Synthesis and biological evaluation of 7-(2-Chlorophenylamino)-5-((2-[ 18F]fluoro-ethyoxy)methyl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile as PET tumor imaging agent

Article abstract of DOI:10.5560/ZNB.2012-0047

An ¹⁸F-labeled pyrazolo[1,5-a]pyrimidine derivative, 7-(2-chlorophenylamino)-5-((2-[¹⁸F] fluoroethyoxy)methyl)pyrazolo[1, 5-a]pyrimidine-3-carbonitrile ([¹⁸F]5), has been designed and prepared as a radio tracer candidate for tumor detection with positron emission tomography (PET). The desired product [¹⁸F]5 was synthesized by nucleophilic substitution of the corresponding tosylated precursor with [ ¹⁸F]KF=Kryptofix 2.2.2 and potassium carbonate in anhydrous DMF at 100 °C for 20 min followed by purification with HPLC. The radiochemical purity was >98%, and the radio-chemical yield was 25% (decay-uncorrected). Compound [¹⁸F]5 was very stable in vitro. The biodistribution study in mice bearing S180 tumors demonstrated that [¹⁸F]5 had a rapid and prolonged accumulation in tumors with moderate washout from other tissues.

Full text of DOI:10.5560/ZNB.2012-0047

Synthesis and Biological Evaluation of 7-(2-Chlorophenylamino)-5-((2-
[18F]fluoro-ethyoxy)methyl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile as
PET Tumor Imaging Agent
Jingli Xu, Hang Liu, Guixia Li, Yong He, Rui Ding, Xiao Wang, Man Feng,
Shuting Zhang, Yurong Chen, Shilei Li, Mingxia Zhao, Yingruo Li, and Chuanmin Qi
Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education,
College of Chemistry, Beijing Normal University, Beijing 100875, PR China
Reprint requests to Prof. C. M. Qi. E-mail: qicmin@sohu.com
Z. Naturforsch. 2012, 67b, 827 – 834 / DOI: 10.5560/ZNB.2012-0047
Received February 10, 2012
An 18F-labeled pyrazolo[1,5-a]pyrimidine derivative, 7-(2-chlorophenylamino)-5-((2-[18F]
fluoroethyoxy)methyl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile ([18F]5), has been designed and
prepared as a radio tracer candidate for tumor detection with positron emission tomography (PET).
The desired product [18F]5 was synthesized by nucleophilic substitution of the corresponding
tosylated precursor with [18F]KF/Kryptofix 2.2.2 and potassium carbonate in anhydrous DMF at
100 ◦C for 20 min followed by purification with HPLC. The radiochemical purity was > 98%, and
the radio-chemical yield was 25% (decay-uncorrected). Compound [18F]5 was very stable in vitro.
The biodistribution study in mice bearing S180 tumors demonstrated that [18F]5 had a rapid and
prolonged accumulation in tumors with moderate washout from other tissues.
Key words: Pyrazolo[1,5-a]pyrimidine Derivative, 7-(2-Chlorophenylamino)-5-((2-
[
18F]fluoroethyoxy)methyl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile, Radiotracer,
Tumor Detection, Positron Emission Tomography
Introduction
longer half-life (110 min), comparable size to H atoms
and lower energy), several new agents radiolabeled
With the increasing availability of PET (positron with 18F that are under investigation or have found
emission tomography) to provide quantitative kinetic clinical use in cancer management [6].
information of metabolic pathways and physiological
Pyrazolo[1,5-a]pyrimidine derivatives display ex-
processes in vivo [1], PET and in particular an in- tensive pharmaceutical activity due to the structural
tegrated PET/CT technique can detect diseases ear- similarity of the pyrazolo[1,5-a]pyrimidine ring sys-
lier and more accurately than any other imaging tech- tem to purine, and the latter exerts antimetabolic ac-
nique. A PET/CT is an effective tool to help to stage tivity in biochemical reactions [7]. Furthermore, the
local, regional, distant diseases and to provide ther- pyrazolo[1,5-a]pyrimidine core structure has several
apeutic guidance for cancer therapy. 2-[18F]Fluoro- positions for introducing a substituent, the C-5 and
2-deoxyglucose (18F-FDG), an analog of glucose, is the C-7 position have commonly been used for intro-
the most widely used compound for cancer detec- ducing substituents in anti-tumor drug design [8 – 13].
tion [2, 3]. In spite of being a very effective PET tracer Recently, a series of novel 3-cyano-5,7-disubstituted
for many types of tumors, with 18F-FDG it is often dif- pyrazolo[1,5-a]pyrimidine derivatives with various C-
ficult to differentiate tumor from inflammatory cells, 7 aniline substituents and C-5 amino alkoxy moieties
especially for brain tumors because of the high back- have been synthesized and showed that pyrazolo[1,5-
ground in the brain, and its low uptake in tumors a]pyrimidine analogs of purine have strong anti-cancer
that are growing slowly can cause false-negative re- activity [9].
sults [4, 5]. Considering the drawbacks of using FDG
We set out to explore a radiolabeled analog of
for tumor imaging and the advantages of fluorine-18 pyrazolo[1,5-a]pyrimidine for use as a PET tumor
as an ideal positron-emitting radionuclide (relatively imaging agent. In this study, a novel 18F-labeled
c
ꢀ 2012 Verlag der Zeitschrift fu¨r Naturforschung, Tu¨bingen · http://znaturforsch.com
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J. Xu et al. · 7-(2-Chlorophenylamino)-5-((2-[18F]fluoro-ethyoxy)methyl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile
Results and Discussion
Chemistry
Fig. 1. Structure of the
18F-labeled pyrazolo[1,5-
a]pyrimidine derivative.
Scheme 1 shows the synthetic procedures for 5 and
its 18F-labeled analog ([18F]5). The synthesis route to
1 followed a procedure described previously [9]. Sub-
stitution of 7-Cl in 1 with 2-chloroaniline produced
pyrazolo[1,5-a]pyrimidine derivative, [18F]5 (Fig. 1), 2. Treatment of 2 with ethylene glycol in DMF so-
has successively been synthesized and tested in Kun- lution using NaH as a base generated 3. Direct inter-
ming Mice bearing S180 tumors. Afterwards, we com- action between 3 and toluene sulfonyl chloride pro-
pared the biodistribution data of [18F]5 with data ob- vided the precursor 4. 19F substitution to obtain 5
tained using the previously described [18F]FDG and was achieved from precursor 4 using TBAF·3H2O in
O-2-[18F]fluoroethyl-L-tyrosine (L-[18F]FET) [14].
dry DMF at 100 ◦C under nitrogen atmosphere for
Scheme 1. The synthetic route to 5 and [18F]5.
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J. Xu et al. · 7-(2-Chlorophenylamino)-5-((2-[18F]fluoro-ethyoxy)methyl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile
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Fig. 2. HPLC profiles of 5 (A) and [18F]5 (B). HPLC conditions: Alltech system; a Grace Alltima C-18 column (250 × 10 mm
i. d.), CH3CN-H2O = 70 : 30 (v/v), 3 mL min−1, 245 nm, tR = 4.70 min (UV), 4.79 min (γ). The slight difference in retention
time between the UV peaks is due to the configuration of the detector system.
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J. Xu et al. · 7-(2-Chlorophenylamino)-5-((2-[18F]fluoro-ethyoxy)methyl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile
40 min with 30% yield. The structure of 5 was char- in the same animal model. The biodistribution data of
1
acterized by H NMR, 13C NMR, ESI-MS, and IR [18F]FDG and L-[18F]FET were taken from our previ-
spectroscopy and elemental analysis. The radiosynthe- ous report [14].
sis of [18F]5 was performed through the correspond-
As shown in Table 1, after a fast distribution, [18F]5
ing tosylate precursor 4 with no-carrier-added potas- was excreted from the body mainly through the liver
sium [18F]fluoride and Kryptofix K2.2.2 in anhydrous and kidney. The radioactivity of [18F]5 in the tu-
DMF at 100 ◦C. [18F]Fluoride incorporation by nu- mor was 2.18 ± 1.02, 3.66 ± 1.41, 2.83 ± 0.18, and
cleophilic displacement of the tosylate leaving group 2.12 ± 0.53% ID/g at 5, 15, 30, and 60 min post-
in 4 gave [18F]5 in a moderate overall radiochem- injection (pi), respectively, showing a remarkably in-
ical yield of about 25% (decay-uncorrected) within creasing uptake of this compound in the tumor. [18F]5
60 min including HPLC separation. The desired prod- reached its peak of 3.66 ± 1.41% ID/g at 15 min pi,
uct [18F]5 was collected by HPLC, and the solvent and then decreased to 2.12 ± 0.53% ID/g after 60 min.
was evaporated using a rotary evaporator. The prod- Nearly 60% radioactivity was still retained in the tu-
uct was dissolved in phosphate-buffered saline solution mor after 60 min indicating good persistence in tu-
(pH = 7.4). The radiochemical purity exceeded 98%. mor uptake. Meanwhile, the radioactivity of [18F]5 in
The structure of [18F]5 was confirmed according to the other organs and tissues decreased rapidly. The tumor
retention of [18F]5 (tR = 4.79 min) which was in good to brain, tumor to muscle and tumor to blood ratios
agreement with that of its corresponding F-19 substi- reached high levels of 2.23, 1.14 and 1.30 at 15 min pi,
tuted 5 (tR = 4.70 min), as shown in Fig. 2.
respectively.
As shown in Table 2, [18F]FDG washed out swiftly
from the blood resulting in high tumor to blood ratios:
11.38 at 30 min pi and 19.47 at 60 min pi. On the other
hand, the high uptake in the brain resulted in low tumor
to brain ratios: 0.61 at 30 min pi and 1.02 at 60 min pi.
Taking this drawback into account, [18F]FDG is lim-
ited in the application as a PET imaging agent of brain
tumors [4]. On the contrary, the tumor to blood ratios
of L-[18F]FET were much lower, and the tumor to brain
ratios were relatively higher: 2.54 at 30 min pi and 2.93
at 60 min pi. Thus, L-[18F]FET is suited as a PET brain
tumor imaging agent [14 – 17].
Measurement of the partition coefficient
The partition coefficient (logP) value of [18F]5 was
0.92, suggesting that this radiotracer is lipophilic.
Stability of [18F]5 in vitro
The radio-HPLC showed that the radiotracer is sta-
ble in both phosphate-buffered saline (pH = 7.4) and
murine plasma at 37 ◦C after 1 h.
Biodistribution of [18F]5 in S180-bearing mice
Compared with [18F]FDG and L-[18F]FET, [18F]5
The biodistribution data of [18F]5 is summarized in exhibited a higher absolute radioactivity uptake in tu-
Table 1. Table 2 shows the comparison of tumor up- mors than [18F]FDG before 30 min pi and L-[18F]FET
take (% ID/g) and the ratios of tumor to brain, mus- before 15 min pi. At 60 min pi all three radiotracers had
cle and blood among [18F]5, [18F]FDGand L-[18F]FET a similar absolute radioactivity uptake in the tumor.
Table 1. Biodistribution of
18F]5 in mice bearing an
Time (min)
Organs
[
5
15
30
60
S 180 tumor, expressed as
percent of injected dose
per gram (% ID/g ± SD),
n = 4.
Heart
Liver
Spleen
Lung
Kidney
Brain
Muscle
Blood
4.79 ± 1.26
13.50 ± 4.63
4.46 ± 2.14
3.77 ± 0.75
6.61 ± 1.50
2.22 ± 0.30
4.88 ± 1.27
3.17 ± 1.21
2.18 ± 1.02
0.98
4.24 ± 1.11
12.20 ± 2.38
3.38 ± 1.15
3.07 ± 0.49
4.74 ± 0.93
1.64 ± 0.50
3.22 ± 1.06
2.81 ± 0.67
3.66 ± 1.41
2.23
3.36 ± 0.47
9.14 ± 2.01
2.63 ± 0.07
2.99 ± 0.59
2.93 ± 0.46
1.47 ± 0.01
2.56 ± 0.55
2.50 ± 0.25
2.83 ± 0.18
1.93
2.68 ± 0.19
3.37 ± 0.23
1.45 ± 0.29
1.77 ± 0.39
1.90 ± 0.08
1.20 ± 0.18
2.47 ± 0.34
1.84 ± 0.39
2.12 ± 0.53
1.77
Tumor
Tumor/Brain
Tumor/Muscle
Tumor/Blood
0.45
0.69
1.14
1.30
1.10
1.13
0.86
1.15
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J. Xu et al. · 7-(2-Chlorophenylamino)-5-((2-[18F]fluoro-ethyoxy)methyl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile
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Table 2.
comparison of
Biodistribution
Time (min)
18F]5,
Organs
Compounds
18F]5
[
5
15
30
60
[
18F]FDG and L-[18F]FET
[
2.18 ± 1.02
1.27 ± 0.54
2.08 ± 0.49
0.98
3.66 ± 1.41
1.56 ± 0.23
2.50 ± 0.23
2.23
2.83 ± 0.18
1.86 ± 0.18
3.28 ± 0.69
1.93
2.12 ± 0.53
2.16 ± 0.34
2.75 ± 0.36
1.77
1.02
2.93
0.86
2.23
1.23
1.15
19.47
1.10
in mice bearing an S 180
tumor, expressed as percent
of injected dose per gram
(% ID/g ± SD), n = 4.
[
18F]FDG
L-[18F]FET
18F]5
18F]FDG
L-[18F]FET
18F]5
18F]FDG
L-[18F]FET
18F]5
18F]FDG
L-[18F]FET
Tumor
[
[
0.53
2.10
0.45
0.81
0.72
0.69
1.17
0.31
0.59
2.43
1.14
1.37
0.75
1.30
3.42
0.76
0.61
2.54
1.10
1.75
0.62
1.13
11.38
1.02
Tumor/Brain
Tumor/Muscle
Tumor/Blood
[
[
[
[
The tumor to brain ratios of [18F]5 were much higher determined on an RY-1capillary tube apparatus without
correction. 1H NMR spectra were recorded on a Bruker
(400 MHz) spectrometer. 13C NMR and 19F NMR spectra
were recorded on a Bruker (100 MHz) spectrometer. Tetra-
methylsilane (TMS) was used as an internal standard. Chem-
ical shift data of the proton resonances (δ) were reported
in parts per million relative to the internal standard TMS
(δ = 0.0). Mass spectra were obtained using a Bruker Apex
IV FTM instrument. Elemental analysis was performed with
a Perkin-Elemer 240-C analyzer. IR data were obtained on
a Nicolet 360 Avatar infrared spectrophotometer.
than those of [18F]FDG at all the selected times and
were close to those of L-[18F]FET between 15 min and
60 min pi. As shown in Table 2, the tumor uptake and
the tumor to brain ratio of [18F]5 reached their peak
at 15 min pi. Meanwhile, the ratio of tumor to muscle
also reached its peak (1.14), 1.5 times the uptake of L-
[18F]FET (0.75) and close to that of [18F]FDG (1.37);
the ratio of tumor to blood also reached its peak (1.3),
1.7 times the uptake of L-[18F]FET (0.76). Taken to-
gether, [18F]5 had the capability to accumulate in the
tumor and could enhance the sensitivity in tumor imag-
ing. As a radioactivity tracer, [18F]5 may be useful for
PET imaging of both central and peripheral tumors as
well as of metastases of tumors.
The [18F]fluoride used for radiosynthesis was produced
by the 18O(p, n)18F nuclear reaction at the Center of Xuanwu
Hospital (Beijing, China). Semipreparative HPLC purifica-
tion was carried out using a semipreparative reversed-phase
Grace AlltimaTM C18 column (250 × 10 mm, particle size:
10 µm) under the indicated conditions. The semipreparative
HPLC system used was an Alltech system with an Alltech
HPLC pump Model 626, a Linear UVIS-201, and Bioscan
flow-counter. Reversed-phase extraction Sep-Pak C18 Plus
cartridges (Waters) were activated with methanol and water
before use.
Conclusion
The new 18F-labeled pyrazolo[1,5-a]pyrimidine
derivative [18F]5 was synthesized by nucleophilic sub-
stitution employing tosylate as leaving group and then
purified by using reverse phase HPLC. The radiochem-
ical purity of the product was higher than 98% with
radiochemical yields of 25% (without decay correc-
tion). The biodistribution was studied in S180 tumor-
bearing mice for comparison of [18F]5 with [18F]FDG
and L-[18F]FET. Compound [18F]5 showed a higher
accumulation in the S180 tumor than [18F]FDG and
L-[18F]FET with promising tumor to muscle and tu-
mor to blood ratios, which indicates that further re-
search is needed to develop [18F]5 and pyrazolo[1,5-
a]pyrimidine derivatives for potential application in
PET tumor imaging.
Synthesis of 5-(chloromethyl)-7-
(2-chlorophenylamino)pyrazolo[1,5-a]pyrimidine-3-
carbonitrile (2)
7-Chloro-5-(chloromethyl)pyrazolo[1,5-a]pyrimidine-3-
carbonitrile (1), shown in Scheme 1, was synthesized as de-
scribed previously [9]. A solution of 1 (7.9 g, 35 mmol) in
2-propanol (5 mL) was mixed with 2-chloroaniline (4.7 mL,
45 mmol) and stirred at 60 ◦C for 3 h. After being cooled
to room temperature, the precipitate was filtered, washed
thoroughly with isopropanol, and then crystallized from
methanol to produce product 2 (9.3 g, 84% yield) as an
orange solid. M. p. 178 – 179 ◦C. – 1H NMR (CDCl3,
500 MHz): δ = 8.38 (s, 1H, -NHAr), 8.35 (s, 1H, pyrazole-
H), 7.64 – 7.60 (m, 2H, Ar-H), 7.50 – 7.47 (m, 1H, Ar-H),
Experimental Section
All commercial reagents and solvents were used with- 7.39 – 7.36 (m, 1H, Ar-H), 6.72 (s, 1H, pyrimidine-H), 4.66
out further purification unless specified. Melting points were (s, 2H, -OCH2 pyrimidine).
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J. Xu et al. · 7-(2-Chlorophenylamino)-5-((2-[18F]fluoro-ethyoxy)methyl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile
Synthesis of 7-(2-chlorophenylamino)-5-(2-
hydroxyethoxymethyl)pyrazolo[1,5-a]pyrimidine-3-
carbonitrile (3)
-CH3). – 13C NMR (CDCl3, 100 MHz): δ = 163.59 (C-7),
150.15 (C-5), 146.68 (C-2), 145.04 (C-3a), 144.96, 132.97,
132.93, 132.68, 130.68, 129.85, 128.54, 128.12, 127.82,
124.17 (Ar), 113.19 (CN), 87.10 (C-6), 81.60 (OCH2), 73.35
(OCH2CH2), 68.80 (C-3), 68.66 (OCH2CH2), 21.60 (CH3).
– MS (EI, 70 eV): m/z (%) = 498 (100) [M+H]+, 500 (50),
501 (10), 326 (5). – C23H20ClN5O4S (497) calcd. C 55.48,
H 4.05, N 14.06; found C 55.53, H 3.90, N 14.22.
To a solution of ethane-1,2-diol (0.24 mL, 4 mmol) in an-
hydrous DMF (5 mL) was added NaH (96 mg, 4 mmol). The
suspension was stirred at ambient temperature for 10 min,
and then 2 (32 mg, 1 mmol) was added. The mixture was
heated at 50 ◦C for another 4 h, then cooled to room temper-
ature and quenched by addition of H2O (5 mL). The solid
was removed by filtration, and a saturated NaHCO3 solu-
tion was added until no more colorless solid was formed.
The colorless solid was collected by filtration and sub-
jected to column chromatography on silica gel eluting with
1 : 2 ethyl acetate-petroleum ether (60 – 90 ◦C) to afford
product 3 (18.2 mg, 53% yield) as a colorless solid. M. p.
157 – 159 ◦C. – IR (KBr): ν = 3513, 3350, 2891, 2237
(CN), 1629, 1593, 1571, 1435, 1138, 1085 cm−1. – 1H
NMR (CDCl3, 500 MHz): δ = 8.36 (s, 1H, pyrazole-H),
8.35 (s, 1H, -NHAr), 7.63 – 7.60 (m, 2H, Ar-H), 7.48 – 7.44
(m, 1H, Ar-H), 7.36 – 7.33 (m, 1H, Ar-H), 6.74 (s, 1H,
pyrimidine-H), 4.72 (s, 2H, –OCH2 pyrimidine), 3.84 – 3.81
(m, 2H, HOCH2CH2-), 3.74 – 3.72 (m, 2H, HOCH2CH2-),
2.18 – 2.15 (m, 1H, HOCH2CH2-). – 13C NMR (CDCl3,
125 MHz): δ = 164.08 (C-7), 150.30 (C-5), 146.80 (C-2),
145.19 (C-3a), 132.67, 130.87, 128.76, 128.17, 128.09,
124.62 (Ar), 113.23 (CN), 86.99 (C-6), 81. 67 (OCH2), 73.21
(OCH2CH2), 72.65 (C-3), 61.80 (OCH2CH2). – MS (EI,
70 eV): m/z (%) = 343 (5) [M]+, 283 (100), 164 (60), 110
(50), 74 (10). – C16H14ClN5O2 (343.1): calcd. C 55.90, H
4.10, N 20.37; found C 56.03, H 4.19, N 20.22.
Synthesis of 7-(2-chlorophenylamino)-5-((2-
fluoroethyoxy)methyl)pyrazolo[1,5-a]pyrimidine-3-
carbonitrile (5)
Tetrabutylammonium fluoride trihydrate (TBAF.3H2O,
0.63 g, 2 mmol) was dissolved in anhydrous CH3CN (1 mL)
and heated rapidly at 120 ◦C under a nitrogen stream, fol-
lowed by additional CH3CN (2 mL). The solvent was evap-
orated, and this process was repeated three times. After
evaporating most of the solvent, a solution of 4 (0.50 g,
1 mmol) in anhydrous DMF (3 mL) was added to the dry
reagent. The reaction mixture was stirred at 100 ◦C for
40 min under a nitrogen stream and cooled to room tem-
perature, then diluted with H2O (20 mL) and extracted with
ethyl acetate (10 mL × 3). The organic layer was dried over
Na2SO4 and concentrated in a vacuum to obtain a yellow
oil. The residue was purified by silica gel chromatography
using petroleum ether (60 – 90 ◦C)-ethyl acetate (6 : 1) as
eluant leading to product 5 (0.10 g, 30% yield) as a color-
less solid. M. p. 171 – 172 ◦C. – IR (KBr): ν = 3444, 3367,
2925, 2231 (CN), 1625, 1599, 1577, 1475, 1311, 1142,
1055 cm−1. – 1H NMR (CDCl3, 400 MHz): δ = 8.34 (s,
1H, pyrazole-H), 7.62 – 7.29 (m, 4H, Ar-H), 6.84 (s, 1H,
pyrimidine-H), 4.71 (s, 2H, -OCH2CH2OCH2-), 4.66 – 4.54
(dm, 2H, J = 47.6 Hz, -CH2CH2F), 3.87 – 3.80 (dm, 2H,
J = 30.0 Hz, -CH2CH2F). – 13C NMR (CDCl3, 100 MHz):
δ = 164.11 (C-7), 150.32 (C-5), 146.76 (C-2), 145.20 (C-3a),
132.77, 130.77, 128.49, 128.17, 127.88, 124.39 (Ar), 113.22
(CN), 87.02 (C-6), 82.68 (d, JC-F = 169.0 Hz, FCH2), 81.64
(OCH2), 73.41 (C-3), 42.57 (d, JC-F = 20.0 Hz, OCH2CH2).
Synthesis of 2-((7-(2-chlorophenylamino)-3-
cyanopyrazolo[1,5-a]pyrimidin-5-yl)methoxy)-ethyl-4-
methylbenzenesulfonate (4)
To a stirred solution of 3 (0.34 mg, 1 mmol) in CH2Cl2
(20 mL) cooled with an ice bath (0 ◦C) was added
Et3N (0.21 mL, 1.5 mmol), p-toluenesulfonyl chloride
(TsCl, 0.29 g, 1.5 mmol) and a catalytic amount of 4-
dimethylaminopyridine (DMAP, 0.03 g, 0.2 mmol). The
reaction was continued at room temperature overnight after
30 min of incubation on ice. 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 (5 : 1) as eluant to give
product 4 (0.32 g, 64% yield) as a colorless solid. M. p.
129 – 131 ◦C. – IR (KBr): ν = 3443, 3310, 2944, 2224 (CN),
1625, 1594, 1573, 1434, 1358, 1191, 1174, 1137, 1026, 925,
–
19F NMR (CDCl3, 100 MHz): δ = –223.10. – MS (EI,
70 eV): m/z (%) = 346 (100) [M+H]+, 348 (50), 350 (17).
– C16H13ClFN5O (345) calcd. C 55.58, H 3.79, N 20.26;
found C 55.53, H 3.93, N 20.32.
Synthesis of 7-(2-chlorophenylamino)-5-
((2-[18F]fluoroethyoxy)methyl)-pyrazolo[1,5-a]pyrimidine-
3-carbonitrile ([18F]5)
[
18F]F− was eluted from the QMA cartridge with 1.3 mL
775, 751 cm−1. – 1H NMR (CDCl3, 400 MHz): δ = 8.32 of a Kryptofix 2.2.2 (K222)/K2CO3 solution (11 mg of
(s, 1H, -NH Ar), 8.26 (s, 1H, pyrazole-H), 7.68 – 7.20 K222 and 3.5 mg of K2CO3 in CH3CN-H2O 1 mL : 0.3 mL).
(m, 8H, Ar-H), 6.74 (s, 1H, pyrimidine-H), 4.52 (s, 2H, The solvent was removed at 120 ◦C under a nitrogen stream.
-OCH2CH2OCH2-), 4.14 – 4.12 (m, 2H, -OCH2CH2OCH2-), The residue was azeotropically dried with 1 mL of anhydrous
3.71 – 3.69 (m, 2H, -OCH2CH2OCH2-), 2.35 (s, 3H, CH3CN at 120 ◦C under a nitrogen stream. A solution of
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J. Xu et al. · 7-(2-Chlorophenylamino)-5-((2-[18F]fluoro-ethyoxy)methyl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile
833
the tosylate precursor 4 (5 mg) in anhydrous DMF (1 mL) acetonitrile and removed by centrifugation. The supernatant
was added. The mixture was sealed and heated at 100 ◦C part was injected into radio-HPLC to determine the stability
for 20 min and subsequently cooled down. After addition of of the compound.
10 mL of water the mixture was passed through a Sep-Pak
Plus C18 cartridge to remove the Kryptofix 2.2.2 and un-
Biodistribution studies in S180 tumor-bearing mice
reacted potassium [18F]fluoride. The Sep-Pak Plus C18 car-
All the animal studies were carried out in compliance with
tridge was washed with water (10 mL × 2), and the labeled
relevant national laws relating to the conduct of animal ex-
compound was eluted with 2 mL of CH3CN. The eluted com-
perimentation. Uptake of [18F]5 in Kunming Mice bearing
pound was purified by semi-preparative HPLC (Grace C18
S180 tumor models were performed according to published
column, 250 × 10 mm i. d.); eluant: CH3CN-H2O = 70 : 30
methods [14].Briefly, S180 tumor cells were trypsinized,
(v/v); flow rate: 3 mL min−1; tR = 4.79 min.
counted, and suspended in a solution containing DMEM and
10% fetal bovine serum. The Kunming Mice were inocu-
Octanol/water partition coefficient
lated subcutaneously into the left forelimb with 5 × 106 tu-
The partition coefficient was measured as previously de- mor cells. Studies of tumor uptake were conducted when the
scribed [14]. Each HPLC-purified radiotracer was added tumors reached a size of 0.5 – 0.8 cm in diameter. The tumor-
to a tube containing 0.5 mL of n-octanol and 0.5 mL of bearing mice were subjected to in vivo biodistribution and
phosphate-buffered saline (pH = 7.4). Each phase had been imaging studies.
presaturated with the opposite phase. The mixture was vor-
Tissue biodistribution studies were conducted by admin-
texed for 1 min and centrifuged at 15 000 rpm for 2 min. istering, via the tail vein, a bolus injection of 370 kBq per
Aliquots of both phases were transferred in triplicate to mouse in a constant volume of 0.1 mL phosphate-buffered
counting tubes and assayed in a γ counter. The partition co- saline solution (pH = 7.4). The animals were sacrificed at 5,
efficient was calculated by dividing the radioactivity of the 15, 30 and 60 min post injection. The tissues and organs of
octanol layer by that of the water layer. This measurement interest were immediately collected, weighed and measured
was repeated three times.
for 18F radioactivity in a γ counter. Values are expressed as
mean ± SD (n = 4).
Stability study
Acknowledgement
Each HPLC-purified radiotracer (3.7 MBq) was mixed
with phosphate-buffered saline (pH = 7.4) at 37 ◦C for a pe-
This project was sponsored by the National Natural Sci-
riod of up to 1 h and was analyzed by radio-HPLC. The sta- ence foundation of China (no. 21071022) and the Fundamen-
bility of each HPLC-purified radiotracer in mouse plasma tal Research Funds for the Central Universities. We also wish
was determined by incubating 0.1 mL of the radiotracer to thank the cyclotron operator team of the PET Centre of
(3.7 MBq) in the solution of 0.5 mL murine plasma at 37 ◦C Xuanwu Hospital for providing the fluoride-18 nuclide and
for 1 h and 2 h. Plasma proteins were precipitated by adding technical assistance.
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