Preparation and bioevaluation of 99mTc nitrido radiopharmaceuticals with pyrazolo[1,5-a]pyrimidine as tumor imaging agents

Article abstract of DOI:10.1007/s00044-011-9558-8

The 7-(2-aminoethylamino)-5-methyl-3-cyanopyrazolo[ 1,5-a]pyrimidine (AMCPP) 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 high tumor-to-muscle (T/M) ratios and rapid clearance from the blood, muscle, liver, kidney, and lung. Among them, the ^99mTcN-MAG-AMCPP showed the most favorable characteristics. The tumor/blood and tumor/muscle ratios reached 1.50 and 1.15 at 30 min post-injection, 2.20 and 1.83 at 60 min post-injection. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9558-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:523–530
DOI 10.1007/s00044-011-9558-8
ORIGINAL RESEARCH
Preparation and bioevaluation of 99mTc nitrido
radiopharmaceuticals with pyrazolo[1,5-a]pyrimidine
as tumor imaging agents
•
Rui Ding Yong He Jingli Xu Hang Liu
•
•
•
•
Xiao Wang Man Feng Chuanmin Qi
•
•
•
Junbo Zhang Cheng Peng
Received: 22 July 2010 / Accepted: 8 January 2011 / Published online: 22 January 2011
Ó Springer Science+Business Media, LLC 2011
Abstract The 7-(2-aminoethylamino)-5-methyl-3-cyano-
pyrazolo[1,5-a]pyrimidine (AMCPP) was synthesized and
conjugated with N-mercaptoacetylglycine (MAG), N-mer-
captoacetylphenylalanine (MAF), and N-mercaptoacetylv-
aline (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
high tumor-to-muscle (T/M) ratios and rapid clearance
from the blood, muscle, liver, kidney, and lung. Among
them, the 99mTcN-MAG-AMCPP showed the most favor-
able characteristics. The tumor/blood and tumor/muscle
ratios reached 1.50 and 1.15 at 30 min post-injection, 2.20
and 1.83 at 60 min post-injection.
much attention (Lane et al., 2001; Sielecki et al., 2000). This
approach to cancer therapy differs from the traditional use
of cytotoxic agents in that the target tissue is the vascula-
ture encompassing the neoplasm, not the tumor cells
(Fraley et al., 2002; Wu et al., 2004; De bondt et al., 1993;
Richardson et al., 2006). A large number of different
structural compounds, including indolin-2-ones, 4-anilino-
pyrazolo[4,3-d]pyrimidines, purines, phthalazines, and
quinazolines were reported as antitumor agents used in
clinics or in clinical trials (Arora and Scholar, 2005; Meijer
and Raymond, 2003; Havlicek et al., 2005; Knockaert
et al., 2002).
Pyrazolo[1,5-a]pyrimidine derivatives are developed as a
new class of CDKs inhibitors. These compounds are cur-
rently being evaluated as antitumor drugs in a growing
number of therapeutic areas. Among them, 3-cyano-5,7-
disubstistituted pyrazolo[1,5-a]pyrimidine derivatives
showed significant antitumor activities (Berman et al., 2000;
Lawrie et al., 1997; Williamson et al., 2005). Gong et al.
discovered that the amino group at position 7 of pyrazolo-
[1,5-a]pyrimidine core structure enhanced the antitumor
activity (Li et al., 2006).
Keywords Pyrazolo[1,5-a]pyrimidine Á Tumor imaging Á
[
99mTcN]2? complexes
Introduction
Tumor is among the most common causes of death in the
world. Recently, the therapeutic potential of small molecule
inhibitors of cyclin-dependent kinases (CDKs) for use as
antitumor agents for the treatment of cancer has come under
Amongst the various chemical forms and preformed
precursors of technetium used for radiolabeling, [99mTcN]2?
has gained interest owing to the presence of the nitrido
N3- ligand as strong p-electron donor that leads to formation
of kinetically inert complexes (Liu and Edwards, 1999). The
[
99mTcN]2? core exhibits a very high chemical stability
R. Ding Á Y. He Á J. Xu Á H. Liu Á X. Wang Á M. Feng Á
C. Qi (&) Á J. Zhang
toward 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 alter its
biological behavior.
Key Laboratory of Radiopharmaceuticals, Ministry of
Education, College of Chemistry, Beijing Normal University,
Beijing 100875, People’s Republic of China
e-mail: qicmin@sohu.com
C. Peng
Center of PET, Xuan Wu Hospital, Beijing 100053,
People’s Republic of China
Benoist et al. reported a novel bifunctional chelating agent
(BFCA), MAG-N-OPh, which formed a stable and inert
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Med Chem Res (2012) 21:523–530
99mTc-complex in high yield under mild conditions (Gal
et al., 2006). To link to the target molecule, it was synthesized
MAG as the precursor of the BFCA according to the previous
modified methods (Qi et al., 2002), and introduced solubi-
lizing basic ethylenediamine at the 7-position to the 3-cyan-
opyrazolo[1,5-a]pyrimidine, thus affording a new N3S type
ligand. This report constitutes the first of its kind in using the
3-cyanopyrazolo[1,5-a]pyrimidine in the preparation of
99mTcN complexes as targeted agents for tumor imaging.
Toward developing new better tumor imaging agents, the
authors introduced hydrophobic groups such as isopropyl
group and benzyl group to MAG, and synthesized
99mTcN-MAG-AMCPP, 99mTcN-MAF-AMCPP, and 99mTcN-
MAA-AMCPP. A series of experiment was carried out in
order to evaluate the bioactivity of the three complexes.
Tritylmercaptoacetic acid N-hydroxysuccinimido ester
(3)
Compound 2 (1.0 g, 3 mmol) and N-hydroxysuccinimide
(NHS) (0.345 g, 3 mmol) were dissolved in anhydrous
THF at -10°C. DCC (0.619 g, 3 mmol) in 5 mL of
anhydrous THF was added dropwise. The clear reaction
mixture was stirred for 2 h at -5°C and then overnight at
room temperature. White precipitate was removed by fil-
tration. Solvent was removed in vacuo to obtain the light
yellow crude product and the crude product was recrys-
tallized in ethyl acetate to obtain the compound 3 (1.06 g,
82%) as white crystal. m.p.: 188–189°C; IR (KBr, cm-1): m
1810, 1782, 1737, 1627, 1445, 1245, 1207, 1064, 742, 699;
1H-NMR (CDCl3, 400 MHz): d 2.74 (4H, s), 3.11 (2H, s),
7.16 (3H, m), 7.25 (6H, m), 7.35 (6H, d, J = 7.8 Hz).
13C-NMR (CDCl3, 100 MHz): d 25.5, 31.4, 68.1, 127.1,
128.2, 129.5, 143.6, 165.1, 168.6.
Materials and methods
99Mo/99mTc generator was obtained from the China insti-
tute of atomic energy (CIAE). All other chemicals were of
analytical grade and were used without further purification.
1H- and 13C-NMR spectra were recorded on a Bruker
spectrometer operating in CDCl3 or DMSO-d6 at 400 and
100 MHz, respectively. The IR spectra were recorded on a
Nicolet-AVATAR 360 FT–IR spectrometer using KBr
pellets in the 4000–400 cm-1 regions. ESI–MS were per-
formed 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 (5u 150 mm 9 4.6 mm)
was used for radiochemical purity analysis.
N-[(Tritylmercapto)acetyl]glycine (Tr-MAG) (4a)
Compound 3 (0.30 g, 0.7 mmol) was dissolved in aceto-
nitrile (15 mL). Glycine (0.105 g, 1.4 mmol) in sodium
hydroxide (0.2 M, 1 mL) was added dropwise. The reac-
tion solution was stirred for 2 h at 50–60°C. 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 recrystal-
lized from ethyl acetate to give compound 4a (0.22 g,
78%); m.p.: 164–166°C; IR (KBr, cm-1): m 3434, 3344,
2960, 2926, 1732, 1619, 1527, 1488, 1445, 1247, 1224,
1
1211, 741, 704; H-NMR (CDCl3): d 3.13 (2H, s), 3.62
(2H, d, 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); 13C-NMR
(CDCl3): d 35.4, 41.6, 67.9, 127.1, 128.2, 129.4, 143.8,
169.0, 172.2.
Ligand synthesis
Tritylmercaptoacetic acid (2)
N-[(Tritylmercapto)acetyl]phenylalanine (Tr-MAF)
(4b)
New distilled mercaptoacetic acid 1 (0.7 mL, 10 mmol)
was dissolved in the mixture of 6 mL of dichloromethane
and 6 mL acetic acid. Trityl chloride (2.79 g, 10 mmol)
was added to the solution. BF3ÁEt2O (2 mL) was added
dropwise as catalyst, and the color of reaction solution
turned yellow-green. After the reaction solution had been
stirred at room temperature for 60 min, the solvent was
removed in vacuo. Water (10 mL) was added to the resi-
due. The white precipitate was collected by filtration and
washed with distilled water, acetonitrile and cool ether to
obtain the compound 2 (3.18 g, 95%). m.p.: 159–160°C; IR
(KBr, cm-1): m 1705, 1488, 1446, 1412, 1292, 749, 701;
1H-NMR (CDCl3, 400 MHz): d 2.98 (2H, s), 7.16 (3H, m),
7.23 (6H, m), 7.35 (6H, d, J = 7.6 Hz). 13C-NMR (CDCl3,
100 MHz): d 34.3, 67.3, 127.0, 128.1, 129.5, 143.8, 173.9.
4b was obtained with the same method of 4a using phen-
ylalanine instead of glycine (yield: 59%); m.p.: 128–130°C;
IR (KBr, cm-1): m 3448, 3340, 3027, 2928, 1727, 1627,
1
1528, 1493, 1443, 1223, 1203, 1184, 742, 700; H-NMR
(CDCl3): d 3.07 (4H, m), 4.48 (1H, dm, J = 6.2 Hz), 6.54
(1H, d, J = 6.2 Hz), 7.10 (2H, m), 7.25 (18H, m); 13C-NMR
(CDCl3): d 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°C; IR (KBr,
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Med Chem Res (2012) 21:523–530
525
cm-1): m 3434, 3339, 3055, 2960, 2929, 1739, 1720, 1636,
1534, 1489, 1441, 1422, 1381, 1304, 1273, 1213, 1082,
After it was cooled to 60°C, chloroform (25 ml) was
added and stirred for 1 h. Cold water (30 ml) was added
to the solution. The insoluble materials was filtered off,
the organic layer of the filtrate was washed with cold
water to pH 7, concentrated, and recrystallized from
1
1035, 1000, 885, 757, 738, 700; H-NMR (CDCl3): d 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): d 17.9, 18.8, 31.0, 36.2, 57.5, 68.2, 127.2, 128.2,
129.6, 144.0, 168.8, 175.4.
ethyl acetate/cyclohexane to give the compound
9
(0.95 g, 87%). m.p.: 213–215°C; MS(ESI): M = 192.0
(Found: 192.9).
2-(Ethoxymethylene)malononitrile (6)
7-(2-Aminoethylamino)-5-methyl-3-
cyanopyrazolo[1,5-a]pyrimidine (AMCPP) (10)
Malononitrile (9.9 g, 0.150 mol), triethoxymethane (37.4 ml,
0.225 mol) and acetic anhydride (35.4 ml, 0.375 mol) were
refluxed for 6 h. Then a little charcoal was added. The solvent
was removed from the mixture by distillation. The hot residue
was filtered and the filter cake was washed with hot ethanol,
the filtrate was placed in a refrigerator overnight. After fil-
tration, the crystalline was washed with ice-cold ethanol and
dried to get the product 6 (17.4 g, 88%). m.p.: 66–68°C; IR
(KBr, cm-1): m 3443, 3031, 2944, 2228, 1611, 1315, 1009,
883.
A mixture of 9 (0.963 g, 5 mmol), ethylenediamine
(0.60 g, 10 mmol), and ethanol (30 ml) was heated to
reflux for 4 h. Solvent was removed in vacuo to obtain
the crude product and the crude product was recrystal-
lized in methanol to obtain the compound 10 (0.86 g,
81%). m.p.: 203–205°C; IR (KBr, cm-1): m 3354, 3293,
3112, 2944, 2861, 2221, 1637, 1578, 1459, 1311, 1193,
944; 1H-NMR (DMSO-d6, 400 MHz): d 2.51 (3H, s),
2.79 (2H, m), 3.17 (2H, m), 6.44 (1H, s), 8.56 (1H, s);
13C-NMR (DMSO-d6, 100 MHz): d 24.5, 40.2, 44.5,
77.8, 88.5, 114.5, 146.1, 147.3, 150.1, 162.7; MS(ESI):
M = 216.2 (Found: 217.3).
3-Amino-4-cyanopyrazole (7)
Compound 6 (15 g, 0.123 mol) was carefully added to 85%
hydrazine hydrate (12 ml, 0.248 mol) at room temperature.
The resulting mixture was refluxed for 1 h. To the resulting
solidified mass was added 10 mL of water. The solution
stay in a refrigerator overnight and the mushy solution was
filtered and the solid was washed with cold water and dried
to get the product 7 (10.7 g, 81%). m.p.: 174–176°C; IR
(KBr, cm-1): m 3416, 3342, 3147, 2955, 2238, 1645, 1570,
1522, 1221, 1034, 716.
Conjugation of Tr-MAG and AMCPP (Tr-MAG-
AMCPP) (11a)
A mixture of 10 (108 mg, 0.5 mmol), triethylamine
(0.105 ml, 0.75 mmol) and anhydrous dichloromethane
(4 ml) was stirred at room temperature for 5 min. Then 4a
(196 mg, 0.5 mmol) and HOBt (75 mg, 0.55 mmol) were
added. DCC (115 mg, 0.55 mmol) in 2 ml of anhydrous
dichloromethane was added dropwise to the solution at
0°C. The mixture was stirred for 30 min at 0°C and then
overnight at room temperature. White precipitate 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 was purified by column chromatography (silica gel;
petroleum ether/ethyl acetate = 1:5) to afford the desired
compound 11a (56 mg, 19%). mp: 166–168°C; IR (KBr,
cm-1): m 3384, 3311, 3096, 3048, 2931, 2844, 2223, 1658,
1637, 1585, 1535, 1467, 1443, 1368, 1310, 1245, 1195,
1116, 1038, 745, 702, 673, 622; 1H-NMR (CDCl3,
400 MHz): d 2.54 (3H, s), 3.18 (2H, s), 3.48 (2H, m), 3.55
(4H, m), 5.98 (1H, s), 6.53 (1H, t, J = 5.6 Hz), 6.73 (1H,
m), 7.09 (1H, m), 7.23 (3H, m), 7.29 (6H, m), 7.39 (6H, m),
8.03 (1H, s); 13C-NMR (DMSO-d6, 100 MHz): d 24.5,
35.9, 40.1, 41.2, 45.7, 65.9, 77.8, 88.3, 114.3, 126.8, 128.0,
129.0, 144.0, 146.1, 147.2, 150.5, 162.7, 167.6, 169.1;
MS(ESI): M = 589.7 (Found: 590.6).
5-Methyl-3-cyano-7-hydroxypyrazolo[1,5-a]pyrimidine
(8)
A mixture of 7 (9.0 g, 0.083 mol), ethyl acetoacetate
(16.25 g, 0.125 mol), and acetic acid (85 ml) was refluxed
for 4 h. After cooling to room temperature, the pale white
solid was filtered and washed with ethanol, dried with
suction, and recrystallized from ethyl acetate to obtain the
solid product 8 (13.01 g, 90%). m.p.:[250°C; MS(ESI):
M = 174.2 (Found: 175.2).
5-Methyl-3-cyano-7-chloropyrazolo[1,5-a]pyrimidine
(9)
Phosphoryl trichloride (0.78 ml, 8.6 mmol) was added
dropwise into a solution of 8 (1.0 g, 5.7 mmol) in
anhydrous pyridine (0.5 g, 6.3 mmol). The mixture was
heated to 85°C slowly, and then stirred at 120°C for 1 h.
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Conjugation of Tr-MAF and AMCPP (Tr-MAF-
AMCPP) (11b)
2.57 (3H, s), 3.19 (2H, s), 3.45 (3H, m), 3.60 (1H, m), 3.87
(1H, t), 5.96 (1H, s), 6.24 (1H, m), 6.43 (1H, m), 6.97 (1H, m),
7.24 (9H, m), 7.33 (6H, m), 7.87 (1H, s); 13C-NMR (DMSO-
d6, 100 MHz): d 17.9, 19.0, 24.5, 30.2, 35.8, 37.5, 41.2, 58.0,
65.9, 77.8, 88.4, 114.4, 126.8, 128.0, 129.0, 144.1, 146.1,
147.1, 150.4, 162.6, 167.4, 171.3; MS(ESI): M = 631.8
(Found: 632.6).
11b was prepared with the same procedure as 11a synthesis
(yield: 23%). m.p.: 208–210°C; IR(KBr, cm-1): m 3377,
3266, 3084, 3058, 2931, 2844, 2221, 1630, 1583, 1536,
1493, 1444, 1374, 1310, 1245, 1192, 1119, 1026, 744, 700,
1
674, 623; H-NMR (CDCl3, 400 MHz): d 2.56 (3H, s),
Preparation of 99mTcN-MAG-AMCPP analogs
2.89 (2H, m), 3.11 (2H, s), 3.33 (3H, m), 3.51 (1H, m), 4.25
(1H, m), 5.93 (1H, s), 6.15 (1H, t), 6.95 (1H, m), 7.09 (1H,
m), 7.12 (5H, m), 7.23 (8H, m), 7.28 (7H, m), 7.98 (1H, s);
13C-NMR (DMSO-d6, 100 MHz): d 24.5, 35.9, 37.5,
37.6, 40.1, 41.2, 54.2, 65.7, 77.8, 88.4, 114.3, 126.2,
126.7, 127.9, 128.0, 129.0, 137.5, 144.0, 146.1, 147.1,
150.5, 162.6, 167.1, 171.3; MS(ESI): M = 679.8 (Found:
680.7).
The tritylated compound 11a (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. 1 ml of saline containing [99mTcO4]-
(15 MBq) was added to a kit containing 0.05 mg of stannous
chloride dehydrate, 5.0 mg of succinic dihydrazide (SDH),
5.0 mg of propylenediamine tetraacetic acid (PDTA). The
mixture was kept at room temperature for 15 min. Succes-
sively, 1 ml of the MAG-AMCPP solution was then added
and the reaction allowed to stand for 15 min at 100°C to
give the final complex 99mTcN-MAG-AMCPP (13a).
99mTcN-MAF-AMCPP (13b) and 99mTcN-MAA-AMCPP
Conjugation of Tr-MAA and AMCPP (Tr-MAA-
AMCPP) (11c)
11c was prepared with the same procedure as 11a synthesis
(yield: 19%). m.p.: 202–203°C; IR(KBr, cm-1):m 3440, 3090,
3048, 2961, 2924, 2223, 1633, 1585, 1536, 1465, 1444, 1368,
1308, 1236, 1193, 1113, 1029, 744, 701; 1H-NMR (CDCl3,
400 MHz): d 0.85 (6H, dd, J = 7.8, 6.8 Hz), 2.04 (1H, m),
Scheme 1 Synthesis of
99mTcN-MAG-AMCPP,
99mTcN-MAF-AMCPP,
and 99mTcN-MAA-AMCPP
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527
Fig. 1 Radio-HPLC chromatograms. a 99mTcN2?. b 99mTcN-MAG-AMCPP (13a). c 99mTcN-MAF-AMCPP (13b). d 99mTcN-MAA-AMCPP
(13c)
Table 1 Partition coefficients of 99mTcN-MAG-AMCPP, 99mTcN-
Partition coefficient
MAF-AMCPP, and 99mTcN-MAA-AMCPP
99mTcN-MAG-
AMCPP
99mTcN-MAF-
AMCPP
99mTcN-MAA-
AMCPP
The partition coefficient was determined by mixing the
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 5000 r/min 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. The partition coefficient, P, was cal-
culated using the following equation:
logP -2.71 0.02
1.13 0.03
-1.55 0.03
(13c) were prepared using the same method as 99mTcN-
MAG-AMCPP (13a).
Characterization of 99mTcN-MAG-AMCPP analogs
P = (cps in octanol-cps in background)/(cps in buffer-
cps in background)
Formation of the complexes and nature of the species
formed was determined by Radio-HPLC analysis. Solvent
system used for elution was water (solvent A) and acetoni-
trile (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.
Usually the final partition coefficient value was
expressed as log P.
Biodistribution
Biodistribution studies were performed in Kunming female
mice (weighing 18–20 g) bearing S 180 tumor, which grew
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Table 2 Biodistribution of 99mTcN-MAG-AMCPP in mice bearing S
Table 4 Biodistribution of 99mTcN-MAA-AMCPP in mice bearing S
180 tumor (%ID/g)a
180 tumor (%ID/g)c
Tissue
5 min
30 min
60 min
120 min
Tissue
5 min
30 min
60 min
120 min
Heart
0.53 0.04 0.05 0.05 0.06 0.01 0.03 0.01
8.24 0.68 2.89 0.09 0.16 0.01 0.16 0.02
0.24 0.04 0.10 0.01 0.06 0.01 0.04 0.01
0.94 0.13 0.12 0.03 0.10 0.01 0.06 0.01
4.09 0.29 0.67 0.01 0.45 0.02 0.61 0.08
0.34 0.05 0.14 0.01 0.08 0.01 0.04 0.01
0.51 0.09 0.13 0.01 0.06 0.01 0.03 0.01
0.37 0.02 0.22 0.01 0.08 0.02 0.02 0.01
1.47 0.07 0.17 0.02 0.18 0.02 0.09 0.01
Heart
0.63 0.05 0.31 0.02 0.20 0.01 0.20 0.02
4.16 0.61 0.75 0.12 0.70 0.10 0.56 0.06
0.41 0.05 0.27 0.03 0.25 0.05 0.16 0.01
1.05 0.10 0.81 0.11 0.43 0.06 0.27 0.03
5.21 0.32 1.71 0.19 1.25 0.17 1.09 0.19
0.45 0.03 0.29 0.03 0.29 0.05 0.22 0.01
0.63 0.06 0.15 0.02 0.12 0.01 0.10 0.01
2.17 0.26 0.42 0.02 0.36 0.04 0.26 0.03
2.06 0.17 0.20 0.02 0.20 0.03 0.54 0.05
Liver
Liver
Spleen
Lung
Spleen
Lung
Kidney
Bone
Kidney
Bone
Muscle
Stomach
Muscle
Stomach
Large
Large
intestine
intestine
Small
1021 0.13 0.89 0.11 0.75 0.10 0.21 0.022
Small
1.09 0.18 0.88 0.15 0.65 0.09 0.23 0.04
intestine
intestine
Blood
1.06 0.14 0.10 0.01 0.05 0.01 0.04 0.01
0.05 0.01 0.01 0.00 0.01 0.00 0.01 0.00
0.52 0.04 0.15 0.02 0.11 0.02 0.06 0.01
Blood
1.46 0.13 1.12 0.12 0.93 0.11 0.60 0.07
0.05 0.01 0.04 0.01 0.04 0.01 0.03 0.01
0.61 0.05 0.35 0.04 0.30 0.03 0.24 0.05
Brain
Brain
Tumor
Tumor
T/M ratio
T/B ratio
1.02
0.49
1.15
1.50
1.83
2.20
2.00
1.50
T/M ratio
T/B ratio
0.97
0.42
2.33
0.31
2.50
0.32
2.40
0.40
T/M tumor to muscle, T/B tumor to blood
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-AMCPP per gram of tissue, the standard deviation
of the mean
All data are the mean percentage (n = 3) of the injected dose of
99mTcN-MAA-AMCPP per gram of tissue, the standard deviation
of the mean
to a leg diameter of 10–15 mm. 99mTcN-MAG-AMCPP,
99mTcN-MAF-AMCPP, and 99mTcN-MAA-AMCPP (0.1 ml,
7.4 9 105 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, 30, 60, and
120 min post-injection. The tumor, other organs of interest
and blood were collected, weighed, and measured for radio-
activity. 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.
Table 3 Biodistribution of 99mTcN-MAF-AMCPP in mice bearing
S 180 tumor (%ID/g)b
Tissue
5 min
30 min
60 min
120 min
Heart
0.05 0.01 0.08 0.03 0.03 0.01 0.04 0.01
16.93 1.15 4.27 0.44 3.89 0.08 3.05 0.19
2.32 .022 1.49 0.27 2.29 0.24 0.84 0.17
1.55 0.21 0.42 0.09 0.20 0.04 0.08 0.01
13.12 1.13 2.46 0.38 1.02 0.12 1.16 0.19
0.51 0.01 0.14 0.02 0.12 0.01 0.15 0.01
0.32 0.02 0.06 0.01 0.04 0.01 0.08 0.02
1.30 0.12 0.32 0.02 0.27 0.03 0.48 0.05
0.64 0.07 0.29 0.01 0.06 0.01 0.08 0.02
Liver
Spleen
Lung
Kidney
Bone
Results and discussion
Muscle
Stomach
Chemistry of 99mTcN-MAG-AMCPP analogs
Large
intestine
Synthesis of 99mTcN-MAG-AMCPP analogs was per-
formed according to the procedure outlined in Scheme 1.
The radiochemical purity of the complexes was routinely
checked by HPLC. The HPLC pattern of 99mTcN-MAG-
AMCPP, 99mTcN-MAF-AMCPP, and 99mTcN-MAA-AM-
Small
intestine
0.80 0.08 0.67 0.06 0.59 0.06 0.54 0.06
Blood
1.53 0.17 0.33 0.02 0.21 0.01 0.13 0.01
0.06 0.01 0.01 0.01 0.02 0.01 0.01 0.00
0.60 0.01 0.21 0.01 0.15 0.01 0.04 0.01
Brain
Tumor
T/M ratio
T/B ratio
1.88
0.39
3.50
0.64
3.75
0.71
0.50
0.31
CPP are shown in Fig. 1. It was observed that the reten-
99mTcN]
was 1.5 min, while that
2?
int
tion time of
[
of 99mTcN-MAG-AMCPP, 99mTcN-MAF-AMCPP, and
99mTcN-MAA-AMCPP were found to be 2.9, 3.5, and
3.3 min, respectively. The radiochemical purity of the three
products was over 90% immediately after the preparation.
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-AMCPP per gram of tissue, the standard deviation
of the mean
123

Med Chem Res (2012) 21:523–530
Partition coefficient
529
were encouraging, the tumor uptake of the three complexes
was comparably low as in case of other 99mTc labeled
tumor imaging agents reported. Attempts to increase the
tumor uptake of the 99mTc labeled 3-cyanopyrazolo[1,5-
a]pyrimidine derivatives by structural modification are
underway.
The partition coefficients (logP) of 99mTcN-MAG-AMCPP,
99mTcN-MAF-AMCPP, and 99mTcN-MAA-AMCPP are
shown in Table 1 and all of them were hydrophilic.
99mTcN-MAG-AMCPP was the highest hydrophilic one
and 99mTcN-MAA-AMCPP was more hydrophilic than
99mTcN-MAF-AMCPP.
Acknowledgments The study was financially supported by
National Natural Science Foundation of China (no 20371009 and
20671014) and Beijing Key Subject Program. We also wish to thank
Beijing Municipal Commission of Education.
Biodistribution study
The results of biodistribution are summarized in Tables 2,
3, and 4. There were significant similarities in biodistri-
bution pattern between these three complexes. The accu-
mulation of the three complexes in all of the organs or
tissue was highest in the 5 min post-injection and then
decreased. Although the uptake in tumor of the three
complexes was comparably low, the clearance rate in the
tumor was slower than in other organs or tissue, as time
lapsed, the activity in the tumor exceeded than that in most
other organs or tissue. For 99mTcN-MAG-AMCPP, the
tumor/blood and tumor/muscle ratios reached 1.50 and
1.15 at 30 min post-injection, 2.20 and 1.83 at 60 min post-
injection. For 99mTcN-MAF-AMCPP, the tumor/blood and
tumor/muscle ratios reached 0.64 and 3.50 at 30 min post-
injection, 0.71 and 3.75 at 60 min post-injection. For
99mTcN-MAA-AMCPP, the tumor/blood and tumor/muscle
ratios reached 0.32 and 2.50 at 60 min post-injection, 0.40
and 2.40 at 120 min post-injection. Early hepatic and renal
activity reflected the three complexes were excreted
through hepatobiliary as well as 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-AMCPP
showed the most favorable characteristics with the highest
tumor/blood ratios. It is deemed worthwhile to modify the
structure of AMCPP suitably to render it less hydrophilic to
enhance the tumor uptake of its 99mTc labeled complex.
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