Synthesis and biological evaluation of pyrazolo[1,5-a]-pyrimidine- containing 99mTc nitrido radiopharmaceuticals as imaging agents for tumors

Article abstract of DOI:10.3390/molecules15128723

The compound 5-((2-aminoethylamino)methyl)-7-(4-bromoanilino)-3- cyanopyrazolo[ 1,5-a]pyrimidine (ABCPP) was synthesized and conjugated with N-mercaptoacetylglycine (MAG), N-mercaptoacetylphenylalanine (MAF) and N-mercaptoacetylvaline (MAA), respectively. These three compounds were labeled successfully with [^99mTcN]²⁺ intermediate in high radiochemical purities. Biodistribution in tumor-bearing mice demonstrated that the three new complexes showed tumor accumulation, high tumor-tomuscle (T/M) ratios and fast clearance from blood and muscle. Among them, the ^99mTcNMAG-ABCPP showed the most favorable characteristics, with tumor/blood and tumor/muscle ratios reaching 1.51 and 2.97 at 30 min post-injection, 1.84 and 2.49 at 60 min post-injection, suggesting it could be further studied as potential tumor imaging agent for single photon emission computed tomography (SPECT).

Full text of DOI:10.3390/molecules15128723

Molecules 2010, 15, 8723-8733; doi:10.3390/molecules15128723
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Evaluation of
Pyrazolo[1,5-a]-pyrimidine-containing 99mTc Nitrido
Radiopharmaceuticals as Imaging Agents for Tumors
Rui Ding, Yong He, Jingli Xu, Hang Liu, Xiao Wang, Man Feng, Chuanmin Qi * and
Junbo Zhang
Key Laboratory of Radiopharmaceuticals, College of Chemistry, Beijing Normal University, Beijing
100875, China; E-Mails: dingrui@mail.bnu.edu.cn (R.D.); heyong@mail.bnu.edu.cn (Y.H.);
xujingli@mail.bnu.edu.cn (J.X.); liuhang@mail.bnu.edu.cn (H.L.);
wangxiao@mail.bnu.edu.cn (X.W.); fengman@mail.bnu.edu.cn (M.F.); zhjunbo@bnu.edu.cn(J.Z.).
* Author to whom correspondence should be addressed; E-Mail: qicmin@sohu.com;
Tel.: +86-136-8138-0097; Fax: +86-010-5880-2075.
Received: 1 November 2010; in revised form: 15 November 2010 / Accepted: 24 November 2010 /
Published: 30 November 2010
Abstract: The compound 5-((2-aminoethylamino)methyl)-7-(4-bromoanilino)-3-cyano-
pyrazolo[1,5-a]pyrimidine (ABCPP) was synthesized and conjugated with N-mercapto-
acetylglycine (MAG), N-mercaptoacetylphenylalanine (MAF) and N-mercaptoacetylvaline
(MAA), respectively. These three compounds were labeled successfully with [99mTcN]2+
intermediate in high radiochemical purities. Biodistribution in tumor-bearing mice
demonstrated that the three new complexes showed tumor accumulation, high tumor-to-
muscle (T/M) ratios and fast clearance from blood and muscle. Among them, the 99mTcN-
MAG-ABCPP showed the most favorable characteristics, with tumor/blood and tumor/
muscle ratios reaching 1.51 and 2.97 at 30 min post-injection, 1.84 and 2.49 at 60 min
post-injection, suggesting it could be further studied as potential tumor imaging agent for
single photon emission computed tomography (SPECT).
Keywords: pyrazolo[1,5-a]pyrimidine; tumor imaging; [99mTcN]2+ complex

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1. Introduction
Tumors are among the most common causes of death in the World. Recently, the use of
pyrazolo[1,5-a]pyrimidine derivatives as antitumor agents for the treatment of cancer has come under
much attention in a growing number of therapeutic areas [1-6]. Among them, 3-cyano-5,7-
disubstistituted pyrazolo[1,5-a]pyrimidine derivatives showed significant antitumor properties [7,8].
The novel nitrido core [99mTcN]2+ is isoelectronic with the [99mTcO]3+ core. The nitrido ligand is a
powerful -electron donor and shows a high capacity to stabilize the Tc(V) oxidation state [9]. The
[
99mTcN]2+ core exhibits a very high chemical stability over a wide range of experimental conditions
(such as oxidation-reduction reactions and pH variations), and high affinity toward chelating ligands
containing sulfur atoms. The presence of the [99mTcN]2+ core in the molecular structure of a
radiopharmaceutical may dramatically affect its physical and biological behaviour [10]. A convenient
preparation of 99mTc nitrido complexes at the tracer level and in sterile and pyrogen-free conditions in
which an intermediate 99mTcN complex of succinic dihydrazide (SDH) is initially formed in the
presence of stannous chloride as reducing agent, has been extensively investigated [11]. So far, the use
of the 3-cyano-5,7-disubstistituted pyrazolo[1,5-a]pyrimidine moiety in the preparation of 99mTcN
complexes as targeted agents for tumor imaging has not been reported. Therefore, it may be of great
interest to probe the 99mTc nitrido chemistry with some ligands involving the ABCPP molecular
structure to explore the biological behaviour of a potential new class of diagnostic agents.
The purpose of this study was to conjugate ABCPP with three chelating agents (MAG, MAF and
MAA), and to evaluate the feasibility of the 99mTc-labeled 3-cyanopyrazolo[1,5-a]pyrimidine
derivatives as useful candidates for tumor imaging.
2. Results and Discussion
2.1. Chemistry
The preparation of N-[(Tritylmercapto)acetyl]glycine (Tr-MAG, 4a) was carried out using the
procedure shown in Scheme 1. After protecting the thiol group with trityl chloride, the resulting
compound 2 was reacted with N-hydroxysuccinimide (NHS) using dicyclohexylcarbodiimide (DCC)
as condensation reagent to obtain the active ester 3. The active ester 3 was reacted with the amine
group of glycine to provide the Tr-MAG (4a). N-[(Tritylmercapto)acetyl]phenylalanine (Tr-MAF, 4b)
and N-[(tritylmercapto)acetyl]valine (Tr-MAA, 4c) were synthesized by the same procedure as 4a.
Scheme 1. Synthesis of Tr-MAG, Tr-MAF and Tr-MAA.

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The synthesis of 5-((2-aminoethylamino)methyl)-7-(4-bromoanilino)-3-cyano-pyrazolo[1,5-a]-
pyrimidine (ABCPP) was performed according to the procedure outlined in Scheme 2. Malononitrile
(5) was reacted with triethyl orthoformate in acetic anhydride to give the intermediate 6. Treatment
with hydrazine generated compound 7. Cyclization of 7 with ethyl chloroacetoacetate gave the
intermediate 8. Chlorination of 8 with phosphoryl trichloride in the presence of anhydrous pyridine
gave the key intermediate 9 [12,13]. Substitution of 7-Cl in 9 with 4-bromoaniline provided the
product 10. Treatment of 10 with ethylenediamine in ethanol generated the desired compound 11.
Scheme 2. Synthesis of ABCPP.
The compound Tr-MAG-ABCPP was synthesized by conjugating Tr-MAG with ABCPP using 1-
hydroxybenzotriazole (HOBt) as nucleophilic catalyst and DCC as condensation agent. The reaction is
shown schematically in Scheme 3. The thiol group was deprotected in trifluoroacetic acid (TFA) to
give 13a. For labeling, 99mTcN-MAG-ABCPP was prepared through a SDH kit. [99mTcO4]- was reacted
with SDH in the presence of stannous chloride as reducing agent to form a technetium-99m nitrido
intermediate. The [99mTcN]2+ is a suitable substrate for the substitution reaction with MAG-ABCPP at
100 ºC for 15 min to give the final complex 99mTcN-MAG-ABCPP (14a). 99mTcN-MAF-ABCPP (14b)
and 99mTcN-MAA-ABCPP (14c) were prepared using the same method.
Scheme 3. Synthesis of 99mTcN-MAG-ABCPP, 99mTcN-MAF-ABCPP and 99mTcN-MAA-ABCPP.
The radiochemical purity of the complexes was routinely checked by HPLC. The HPLC pattern of
99mTcN-MAG-ABCPP, 99mTcN-MAF-ABCPP and 99mTcN-MAA-ABCPP are shown in Figure 1. It
was observed that the retention time of [99mTcN]2+ was 1.5 min, while those of 99mTcN-MAG-
int
ABCPP, 99mTcN-MAF-ABCPP and 99mTcN-MAA-ABCPP were found to be 2.7 min, 3.6 min and

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3.4 min, respectively. The radiochemical purity of the three products was over 90% immediately after
the preparation.
Figure 1. Radio-HPLC chromatograms.
(A) [99mTcN]2+; (B) 99mTcN-MAG-ABCPP (14a); (C) 99mTcN-MAF-ABCPP (14b); (D) 99mTcN-MAA-ABCPP (14c).
2.2. Measurement of partition coefficients
The partition coefficients were determined by mixing the complex with an equal volume of 1-
octanol and phosphate buffer (0.025 mol/L, pH 7.4) in a centrifuge tube. The partition coefficients
(logP) of 99mTcN-MAG-ABCPP, 99mTcN-MAF-ABCPP and 99mTcN-MAA-ABCPP are shown in
Table 1. All of them were hydrophilic. 99mTcN-MAG-ABCPP was the most hydrophilic one and
99mTcN-MAA-ABCPP was more hydrophilic than 99mTcN-MAF-ABCPP.
Table 1. Partition coefficients of 99mTcN-MAG-ABCPP, 99mTcN-MAF-ABCPP and
99mTcN-MAA-ABCPP.
99mTcN-MAG-ABCPP 99mTcN-MAF-ABCPP 99mTcN-MAA-ABCPP
logP -1.54 ± 0.02
-1.18 ± 0.02
-1.35 ± 0.01
2.3. In vitro stability study
As the HPLC analysis results for the three complexes indicate, they were all stable in PBS after
incubation for 2 h.
2.4. Biodistribution study
The biodistribution results are summarized in Tables 2-4. There were significant similarities in the
biodistribution patterns of these three complexes, which all demonstrated tumor accumulation and high

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tumor-to-muscle (T/M) ratios. The blood and muscle clearance was faster than that of the tumor so that
the tumor/blood and tumor/muscle ratios increased with time. For 99mTcN-MAG-ABCPP, the
tumor/blood and tumor/muscle ratios reached 1.51 and 2.97 at 30 min post-injection, 1.84 and 2.49 at
60 min post-injection. For 99mTcN-MAF-ABCPP, the tumor/blood and tumor/muscle ratios reached
0.51 and 2.56 at 30 min post-injection. For 99mTcN-MAA-ABCPP, the tumor/blood and tumor/muscle
ratios reached 0.72 and 2.32 at 30 min post-injection. Early hepatic and renal activity reflected the fact
that the three complexes were excreted through the hepatobiliary as well as the renal system. The three
complexes were hydrophilic, so that they were unable to cross the blood brain barrier, thus making
their brain uptake much lower. Among them, 99mTcN-MAG-ABCPP showed the most favorable
characteristics with highest uptake at the tumor site and fast clearance from blood and muscle.
Table 2. Biodistribution of 99mTcN-MAG-ABCPP in mice bearing S 180 tumor (%ID/g)a.
Tissue
Heart
5 min
0.93 ± 0.11
30 min
0.32 ± 0.06
60 min
0.35 ± 0.02
120 min
0.37 ± 0.08
Liver
23.32 ± 1.32 23.88 ± 2.83 14.67 ± 0.87 12.84 ± 0.91
Spleen
Lung
Kidney
Bone
Muscle
Stomach
Large Intestine 1.62 ± 0.20
Small Intestine 8.82 ± 1.29
Blood
Brain
Tumor
T/M ratio
T/B ratio
19.90 ± 1.70 14.38 ± 1.64 8.16 ± 0.10
8.32 ± 1.05
0.73 ± 0.41
3.35 ± 0.29
0.86 ± 0.09
0.22 ± 0.03
0.09 ± 0.02
0.67 ± 0.06
0.62 ± 0.15
0.38 ±0.01
0.07 ± 0.01
0.45 ± 0.07
2.01
4.53 ± 0.99
11.08 ± 1.13 5.06 ± 1.16
1.25 ± 0.74
0.72 ± 0.05
0.69 ± 0.12
2.30 ± 0.60
0.86 ± 0.07
3.58 ± 0.21
0.73 ± 0.28
0.34 ± 0.07
0.32 ± 0.04
0.68 ± 0.02
0.64 ± 0.08
0.46 ± 0.04
0.95 ± 0.27
0.39 ± 0.08
0.54 ± 0.11
0.62 ± 0.08
3.42 ± 0.42
0.77 ± 0.01
0.04 ± 0.004 0.08 ± 0.01
1.16 ± 0.14
2.97
1.51
a
2.38 ± 0.17
0.13 ± 0.01
1.23 ± 0.13
1.71
0.86 ± 0.14
2.49
0.52
1.84
1.17
T/M = tumor-to-muscle, T/B = tumor-to-blood; All data are the mean percentage (n = 3) of the
injected dose of 99mTcN-MAG-ABCPP; per gram of tissue, ± the standard deviation of the mean.
Table 3. Biodistribution of 99mTcN-MAF-ABCPP in mice bearing S 180 tumor (%ID/g)b.
Tissue
Heart
5 min
0.82 ± 0.02
30 min
0.53 ± 0.09
120 min
0.35 ± 0.02
180 min
0.29 ± 0.04
Liver
20.12 ± 1.03 15.55 ± 1.33 14.46 ± 0.41 14.42 ± 0.89
Spleen
Lung
Kidney
Bone
Muscle
Stomach
Large Intestine 1.03 ± 0.10
Small Intestine 1.45 ± 0.08
Blood
Brain
Tumor
T/M ratio
T/B ratio
18.62 ± 1.11 16.39 ± 1.21 3.09 ± 0.41
4.25 ± 0.37
0.89 ± 0.08
2.05 ± 0.30
0.62 ± 0.07
0.17 ± 0.02
2.38 ± 0.05
0.50 ± 0.07
0.37 ± 0.06
0.69 ± 0.05
4.84 ± 0.56
10.25 ± 0.92 5.36 ± 0.38
1.02 ± 0.09
0.68 ± 0.02
5.12 ± 0.22
4.11 ± 0.21
1.18 ± 0.25
3.42 ± 0.22
0.62 ± 0.06
0.16 ± 0.06
2.62 ± 0.33
0.25 ± 0.03
0.31 ± 0.08
0.94 ± 0.07
0.79 ± 0.08
0.28 ± 0.04
4.65 ± 0.42
0.33 ± 0.01
1.39 ± 0.04
1.44 ± 0.05
0.06 ± 0.006 0.05 ± 0.007 0.06 ± 0.01
0.74 ± 0.08
2.56
0.51
b
2.45 ± 0.12
0.12 ± 0.01
0.89 ± 0.07
1.31
0.41 ± 0.01
2.63
0.45 ± 0.02
2.57
0.66
0.36
0.44
T/M = tumor-to-muscle, T/B = tumor-to-blood; All data are the mean percentage (n = 3) of the
injected dose of 99mTcN-MAF-ABCPP; per gram of tissue, ± the standard deviation of the mean.

Molecules 2010, 15
Table 4. Biodistribution of 99mTcN-MAA-ABCPP in mice bearing S 180 tumor (%ID/g)c.
8728
Tissue
Heart
5min
1.09 ± 0.13
30min
0.43 ± 0.08
60min
120min
0.32 ± 0.005 0.29 ± 0.02
Liver
24.76 ± 1.18 20.46 ± 2.20 22.47 ± 1.73 24.19 ± 3.07
Spleen
Lung
Kidney
Bone
Muscle
Stomach
Large Intestine 0.97 ± 0.16
Small Intestine 5.17 ± 0.39
Blood
Brain
Tumor
T/M ratio
T/B ratio
15.81 ± 1.73 6.87 ± 1.29
7.31 ± 0.58
2.30 ± 0.10
2.33 ± 0.11
0.57 ± 0.07
0.30 ± 0.08
3.43 ± 0.55
0.33 ± 0.02
1.79 ± 0.09
0.74 ± 0.02
0.05 ± 0.01
0.51 ± 0.03
1.72
7.85 ± 0.76
2.67 ± 0.34
2.08 ± 0.27
0.50 ± 0.03
0.17 ± 0.04
2.79 ± 0.41
0.43 ± 0.06
1.00 ± 0.14
0.67 ± 0.07
0.05 ± 0.008
0.42 ± 0.05
2.47
8.31 ± 0.59
5.58 ± 0.55
0.89 ± 0.08
0.63 ± 0.05
3.74 ± 0.51
2.94 ± 0.57
2.59 ± 0.24
1.11 ± 0.13
0.34 ± 0.07
4.53 ± 0.31
0.48 ± 0.08
2.92 ± 0.22
1.12 ± 0.11
0.07 ± 0.01
0.81 ± 0.05
2.32
2.85 ± 0.15
0.12 ± 0.01
0.92 ± 0.03
1.47
0.33
0.72
0.70
0.63
c
T/M = tumor-to-muscle, T/B = tumor-to-blood; All data are the mean percentage (n = 3) of the
injected dose of 99mTcN-MAA-ABCPP; per gram of tissue, ± the standard deviation of the mean.
3. Experimental
3.1. General
99Mo/99mTc generator was obtained from the China Institute of Atomic Energy (CIAE). All other
1
13
chemicals were of analytical grade and were used without further purification. H- and C-NMR
spectra were recorded in CDCl3 or DMSO-d6 on a Bruker spectrometer operating at 400 and 100 MHz,
respectively. The IR spectra were recorded on a Nicolet-AVATAR 360 FT-IR spectrometer using KBr
pellets in the 4,000-400 cm-1 region. ESI-MS were performed on Waters LCT Premier XE. HPLC
analyses were performed on a Shimadzu SCL-10 AVP equipped with a Packard 500 TR series flow
scintillation analyzer. A C-18 reversed-phase Alltima column (5um, 150 mm × 4.6 mm) was used for
radiochemical purity analysis.
3.2. Ligand synthesis
The synthesis of compounds 2 and 3 was carried out by the same procedure described in ref. [14].
The preparation of compounds 6 and 7 was carried out by the same method given in ref. [15] and the
preparation of compounds 8 and 9 was carried out by the same methods as in ref. [12] and ref. [13].
The preparation and the analysis data of the other compounds are shown below.
N-[(Tritylmercapto)acetyl]glycine (Tr-MAG) (4a). Compound 3 (0.30 g, 0.7 mmol) was dissolved in
acetonitrile (15 mL). Glycine (0.105 g, 1.4 mmol) in sodium hydroxide (0.2 M, 1 mL) was added
dropwise. The reaction solution was stirred for 2 h at 50-60 ºC. Distilled water (1 mL) was added to
the reaction mixture and the pH was adjusted to 1. The acetonitrile was removed in vacuo and a white
solid precipitated. The white residue was recrystallized from ethyl acetate to give compound 4a (0.22 g,
78%); m.p.: 164-166 ºC; IR (KBr, cm-1): ν 3434.4, 3344.0, 2960.3, 2926.5, 1732.4, 1619.0, 1526.7,
1
1487.9, 1445.3, 1247.6, 1224.1, 1210.7, 740.7, 703.9; H-NMR (CDCl3): δ 3.13 (2H, s), 3.62 (2H, d,

Molecules 2010, 15
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13
J = 5.1 Hz), 6.46 (1H, t, J = 4.9 Hz), 7.15(3H, m), 7.22 (6H, m), 7.36 (6H, d, J = 7.6 Hz); C-NMR
(CDCl3): δ 35.4, 41.6, 67.9, 127.1, 128.2, 129.4, 143.8, 169.0, 172.2.
N-[(Tritylmercapto)acetyl]phenylalanine (Tr-MAF) (4b). 4b was obtained with the same method of 4a
using phenylalanine instead of glycine (yield: 59%); m.p.: 128-130 ºC; IR (KBr, cm-1): ν 3448.2,
3340.0, 3026.9, 2928.0, 1727.1, 1626.7, 1528.3, 1492.8, 1443.4, 1222.9, 1203.4, 1183.6, 741.9, 700.4;
1H-NMR (CDCl3): δ 3.07 (4H, m), 4.48 (1H, dm, J = 6.2 Hz), 6.54 (1H, d, J = 6.2 Hz), 7.10 (2H, m),
13
7.25 (18H, m); C-NMR (CDCl3): δ 36.0, 37.0, 53.8, 68.1, 127.2, 127.3, 128.2, 128.7, 129.4, 129.5,
135.5, 143.9, 169.1, 173.6.
N-[(Tritylmercapto)acetyl]valine (Tr-MAA) (4c). 4c was obtained with the same method of 4a using
valine instead of glycine (yield: 65%); m.p.: 146-148 ºC; IR (KBr, cm-1): ν 3433.5, 3339.4, 3055.4,
2960.3, 2928.7, 1738.7, 1720.0, 1635.8, 1534.3, 1488.8, 1441.3, 1422.1, 1380.8, 1304.3, 1273.4,
1
1213.3, 1081.8, 1035.1, 1000.3, 884.9, 757.5, 738.1, 700.5; H-NMR (CDCl3): δ 0.88 (6H, d,
J = 6.8 Hz), 2.10 (1H, m), 3.13 (2H, s), 4.31 (1H, dd, J = 4.9, 3.2 Hz), 6.62 (1H, d, J = 8.2 Hz), 7.22
(3H, m), 7.28 (6H, m), 7.38 (6H, d, J = 7.6 Hz); 13C-NMR (CDCl3): δ 17.9, 18.8, 31.0, 36.2, 57.5, 68.2,
127.2, 128.2, 129.6, 144.0, 168.8, 175.4.
5-Chloromethyl-7-(4-bromoanilino)-3-cyanopyrazolo[1,5-a]pyrimidine (10). A mixture of 9 (0.8 g,
3.5 mmol), 4-bromoaniline (0.8 g, 4.6 mmol), and isopropanol (10 mL) was heated at 60 ºC for 2 h.
After cooling to room temperature, a solid mass precipitated. Recrystallization from methanol gave 10
as a light yellow powder (0.81 g, 66%); m.p.: 206-208 ºC; IR (KBr, cm-1): ν 3439.1, 3328.8, 3275.4,
3085.8, 2973.2, 2233.0, 1626.9, 1600.0, 1572.0, 1536.4, 1483.7, 1456.9, 1416.1, 1406.5, 1317.6,
1
1265.1, 1220.6, 1193.6, 1073.8, 1009.7, 829.9, 780.5, 744.6; H-NMR (DMSO-d6): δ 4.73 (2H, s),
6.67 (1H, s), 7.41 (2H, d, J = 8.7 Hz), 7.68 (2H, d, J = 8.7 Hz), 8.79 (1H, s), 10.69 (1H, s); 13C-NMR
(DMSO-d6): δ 45.8, 79.5, 89.4, 113.9, 119.0, 127.0, 132.5, 135.7, 146.6, 147.1, 150.6, 160.5.
5-((2-Aminoethylamino)methyl)-7-(4-bromoanilino)-3-cyanopyrazolo[1,5-a]pyrimidine (ABCPP) (11).
A mixture of 10 (0.725 g, 2 mmol), ethylenediamine (0.30 g, 5 mmol), and ethanol (15 mL) was
heated to reflux for 4 h. Solvent was removed in vacuo to afford the yellow crude product which was
recrystallized from methanol to obtain compound 11 (0.56 g, 75%); IR (KBr, cm-1): ν 3447.0, 2922.2,
2221.1, 1621.1, 1595.5, 1565.1, 1536.8, 1479.7, 1288.4, 1192.1, 1071.1, 1009.7; 1H-NMR (DMSO-d6):
δ 2.65 (2H, t, J = 5.9 Hz), 2.76 (2H, t, J = 5.9 Hz), 3.67 (2H, s), 6.33 (1H, s), 7.22 (2H, d, J = 8.6 Hz),
13
7.59 (2H, d, J = 8.6 Hz), 8.49 (1H, s); C-NMR (DMSO-d6): δ 46.5, 48.8, 53.9, 77.0, 88.0, 115.1,
115.6, 125.7, 132.0, 142.4, 145.3, 147.1, 151.9, 163.3; MS (ESI): M = 386.2 (Found: 386.3).
Conjugation of Tr-MAG and ABCPP (Tr-MAG-ABCPP) (12a). A mixture of 11 (386mg, 1mmol),
triethylamine (0.21 mL, 1.5 mmol) and anhydrous dichloromethane (8 mL) was stirred at room
temperature for 5 min. Then 4a (407 mg, 1 mmol) and HOBt (150 mg, 1.1 mmol) were added. DCC
(230 mg, 1.1 mmol) in 4 mL of anhydrous dichloromethane was added dropwise to the solution at 0 ºC.
The mixture was stirred for 30 min at 0 ºC and then overnight at room temperature. White precipitate

Molecules 2010, 15
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was removed by filtration. The organic phase was washed with saturated aqueous sodium bicarbonate,
brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified
by column chromatography (silica gel; petroleum ether-ethyl acetate = 1:5) to afford the desired
compound 12a (220 mg, 29%); m.p.: 185-187 ºC; IR (KBr, cm-1): ν 3430.5, 3083.8, 3056.1, 3018.0,
2928.1, 2844.3, 2230.2, 1624.0, 1596.2, 1567.0, 1541.6, 1485.7, 1444.3, 1315.3, 1295.1, 1193.6,
1
1110.1, 1074.1, 1011.0, 822.5, 743.9, 701.6, 614.9; H-NMR (CDCl3): δ 3.01 (2H, s), 3.14 (2H, s),
3.51 (2H, s), 3.68 (2H, s), 4.22 (2H, s), 6.68 (1H, s), 6.89 (1H, s), 7.18 (11H, m), 7.32 (6H, m), 7.50
(2H, d, J = 6.4 Hz), 8.18 (1H, s); 13C-NMR (DMSO-d6): δ 35.9, 42.4, 45.4, 47.2, 53.6, 65.9, 79.2, 88.5,
114.0, 118.7, 126.5, 126.7, 126.8, 128.0, 129.0, 132.4, 135.9, 144.0, 146.1, 146.9, 150.5, 167.6, 168.9;
MS (ESI): M = 759.7 (Found: 759.5).
Conjugation of Tr-MAF and ABCPP (Tr-MAF-ABCPP) (12b). 12b was prepared with the same
procedure as 12a; yield: 22%; m.p.: 188-189 ºC; IR (KBr, cm-1): ν 3443.9, 3363.6, 3297.4, 3059.9,
3024.0, 2916.3, 2851.0, 2229.8, 1678.9, 1664.1, 1619.5, 1593.2, 1563.7, 1534.9, 1481.4, 1444.9,
1316.1, 1286.9, 1190.1, 1014.3, 830.9, 743.1, 702.0, 672.1, 623.5; 1H-NMR (CDCl3): δ 2.83 (1H, m),
2.92 (1H, m), 3.02 (2H, s), 3.16 (1H, m), 3.24 (1H, m), 3.48 (2H, m), 3.73 (2H, s), 4.25 (1H, m), 5.89
(1H, t), 6.46 (1H, s), 6.59 (1H, d, J = 7.0 Hz), 7.22 (12H, m), 7.28 (4H, m), 7.32 (6H, m), 7.63 (2H, d,
J = 8.6 Hz), 8.28 (1H, s); 13C-NMR (DMSO-d6): δ 37.7, 47.5, 47.7, 54.0, 54.2, 65.8, 78.6, 88.1, 114.2,
118.2, 126.1, 126.4, 126.7, 127.9, 128.0, 129.0, 129.1, 132.3, 137.6, 144.0, 146.1, 146.5, 150.8, 165.9,
167.0, 170.5; MS (ESI): M = 849.8 (Found: 849.6).
Conjugation of Tr-MAA and ABCPP (Tr-MAA-ABCPP) (12c). 12c was prepared with the same
procedure as 12a; yield: 19%; m.p.: 165-167 ºC; IR (KBr, cm-1): ν 3446.2, 3083.8, 3047.9, 2960.8,
2926.1, 2226.1, 1647.4, 1622.3, 1593.1, 1563.6, 1484.3, 1444.7, 1308.8, 1274.2, 1188.0, 1065.2,
1
1008.2, 802.2, 743.0, 700.6, 621.9; H-NMR (CDCl3): δ 0.85 (6H, dd, J = 6.8, 2.2Hz), 2.78 (1H, m),
3.03 (2H, s), 3.35 (4H, m), 3.83 (2H, s), 3.95 (1H, m), 6.29 (1H, t), 6.48 (1H, s), 7.21 (5H, m), 7.28
(6H, m), 7.36 (6H, m), 7.62 (2H, m), 8.28 (1H, s); 13C-NMR (DMSO-d6): δ 18.1, 19.0, 30.3, 35.9, 38.6,
42.1, 47.8, 54.0, 58.1, 65.9, 78.7, 88.2, 114.2, 118.3, 126.4, 126.7, 128.0, 129.1, 132.4, 144.1, 146.0,
146.6, 150.7, 167.3, 170.6; MS (ESI): M = 801.8 (Found: 801.6).
3.3. Radiochemistry
The tritylated compound 12a (5 mg) was treated with trifluoroacetic acid under cation trapping
conditions (5% triethylsilane) at room temperature. After removing the solvent under a stream of
nitrogen, the residue was neutralized with 0.1 M NaOH. The solution was extracted with
dichloromethane, and the aqueous phase was under nitrogen protection. Saline containing [99mTcO4]-
(1 mL, 15MBq) was added to a kit containing stannous chloride dihydrate(0.05 mg), succinic
dihydrazide (SDH, 5.0 mg) and propylenediamine tetraacetic acid (PDTA, 5.0 mg). The mixture was
kept at room temperature for 15 min. Next, the MAG-ABPP solution (1 mL) was added and the
reaction allowed to stand for 15 min at 100 ºC to give the final complex 99mTcN-MAG-ABCPP (14a).
99mTcN-MAF-ABCPP (14b) and 99mTcN-MAA-ABCPP (14c) were prepared using the same method.
Formation of the complexes and nature of the species formed was determined by Radio-HPLC

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analysis. Solvent system used for elution was water (solvent A) and acetonitrile (solvent B). The
HPLC gradient system for analysis of the product started with 100% A/0% B with a linear gradient to
0% A/100% B from 0 to 30 min. The flow rate was 1.0 mL/min. Five microliters of the sample was
used for analysis. Recovery was determined by summing the total counts in all fractions and
comparing them to the total injected activity.
3.4. Measurement of partition coefficients
The partition coefficients of the three complexes were determined according to the published
method [16] by mixing each complex with an equal-volume of 1-octanol and phosphate buffer
(0.025 M, pH 7.4) in a centrifuge tube. The mixture was vortexed at room temperature for 1 min and
then centrifuged at 5,000 rpm for 5 min. From each phase, 0.1 mL of the aliquot was pipetted and
counted in a well-type NaI(Tl) detector. The measurement was repeated three times. Care was taken to
avoid cross contamination between the phases. Usually the final partition coefficient value was
expressed as log P, where the partition coefficient, P, was calculated using the following equation:
P = (cps in octanol-cps in background)/(cps in buffer-cps in background)
3.5. In vitro stability study
The stability of the complexes was studied by measuring the radiochemical purity using HPLC
analysis at different time intervals after preparation. The complexes were added to test tubes
containing PBS solution (0.025 M, pH 7.4). The mixtures were incubated by shaking them at 37 ºC in
a thermomixer. The radiochemical purity was measured at 15 min, 30 min, 60 min and 120 min by
Radio-HPLC.
3.6. Biodistribution study
Biodistribution studies were performed in Kunming female mice (weighing 18–20 g) bearing S 180
tumors, which grew to a leg diameter of 10-15 mm. 99mTcN-MAG-ABCPP, 99mTcN-MAF-ABCPP and
99mTcN-MAA-ABCPP (0.1 mL, 7.4 × 105 Bq) were injected via a tail vein and the injected
radioactivity was measured with a well-type NaI(Tl) detector, respectively. The mice were sacrificed at
5 min, 30 min, 60 min and 120 min post-injection. The tumor, other organs of interest and blood were
collected, weighed and measured for radioactivity. The results were expressed as the percent uptake of
injected dose per gram of tissue (%ID/g). All biodistribution studies were carried out in compliance
with the national laws related to the conduct of animal experimentation.
4. Conclusion
In summary, the new 3-cyanopyrazolo[1,5-a]pyrimidine ABCPP was successfully synthesized and
conjugated with MAG, MAF and MAA, respectively. The three compounds were labeled with 99mTc in
high radiochemical purities through a ligand exchange reaction, which can be easily used for the
preparation of a radiopharmaceutical through a freeze-dried kit formulation. The three complexes
demonstrated tumor accumulation, high tumor-to-muscle (T/M) ratios and fast clearance from blood

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and muscle. Among them, 99mTcN-MAG-ABCPP showed the most favorable characteristics and could
be further studied as potential tumor imaging agent.
Acknowledgement
The work was financially supported by National Natural Science foundation of China (No
21071022) and Beijing Key Subject Program. We also wish to thank Beijing Municipal Commission
of Education.
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Sample Availability: Samples of the compounds mentioned above are available from the authors.
© 2010 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/).