-bearing mouse acquired at 30 min post-injection
illustrated that the uptake in S180 tumor was obvious. These results suggest that compound [18F]1 could
be a new probe for PET tumor imaging.
Ó 2011 Elsevier Ltd. All rights reserved.
Pyrazolopyrimidines have multiple pharmacological activities
including hypnotic,1 anti-inflammatory,2 anti-tumor,3–7 antimyco-
bacterial8 and anti-viral.9,10 Owing to the similarity in structure to
purine, pyrazolo[1,5-a]pyrimidine and its derivatives have at-
tracted broad interest for the years.11 They were found to have
antitrypanosomal12 and antischistosomal activity,13 and most
importantly, anti-tumor activity.14–17 It has been found that many
pyrazolo[1,5-a]pyrimidine derivatives may block proliferation of
various cancer cell lines.15,18,19 As a result, pyrazolo[1,5-a]pyrimi-
dine was widely applied as an important pharmacophore or
building block in anti-tumor drug design, which was motivating
further study on physiological and biological property of this kind
of compounds, including in vitro stability, in vivo distribution,
metabolism and elimination. With the increasing availability of
PET (positron emission topography) to provide quantitative kinetic
information of metabolic pathways and physiological processes in
vivo,20 PET and in particular integrated PET/CT technique can de-
tect diseases earlier and more accurately than any other imaging
technique. A PET/CT is an effective tool to help stage local, regional,
distant disease and provide therapeutic guidance for cancer ther-
apy. Of all the most commonly used positron emission isotopes,
18F is the most favorable mainly due to its longer physical half-life
of 110 min,21 the comparable size to H atom and lower positron
energy. 2-[18F]fluoro-2-deoxyglucose (18F-FDG), an analog of glu-
cose, is the most widely used compound for cancer detection.22
In spite of being a very effective PET tracer for many types of
tumors, 18F-FDG can suffer from certain limitations: because high
⇑
Corresponding author.
E-mail address: qicmin@sohu.com (C. Qi).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.072

J. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4736–4741
4737
accumulation in inflammation and infection can lead to false-posi-
tive results and low uptake in tumors that are growing slowly can
cause false-negative results.23,24
35% radiochemical yield (without decay correction). While [18F]2
18
was prepared through a 25 min F fluorination at 20-nitryl in dry
DMF under N2 at 120 °C. The radiochemical yield was about 30%
(without decay correction). Fixation of K[18F]F on Kryptofix
2.2.2.was used in both synthetic processes as 18F fluridizer. The
mixed product was highly diluted with water, adsorbed on a C18
Sep-Pak cartridge and almost quantitatively desorbed with aceto-
nitrile. The crude products were purified by semi-preparative HPLC
with reversed-phase Grace Alltima™ C18 Column (250ꢁ10 mm,
Consequently, more radiopharmaceuticals are needed to be em-
ployed in the clinical cancer management. We are focusing on
designing and developing novel 18F labeled radioactive tracers for
PET imaging and we are especially interested in small molecular
PET tracers for tumor detection. Although the anti-tumor activities
and the corresponding mechanisms of pyrazolo[1,5-a]pyrimidines
have been explored extensively, the application of 18F labeled
pyrazolo[1,5-a]pyrimidine derivatives in PET imaging has little
been reported. Pyrazolo[1,5-a]pyrimidine core structure has sev-
eral positions for introducing substituent, the C-5 position and
the C-7 position were commonly used for introducing substituent
in anti-tumor drug design.14,18,19,25,26 As far as molecular design of
radiopharmaceuticals is concerned, we should consider not only
structural variety, but also radiochemosynthetic factors (which
means faster synthesis of 18F-labeled compound for production, a
higher yield by avoiding side reactions and higher reproducibility).
We previously explored a novel one-step 18F fluorination synthetic
method using 2,4-dinitrobenzoic acid modified targeting mole-
cules as precursors.27 This method was improved by us to modify
the pyrazolo[1,5-a]pyrimidine and designed precursor 9 (Fig. 1).
By labeling 9 with 18F, we got [18F]2 (Fig. 1). In addition, we also
used tosylate as leaving group and designed precursor 7 (Fig. 1),
which can be labeled with 18F to get [18F]1 (Fig. 1). The biological
properties of these new radiotracers were evaluated in vitro and
in vivo in comparison with [18F]FDG and L-[18F]FET.
particle size: 10 lm). The column was eluted with isocratic solvent
system as follows: 30% water and 70% acetonitrile; flow rate: 5 L/
min. The retention time of [18F]1 was 3.061 min and the retention
time of [18F]2 was 3.160 min in our gradient, respectively. The
desired product was collected from HPLC and the solvent was
evaporated using a rotary evaporator. The product was dissolved
in phosphate-buffered saline solution (pH 7.4). The nondecay-
corrected radiochemical yields were approximately 30–35% and
with specific activity between 41–50 GBq/lmol. The radiochemical
purity and chemical purity were above 99%. The total reaction time
for either [18F]1 or [18F]2 was about 60 min, with final high-
performance liquid chromatography purification included.
The lipophilicity of both radiotracers was measured according
to the method previously reported by us.27 The partition coefficient
(log P) values of [18F]1 and [18F]2 were 0.68 0.02 and 0.69 0.02,
respectively. The similar partition coefficient of both radiotracers
has demonstrated that the different substituents of [18F]1 and
[
18F]2 at C-7 position of pyrazolo[1,5-a]pyrimidine core structure
did not lead to significant change in lipophilicity. The stability of
each 18F labeled compound in mouse plasma was determined by
incubating 0.1 mL of [18F]1 and [18F]2 (3.7 MBq) in the solution
of 0.5 mL murine plasma at 37 °C for 1 h and 2 h. Plasma proteins
were precipitated by adding acetonitrile and removed by centrifu-
gation. The supernatant part was injected into HPLC to determine
the stability of each compound. The HPLC patterns indicated that
both [18F]1 and [18F]2 were stable in mouse plasma.
The [19F]1 and [19F]2 were prepared from the same intermedi-
ate 5 which was synthesized from malononitrile through an easy
four-step procedure (Scheme 1).26
The tosylate modified precursor 7 (Scheme 2) was prepared
from intermediate 5 by the introducing of 4-methylbenzene-1-
sulfonyl chloride bridged by 2-aminoethanol at C-7 position of
pyrazolo[1,5-a]pyrimidine core structure, 19F substitution [19F]1
(Scheme 2) was obtained from precursor 7 using TBAFꢀ3H2O in
dry acetonitrile at 80 °C under nitrogen atmosphere for 5 h with
38% yield. The reaction is shown in Scheme 2.
Uptake kinetic of [18F]1, [18F]2, [18F]FDG and L-[18F]FET in S180
tumor cells28,29 are shown in Figure 2. As shown, the uptake of
[
18F]1 and [18F]FDG is rapid for the first 30 min, followed by a
The 2,4-dinitrobenzoic acid modified precursor 9 (Scheme 3)
was prepared from intermediate 5 by the introducing of 2,4-
dinitrobenzoic acid bridged by ethane-1,2-diamine at C-7 position
of pyrazolo[1,5-a]pyrimidine core structure, 19F substitution [19F]2
was obtained from 9 using TBAFꢀ3H2O in dry DMF at 120 °C for
40 min with 29% yield. The reaction is shown in Scheme 3.
The radiosynthesis of [18F]1 and [18F]2 was completed through a
simple substitution (Scheme 4). [18F]1 was obtained under a mild
condition in dry acetonitrile refluxing under N2 for 20 min with a
nearly steady state from 30 min onward. The maximum uptake
of [18F]1 is about two-fold of [18F]FDG. Radioactivity uptake into
S180 tumor cells after a 60-min incubation at 37 °C (pH 7.4) varied
from 9% to 23% of the total cell radioactivity uptake. On balance, a
greater percentage of [18F]1 relative to [18F]FDG was taken up into
the cell at any given time. In comparison, the cell uptake of [18F]2
and L-[18F]FET remained quantitatively low (<7%).
The biodistribution studies and tumor uptake were investigated
in Kunming Mice bearing S180 tumor.30 An approximately
370 kBq/0.1 mL phosphate-buffered saline solution (pH 7.4) of
18F labeled compound purified by HPLC as described above was in-
jected into the tail vein. The animals were sacrificed at 5 min,
15 min, 30 min, 60 min and 120 min post-injection. The tissues
and organs of interest were immediately removed, weighed and
O
NH
TsO
NH
NH
O2N
N
N
N
N
measured for 18F radioactivity in a
c counter. Values are expressed
N
as mean SD (n = 4). The biodistribution data of [18F]1 and [18F]2
were summarized in Table 1 and Table 2 above, respectively. All
the animal studies were carried out in compliance with relevant
national laws relating to the conduct of animal experimentation.
Table 1 showed the biodistribution of [18F]1. After a fast distri-
bution, [18F]1 was excreted from the body mainly through the kid-
ney. Radioactivity of [18F]1 in tumor was 1.88 0.63, 4.37 0.30,
5.51 0.31, 2.95 0.36 and 2.88 0.34%ID/g at 5 min, 15 min,
N
CN
CN
NO2
9
7
H
N
18F
O
NH
NH
18F
N
N
N
N
N
N
CN
18F]1
CN
30 min, 60 min, and 120 min post-injection, respectively,
a
NO2
remarkable increasing uptake of this compound in tumor. Mean-
while, radioactivity of [18F]1 in other organs and tissues decreased
rapidly. The tumor/brain, tumor/muscle and tumor/blood ratios
reached high levels of 2.23, 1.81 and 1.23 at 30 min post-injection.
[
[
18F]2
Figure 1. Structure of 18F labeled pyrazolo[1,5-a]pyrimidine derivatives and their
corresponding precursors.

4738
J. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4736–4741
OH
Cl
N
N
OEt
CN
CN
CN
a
HN
H2N
b
N
d
N
c
N
N
NC
N
CN
CN
CN
2-(ethoxymethylene)
malononitrile
malononitrile
5
3
4
Scheme 1. Synthesis of intermediate 5. Experiment reagents and conditions: (a) HC(OC2H5)3, Ac2O, refluxing, 6 h, then 0 °C, 12 h, 84%; (b) 85% H2NNH2ꢀH2O, 100 °C, 1 h, then
10 mL H2O, 0 °C, 12 h, 81%; (c) CH3COCH2COOCH2CH3, AcOH, refluxing 4 h, 78%; (d) POCl3, Py, stirred at 85 °C, 120 °C, 1 h, then added CHCl3 at 60 °C, refluxing 1 h, followed by
adding water at 0 °C, 87%.
HO
TsO
Cl
N
F
NH
NH
NH
N
j
h
i
N
N
N
N
N
N
N
N
N
N
CN
CN
CN
CN
1
F]
19
5
6
7
[
Scheme 2. Synthesis of [19F]1. Experiment reagents and conditions: (h) HOCH2CH2NH2, CH3CH2OH, refluxing 4 h, 54%; (i) TsCl, TEA, DMAP, CH2Cl2, 0 °C, 0.5 h, then rt, 12 h,
45%; (j) TBAFꢀ3H2O, CH3CN, N2, refluxing 5 h, 38%.
O
O
NH
NH
NH2
Cl
N
NH
NH
NH
f
e
O2N
F
g
N
N
N
N
N
N
N
N
N
N
N
CN
CN
CN
CN
NO2
NO2
5
8
9
[
19F]2
Scheme 3. Synthesis of [19F]2. Experiment reagents and conditions: (e) H2NCH2CH2NH2, CH3CH2OH, refluxing 4 h, 81%; (f) 2,4-dinitrobenzoic acid, TEA, HOBt, DCC, CH2Cl2,
0 °C, 0.5 h, then rt, 12 h, 51%; (g) TBAFꢀ3H2O, DMF, 120 °C, 40 min, 29%.
18F
TsO
NH
NH
g'
N
N
N
N
N
N
CN
NH
CN
[18F]1
7
O
NH
NH
O
NH
18F
j'
O2N
N
N
N
N
N
N
CN
CN
NO2
NO2
[18F]2
9
Scheme 4. Radiochemical synthesis of [18F]1 and [18F]2. Experiment reagents and
conditions: (g0) K [18F] F, Kryptofix 2.2.2., CH3CN, 80 °C, 20 min, 35%; (j0) K [18F] F,
Kryptofix 2.2.2., DMF, 120 °C, 25 min, 30%.
Figure 2. Uptake kinetics of [18F]FDG, L-[18F]FET, [18F]1 and [18F]2 into S180 tumor
cells at 37 °C (pH 7.4).
Table 2 showed a rapid distribution of [18F]2 into all organs and
tissues after injection. Radioactivity uptake in all organs and tis-
sues reached their peak values at the initial time and then sharp
decline occurred as time elapsed. The relatively low uptake of
Comparing the biodistribution data of [18F]1 and [18F]2 in mice
bearing S180 tumor as shown in Table 1 and Table 2, there were
significant differences in initial uptake, accumulation and clear-
ance in brain and tumor. Meanwhile, we noticed that [18F]1 and
[
18F]2 in brain which decreased through the course of study from
0.38 0.07%ID/g at 5 min pi. to 0.04 0.00%ID/g at 120 min pi. gave
the high tumor/brain ratios: 5.87, 12.35, 10.44, 8.69 and 5.13 at
5 min, 15 min, 30 min, 60 min and 120 min, respectively. More-
over, the tumor had a proportion of 2.26 0.48%ID/g at 5 min pi.,
not preponderating over other organs or tissues. However, as a re-
sult of a relatively slower elimination in tumor, both the tumor/
muscle and tumor/blood ratios of [18F]2 had a continual increasing
for a long time post-injection, and reached their maximum 1.57
and 2.50 at 60 min and 120 min, respectively.
[
18F]2 had a similar lipophilicity, as it was characterized by both
the much close log P values (0.69 0.02 for [18F]1 and 0.68 0.02
for [18F]2) and retention time (3.061 min for [18F]1 and 3.160 min
for [18F]2) on the same analytical HPLC system. Therefore, the abil-
ity to penetrate BBB (blood–brain barrier) for both radioactivity
tracers is supposed to be comparable. However, the brain uptake
of
[ [
18F]1 is much higher than that of 18F]2: 3.17 0.48,

J. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4736–4741
4739
Table 1
Biodistribution of [18F]1 in mice bearing S180 tumor. Expressed as% injected dose per gram (% ID/g SD), n = 4
Organs
Time (min)
30
5
15
60
120
Heart
Liver
Spleen
Lung
Kidney
Brain
Muscle
Blood
Tumor
5.53 0.61
6.99 1.00
4.66 0.26
6.61 0.54
7.93 1.27
3.17 0.48
3.99 1.05
4.83 0.45
1.88 0.63
0.59
3.57 0.65
4.02 0.20
3.42 0.52
3.79 0.93
5.04 0.48
2.70 0.07
3.84 0.45
4.65 0.05
4.37 0.30
1.62
4.06 0.09
3.17 0.16
3.43 0.39
3.77 0.31
3.28 0.20
2.47 0.24
3.05 0.22
4.49 0.31
5.51 0.31
2.23
3.46 0.78
2.45 0.30
2.82 0.31
2.78 0.19
2.58 0.16
1.93 0.25
2.31 0.48
3.63 0.33
2.95 0.36
1.53
2.59 0.18
1.84 0.22
2.15 0.94
2.54 0.96
1.92 0.40
1.79 0.27
2.64 0.60
2.95 0.30
2.88 0.34
1.61
Tumor/brain
Tumor/muscle
Tumor/blood
0.47
0.39
1.14
0.94
1.81
1.23
1.28
0.81
1.09
0.98
Table 2
Biodistribution of [18F]2 in mice bearing S180 tumor. Expressed as% injected dose per gram (% ID/g SD), n = 4
Organs
Time (min)
30
5
15
2.78 0.16
60
120
Heart
Liver
Spleen
Lung
Kidney
Brain
Muscle
Blood
Tumor
5.77 0.77
1.16 0.23
7.30 1.06
0.99 0.24
1.19 0.08
5.63 0.58
0.12 0.02
0.89 0.17
1.42 0.43
1.24 0.39
10.44
0.63 0.01
4.07 0.60
0.53 0.08
0.56 0.26
1.83 0.50
0.08 0.02
0.47 0.16
0.45 0.12
0.74 0.12
8.69
0.12 0.02
2.05 0.17
0.14 0.03
0.19 0.07
0.74 0.03
0.04 0.00
0.28 0.07
0.09 0.04
0.21 0.02
5.13
17.90 1.70
4.54 0.86
5.60 0.66
17.70 1.04
0.38 0.07
3.82 0.39
5.23 0.16
2.26 0.48
5.87
10.12 0.34
1.96 0.25
2.60 0.24
8.45 0.35
0.16 0.01
2.07 0.83
2.50 0.37
1.99 0.21
12.35
Tumor/brain
Tumor/muscle
Tumor/blood
0.59
0.43
0.96
0.80
1.40
0.88
1.57
0.77
1.66
2.50
2.70 0.07, 2.47 0.24, 1.93 0.25 and 1.79 0.27%ID/g for [18F]1
and 0.38 0.07, 0.16 0.01, 0.12 0.02, 0.08 0.02 and
0.04 0.00%ID/g for [18F]2 at 5 min, 15 min, 30 min, 60 min and
120 min post-injection. It was reported that the ideal log P value
for molecules to penetrate the BBB is between 1 and 3.31 The log P
values of both radioactivity tracers are less than 1, thus it is reason-
able for the low brain uptake for [18F]2, whereas, it could not ex-
plain the high uptake of [18F]1 in brain. In addition, as the time
lapsed, the tumor uptake of [18F]1 and [18F]2 behaved differently:
tumor/blood ratios. However, it had higher tumor/brain ratios (the
maximum value was 2.95 at 120 min pi.), which was suitable for
the application in PET imaging agent of brain tumors and made it
a necessary complement to [18F]FDG.
By comparison with [18F]FDG and L-[18F]FET, [18F]1 displayed
some peculiarities: [18F]1 had significantly higher absolute radio-
activity uptake in tumor than those of [18F]FDG and L-[18F]FET at
all time points as shown in Figure 3. The maximum uptake of
[
18F]1 in tumor was 5.51 0.31%ID/g, nearly three times as much
[
18F]1 reached its peak of 5.51 0.31%ID/g at 30 min pi. and then
as that of [18F]FDG and two times as much as that of L-[18F]FET
at 30 min post-injection.
decreased to 2.88 0.34%ID/g at 120 min post-injection, 52% radio-
activity still retained in the body after 120 min indicating good
persistence in tumor uptake. On the other hand, [18F]2 had a rela-
tive high initial tumor uptake 2.26 0.48%ID/g at 5 min but the
rate of elimination from tumor was rapid with only
0.21 0.02%ID/g at 120 min post-injection. From the uptake re-
sults, we could make some inference that the distribution of
At the same time (30 min post-injection), as shown in Figure 4,
the tumor to brain ratio reached the peak 2.23 which was similar
to that of L-[18F]FET (2.54) and much higher than that of [18F]
FDG (0.61); the tumor to muscle ratio was 1.81 which was similar
to that of [18F]FDG (1.75) and much higher than that of L-[18F] FET
(0.62); the tumor to blood ratio also reached the maximum of 1.23,
slightly higher than that of L-[18F]FET (1.03). Taken together, [18F]1
had the capability to accumulate in tumor and could greatly en-
hance the sensitivity in tumor imaging. As a radioactivity tracer,
[
18F]1 underwent a special transport mechanism and apparently
much more suitable for PET tumor imaging.
In order to confirm this, we compared [18F]FDG and L-[18F] FET
with [18F]1 in the same animal model, the results were shown in
Table 3.
[
18F]1 may be useful for PET imaging of both central and peripheral
tumors as well as metastasis of tumors.
As shown in Table 3, [18F]FDG had high tumor/blood ratios after
30 min post-injection due to fast clearance from the blood. The
tumor/blood ratios were 11.38, 19.47 and 15.85 after 30 min,
60 min and 120 min post-injection, respectively. The high uptake
in brain resulted in low tumor/brain ratios. The maximum value
of tumor/brain ratio was 1.33 at 120 min post-injection. Therefore,
In order to visualize the distribution of [18F]1, small animal PET
imaging studies was performed in the mouse bearing S180 tumor.
The mouse was injected intravenously with approximately
3.7 MBq of [18F]1 in saline and anesthetized with 1.5% isoflurane
for imaging. Whole body image was acquired by micro PET at
30 min post-injection is shown in Figure 5. As expected from the
ex vivo biodistribution data, tracer uptake in excretory organs
was predominant, but tumor (pointed to by the arrowhead) in
[
18F]FDG is not an ideal PET imaging agent for brain tumors. As
show in Table 3, in contrast to [18F]FDG, L-[18F]FET had much lower

4740
J. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4736–4741
Table 3
The biodistribution comparison of [18F] 1, [18F]FDG and L-[18F]FET in mice bearing S180 tumor. Expressed as% injected dose per gram (% ID/g SD), n = 4a
Organs
Tumor
Compounds
Time (min)
30
5
15
60
120
[
[
18F]1
1.88 0.63
1.27 0.54
2.08 0.49
4.37 0.30
1.56 0.23
2.50 0.23
5.51 0.31
1.86 0.18
3.28 0.69
2.95 0.36
2.16 0.34
2.75 0.36
2.88 0.34
1.70 0.06
2.15 1.36
18F]FDG
L-[18F]FET
Tumor/brain
Tumor/muscle
Tumor/blood
[
[
18F]1
0.59
0.53
2.10
1.62
0.59
2.43
2.23
0.61
2.54
1.53
1.02
2.93
1.61
1.33
2.95
18F]FDG
L-[18F]FET
[
[
18F]1
0.47
0.81
0.72
1.14
1.37
0.75
1.81
1.75
0.62
1.28
2.23
1.23
1.09
2.20
1.04
18F]FDG
L-[18F]FET
[
[
18F]1
0.39
1.17
0.31
0.94
3.42
0.76
1.23
11.38
1.02
0.81
19.47
1.10
0.98
15.85
1.92
18F]FDG
L-[18F]FET
a
The data of [18F]FDG and L-[18F]FET were quoted from Ref. 27.
6.0
18F
18F
1
2
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
18F FDG
L- 18F FET
0
20
40
60
80
100
120
Time (min)
Figure 3. Comparison of tumor uptake rates of [18F]1 and [18F]2 with those of
18F]FDG and L-[18F]FET in the same animal model bearing S180 tumor.
[
Figure 5. Coronal PET image of Kunmin mice with S180 tumor (arrow) in the left
front flank at 30 min post-injection of [18F]1.
cells. Preliminary biodistribution experiments were performed in
S180 tumor-bearing mice to evaluate the potential of [18F]1 and
[
18F]2 for application in PET tumor imaging. The comparative in
ex vivo analysis of [18F]1, [18F]2, [18F]FDG and L-[18F]FET showed
superior characteristics for [18F]1 with respect to the total tracer
accumulation and retention in tumor. PET imaging revealed that
the accumulation of radioactivity in S180 tumor was visualized.
The positive in vitro and in vivo results of [18F]1 suggest that this
compound represents a promising PET tracer candidate for tumor
detection. On the other hand, on-going effort to optimize the struc-
ture of [18F]1 aiming at enhancing the tumor-to-nontarget ratios
in vivo is under way.
Figure 4. Comparison of tumor to brain (T/Br), tumor to muscle (T/M) and tumor to
blood (T/Bl) uptake ratios of [18F]1 with those of [18F]FDG and L-[18F]FET at 30 min
post-injection in the same animal model bearing S180 tumor.
the left forelimb was also obvious, while no accumulation in the
right forelimb was found. The imaging results clearly confirmed
[
Acknowledgments
18F]1 could be accumulated in tumor .
In conclusion, we successfully labeled pyrazolo[1,5-a]pyrimi-
This project was sponsored by the National Natural Science
foundation of China (No. 21071022). We also thank the cyclotron
operator team of the PET Centre of Xuanwu Hospital for providing
fluoride-18 nuclide and technical assistance.
dine derivatives by two time-saving and high-yield methods and
got two novel 18F labeled compounds, [18F]1 and [18F]2. The
in vitro studies showed a high uptake of [18F]1 by the S180 tumor

J. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4736–4741
4741
Freemont, P. S.; Aboagye, E. O.; Coombes, R. C.; Barrett, A. G. M.; Ali, S. J. Med.
Chem. 2010, 53, 8508.
Supplementary data
17. Fraley, M. E.; Rubino, R. S.; Hoffman, W. F.; Hambaugh, S. R.; Arrington, K. L.;
Hungate, R. W.; Bilodeau, M. T.; Tebben, A. J.; Rutledge, R. Z.; Kendall, R. L.;
McFall, R. C.; Huckle, W. R.; Coll, K. E.; Thomas, K. A. Bioorg. Med. Chem. Lett.
2002, 12, 3537.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.06.072.
18. Powell, D.; Gopalsamy, A.; Wang, Y. D.; Zhang, N.; Miranda, M.; McGinnisb, J.
P.; Rabindranb, S. K. Bioorg. Med. Chem. Lett. 2007, 17, 1641.
19. Gopalsamy, A.; Yang, H.; Ellingboe, J. W.; Tsou, H.-R.; Zhang, N.; Honores, E.;
Powell, D.; Miranda, M.; McGinnisb, J. P.; Rabindranb, S. K. Bioorg. Med. Chem.
Lett. 2005, 15, 1591.
20. Koehler, L.; Graf, F.; Bergmann, R.; Steinbach, J.; Pietzsch, J.; Wuest, F. Eur. J.
Med. Chem. 2010, 45, 727.
21. Miller, P. W.; Long, N. J.; Vilar, R.; Gee, A. D. Angew. Chem., Int. Ed. 2008, 47,
8998.
References and notes
1. George, C. F. P. Lancet 2001, 357, 1623.
2. Stefano, A.; Anna, A.; Maurizio, B.; Alessandra, T.; Francisco, O.; Francesco, O.;
Silvia, S.; Chiara, B.; Matilde, Y. ChemMedChem 2010, 5, 1242.
3. Curran, K. J.; Verheijen, J. C.; Kaplan, J.; Richard, D. J.; Toral-Barza, L.; Hollander,
I.; Lucas, J.; Ayral-Kaloustian, S.; Yu, K.; Zask, A. Bioorg. Med. Chem. Lett. 2010,
20, 1440.
22. Varagnolo, L.; Stokkel, M. P. M.; Mazzi, U.; Pauwels, E. K. J. Nucl. Med. Biol. 2000,
27, 103.
4. Zask, A.; Verheijen, J. C.; Curran, K.; Kaplan, J.; Richard, D. J.; Nowak, P.;
Malwitz, D. J.; Brooijmans, N.; Bard, J.; Svenson, K.; Lucas, J.; Toral-Barza, L.;
Zhang, W.-G.; Hollander, I.; Gibbons, J. J.; Abraham, R. T.; Ayral-Kaloustian, S.;
Mansour, T. S.; Yu, K. J. Med. Chem. 2009, 52, 5013.
5. Manetti, F.; Santucci, A.; Locatelli, G. A.; Maga, G.; Spreafico, A.; Serchi, T.;
Orlandini, M.; Bernardini, G.; Caradonna, N. P.; Spallarossa, A.; Brullo, C.;
Schenone, S.; Bruno, O.; Ranise, A.; Bondavalli, F.; Hoffmann, O.; Bologna, M.;
Angelucci, A.; Botta, M. J. Med. Chem. 2007, 50, 5579.
23. Yu, W.; McConathy, J.; Williams, L.; Camp, V. M.; Malveaux, E. J.; Zhang, Z.;
Olson, J. J.; Goodman, M. M. J. Med. Chem. 2010, 53, 876.
24. Wood, K. A.; Hoskin, P. J.; Saunders, M. I. Clin. Oncol. 2007, 19, 237.
25. Gopalsamy, A.; Ciszewski, G.; Hu, Y.; Lee, F.; Feldberg, L.; Frommer, E.; Kim, S.;
Collins, K.; Wojciechowicz, D.; Mallon, R. Bioorg. Med. Chem. Lett. 2009, 19,
2735.
26. Li, J.; Zhao, Y.; Zhao, X.; Yuan, X.; Gong, P. Arch. Pharm. Chem. Life Sci. 2006, 339,
593.
27. Qiao, Y.; He, Y.; Zhang, S.; Li, G.; Liu, H.; Xu, J.; Wang, X.; Qi, C.; Peng, C. Bioorg.
Med. Chem. Lett. 2009, 19, 4873.
28. Sun, X.; Chu, T.; Wang, X. Nucl. Med. Biol. 2010, 37, 117.
29. S180 tumor cells were suspended in fresh DMEM medium supplemented with
10% fetal bovine serum at a cell concentration of 2 ꢁ 106 cells/mL. Aliquots of
10 mL were placed in glass vials and incubated at 37 °C with gentle stirring
under an atmosphere of 95% air plus 5% carbon dioxide. After a 30- to 45-min
equilibration period, 0.15 mL of cell culture medium with about 0.25 MBq/mL
radiotracer was added and incubated at 37 °C. At 5, 15, 30, 60 and 120 min,
6. Schenone, S.; Bruno, O.; Ranise, A.; Bondavalli, F.; Brullo, C.; Fossa, P.; Mosti, L.;
Menozzi, G.; Carraro, F.; Naldini, A.; Bernini, C.; Manettic, F.; Botta, M. Bioorg.
Med. Chem. Lett. 2004, 14, 2511.
7. Traxler, P.; Bold, G.; Frei, J.; Lang, M.; Lydon, N.; Mett, H.; Buchdunger, E.;
Meyer, T.; Mueller, M.; Furet, P. J. Med. Chem. 1997, 40, 3601.
8. Ballell, L.; Field, R. A.; Chungc, G. A. C.; Youngc, R. J. Bioorg. Med. Chem. Lett.
2007, 17, 1736.
9. Gudmundsson, K. S.; Johns, B. A.; Weatherhead, J. Bioorg. Med. Chem. Lett. 2009,
19, 5689.
10. Rashad, A. E.; Hegab, M. I.; Abdel-Megeid, R. E.; Micky, J. A.; Abdel-Megeid, F. M.
E. Bioorg. Med. Chem. 2008, 16, 7102.
11. Frizzo, C. P.; Scapin, E.; Campos, P. T.; Moreira, D. N.; Martins, M. A. P. J. Mol.
Struct. 2009, 933, 142.
12. Novinson, T.; Bhooshan, B.; Okabe, T.; Revankar, G. R.; Wilson, H. R.; Robins, R.
K.; Senga, K. J. Med. Chem. 1976, 19, 512.
13. Senga, K.; Novinson, T.; Wilson, H. R.; Robins, R. K. J. Med. Chem. 1981, 24, 610.
14. Di Grandi, M. J.; Berger, D. M.; Hopper, D. W.; Zhang, C.; Dutia, M.; Dunnick, A.
L.; Torres, N.; Levin, J. I.; Diamantidis, G.; Zapf, C. W.; Bloom, J. D.; Dennis
Powell, Y. B. H.; Wojciechowicz, D.; Collins, K.; Frommer, E. Bioorg. Med. Chem.
Lett. 2009, 19, 6957.
200
centrifuged at 1500 rpm for 5 min. A 180-
removed for counting, and the left sample containing cells and 20
was also counted. Percent cell uptake is calculated as [residue counts
l
L samples of the suspension were transferred to microfuge tubes and
L sample of each supernatant was
L medium
l
l
ꢂ
(supernatant counts/9)]/(residue counts + supernatant counts) ꢁ 100%. The
data are expressed as mean S.D. (n = 5). During all the experiments, cell
viability was always greater than 90%.
30. The S180 tumor model was established by injecting subcutaneously 5 ꢁ 106
tumor cells into the left forelimb of Kunming mice weighing 18 2 g on
average, the biodistribution studies were performed after the tumor cells
were inoculated for 7–14 days when the tumors were 0.5–0.8 cm in
diameter.
15. Ahmeda, O. M.; Mohamedc, M. A.; Ahmedb, R. R.; Ahmedd, S. A. Eur. J. Med.
Chem. 2009, 44, 3519.
16. Heathcote, D. A.; Patel, H.; Kroll, S. H. B.; Hazel, P.; Periyasamy, M.; Alikian, M.;
Kanneganti, S. K.; Jogalekar, A. S.; Scheiper, B.; Barbazanges, M.; Blum, A.;
Brackow, J.; Siwicka, A.; Pace, R. D. M.; Fuchter, M. J.; Snyder, J. P.; Liotta, D. C.;
31. Evensa, N.; Mucciolib, G. G.; Houbrechtsa, N.; Lambertb, D. M.; Verbruggena, A.
M.; Laerec, K. V.; Bormansa, G. M. Nucl. Med. Biol. 2009, 36, 455.