18F-labeled pyrazolo[1,5-a]pyrimidine derivatives: Synthesis from 2,4-dinitrobenzamide and tosylate precursors and comparative biological evaluation for tumor imaging with positron emission tomography

Article abstract of DOI:10.3390/molecules17043774

We previously reported ¹⁸F-labeled pyrazolo[1,5-a]pyrimidine derivatives: 7-(2-[¹⁸F]fluoroethylamino)-5-methylpyrazolo[1,5-a] pyrimidine-3-carbonitrile ([¹⁸F]1) and N-(2-(3-cyano-5- methylpyrazolo[1,5-a]pyrimidin-7-ylamino)ethyl)-2-[¹⁸F]fluoro-4- nitrobenzamide ([¹⁸F]2). Preliminary biodistribution experiments of both compounds showed s slow clearance rate from excretory tissues which warranted further investigation for tumor imaging with PET. Here we modified [¹⁸F]1 and [¹⁸F]2 by introducing polar groups such as ester, hydroxyl and carboxyl and developed three additional ¹⁸F-18 labeled pyrazolo[1,5-a] pyrimidine derivatives: (3-Cyano-7-(2-[ ¹⁸F]fluoroethylamino)pyrazolo[1,5-a]-pyrimidin-5- yl)methyl acetate ([¹⁸F]3), 7-(2-[¹⁸F]fluoroethylamino)-5-(hydroxymethyl) pyrazolo[1,5-a]- pyrimidine-3-carbonitrile ([¹⁸F]4) and (S)-6-(3-cyano-5-methylpyrazolo[1,5-a]pyrimidin-7- ylamino)-2-(2-[ ¹⁸F]fluoro-4-nitrobenzamido)hexanoic acid ([¹⁸F]5). The radiolabeled probes were synthesized by nucleophilic substitution of the corresponding tosylate and nitro precursors with ¹⁸F-fluoride. In Vitro studies showed higher uptake of [¹⁸F]3 and [¹⁸F]4 than that of [¹⁸F]5 by S180 tumor cells. In Vivo biodistribution studies in mice bearing S180 tumors showed that the uptake of both [ ¹⁸F]3 and [¹⁸F]4 in tumors displayed an increasing trend while the uptake of [¹⁸F]5 in tumor decreased through the course of the 120 min study. This significant difference in tumor uptake was also found between [¹⁸F]1 and [¹⁸F]2. Thus, we compared the biological behavior of the five tracers and reported the tumor uptake kinetic differences between 2-[¹⁸F]fluoroethylamino- and 2-[ ¹⁸F]fluoro-4-nitro- benzamidopyrazolo[1,5-a] pyrimidine derivatives.

Full text of DOI:10.3390/molecules17043774

Molecules 2012, 17, 3774-3793; doi:10.3390/molecules17043774
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
18
F-labeled Pyrazolo[1,5-a]pyrimidine Derivatives: Synthesis
from 2,4-Dinitrobenzamide and Tosylate Precursors and
Comparative Biological Evaluation for Tumor Imaging with
Positron Emission Tomography
1
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Jingli Xu , Hang Liu , Guixia Li , Yong He , Rui Ding , Xiao Wang , Man Feng ,
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Shuting Zhang , Yurong Chen , Shilei Li , Mingxia Zhao , Yingruo Li , Chuanmin Qi *
2
and Yonghong Dang
1
Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education,
College of Chemistry, Beijing Normal University, Beijing 100875, China
2
Department of Nuclear Medicine, PUMC Hospital, Beijing 100730, China
*
Author to whom correspondence should be addressed; E-Mail: qicmin@sohu.com;
Tel.: +86-136-8138-0097; Fax: +86-010-5880-2075.
Received: 22 February 2012; in revised form: 14 March 2012 / Accepted: 19 March 2012 /
Published: 27 March 2012
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Abstract: We previously reported F-labeled pyrazolo[1,5-a]pyrimidine derivatives:
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7
-(2-[ F]fluoroethylamino)-5-methylpyrazolo[1,5-a]pyrimidine-3-carbonitrile ([ F]1) and
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N-(2-(3-cyano-5-methylpyrazolo[1,5-a]pyrimidin-7-ylamino)ethyl)-2-[ F]fluoro-4-nitro-
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benzamide ([ F]2). Preliminary biodistribution experiments of both compounds showed s
slow clearance rate from excretory tissues which warranted further investigation for tumor
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imaging with PET. Here we modified [ F]1 and [ F]2 by introducing polar groups such as
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ester, hydroxyl and carboxyl and developed three additional F-18 labeled pyrazolo[1,5-a]
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pyrimidine derivatives: (3-Cyano-7-(2-[ F]fluoroethylamino)pyrazolo[1,5-a]-pyrimidin-5-
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yl)methyl acetate ([ F]3), 7-(2-[ F]fluoroethylamino)-5-(hydroxymethyl)pyrazolo[1,5-a]-
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pyrimidine-3-carbonitrile ([ F]4) and (S)-6-(3-cyano-5-methylpyrazolo[1,5-a]pyrimidin-7-
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ylamino)-2-(2-[ F]fluoro-4-nitrobenzamido)hexanoic acid ([ F]5). The radiolabeled probes
were synthesized by nucleophilic substitution of the corresponding tosylate and nitro
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precursors with F-fluoride. In Vitro studies showed higher uptake of [ F]3 and [ F]4
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than that of [ F]5 by S180 tumor cells. In Vivo biodistribution studies in mice bearing
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S180 tumors showed that the uptake of both [ F]3 and [ F]4 in tumors displayed an
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increasing trend while the uptake of [ F]5 in tumor decreased through the course of the

Molecules 2012, 17
20 min study. This significant difference in tumor uptake was also found between [ F]1
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and [ F]2. Thus, we compared the biological behavior of the five tracers and reported the
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tumor
uptake
kinetic
differences
between
2-[ F]fluoroethylamino-
and
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2
-[ F]fluoro-4-nitro- benzamidopyrazolo[1,5-a] pyrimidine derivatives.
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Keywords: F-labeled; pyrazolo[1,5-a]pyrimidine derivatives; 2,4-dinitrobenzamide and
tosylate precursors; PET imaging agents; tumor detection
1
. Introduction
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2
-[ F]Fluoro-2-deoxyglucose ( F-FDG) positron emission tomography (PET) is generally
accepted as a routine method in clinical diagnosis and evaluation of cancer. However, this tracer has
inherent drawbacks. For example, its high accumulation in inflammed and infected tissues can lead to
false-positive results and its low uptake in tumors that are growing slowly can cause false-negative
results [1,2]. Consequently, there is a need to develop new PET tumor-imaging agents. Pyrazolo-
pyrimidines have multiple pharmacological activities including hypnotic [3], anti-inflammatory [4],
anti-tumor [5–9], antimycobacterial [10] and anti-viral properties [11,12]. Pyrazolo[1,5-a]pyrimidine
and its derivatives have attracted broad interest over the years due to their structural resemblance
to purine [13]. They were found to have antitrypanosomal [14] and antischistosomal activity [15],
and most importantly, anti-tumor activity [16–19]. Furthermore, many pyrazolo[1,5-a]pyrimidine
derivatives were proved to block proliferation of various cancer cell lines [17,20,21]. As a result,
pyrazolo[1,5-a]pyrimidine has been widely applied as an important pharmacophore or building block
in anti-tumor drug design, which has motivated further study on the physiological and biological
properties of this kind of compounds, including in Vitro stability, in Vivo distribution, metabolism
and elimination.
In a previous paper, we reported our initial efforts to design and label the pyrazolo[1,5-a]pyrimidine
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pharmacophore from the corresponding tosylate and nitro precursors to get compounds [ F]1 and
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[
F]2 with the aim of developing new radiotracers for tumor imaging by means of PET. The studies of
in Vitro cell uptake and in Vivo biodistribution demonstrated that both tracers had more accumulation
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in the tumors than L-[ F]FET and [ F]FDG. Unfortunately, their low tumor-to-non target ratios
limited their application in PET tumor imaging. These data suggested that our important design
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consideration for labeling pyrazolo[1,5-a]pyrimidine pharmacophore with F-fluoride is to maintain
the high concentration of radioactivity in the tumor and improve the clearance rate from other organs.
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What’s more, in our previous work we noted that [ F]1 and [ F]2, which had similar lipophilicity,
showed quite different biodistribution.
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The objective of this study was to lower the lipophilicity of [ F]1 and [ F]2 by the introduction of
ester, hydroxyl and carboxyl groups, which have been extensively used to modify the lipophilicity
properties of other tracers and therefore their biodistribution [22] to produce the compounds with
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potential applicability in PET imaging. We thus designed [ F]3, [ F]4 and [ F]5 (Figure 1) and
intended to find some relationship among the structure, the physical property (e.g., lipophilicity) and
the biological property (e.g., accumulation in tumor). In addition, we attempted to label

Molecules 2012, 17
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pyrazolo[1,5-a]pyrimidine pharmacophore with F-fluoride employing tosylate and nitro compounds
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as precursors and identify the influences of two kinds of F labeled pyrazolo[1,5-a]pyrimidine
pharmacophore on the tumor uptake.
18
Figure 1. Structures of F labeled pyrazolo[1,5-a]pyrimidine derivatives.
18
In view of the above mentioned facts we successively synthesized three novel F labeled
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pyrazolo[1,5-a]pyrimidine derivatives: [ F]3, [ F]4 and [ F]5 and investigated their radiopharmacological
characterization: Cellular uptake and biological activities in mice bearing S180 tumor (sarcoma S180
has been widely used to evaluate the antitumor activities of drugs due to its growth advantage [23–26]).
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Furthermore, we compared the cellular uptake of [ F]3, [ F]4 and [ F]5 with that of [ F]FDG,
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biodistribution data of [ F]3, [ F]4 and [ F]5 with those of [ F]1 and [ F]2. The data of [ F]FDG,
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[
F]1 and [ F]2 were obtained using the previously described [27].
2
2
. Results and discussion
.1. Chemistry
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The synthesis route of [ F]3 and [ F]4, shown in Scheme 1, is a modification of the synthesis of
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9
[
2
F]1 described by us previously [27]. Treatment of 9 with sodium acetate in a basic mixture of H O
and DMSO solution generated the desired product 10. Subsequent reaction with p-toluenesulfonyl
chloride (1.5 eq.) in dry dichloromethane containing triethylamine (1.5 eq.) and a catalytic amount of
4
-(N,N-dimethylamino)pyridine (DMAP) at room temperature overnight gave the target precursor 11.
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19
F substituted [ F]3 was obtained from precursor 11 using TBAF·3H
2
O in dry acetonitrile at 75 °C
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19
under nitrogen atmosphere for 2 h. Preparation of [ F]4 was accomplished by hydrolysis of [ F]3
with NaOH in MeOH, subsequent acidification with 1 N HCl and purification by recrystallization. The
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synthesis procedure of [ F]5, as illustrated in Scheme 2, is the modification of the route to [ F]2
described by us previously [27]. The 2,4-dinitrobenzaldehyde group was linked through L-lysine to
the pyrazolo[1,5-a]pyrimidine core. L-Lysine methyl ester hydrochloride was reacted with 12 in
the presence of triethylamine to give the lysine pyrazolo[1,5-a]pyrimidine and coupled to

Molecules 2012, 17
3777
1
9
2
,4-dinitrobenzoic acid using HOBt and DCC to provide the precursor 13. Finally [ F]5 was obtained
1
9
2
by hydrolysis of [ F]14 with LiOH·2H O in MeOH, subsequent acidification with 1 N HCl and
purification by recrystallization.
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Scheme 1. Synthesis of [ F]3 and [ F]4.
Reagents and conditions: (a) HC(OC H ) , Ac O, refluxing, 6 h, then 0 °C, 12 h, 84%; (b) 85%
2
5 3
2
H NNH ·H O, 100 °C, 1 h, then 10 mL H O, 0 °C, 12 h, 81%; (c) ClCH COCH COOCH CH ,
2
2
2
2
2
2
2
3
AcOH, refluxing 4 h, 92%; (d) POCl , Py, stirred at 85 °C, 120 °C, 1 h, then added CHCl at 60 °C,
3
3
i
refluxing 1 h, followed by adding water at 0 °C, 64%; (e) HOCH CH NH , PrOH, refluxing 4 h,
2
2
2
7
0%; (f) CH COONa·3H O, NaOH, H O, DMSO, 120 °C, 1.5 h, 60%; (g) TsCl, TEA, DMAP,
3 2 2
CH Cl , 0 °C, 0.5 h, then rt, 12 h, 85%; (h) TBAF·3H O, CH CN, N , refluxing 2 h, 20%;
2
2
2
3
2
(
i) CH OH, NaOH, 80 °C, 20 min, HCl. 85%.
3
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Scheme 2. Synthesis of [ F]5.
3
Reagents and conditions: (j) L-Lys·2HCl, CH CN, TEA, refluxing, 15 min; (k) 2,4-dinitrobenzoic
acid, TEA, HOBt, DCC, CH Cl , 0 °C, 0.5 h, then rt, 12 h, 42%; (l) TBAF·3H O, DMF, 120 °C,
2
2
2
4
0 min, 29%. (m) LiOH·H O, CH OH, HCl, 0 °C, 0.5 h, then rt, 12 h, 95%.
2 3
2
.2. Radiochemistry
The radiosynthesis from 2,4-dinitrobenzamide and tosylate precursors followed the conditions as
shown in Scheme 3. The synthesis of all the three radiotracers required about 2 h with overall

Molecules 2012, 17
3778
radiochemical yields of 20–30% non decay-corrected, radiochemical purity >98% and specific activity
between 31–40 GBq/μmol. The identity of the radiolabeled products was confirmed by comparing the
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retention time of radioactive product with that of F-compound by HPLC. The retention time of [ F]3,
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[
(
F]4 and [ F]5 were 11.3 min, 6.6 min and 2.3 min, respectively, which matched well with [ F]3
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19
11.0 min), [ F]4 (6.3 min) and [ F]5 (2.1 min) within admissible error. All dry radiolabeled products
then formulated in physiological phosphate buffer solution (pH = 7.4).
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Scheme 3. Radiochemical synthesis of [ F]3, [ F]4 and [ F]5.
COOCH3
COOCH3
18F
HN
NH
HN
NH
l'
O N
2
N
N
O
N
O
N
O N
N
O N
N
2
2
CN
CN
13
14
COOH
O
18F
HN
NH
m'
N
N
O N
N
2
CN
5
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Reagents and conditions: (h') K [ F] F, Kryptofix 2.2.2., CH
3
CN, 80 °C, 20 min, 30%;
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(
i') LiOH·H O, CH OH, HCl, 80 °C, 20 min, (l') K [ F] F, Kryptofix 2.2.2., DMF, 120 °C, 25 min,
2
3
2
0%. (m') LiOH·H O, CH OH, HCl, 80 °C, 20 min.
2 3
2
.3. Measurement of Partition Coefficient
The partition coefficient (logP) values of [ F]3, [ F]4 and [ F]5 were −0.04, −0.09 and −1.78
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18
18
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respectively, much lower than those of [ F]1(0.68) and [ F]2(0.69), suggesting those radiotracers
were hydrophilic. Clearly, the introduction of ester, hydroxyl and carboxyl groups at the C-5 or C-7
positions of the pyrazolo[1,5-a]pyrimidine core structure significantly decreased the lipophilicity of
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18
[
F]1 and [ F]2.
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18
2
.4. Stability of [ F]3, [ F]4 and [ F]5
The Radio-HPLC showed that all the three radiotracers were stable in both phosphate-buffered
saline (pH 7.4) and murine plasma at 37 °C after 2 h.

Molecules 2012, 17
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2
.5. Cellular Accumulation of [ F]3, [ F]4 and [ F]5
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Cell uptake [27] values of [ F]3, [ F]4 and [ F]5 at 37 °C over incubation time of 5, 15, 30, 60
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and 120 min are shown in Figure 2. [ F]3 exhibited rapid accumulation in S180 tumor cells, reached
the peak value of 0.17 ± 0.1% at 30 min and maintained the high uptake value at 2 h (0.16 ± 0.1%). A
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similar cell uptake pattern was observed for [ F]4.
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Figure 2. Uptake kinetics of [ F]3, [ F]4, [ F]5 and [ F]FDG into S180 tumor cells at
7 °C (pH = 7.4). Data are expressed as % uptake (mean with S.D. n = 5 each). P < 0.05.
3
1
1
1
8
8
8
[
[
F]3
F]4
0.20
0.19
0.18
0.17
0.16
0.15
0.14
0.13
0.12
0.11
0.10
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
[ F]5
18
[
F]FDG
0
20
40
60
80
100
120
Time (min)
The peak value occurred at 30 min (0.2 ± 0.08%) and slightly decreased at 2 h (0.14 ± 0.2%). The
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uptake of [ F]FDG was rapid for the first 30 minutes, followed by a nearly steady state from
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18
3
0 minutes onward. Compared to [ F]FDG, the cell uptake of [ F]3 and [ F]4 was much higher at all
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the selected time points. Uptake of [ F]5 was significantly lower than [ F]3 and [ F]4, but similar to
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[
F]FDG at all time points. The results were consistent with ex vivo biodistribution studies. Moreover,
uptake of all the three radiotracers showed only slight temperature dependence in our study (data of
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18
[
F]3 shown in Figure 3, data of [ F]4 and [ F]5 not shown). As no significant difference between
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18
the in Vitro uptake kinetics of [ F]3, [ F]4 or [ F]5 at 4 °C and at 37 °C could be observed, it can be
concluded that said compounds enter the cell by passive diffusion and not by an active transport
mechanism, in agreement with ex vivo biodistribution data.

Molecules 2012, 17
Figure 3. Comparison of uptake kinetics of [ F]3 into S180 tumor cells at 37 °C and 4 °C
3780
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(
pH = 7.4). Data are expressed as % uptake (mean with S.D. n = 5 each). P < 0.05.
o
3
7 C
o
4
C
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
30
60
120
Time (min)
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18
2
.6. Biodistribution of [ F]3, [ F]4 and [ F]5 in S180 Bearing Mice
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The biodistribution data of [ F]3, [ F]4 and [ F]5 were summarized in Table 1, Table 2 and Table 3,
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respectively. Figure 3 showed the direct comparison of tumor uptake (%ID/g) among [ F]1, [ F]2,
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[
F]3, [ F]4 and [ F]5 in the same animal model. The biodistribution data of [ F]1 and [ F]2 were
obtained from our previous report [27].
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Table 1. Biodistribution of [ F]3 in mice bearing S 180 tumor. Expressed as % injected
dose per gram (% ID/g ± SD), n = 4.
Time (min)
Organs
5
15
30
60
120
Heart
Liver
Spleen
Lung
Kidney
Brain
Muscle
Blood
Tumor
6.97 ± 0.47 5.08 ± 1.50 4.78 ± 0.36 3.63 ± 0.65 2.50 ± 0.67
10.3 ± 0.44 6.03 ± 0.48 4.72 ± 0.81 2.97 ± 0.41 1.82 ± 0.079
6.92 ± 0.95 4.02 ± 0.90 3.14 ± 0.096 3.09 ± 0.69 1.66 ± 0.080
6.21 ± 1.12 4.96 ± 1.08 4.02 ± 0.48 3.38 ± 0.38 1.88 ± 0.32
9.36 ± 0.25 4.97 ± 0.66 4.26 ± 0.29 2.94 ± 0.43 2.08 ± 0.26
2.38 ± 0.17 2.30 ± 0.62 2.27 ± 0.69 2.18 ± 0.29 1.47 ± 0.14
6.64 ± 0.91 4.02 ± 1.03 3.79 ± 0.31 3.59 ± 0.43 2.12 ± 0.69
6.09 ± 0.25 4.46 ± 0.18 4.43 ± 0.30 3.78 ± 0.56 2.59 ± 0.14
3.02 ± 0.36 3.45 ± 0.46 5.03 ± 0.68 3.96 ± 1.10 2.94 ± 0.27
Tumor/Brain
Tumor/Muscle
Tumor/Blood
1.27
0.45
0.49
1.50
0.86
0.77
2.21
1.33
1.14
1.82
1.10
1.05
2.00
1.39
1.14

Molecules 2012, 17
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Table 2. Biodistribution of [ F]4 in mice bearing S 180 tumor. Expressed as % injected
dose per gram (% ID/g ± SD), n = 4.
Time (min)
Organs
5
15
30
60
120
Heart
Liver
Spleen
Lung
Kidney
Brain
Muscle
Blood
Tumor
6.67 ± 0.92 4.20 ± 1.0 5.07 ± 0.96 4.09 ± 0.59 3.61 ± 1.1
10.7 ± 0.65 6.05 ± 0.56 4.61 ± 1.0 3.07 ± 0.35 2.32 ± 0.18
6.97 ± 0.34 3.87 ± 0.13 4.33 ± 1.3 2.99 ± 0.66 2.34 ± 0.69
6.33 ± 0.87 4.06 ± 0.41 4.07 ± 0.18 3.24 ± 0.73 2.58 ± 0.30
11.1 ± 1.4 5.86 ± 0.14 4.2 ± 0.52 3.03 ± 0.55 2.58 ± 0.48
1.75 ± 0.18 2.33 ± 0.26 2.95 ± 0.56 2.26 ± 0.49 1.6 ± 0.16
6.48 ± 1.0 3.75 ± 0.32 4.19 ± 0.65 3.28 ± 0.71 1.54 ± 0.39
6.33 ± 0.45 4.91 ± 0.46 4.57 ± 0.59 3.64 ± 0.40 3.30 ± 0.22
1.91 ± 1.2 3.84 ± 0.46 4.41 ± 1.3 4.53 ± 0.90 3.75 ± 0.21
Tumor/Brain
Tumor/Muscle
Tumor/Blood
1.09
0.29
0.30
1.65
1.03
0.78
1.5
1.05
0.96
2.01
1.38
1.25
2.35
2.44
1.14
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Table 3. Biodistribution of [ F]5 in mice bearing S 180 tumor. Expressed as % injected
dose per gram (% ID/g ± SD), n = 4.
Time (min)
Organs
5
15
30
60
120
Heart
Liver
Spleen
Lung
Kidney
Brain
Muscle
Blood
Tumor
3.02 ± 0.51
10.39 ± 1.86 2.90 ± 0.86 2.29 ± 0.38
3.10 ± 0.12 2.76 ± 0.11 0.54 ± 0.07
16.34 ± 1.80 15.13 ±1.57 6.35 ± 1.28
12.70 ± 0.90 3.71 ± 0.69 2.21 ± 0.29
0.22 ± 0.00
2.15 ± 0.66
6.68 ± 0.05
2.55 ± 0.42
11.59
1.19 ± 0.21 0.97 ± 0.19
0.42 ± 0.07
1.79 ± 0.38
0.55 ± 0.19
2.45 ± 0.55
0.94 ± 0.28
0.15 ± 0.00
0.54 ± 0.00
0.45 ± 0.09
0.71 ± 0.11
4.73
0.52 ± 0.05
1.63 ± 0.22
0.53 ± 0.02
4.09 ± 0.19
0.58 ± 0.01
0.14 ± 0.00
0.18 ± 0.04
0.35 ± 0.02
0.83 ± 0.17
5.93
0.14 ± 0.00 0.15 ± 0.02
1.07 ± 0.35 0.49 ± 0.09
2.24 ± 0.04 0.95 ± 0.10
1.75 ± 0.54 1.30 ± 0.15
Tumor/Brain
Tumor/Muscle
Tumor/Blood
12.5
1.64
0.78
8.67
2.65
1.37
1.19
0.38
1.31
1.58
4.61
2.37
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In general, as shown in Table 1 and Table 2, [ F]3 and [ F]4 accumulated to high levels in the
tumor, 5.03 ± 0.68 and 4.41 ± 1.3 %ID/g at 30 min post-injection, respectively, and stayed at similarly
high levels 3.96 ± 1.10 and 4.53 ± 0.90 %ID/g at 60 min post-injection, 2.94 ± 0.27 and 3.75 ± 0.21 %ID/g
at 120 min post-injection, respectively, demonstrating persistent retention over time. Meanwhile, they
also showed moderate clearance from other organs and tissues, resulting in tumor/brain, tumor/muscle
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18
and tumor/blood ratios of 2.00, 1.39 and 1.14 for [ F]3, 2.35, 2.44 and 1.14 for [ F]4 at 120 min
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post-injection. [ F]5, which contains a carboxyl group, exhibited high liver and kidney uptake,
1
1
[
0.39 ± 1.86 and 12.70 ± 0.90 %ID/g at 5 min post-injection, respectively, and rapid clearance of
.63 ± 0.22 and 0.58 ± 0.01 %ID/g at 120 min post-injection, respectively. Surprisingly, The uptake of
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8
F]5 in lung was relatively higher than that in other tissues at all time points studied. This

Molecules 2012, 17
3782
phenomenon might result from a passive process of the radiotracers in lung. Furthermore, this tracer
displayed 2.55 ± 0.42 %ID/g in tumor at the earliest (5 min) and then it was gradually reducing over the
18
2
h time period of the study, which indicated that [ F]5 had insignificant retention in tumor. In
addition, very low brain accumulation afforded high tumor/brain ratios: 11.59, 12.5, 8.67, 4.73 and
.93. The ratios of tumor to other tissues were over 1 except for the ratios of tumor to liver (0.5) and
5
tumor to kidney (0.02), on the other hand, the tumor/muscle and tumor/blood ratios reached their peak
value of 4.61 and 2.37 respectively at 120 min post-injection.
1
8
18
18
Biodistribution studies revealed generally similar uptake patterns of [ F]1, [ F]3 and [ F]4 in
most organs and the tumor. High initial activity accumulations but slow reduction in the excretory
tissues for all the three radiotracers were observed, which may be the result of the relatively slow
clearance from blood. However, all the three radiotracers displayed a remarkable increasing uptake in
1
8
18
tumor during in Vivo animal studies as shown in Figure 4. [ F]1 and [ F]3 reached their peak tumor
1
8
18
uptake at 30 min post-injection (5.51 ± 0.31 %ID/g for [ F]1 and 5.03 ± 0.68 %ID/g for [ F]3) while
1
8
18
[
F]4 did at 60 min post-injection (4.53 ± 0.90 %ID/g for [ F]4). After the peak, the uptake of all the
three radiotracers started to decline. After 120 minutes post injection, the radioactivity was about
1
8
18
half of the peak value. They were 2.88 ± 0.34 %ID/g for [ F]1, 2.94 ± 0.27 %ID/g for [ F]3 and
1
8
3
.75 ± 0.21 %ID/g for [ F]4 respectively. These data indicated that all the three tracers remained
in tumor for a relatively long time. What’s more, the ratios of tumor to brain, muscle and blood for
all three tracers at each selected time points were close. These findings demonstrated that the
decreased lipophilicity caused by introduction of ester and hydroxyl groups at the C-5 position of the
pyrazolo[1,5-a]pyrimidine core structure did not affect the in Vivo biological behavior of
these compounds.
1
8
18
18
Figure 4. Comparison of tumor uptake rates of [ F]1, [ F]3 and [ F]4 with those of
1
8
18
[
F]2 and [ F]5 in the same animal model bearing S180 tumor.
1
8
[
[
[
[
[
F
]
1
3
4
2
5
1
1
1
1
8
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
F]
F]
F]
F]
8
8
8
0
20
40
60
80
100
120
Time (min)

Molecules 2012, 17
3783
1
8
18
On the other hand, [ F]5 showed similar distribution patterns as [ F]2 in most organs, including
the tumor. For both radiotracers, peak uptake in all organs and tissues occurred at the initial time and
then they washed out swiftly from these organs and tissues as time elapsed. Additionally, radioactivity
1
8
18
concentrations in tumor was 2.26 ± 0.48 %ID/g for [ F]2 and 2.55 ± 0.42 %ID/g for [ F]5 at 5 min p.i.
1
8
18
and continually decreased to 0.21 ± 0.02 %ID/g for [ F]2 and 0.83 ± 0.17 %ID/g for [ F]5 at
0 min p.i., indicating temporary retention in tumor. Due to relatively low uptake into brain, both
radiotracers afforded high tumor/brain ratios at each selected time point. The tumor/blood ratios of
6
1
8
both radiotracers were close at each selected time points while the tumor/muscle ratios of [ F]5 were
18
higher than those of [ F]2 at 5, 15, 30 and 120 min. These data confirmed that the decreased lipophilicity
caused by introduction of the carboxyl group at the C-7 position of the pyrazolo[1,5-a]pyrimidine core
structure gave rise to insignificant biological behavior of these compounds in Vivo.
Overall, the total yields of the radiolabeled products obtained from 2,4-dinitrobenzamide and
tosylate precursors were comparable, but statistically significant differences in tumor uptakes were
1
8
18
18
observed: all tracers [ F]1, [ F]3 and [ F]4 had higher uptake and longer retention in tumor than
1
8
18
those of both tracers [ F]2 and [ F]5. These differences are noteworthy, as they suggest that the
1
8
2
-[ F]fluoro-4-nitrobenzamide group on position 7 seems to have negative effect on the tumor uptake
1
8
kinetic when compared to a 2-[ F]fluoroethylamino group in this position. The findings could not be
1
8
18
18
explained simply by the differential lipophilicity. [ F]1, [ F]3 and [ F]4 showed the similar
biological behavior including the tumor uptake kinetic despite they possess the distinct lipophilicity.
1
8
18
18
18
18
[
F]2(0.68) and [ F]5(−1.78) exhibited the same findings as [ F]1, [ F]3 and [ F]4. The different
metabolic fate of these compounds could be the possible answer for this finding. By comparing the
1
8
18
parallel biodistribution of 2-[ F]fluoroethylaminopyrazolo[1,5-a]pyrimidine derivatives and 2-[ F]-
fluoro-4-nitrobenzamideopyrazolo[1,5-a]pyrimidine derivatives, we found that the tumor uptake
is related to the amount of radioactivity present in the blood. For both kinds of radiotracers, the
initial radioactivity in the blood was similar high, but the clearance rate by 120 min was significantly
1
8
different. The slow blood clearance rate of 2-[ F]fluoroethylaminopyrazolo[1,5-a]pyrimidine
18
derivatives was along with higher uptake and longer retention in tumor. 2-[ F]fluoro-4-nitrobenzamide-
pyrazolo[1,5-a] pyrimidine derivatives did the contrary. One of the disadvantages of this high
circulating radioactivity in the blood would be not very clear visualization of tumor in Vivo using this
kind of probe. The metabolic tissues such as liver and kidney showed initial high uptake but continued
1
8
to clear out further with time. Meanwhile 2-[ F]fluoro-4-nitrobenzamidopyrazolo[1,5-a] pyrimidine
1
8
derivatives washed out faster than 2-[ F]fluoroethylaminopyrazolo[1,5-a]pyrimidine derivatives did,
which may derive from the distinct clearance rates from blood for both kinds of radiotracers.
1
8
2
.7. MicroPET Imagings of [ F]3 in S180 Bearing Mouse
Coronal microPET images of a mouse bearing S180 tumor at 30 and 60 min after tail vein injection
1
8
of [ F]3 are shown in Figures 5A,B, respectively. The tumor in the left forelimb was visible while no
1
8
accumulation in the right forelimb was found. The regions of interest (ROI) ratio of [ F]3 uptake
for tumor site versus the corresponding non-tumor region (T/NT ratio) was 1.1 at 30 min and 1.5 at
6
0 min, suggesting that it would be able to accumulate in tumor for a relatively long time. The
tumor-to-background contrast at 30 min p.i. was low, but it became a slightly better at 60 min p.i.

Molecules 2012, 17
3784
1
8
These results were consistent with ex vivo biodistribution studies. Furthermore, [ F]3 had low
lipophilicity (logP = −0.04), which would expect to have less brain uptake [28]. By the comparison of
the PET images at 30 min and 60min post-injection, the persistent retention of radiotracer in the brain
was observed.
Figure 5. Coronal PET images of Kunmin mice with S180 tumor (arrow) in the left front
1
8
flank at 30 min (A) and 60 min (B) post-injection of [ F]3.
3. Experimental
3.1. General
All chemicals were commercially obtained and used without further purification. Melting points
1
were measured in capillary tubes using a RY-1 apparatus without correction. H-NMR spectra were
1
3
19
recorded on a Bruker (400 MHz) spectrometer. C-NMR, F NMR spectra were recorded on a Bruker
100 MHz) spectrometer. Tetramethylsilane (TMS) was used as an internal standard for both spectra.
(
Chemical shift data of the proton resonances were reported in parts per million (δ) relative to internal
standard TMS (δ 0.0). Mass spectra were obtained using a Brucker Apex  FTM instrument. Elemental
analysis was performed on a Pekin-Elmer 240-C instrument. IR data were obtained on a Nicolet 360
1
8
Avatar infrared spectrophotometer. The [ F]fluoride used for radiosynthesis was produced by the
1
8
18
O(p, n) F nuclear reaction at Center of Xuanwu Hospital (Beijing, China). Semipreparative HPLC
purification was carried out using a semipreparative reversed-phase Grace Alltima™ C18 Column
250 × 10 mm, particle size: 10 μm) under the indicated conditions. The Semipreparative HPLC
(
system used was an Alltech system with Alltech HPLC pump Model 626, LINEAR UVIS-201, and
BIOSCAN flow-counter. Reversed-phase extraction Sep-Pak C18 Plus cartridges (Waters) were
activated with methanol and water before use. Compounds 9 and 12 shown in Schemes 1 and 2 were
synthesized as described previously [27,29].

Molecules 2012, 17
.2. Synthesis
3785
3
(
3-Cyano-7-(2-hydroxyethylamino)pyrazolo[1,5-a]pyrimidin-5-yl)methyl acetate (10). To a solution of
(0.25 g, 1.0 mmol) in DMSO (7.5 mL) and water (5 mL) at room temperature were added acetic acid
0.15 g, 1.1 mmol) and NaOH (1.2 mg, 0.03 mmol). The reaction mixture was heated to reflux at
20 °C for 1.5 h and then cooled to room temperature. The solvent was removed under reduced
9
(
1
pressure and the crude product was purified by column chromatography on silica gel eluted with 1:1
ethyl acetate/petroleum ether (60–90 °C) to afford 10 (0.16 g, 60%) as yellow solid, mp: 215 °C; IR
−
1
(
KBr, cm ) υ: 3308.0, 3093.3, 2939.8, 2224.1, 1741.6, 1630.6, 1587.8, 1533.9, 1446.1, 1306.3, 1263.8,
1
1
234.1, 1190.8, 1068.6; H-NMR (DMSO) δ 8.64 (s, 1H, Pyrazole-H), 6.50 (s, 1H, Pyrimidine-H),
1
,2
5
.09 (s, 2H, -CH O-), 3.66–3.61 (m, 2H, OHCH CH -), 3.50 (dd, J = 11.2 Hz, 5.56 Hz, 2H,
2
2
2
1
3
-CH
2
CH
2
NH-), 2.17 (s, 3H, CH
3
CO-); C-NMR (DMSO, 100 MHz) δ 170.00, 159.86, 150.28, 148.07,
+
1
46.57, 114.11, 86.28, 78.47, 65.41, 59.31, 44.31, 20.59; MS (EI) m/z: 275.1, found: 276.3 [M+H] ;
Anal. calcd for C H N O : C, 52.36; H, 4.76; N, 25.44; found: C, 52. 54; H, 4.77; N, 25.36.
1
2
13
5
3
(
3-Cyano-7-(2-(tosyloxy)ethylamino)pyrazolo[1,5-a]pyrimidin-5-yl)methyl acetate (11). To a stirred
solution of 10 (0.28 mg, 1 mmol) in CH Cl (20 mL) cooled with ice bath (0 °C) was added to Et
0.21 mL, 1.5 mmol), p-toluenesulfonyl chloride (TsCl, 0.29 g, 1.5 mmol) and a catalytic amount of
2
2
3
N
(
4-dimethylaminopyridine (DMAP, 0.03 g, 0.2 mmol). After 30 minutes incubation on ice, the reaction
was kept at room temperature overnight. The reaction was washed with water (1 × 12 mL), saturated
sodium bicarbonate (1 × 10 mL), and brine (1 × 15 mL) successively. After drying over sodium sulfate,
the volatile materials were evaporated under vacuum. The crude oily residue was further purified by
column chromatography using petroleum ether (60–90 °C)/ethyl acetate (2:1) as eluant to give 11
−1
(
0.36 g, 85%) as white solid, mp: 150–152 °C. IR (KBr, cm ) υ: 3329.0, 3101.8, 2221.8, 1735.6,
1
628.7, 1586.4, 1451.2, 1361.7, 1320.8, 1231.0, 1188.3, 1175.7, 1032.8, 907.2, 807.5, 762.5, 665.0,
1
5
78.0, 550.8; H-NMR (DMSO) δ 8.63 (s, 1H, Pyrazole-H), 7.49 (d, J = 8.2 Hz, 2H, Ar-H), 6.98 (d,
J = 8.1 Hz, 2H, Ar-H), 6.31 (s, 1H, Pyrimidine-H), 5.04 (s, 2H, -CH O-), 4.28–4.30 (m, 2H,
2
1
3
-
2 2 2 2 3 3
CH CH NH-), 3.67–3.71 (m, 2H, OHCH CH -), 2.21 (s, 3H, CH CO-), 2.19 (s, 3H, CH -); C-NMR
(
DMSO, 100 MHz) δ 169.98, 159.78, 149.99, 147.01, 146.41, 144.39, 131.56, 129.20, 127.10, 114.09,
+
8
6.10, 78.68, 68.12, 65.27, 45.71, 20.84, 20.60; MS (EI) m/z: 429.1, found: 429.9 [M+H] ; Anal. calcd
19 5 5
for C19H N O S: C, 53.14; H, 4.46; N, 16.31; found: C, 53. 32; H, 4.47; N, 16.26.
1
9
(
3-Cyano-7-(2-fluoroethylamino)pyrazolo[1,5-a]pyrimidin-5-yl)methylacetate ([ F]3). Tetrabutyl-
.
ammonium fluoride trihydrate (TBAF 3H O, 0.63 g, 2 mmol) was dissolved in anhydrous CH CN
2
3
(
1 mL) and heated rapidly at 120 °C under a nitrogen stream, followed by additional CH
3
CN (2 mL),
the solvent was evaporated, this process was repeated three times. After evaporating most of the
solvent, solution 11 (0.43 g, 1 mmol) in anhydrous CH CN (3 mL) was added to the dry reagent. The
3
reaction mixture was stirred at 75 °C for 2 h under a nitrogen steam and cooled to room temperature.
The solvent was removed under reduced pressure. The residue was purified by silica gel
1
9
chromatography using petroleum ether (60–90 °C)/ethyl acetate (1:1) as eluant to afford [ F]3 (0.06 g,
−
1
2
1
0%) as a yellow solid. IR (KBr, cm ) υ: 3379.9, 3093.8, 2940.1, 2225.0, 1730.3, 1627.3, 1597.0,
1
9
1
3
548.3, 1442.7, 1379.8, 1258.0, 1062.2; F-NMR (CDCl , 100 MHz) δ −223.00 (1F); H-NMR

Molecules 2012, 17
3786
(
-
CDCl ) δ 8.16 (s, 1H, Pyrazole-H), 6.73 (s, 1H, -NH-), 6.18 (s, 1H, Pyrimidine-H), 5.13 (s, 2H,
3
1
,2
1,2
CH
2
O-), 4.68 (dt, J = 46.9 Hz, 4.8 Hz, 2H, -CH
2
F), 3.73 (dq, J = 25.9 Hz, 5.2 Hz, 2H, -CH
2 2
CH F),
1
3
2
.14 (s, 3H, CH -); C-NMR (CDCl , 100 MHz) δ 170.33, 160.12, 150.29, 148.00, 146.72, 114.22,
3 3
8
6.20, 82.05 (d, J = 173.0 Hz), 78.66, 65.37, 42.11 (d, J = 20.0 Hz), 20.61; MS (EI) m/z: 277.10, found:
+
277.9 [M] ; Anal. calcd for C H FN O : C, 51.98; H, 4.36; N, 25.26; found: C, 52.16; H, 4.37;
1
2
12
5
2
N, 25.18.
1
9
7
-(2-Fluoroethylamino)-5-(hydroxymethyl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile ([ F]4). The
1
9
compound [ F]3 (0.28 g, 1 mmol) was dissolved in CH
mmol) in 2 mL H O was added. The reaction mixture was heated at 80 °C for 20 min at which point
TLC indicated the completion of the reaction. The yellow precipitate was formed after adjusting the
3
OH (10 mL) and a solution of NaOH (0.08 g,
2
2
1
9
−1
pH to 7 with 1 N HCl aqueous solution and then filtered to afford [ F]4 (0.2 g, 85%). IR (KBr, cm ) υ:
1
9
3
398.3, 2916.2, 2850.3, 2224.3, 1628.3, 1604.6, 1463.7, 1427.7, 1128.5, 1020.2, 624.7; F-NMR
1
(
DMSO) δ −221.66 (1F); H-NMR (DMSO) δ (ppm): 8.24 (s, 1H, Pyrazole-H), 6.75 (s, 1H, -NH-),
1
,2
6
.21 (s, 1H, Pyrimidine-H), 4.78 (d, 2H, J = 5.2 Hz, -CH
2
O-), 4.74 (dt, 2H, J = 46.9 Hz, 4.8 Hz,
1
3
-
(
CH
2
F), 3.84–3.74 (dm, 2H, J = 25.9 Hz, -CH CH F), 3.34 (t, 1H, J = 5.2 Hz, -OH); C-NMR
2
2
DMSO, 100 MHz) δ 166.83, 150.35, 147.74, 146.55, 114.22, 85.41, 82.08 (d, 167 Hz), 78.04, 63.90,
+
4
4
2.97 (d, 22 Hz); MS (EI) m/z: 235.09, found: 235.1 [M] ; Anal. calcd for C10
H10FN
5
O: C, 51.06; H,
.29; N, 29.77; found: C, 51.24; H, 4.30; N, 29.68.
(
S)-Methyl 6-(3-cyano-5-methylpyrazolo[1,5-a]pyrimidin-7-ylamino)-2-(2,4-dinitrobenzamido)hexanoate
13). To a stirred solution of L-lysine methyl ester hydrochloride (0.24 g, 1 mmol) in anhydrous
CH CN was successively added TEA (0.42 mL, 3 mmol) and the compound 12 (0.19 g, 1 mmol), and
the reaction mixture heated to reflux for 15 min. The resulting mixture was cooled and concentrated in
vacuo. Thereafter, the residue was then dissolved in anhydrous CH Cl (20 mL), cooled to 0 °C and
treated successively with 2,4-dinitrobenzoic acid (0.21 g, 1 mmol), HOBt (0.12 g, 1 mmol) and DCC
0.23 g, 1 mmol) in CH Cl (5 mL). The reaction mixture was stirred at 0 °C for 30 min and then at
(
3
2
2
(
2
2
room temperature overnight. Following filtration the organic phase was washed with saturated sodium
bicarbonate (1 × 10 mL), brine (1 × 15 mL), dried over sodium sulfate and evaporated to give a glassy
residue, which was purified on silica gel eluted with petroleum ether (60–90 °C)/ethyl acetate (3:2) to
−1
give 13 (0.21 g, 42%) as yellow solid, mp: 157–158 °C; IR (KBr) υ (cm ): 3372.7, 3295.3, 3099.5,
2
1
941.2, 2863.5, 2222.9, 1752.4, 1638.0, 1612.0, 1588.8, 1540.7, 1465.3, 1449.1, 1351.7, 1303.1,
1
230.0, 1230.0, 1172.5, 1158.4, 836.9, 739.5, 729.4, 671.4; H-NMR (CDCl
3
) δ 8.91 (d, 1H,
1
,2
J = 2.1 Hz, Ar-H), 8.52 (dd, 1H, J = 8.3, 2.2 Hz, Ar-H), 8.09 (s, 1H, Pyrazole-H), 7.78 (d, 1H,
J = 8.3 Hz, Ar-H), 6.78 (d, 1H, J = 7.9 Hz, -NH(CO)), 6.42 (t, 1H, J = 5.7 Hz, -NHCH ), 6.04 (s, 1H,
Pyrimidine-H), 4.91–4.86 (m, 1H, -(CO)CHCH -), 3.83 (s, 3H, -OCH ), 3.49–3.44 (m, 2H,
HNCH CH ), 2.56 (s, 3H, -CH ), 2.16–2.12 (m, 1H, -(CO)CHCH -), 1.96–1.91 (m, 2H, -HNCH CH ),
.85–1.85 (m, 1H, -(CO)CHCH -), 1.69–1.58 (m, 2H, -CHCH CH , 100 MHz) δ
2
2
3
-
2
2
3
2
2
2
1
3
1
1
1
2
2
2 3
); C-NMR (CDCl
72.03, 164.06, 163.63, 150.38, 148.44, 146.74 (2C, Py-C, Ar-C), 146.03, 137.36, 130.30, 128.26,
20.21, 113.73, 88.17, 80.10, 52.97, 52.45, 41.98, 31.75, 28.14, 25.25, 22.27; MS (EI) m/z: 510.2,
+
found: 510.5 [M] ; Anal. calcd for C22
N 21.88.
22 8 7
H N O : C 51.76, H 4.34, N 21.95, found: C 51.94, H 4.35,

Molecules 2012, 17
3787
(
S)-Methyl 6-(3-cyano-5-methylpyrazolo[1,5-a]pyrimidin-7-ylamino)-2-(2-fluoro- 4-nitrobenzamido)hexanoate
1
9
.
(
[ F]14). Tetrabutylammonium fluoride trihydrate (TBAF 3H O, 0.63 g, 2 mmol) was dissolved in
2
3
anhydrous CH CN (1 mL) and heated rapidly at 120 °C under a nitrogen stream, followed by
additional CH CN (2 mL), the solvent was evaporated, this process was repeated three times. After
3
evaporating most of the solvent, solution 13 (0.51 g, 1 mmol) in anhydrous DMF (3 mL) was added to
the dry reagent. The reaction mixture was stirred at 120 °C for 40 min under a nitrogen steam and
cooled to room temperature, then diluted with H O (20 mL), extracted with ethyl acetate (10 mL × 3).
2
The combined organic phases were dried over Na
2 4
SO , filtered and concentrated in vacuum. The
residue was subjected to silica gel column chromatography (20% ethyl acetate in petroleum ether
−1
(
60–90 °C)) to afford 14 (0.14 g, 29%) as yellow solid, mp: 62–63 °C; IR (KBr) υ (cm ): 3384.1,
3
1
104.7, 2953.2, 2866.3, 2223.7, 1742.5, 1670.1, 1625.8, 1586.1, 1532.3, 1457.3, 1419.8, 1353.1,
1
306.5, 1226.0, 1192.1, 1154.8, 1092.1, 809.8, 738.0, 668.1; H-NMR (CDCl
3
) δ 8.30–8.27 (m, 1H,
Ar-H), 8.15 (d, 1H, J = 7.5 Hz, Ar-H), 8.14 (s, 1H, Pyrazole-H), 8.05 (d, 1H, J = 11.0 Hz, Ar-H),
.34 (m, 1H, -NH(CO)), 6.36 (m, 1H, -NHCH ), 6.02 (s, 1H, Pyrimidine-H), 4.92–4.87 (m, 1H,
7
2
-
(
(CO)CHCH -), 3.82 (s, 3H, -OCH ), 3.46–3.41 (m, 2H, -HNCH CH ), 2.58 (s, 3H, -CH ), 2.12–2.05
2
3
2
2
3
m, 1H, -CH(CO)CH
2
2 2 2
), 1.96–1.87 (m, 2H, -HNCH CH ), 1.87–1.79 (m, 1H, -(CO)CHCH -),
13
1
2 2 3
.63–1.56 (m, 2H, -CHCH CH ); C-NMR (CDCl , 100 MHz) δ 172.01, 163.51, 161.10 (d, J = 12.0 Hz,
-
HNCO), 159.83 (d, J = 236.0 Hz, Ar-C), 150.46, 150.44 (d, J = 4.0 Hz, Ar-C), 146.69, 146.08, 133.37
d, J = 2.0 Hz, Ar-C), 126.10 (d, J = 12.0 Hz, Ar-C), 119.67 (d, J = 4.0 Hz, Ar-C), 113.52, 112.24 (d,
(
19
J = 30.0 Hz, Ar-C), 87.99, 80.49, 52.82, 52.52, 41.94, 32.23, 28.14, 25.28, 22.58; F-NMR (CDCl
3
)
+
δ −109.18 (1F); MS (EI) m/z: 483.2, found: 483.2 [M] ; Anal. calcd for C22
H22FN
7
O
5
:
C 54.66, H 4.59, N 20.28, found: C 54.74, H 4.60, N 20.22.
(
S)-6-(3-Cyano-5-methylpyrazolo[1,5-a]pyrimidin-7-ylamino)-2-(2-fluoro-4-nitrobenzamido)hexanoic acid
1
9
19
(
3
[ F]5). To a solution of [ F]14 (48 mg, 0.1 mmol) in CH OH (3 mL) cooled to 0 °C was added
LiOH·H O (21 mg, 0.5 mmol) in H O (0.6 mL). After 30 min, the cold bath was removed, and the
2
2
reaction was continued at ambient temperature overnight. The reaction mixture was concentrated in
vacuo and the pH was adjusted to 2 with 35% HCl at which point a yellow precipitate was formed. The
precipitate was filtered, washed thoroughly with water, and then crystallized from methanol/ether to
1
9
−1
produce [ F]5 (0.45 mg, 95%) as yellow solid, mp: 224–226 °C; IR (KBr) υ (cm ): 3394.4, 3103.7,
2
934.8, 2226.3, 1656.4, 1619.4, 1581.6, 1531.8, 1417.1, 1353.5, 1311.0, 1192.7, 1014.2, 810.1,
1
7
39.5, 650.1; H-NMR (DMSO) δ 8.54 (s, 1H, Pyrazole-H), 8.39 (m, 1H, -NH(CO)), 8.28 (t, 1H,
J = 5.8 Hz, -NHCH
2
), 8.18 (dd, 1H, J = 10.5, 2.0 Hz, Ar-H), 8.13 (dd, 1H, J = 8.6, 2.0 Hz, Ar-H),
.99–7.95 (m, 1H, Ar-H), 6.39 (s, 1H, Pyrimidine-H), 3.99–3.95 (m, 1H, -CH(CO)CH -), 3.36–3.33 (m,
H, -NHCH CH ), 2.43 (s, 3H, -CH ), 1.88–1.84 (m, 1H, -CH(CO)CH -), 1.73–1.70 (m, 1H,
), 1.61–1.59 (m, 2H, -NHCH CH ), 1.39–1.34 (m, 2H, -CHCH CH
00 MHz) δ 173.64, 162.62, 160.04 (d, J = 9.0 Hz, -NHCO), 158.70 (d, J = 251.0 Hz, Ar-C), 150.50,
49.01 (d, J = 10.0 Hz, Ar-C), 146.82, 145.99, 131.66 (d, J = 3.0 Hz, Ar-C), 129.21 (d, J = 15.0 Hz,
7
2
2
2
2
3
2
1
3
-CH(CO)CH
2
2
2
2
2
); C-NMR (DMSO,
1
1
Ar-C), 119.49 (d, J = 3.0 Hz, Ar-C), 114.39, 111.98 (d, J = 29.0 Hz, Ar-C), 88.22, 77.61, 54.68, 41.29,
1
9
3
1.76, 28.01, 24.39, 22.18; F-NMR (DMSO) δ −110.47 (1F); MS (EI) m/z: 469.2, found: 470.4
+
[
M+H] ; Anal. calcd for C H FN O : C 53.73, H 4.29, N 20.89, found: C 53.92, H 4.30, N 20.83.
2
1
20
7
5

Molecules 2012, 17
3788
1
8
18
(
3-Cyano-7-(2-[ F]fluoroethylamino)pyrazolo[1,5-a]pyrimidin-5-yl)methyl
acetate
([ F]3),
1
8
18
7
-(2-[ F]fluoroethylamino)-5-(hydroxymethyl)pyrazolo[1,5-a]pyrimidine- 3-carbonitrile ([ F]4) and
18
(
S)-6-(3-cyano-5-methylpyrazolo[1,5-a]pyrimidin- 7- ylamino)- 2-(2-[ F]fluoro-4-nitrobenzamido)hexanoic
1
8
18
18
18
acid ([ F]5). [ F]Fluoride was generated from a [ O(p,n) F] nuclear reaction by irradiation on 97%
1
8
18
2
enriched H O (Beijing PET Center of Xuanwu Hospital). [ F]fluoride was trapped on an anion
exchange cartridge and directly eluted into a 10 mL sealed reaction vessel using a solution of
potassium carbonate (3–5 mg) and Kryptofix 2.2.2 (15–20 mg) in 1.5 mL acetonitrile/water (3:1). The
solvents were removed under a stream of nitrogen at 110 °C. Subsequently, 0.8–1.0 mL of dry
1
8
acetonitrile was added three times and evaporated to dryness. To the obtained dry [ F] K2.2.2/KF
complex, 11 (3 mg, 7 μmol) in dry CH CN (0.5 mL) was added. The solution was heated at 55–80 °C
3
for 40 min and diluted with H
Sep-Pak cartridge was washed with H
with 2 mL of CH CN, which was injected into a semi-preparative HPLC performed on Grace Alltima™
2
O (10 mL) and loaded onto an activated C18 Sep-Pak cartridge. The
18
18
2
O (10mL) to remove unlabeled F and the [ F]3 was then eluted
3
3 2
C18 Column (250 × 10 mm, particle size: 10 μm) isocratically eluted with CH CN/H O = 40:60 at a
1
8
flow rate of 3 mL/min. The HPLC solvents were removed under reduced pressure to give [ F]3.
Hydrolysis was rapid following the addition of LiOH (10 mg) in MeOH (2 mL) at 80 °C for
1
8
2
0 min and then neutralized with 1 N HCl. The solvent was evaporated in vacuo to give [ F]4.
1
8
To synthesize [ F]5, 13 (5 mg, 10 μmol) in dry DMF (0.5 mL) was added to the vial containing dry
1
8
[
F] K2.2.2/KF complex and heated at 110 °C for 40 min, diluted with H
2
O (10 mL) and loaded onto
O (10 mL) to remove
CN, which was injected into a
semi-preparative HPLC performed on Grace Alltima™ C18 Column (250 × 10 mm, particle size:
0 μm) isocratically eluted with CH CN/H O = 40:60 at a flow rate of 3 mL/min. The elution was
an activated C18 Sep-Pak cartridge. The Sep-Pak cartridge was washed with H
2
18
18
unlabeled F and the [ F]14 was then eluted with 2 mL of CH
3
1
3
2
1
8
collected after purification and the solvents were removed under reduced pressure to provide [ F]14,
subsequently hydrolyzed using LiOH (10 mg) in MeOH (2 mL) at 80 °C for 20 min and then adjusted
1
8
pH to 7 with 1 N HCl. The solvent was evaporated in vacuo to give [ F]5. The product was dissolved
in certain amount of phosphate-buffered saline (pH = 7.4) (370 kBq/0.1 mL) for further use.
3
.3. Octanol/Water Partition Coefficient
The partition coefficient was measured as previously described [30]. Each HPLC-purified
radiotracer was added to a tube containing 0.5 mL of n-octanol and 0.5 mL of phosphate-buffered
saline (pH 7.4). Each phase had been presaturated with the opposite phase. The mixed solution
vortexed for 1 min and centrifuged at 15,000 rpm for 2 min. Aliquots of both phases were transferred
in triplicate to counting tubes and assayed in a γ counter. The partition coefficient was calculated by
dividing the radioactivity of the octanol layer with that of the water layer. This measurement was
repeated three times.
3
.4. Quality Control of Purified Radiotracers
1
8
18
18
19
19
19
Quality control for [ F]3, [ F]4 and [ F]5 using authentic [ F]3, [ F]4 or [ F]5 as a reference
was done by Radio-HPLC on Grace Alltima™ C18 Column (250 × 10 mm, particle size: 10 μm)

Molecules 2012, 17
3789
isocratically eluted with CH CN/H O = 40:60 at a flow rate of 3 mL/min. Using this system, specific
3
2
activity was calculated by the UV peak area of the purified radiotracers as compared with standard
curves of a reference nonradioactive compound.
3.5. Stability Study
Each HPLC-purified radiotracer (3.7 MBq) was mixed with phosphate-buffered saline (pH 7.4) at
7 °C for a period up to 2 h and was analyzed by radio-HPLC. The stability of each HPLC-purified
3
radiotracer in mouse plasma was determined by incubating 0.1 mL of radiotracer (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 centrifugation. The supernatant part was injected into radio-HPLC
on Grace Alltima™ C18 Column (250 × 10 mm, particle size: 10 μm) isocratically eluted with
1
9
19
CH CN/H O = 40:60 at a flow rate of 3 mL/min using the corresponding non-radioactive [ F]3, [ F]4
3
2
1
9
and [ F]5 as references to determine the stability of each compound.
3.6. Cellular Accumulation Studies
Cellular accumulation studies were performed with S180 tumor cells, which were suspended in
fresh DMEM medium supplemented with 10% fetal bovine serum at a cell concentration of
6
2
5
0
× 10 cells/mL and incubated at 37 or 4 °C with gentle stirring under an atmosphere of 95% air plus
% carbon dioxide. The cells were incubated together with each purified radiotracer (0.25 MBq in
.15 mL of cell culture medium) at 37 or 4 °C. At 5, 15, 30, 60 and 120 min, 200 μL samples of the
suspension were transferred to microfuge tubes and centrifuged at 1500 rpm for 5 min. A 180-μL
sample of each supernatant was removed for counting, and the left sample containing cells and 20 μL
medium was also counted. Percent cell uptake is calculated as [residue counts-(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%. The method is validated and the
relevant references have been listed [25,31].
3
.7. Biodistribution and MicroPET Imaging Studies in S180 Tumor Bearing Mice
All the animal studies were carried out in compliance with relevant national laws relating to the
1
8
18
18
conduct of animal experimentation. Uptake of [ F]3, [ F]4 and [ F]5 in Kunming Mice bearing S180
tumor models were performed according to published methods [27]. Briefly, S180 tumor cells were
trypsinized, counted, and suspended in a solution containing DMEM and 10% fetal bovine serum.
6
Kunming Mice were inoculated subcutaneously into the left forelimb with 5 × 10 tumor cells. Studies
of tumor uptake were conducted when the tumors reached a size of 0.5–0.8 cm in diameter. The
tumor-bearing mice were subjected to in Vivo biodistribution and imaging studies.
Tissue biodistribution studies were conducted by administering, via the tail vein, a bolus injection
of 370 kBq/mouse in a constant volume of 0.1 mL phosphate-buffered saline solution (pH = 7.4). The
animals were sacrificed at 5, 15, 30, 60 and 120 min post injection. The tissues and organs of interest
1
8
were immediately collected, weighed and measured for F radioactivity in a γ counter. Values are
expressed as mean ± SD (n = 4).

Molecules 2012, 17
3790
For small-animal PET (Siemens Inveon dedicated microPET), the mouse was injected intravenously
1
8
with approximately 3.7 MBq of [ F]3 in saline, anesthetized with 1.5% isoflurane and fixed near the
center of microPET scanner. Static scans (for 30 and 60 min, 10-min scans) were obtained, the data
were histogrammed into 25 consecutive frames and the images were reconstructed by a three
dimensional ordered subsets expectation maximum (OSEM) algorithm. The manually selected region
of interest (ROI) was applied to each image to estimate the counts present in the tumor and the
corresponding nontumor region by Amide’s a Medical Image Data Examiner (AMIDE) software.
3
.8. Statistical Analysis
Data are expressed as the mean ± S.D. Mean values were compared using Student's t test. Differences
were considered statistically significant for P ≤ 0.05.
4
. Conclusions
In conclusion, on the basis of our previous studies, we designed, synthesized and evaluated the
1
8
18
biological characteristics of three novel F-labeled pyrazolo[1,5-a]pyrimidine derivatives: [ F]3,
1
8
18
[
F]4 and [ F]5. During in Vivo animal studies, we observed that biodistribution studies revealed
1
8
18
18
generally similar uptake patterns of [ F]1, [ F]3 and [ F]4 for most organs and the tumor, while
1
8
18
[
F]5 showed similar distribution patterns as for [ F]2 in the most organs and the tumor. PET
1
8
images indicated that the persistent tumor uptake of [ F]3 was visible. By comparison among the
1
8
18
18
five compounds, 2-[ F]fluoroethylaminopyrazolo[1,5-a]pyrimidine derivatives: [ F]1, [ F]3 and
1
8
18
[
F]4 offered significant advantages over 2-[ F]fluoro-4-nitrobenzamidopyrazolo[1,5-a]pyrimidine
1
8
18
derivatives: [ F]2 and [ F]5 with regard to notably and persistent tumor uptake. These results implied
that tosylate precursor for F-18 labeled pyrazolo[1,5-a] pyrimidine core structure was more efficient
1
8
than a 2,4-dinitrobenzamide precursor and the fact that neither 2-[ F]fluoroethylaminopyrazolo
1
8
18
18
18
[
1,5-a]pyrimidines ([ F]1, [ F]3 and [ F]4) nor 2-[ F]fluoro-4-nitrobenzamidopyrazolo[1,5-a]
1
8
18
pyrimidines ([ F]2 and [ F]5) showed any relationship between their lipophilicity and biodistribution
behavior. These results are interesting enough to be merit more effort in further research.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/17/4/3774/s1.
Acknowledgments
This project was sponsored by the National Natural Science foundation of China (No.21071022)
and the Fundamental Research Funds for the Central Universities. We also wish to thank the cyclotron
operator team of the PET Centre of Xuanwu Hospital for providing fluoride-18 nuclide and
technical assistance.

Molecules 2012, 17
3791
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Sample Availability: Samples of the compounds [ F]1–5 are available from the authors.
©
2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/3.0/).
(