Novel [99mTcN]2+ labeled EGFR inhibitors as potential radiotracers for single photon emission computed tomography (SPECT) tumor imaging

Article abstract of DOI:10.3390/molecules19055508

The epidermal growth factor receptor (EGFR) is overexpressed in many cancers, including breast, ovarian, endometrial and non-small cell lung cancer. An EGFR-specific imaging agent could facilitate clinical evaluation of primary tumors or metastases. To achieve this goal, 4-(2-aminoethylamino)-6,7- dimethoxyquinazoline (ADMQ) was synthesized based on a 4-aminoquinazoline core and then conjugated with N-mercapto-acetylglycine (MAG) and N- mercaptoacetyltriglycine (MAG3), respectively, to give compounds 1 and 2. The final complexes [^99mTcN]-1 and [^99mTcN]-2 were successfully obtained with radiochemical purities of >99% and >98% as measured by radio-HPLC. No decomposition of the two complexes at room temperature was observed over a period of 2 h. Their partition coefficients indicated they were hydrophilic and the electrophoresis results showed they were negatively charged. Biodistribution in tumor-bearing mice demonstrated that the two new complexes showed tumor accumulation, high tumor-tomuscle (T/M) ratios and fast clearance from blood and muscle. Between the two compounds, the ^99mTcN-MAG3-ADMQ ([^99mTcN]-2) showed the better characteristics, with the tumor/muscle and tumor/blood ratios reached 2.11 and 1.90 at 60 min post-injection, 4.20 and 1.10 at 120 min post-injection, suggesting it could be a promising radiotracer for SPECT tumor imaging.

Full text of DOI:10.3390/molecules19055508

Molecules 2014, 19, 5508-5521; doi:10.3390/molecules19055508
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Novel [99mTcN]2+ Labeled EGFR Inhibitors as Potential
Radiotracers for Single Photon Emission Computed
Tomography (SPECT) Tumor Imaging
Mingxia Zhao, Hongyu Ning, Man Feng, Shilei Li, Jin Chang and Chuanmin Qi *
Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing
Normal University, Beijing 100875, China
* Author to whom correspondence should be addressed; E-Mail: qicmin@sohu.com;
Fax: +86-10-5880-2075.
Received: 17 February 2014; in revised form: 21 April 2014 / Accepted: 21 April 2014 /
Published: 29 April 2014
Abstract: The epidermal growth factor receptor (EGFR) is overexpressed in many
cancers, including breast, ovarian, endometrial and non-small cell lung cancer. An
EGFR-specific imaging agent could facilitate clinical evaluation of primary tumors or
metastases. To achieve this goal, 4-(2-aminoethylamino)-6,7-dimethoxyquinazoline
(ADMQ) was synthesized based on a 4-aminoquinazoline core and then conjugated with
N-mercapto- acetylglycine (MAG) and N-mercaptoacetyltriglycine (MAG3), respectively,
to give compounds 1 and 2. The final complexes [99mTcN]-1 and [99mTcN]-2 were
successfully obtained with radiochemical purities of >99% and >98% as measured by
radio-HPLC. No decomposition of the two complexes at room temperature was observed
over a period of 2 h. Their partition coefficients indicated they were hydrophilic and the
electrophoresis results showed they were negatively charged. Biodistribution in tumor-bearing
mice demonstrated that the two new complexes showed tumor accumulation, high
tumor-tomuscle (T/M) ratios and fast clearance from blood and muscle. Between the two
compounds, the 99mTcN-MAG3-ADMQ ([99mTcN]-2) showed the better characteristics,
with the tumor/muscle and tumor/blood ratios reached 2.11 and 1.90 at 60 min
post-injection, 4.20 and 1.10 at 120 min post-injection, suggesting it could be a promising
radiotracer for SPECT tumor imaging.

Molecules 2014, 19
Keyword: [99mTcN]2+ complex; 4-aminoquinazoline; EGFR; tumor imaging
5509
1. Introduction
Epidermal growth factor receptor (EGFR) is a member of the epidermal growth factor (EGF) family
of tyrosine kinase receptors (which also includes ErbB2, ErbB3 and ErbB4). Ligand binding to the
extracellular domain of the receptor results in the activation of the receptor [1,2]. The activated
receptor can dimerize with other EGFRs followed by phosphorylation of tyrosine residues on the
receptors. This active conformation makes it possible for TK domain of the receptors both to bind ATP
and to transphosphorylate each other [3,4]. Receptor autophosphorylation on tyrosine residues both
enhances the activity of kinases and provides docking sites for downstream signal transduction
molecules that habor SH2 or PTB domains. These interaction activate signal transduction pathways,
which ultimately lead to multiple cellular processes, such as proliferation, differentiation, apotosis,
angiogenesis, cell adhesion and movement [5–7].
Correlation between EGFR overexpression and metastasis foramation, therapy resistance, poor
prognosis, and short survival has prompted the design and development of various anti-EGFR-targeted
therapies [8–12]. Several quinazoline derivatives have been used as tyrosine kinase inhibitors targeting
EGFR, such as gefitinib, erlotinib and lapatinib [13–16]. However, analysis of the expression of EGFR and
the presence of mutations requires a tumor biopsy, which is not possible to get in all situations [17–20].
Thus, the ability to noninavasively quantitate EGFR content in tumors would aid in selecting patients
who are likely to benefit from anti-EGFR-targeted therapy and in monitoring such treatment. Therefore,
there has been a growing interest in the use of EGFR-TK inhibitors as radiotracers for molecular imaging
of EGFR overexpressing tumors via nuclear medicine modalities such as SPCET and PET [21–25].
The [99mTcN]2+ core exhibits a very high chemical stability and high affinity toward chelating ligands
containing sulfur atoms which when present in the molecular structure of a radiopharmaceutical may
dramatically affect its physical and biological behaviour [23,26]. Based on the good performance of
quinazoline derivatives in anticancer applications, we have designed and synthesized 4-(2-aminoethyl-
amino)-6,7-dimethoxyquinazoline (ADMQ, 8) by introducing a solubilizing basic ethylenediamine
moiety at the 4-position of 4-chloro-6, 7-dimethoxyquinazoline (7) to link to the target molecules with
MAG or MAG3, thus affording a new N3S type ligand. So far, the use of the ADMQ moiety 8 in the
preparation of 99mTcN complexes as targeted agents for tumor imaging has not been reported. Herein,
99mTcN-MAG-ADMQ ([99mTcN]-1) and 99mTcN-MAG3-ADMQ ([99mTcN]-2) were designed and
synthesized to evaluate the feasibility of the 99mTc-labeled 4-aminoquinazoline derivatives as useful
candidates for tumor imaging.
2. Results and Discussion
2.1. Chemistry
The synthesis of 4-(2-aminoethylamino)-6,7-dimethoxyquinazoline was carried out using the
procedure shown in Scheme 1. A solution of compound 3 in AcOH was added to nitric acid solution

Molecules 2014, 19
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under stirring at 0–5 °C for 30 min and then at room temperature for 24 h to obtain 4. Compound 4
was hydrogenated with Pd/C in methanol at room temperature to obtain 5. Compound 5 was reacted
with formamide at 165–170 °C under N2 to obtain 6, which was treated with thionyl chloride and DMF
to get chloro derivative 7 and then 7 coupled with ethylenediamine in i-PrOH at 80 °C to obtain 8. The
synthesis of compounds 9–12 is shown in Scheme 2. Compound 9 in which the thiol group was
protected by trityl chloride in advance was reacted with N-hydroxysuccinimide (NHS) using
dicyclohexylcarbodiimide (DCC) as condensation reagent to obtain the active ester 10. The active ester
10 was reacted with the amine group of glycine to provide the Tr-MAG (11).
Scheme 1. Synthesis of ADMQ.
MeO
MeO
COOEt
NO2
MeO
MeO
COOEt
MeO
MeO
COOEt
NH2
b
c
a
5
4
3
6
NH2
O
N
Cl
N
N
HN
N
MeO
MeO
MeO
MeO
MeO
MeO
NH
d
e
N
7
8
Reagents and conditions: (a) AcOH, HNO3, 0–5 °C to rt, 24 h, 90%; (b) Pd/C, H2, rt, 8 h, 86%; (c) HCONH2,
165–170 °C, 6 h, 73%; (d) SOCl2, DMF, reflux, 4 h, 82%; (e) NH2CH2CH2NH2, i-PrOH, 80 °C, 4 h, 81%.
Scheme 2. Synthesis of Tr-MAG and Tr-MAG3.
O
O
O
O N
O
NH
O
OH
OH
a
b
O
STr
STr
STr
9
10
11
c
O
HN
HN
O
NH
O
STr
COOH
12
Reagents and conditions: (a) NHS, DCC, anhydrous THF, −5 °C to rt, 10 h, 82%; (b) CH3CN, glycine,
50–60 °C, 2 h, 78%; (c) CH3CN, triglycine, 50–60 °C, 2 h, 71%.
Tr-MAG3 (12) was synthesized by the same procedure as 11, except using triglycine instead of
glycine. Tr-MAG (11) was conjugated with compound 8 with DCC as condensation agent and
4-dimethylaminopyridine (DMAP) as nucleophilic agent to provide the compound 13. Tr-MAG3 (12)
was conjugated with compound 8 under the same conditions to give compound 14. Next, the thiol
groups were deprotected in trifluoroacetic acid (TFA) to obtain 1. Compound 2 was prepared using the
same method as product 1.

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2.2. Radiochemistry
2.2.1. Synthesis of 99mTcN-MAG-ADMQ and 99mTcN-MAG3-ADMQ
Synthesis of the two compounds [99mTcN]-1 and [99mTcN]-2 was performed according to the
procedure shown in Schemes 3 and 4.
Scheme 3. Synthesis of 99mTcN-MAG-ADMQ.
O
O
O
O
NH
SH
O
N
S
O
NH
N
O
HN
N
HN
NH2
O
HN
N
Tc
NH
OH
MeO
MeO
b
c
STr
HN
N
N
HN
N
a
N
MeO
MeO
MeO
MeO
+
MeO
MeO
N
N
N
STr
N
[
99mTcN]-1
11
13
1
8
Reagents and conditions: (a) HOBt, DCC, Et3N, anhydrous CH2Cl2, 0 °C to rt, 10 h, 61%; (b) Et3SiH, TFA,
rt, 5 min; (c) 99mTc-SDC, SnCl2, PDTA, rt to 100 °C, 30 min.
Scheme 4. Synthesis of 99mTcN-MAG3-ADMQ.
O
O
NH
HN
HN
O
HN
HN
NH2
STr
O
HN
N
H
N
O
NH
MeO
MeO
a
N
HN
N
+
O
O
MeO
MeO
STr
N
COOH
8
14
12
O
O
O
O
NH
SH
H
N
N
S
H
N
HN
HN
N
N
Tc
O
N
O
b
c
HN
HN
O
O
MeO
MeO
MeO
MeO
N
N
N
N
[
99mTc-N]-2
2
Reagents and conditions: (a) HOBt, DCC, Et3N, anhydrous CH2Cl2, 0 °C to rt, 10 h, 60%; (b) Et3SiH, TFA,
rt, 5 min; (c) 99mTc-SDC, SnCl2, PDTA, rt to 100 °C, 30 min.
For labeling, [99mTcN]-1 was prepared through a SDH kit. [99mTcO4]− 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 compound 1 at 100 °C for 15 min to
give the final complex [99mTcN]-1. The preparation of [99mTcN]-2 was analogous to that for [99mTcN]-1
given above, except that the ligand used was compound 2. Early experimental anionic compounds such
as [99gTcO(MAG3)]−, [99gTcO(MAG3OMe)] and [ReO(MAG2-pABAH)]− established the structure

Molecules 2014, 19
5512
model using the N3S ligand system [27,28]. It is reasonable to suppose that the structure of the two
complexes in this paper might be similar to those that have been reported in the previous work for the
same N3S ligand system was employed to coordinate to 99mTc. The radiochemical purity of the two
complexes was routinely checked by radio-HPLC. The HPLC patterns of [99mTcN]-1 and [99mTcN]-2
are shown in Figure 1. It was observed that the retention time of [99mTcN]2+int was 1.8 min, while those
of [99mTcN]-1 and [99mTcN]-2 were found to be 4.4 min and 3.0 min, respectively. The mean
radiochemical purity of the two products was over 90% immediately after the preparation.
Figure 1. Radio-HPLC chromatograms.
A
B
C
(A) 99mTcN2+, tR = 1.8 min; (B) [99mTcN]-1, tR = 4.4 min; (C) [99mTcN]-2, tR = 3.0 min.
2.2.2. In Vitro Stability
The HPLC analysis results for the two complexes indicated that they were all stable in PBS after
incubation for 2 h.
2.2.3. Paper Electrophoresis
Paper electrophoresis showed that about 90% of the initial activity remained in the positive
electrode, indicating that they are negatively charged complexes.

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2.2.4. Partition coefficients
As far as passive diffusion into tissues and cells is concerned, the lipophilicity of the molecule
(generally denoted by log P) should be sufficiently high to allow penetration through the cell
membrane. However, high log P values usually lead to slow clearance from blood, accumulation in
metabolic tissue, and non-specific binding in tumors. The partition coefficients (log P) of [99mTcN]-1
and [99mTcN]-2 were measured according to the published method [29] (Table 1) and indicated that
both complexes were hydrophilic. [99mTcN]-2 was less hydrophilic than [99mTcN]-1.
Table 1. Partition coefficients of [99mTcN]-1 and [99mTcN]-2.
[
99mTcN]-1
−1.81 ± 0.02
[
99mTcN]-2
−1.57 ± 0.01
log P
2.2.5. Biological Evaluation
Mice were sacrificed and major organs such as brain, liver, lungs, kidneys, spleen, stomach and
heart, as well as the tumors and blood, were isolated to evaluate the tissue distribution of the two
complexes. The biodistribution results are summarized in Tables 2 and 3.
Table 2. Biodistribution of [99mTcN]-1 in mice bearing S180 tumor (%ID/g) a.
Tissue
Heart
30 min
60 min
120 min
0.27 ± 0.02
2.71 ± 0.16
0.31 ± 0.03
1.36 ± 0.13
2.42 ± 0.10
0.27 ± 0.01
0.19 ± 0.02
0.71 ± 0.22
0.31 ± 0.01
0.43 ± 0.05
0.04 ± 0.01
0.35 ± 0.03
1.09 ± 0.11
1.29 ± 0.14
1.84 ± 0.07
0.11 ± 0.01
1.01 ± 0.08
0.15 ± 0.01
0.94 ± 0.06
2.3 ± 0.07
0.05 ± 0.01
0.55 ± 0.11
0.07 ± 0.02
0.44 ± 0.09
1.35 ± 0.11
0.06 ± 0.02
0.04 ± 0.01
0.51 ± 0.10
0.18 ± 0.03
0.07 ± 0.03
0.01 ± 0.01
0.07 ± 0.01
0.11 ± 0.04
1.17 ± 0.13
1.75 ± 0.16
Liver
Spleen
Lung
Kidney
Muscle
0.07 ± 0.01
0.16 ± 0.04
0.79 ± 0.12
0.25 ± 0.03
0.04 ± 0.01
0.03 ± 0.02
0.31 ± 0.04
0.82 ± 0.03
1.82 ± 0.33
1.94 ± 0.24
Bone
Large Intestine
Small Intestine
Stomach
Brain
Tumor
Blood
T/M ratio
T/B ratio
T/M = tumor-to-muscle, T/B = tumor-to-blood; a All data are the mean percentage (n = 3) of the injected dose
of [99mTcN]-1; per gram of tissue, ± the standard deviation of the mean.
There were apparent similarities in the biodistribution patterns of the two compounds that
demonstrated tumor accumulation, high tumor-to-muscle (T/M) ratio, tumor-to-blood (T/B) ratio and
rapid washout from blood. Early hepatic and renal activity reflected the two compounds were excreted
through the hepatobiliary as well as the renal system. The clearance rate in the tumor was slower than
in other tissues or organs with time, so the activity in the tumor exceeded than that in most other tissue
or organs. For [99mTcN]-1, the tumor-to-muscle (T/M) and tumor-to-blood (T/B) ratios reached 1.82

Molecules 2014, 19
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and 1.94 at 60 min, 1.17 and 1.75 at 120 min post-injection, respectively. For [99mTcN]-2, the
tumor-to-muscle (T/M) and tumor-to-blood (T/B) ratios reached 2.11 and 1.90 at 60 min, 4.20 and
1.10 at 120 min, respectively. Between them, [99mTcN]-2 showed the better characteristics with the
higher tumor/muscle ratio that reached to 4.2 at 120 min. The two compounds were hydrophilic so that
they were unable to cross the blood brain barrier, thus making their brain uptake much lower. It is
deemed worthwhile to modify the structure of ADMQ suitably to render it less hydrophilic to enhance
the tumor uptake of its 99mTc labeled complex.
Table 3. Biodistribution of [99mTcN]-2 in mice bearing S180 tumor (%ID/g) b.
Tissue
Heart
30 min
60 min
120 min
0.08 ± 0.01
0.39 ± 0.05
0.05 ± 0.01
0.14 ± 0.04
1.65 ± 0.16
0.05 ± 0.02
0.20 ± 0.01
1.22 ± 0.24
0.20 ± 0.05
0.04 ± 0.02
0.01 ± 0.005
0.21 ± 0.08
0.13 ± 0.06
4.20 ± 0.25
1.10 ± 0.08
0.40 ± 0.05
0.78 ± 0.09
0.16 ± 0.01
0.50 ± 0.11
1.64 ± 0.15
0.25 ± 0.06
0.55 ± 0.15
0.39 ± 0.17
0.99 ± 0.14
0.15 ± 0.03
0.02 ± 0.01
0.21 ± 0.07
0.96 ± 0.23
0.84 ± 0.09
0.38 ± 0.05
0.12 ± 0.03
0.79 ± 0.04
0.08 ± 0.02
0.30 ± 0.08
1.97 ± 0.21
0.19 ± 0.02
0.21 ± 0.06
0.51 ± 0.04
0.56 ± 0.09
0.14 ± 0.07
0.01 ± 0.01
0.40 ± 0.05
0.87 ± 0.16
2.11 ± 0.18
1.90 ± 0.16
Liver
Spleen
Lung
Kidney
Muscle
Bone
Large Intestine
Small Intestine
Stomach
Brain
Tumor
Blood
T/M ratio
T/B ratio
b
T/M = tumor-to-muscle, T/B = tumor-to-blood; All data are the mean percentage (n = 3) of the injected
dose of [99mTcN]-2; per gram of tissue, ± the standard deviation of the mean.
3. Experimental
3.1. General Information
Succinic dihydrazide (SDH) kit was obtained from BeijingShihong Pharmaceutical Center, Beijing
99
Normal University (Beijing, China). Mo/99mTc generator was obtained from the China Institute of
Atomic Energy (CIAE) (Beijing, China). THF was refluxed over sodium/benzophenone and CH2Cl2
was refluxed over phosphorous CaH2. Other solvents were purchased as anhydrous. 1H- and 13C-NMR
spectra were recorded on a Bruker model (Bruker, Karlsruhe, Germany) spectrometer operating in
DMSO-d6 or CDCl3 at 400 MHz and 100 MHz, respectively. 1H signals were reported in ppm. The IR
spectra were recorded using KBr pellets in the 4,000-400 cm−1 region on a Nicolet-AVATAR 360
FT-IR spectrometer (Nicolet-AVATAR, Belmont, MA, USA). ESI-MS spectra were obtained on
Waters LCT Premier XE (Waters, Milford, MA, USA). HPLC analyses were performed on a
Shimadzu SCL-10 AVP (Shimadzu, Kyoto, Japan) equipped with a Packard 500 TR series flow
scintillation analyzer (Shimadzu) A C-18 reversed-phase Alltima column (5 μm, 150 mm × 4.6 mm)
was used for radiochemical purity analysis.

Molecules 2014, 19
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3.2. Synthesis
The preparation of compounds 3–8 was carried out by the similar procedure described in [30,31]
with some modifications. The synthesis of compounds 9–12 was carried out by the same procedure
described in [32]. The preparation and the analysis data of the compounds are shown below.
Ethyl 4,5-dimethoxy-2-nitrobenzoate (4). A solution of ethyl 3,4-dimethoxybenoate (6.0 g, 0.026 mol)
in AcOH (23 mL) was added dropwise to nitric acid (6 mL, 65%) at 0–5 °C, and stirred at the same
temperature for 30 min and then for 24 h at room temperature. Reaction progress was monitored by
TLC and it was found to be complete after this time. The reaction mixture was poured onto ice/water
to afford the yellow precipitate which was filtered and washed with ice water. The precipitate dried
over P2O5 to afford the product 4 (5.91 g, yield 90.3%), m.p.: 78–79 °C.; IR (cm−1): ν 3451, 2983,
1
1722, 1609, 1536, 1389, 1361, 1294, 1219, 1016, 877; H-NMR (CDCl3) δ (ppm): 7.46 (1H, s, 6-H),
7.08 (1H, s, 3-H), 4.39 (2H, q, J = 7.08 Hz, -CH2), 3.98 (6H, s, -OCH3) 1.35 (3H, t, J = 7.16 Hz,
13
-CH3). C-NMR (CDCl3) δ (ppm): 165.78 (C=O), 152.50 (5-C), 150.33 (4-C), 141.25 (2-C), 122.03
(1-C), 110.85 (6-C), 106.98 (3-C), 62.42 (-CH2), 56.61 (-OCH3), 56.57 (-OCH3), 13.74 (-CH3).
Ethyl 2-amino-4,5-dimethoxybenzoate (5). A solution of 4 (5.0 g, 0.020 mol) in methanol (30 mL) was
hydrogenated with Pd/C (1.15 g) at room temperature. Reaction development was monitored by TLC
and the reduction continued until no more hydrogen was consumed. The catalyst was filtered off and
methanol removed to afford a brown precipitate 5 which was filtered through a glass funnel and dried
under reduced pressure (3.88 g, yield 85.5%), m.p.: 76–78 °C. IR (cm−1): ν 3489, 3375, 2978, 2829,
1
1680, 1591, 1516, 1398, 1300, 1157, 1029, 785, 592; H-NMR (CDCl3) δ (ppm): 7.32 (1H, s, 6-H),
6.14 (1H, s, 3-H), 5.57 (2H, br, -NH2), 4.32 (2H, q, J = 7.08 Hz, -CH2), 3.87 (3H, s, -OCH3), 3.87 (3H,
13
s, -OCH3), 1.38 (3H, t, J = 7.12 Hz, -CH3). C-NMR (CDCl3) δ (ppm): 167.79 (C=O), 154.85 (4-C),
147.06 (2-C), 140.64 (5-C), 113.06 (6-C), 102.52 (1-C), 99.46 (3-C), 60.09 (-CH2), 56.55 (-OCH3),
55.76 (-OCH3), 14.50 (-CH3).
6,7-Dimethoxyquinazoline-4-one (6). A solution of 5 (2.5 g, 0.011 mol) in formamide (50 mL) was
heated to 165–170 °C under N2 for 6 h. When TLC indicated the absence of starting material, the
reaction mixture was cooled and the amber sticky precipitate was filtered using a sintered glass frit and
dried on the air (1.90 g, yield 73.2%), m.p.: 295–296 °C. IR (cm−1): ν 3159, 3015, 2843, 2768, 1658,
1
1610, 1504, 1439, 1357, 1271, 1078, 877, 642; H-NMR (DMSO-d6) δ (ppm): 12.03 (1H, s, -NH),
7.99 (1H, s, 2-H), 7.42 (1H, s, 5-H), 7.11 (1H, s, 8-H), 3.88(3H, s, -OCH3), 3.85 (3H, s, -OCH3);
13C-NMR (DMSO-d6) δ (ppm): 159.99 (4-C), 154.43 (7-C), 148.53 (6-C), 144.84 (2-C) 143.77 (9-C),
115.58 (10-C), 108.00 (5-C), 104.93 (8-C), 55.88 (-OCH3), 55.66 (-OCH3).
4-Chloro-6, 7-dimethoxyquinazoline (7). A stirred mixture of 6 (2.0 g, 0.010 mol), thionyl chloride
(30 mL) and N,N-dimethylformamide (0.6 mL) was heated under reflux for 4 h. The solvent was
removed in vacuo to obtain the off-white crude product and the crude product was recrystallized in
DMF to obtain the compound 7 (1.81g, yield 81.7%), m.p.: 178 °C. IR (cm−1): ν 3431, 1618, 1560,
1
1508, 1412, 1348, 1234, 1161, 968, 850, 698; H-NMR (DMSO-d6) δ (ppm): 8.88(1H, s, 2-H),
7.46(1H, s, 5-H), 7.41 (1H, s, 8-H), 4.00 (6H, s, -OCH3); 13C-NMR (DMSO-d6) δ (ppm): 158.82 (2-C),

Molecules 2014, 19
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155.05 (7-C), 149.34 (6-C), 145.86 (9-C), 138.85 (10-C), 114.73 (5-C), 105.52 (8-C), 104.11 (C-4),
56.16 (-OCH3), 55.94 (-OCH3).
4-(2-Aminoethylamino)-6, 7-dimethoxyquinazoline (ADMQ, 8). To a solution of 7 (1.0 g, 4.5 mmol) in
i-PrOH (30 mL) ethylenediamine (0.45 mL, 6.75 mmol) was added. The reaction mixture was heated
to 80 °C and stirred for 4 h and then was quenched in ice water and filtered to afford the solid product
8 (0.9 g, yield 80.6%). IR (cm−1): ν 3504, 3419, 3340, 2986, 2071, 1625, 1598, 1541, 1511, 1421, 1251,
843; 1H-NMR (DMSO-d6) δ (ppm): 8.37 (1H, s, 2-H), 8.21 (1H, s, -NH), 7.67(1H, s, 5-H), 7.11(1H, s,
8-H), 3.90 (6H, s, -OCH3), 3.75 (2H, t, J = 5.96 Hz, -CH2), 3.09 (2H, t, J = 5.96 Hz, -CH2); 13C-NMR
(DMSO-d6) δ (ppm): 158.34 (7-C), 153.82 4-C), 153.13 (2-C), 148.30 (6-C), 145.97 (9-C), 108.60
(10-C), 106.90 (8-C), 102.48 (5-C), 56.18 (-OCH3), 55.61 (-OCH3), 38.50 (2CH2); MS (ESI):
M = 248.1 (Found: 247.2); Anal. Calcd. for C12H16N4O2: C, 58.05; H, 6.50; N, 22.57%. Found: C,
57.88; H, 5.92; N, 22.62%.
Conjugation of Tr-MAG and ADMQ (Tr-MAG-ADMQ, 13). Triethylamine (0.13 mL, 0.9 mmol ) was
added dropwise into a solution of 8 (148 mg, 0.6 mmol) in anhydrous dichloromethane (5 mL) and the
mixture was stirred at room temperature for 5 min. After 11 and HOBt (90 mg, 0.66 mmol) were added
to the mixture, DCC (138 mg, 0.66 mmol) in anhydrous dichloromethane (2 mL) 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 to give a white precipitate which was removed by filtration. The organic phase was
washed with saturated aqueous sodium bicarbonate, brine and dried over anhydrous sodium sulfate.
After filtered and concentrated, the crude product was purified by column chromatography (petroleum
ether/ethyl acetate = 1:3) to afford the desired compound 13 (yield: 60.5%). m.p. 188 °C; IR (cm−1): ν
3358, 3326, 2928, 2850, 1625, 1574, 1535, 1307, 1247, 1088; 1H-NMR (DMSO-d6) δ (ppm): 8.38 (1H,
s, 2-H), 8.14 (1H, m, -NH), 8.09 (1H, m, -NH), 8.02–8.03 (1H, m, -NH), 7.54 (1H, s, 5-H), 7.24–7.26
(15H, m, Ar), 7.08 (1H, s, 8-H), 3.89 (3H, s, -OCH3), 3.86 (3H, s, -OCH3), 3.63(2H, m, -CH2),
13
3.52-3.59 (4H, m, -CH2), 2.86 (2H, s, -CH2); C-NMR (DMSO-d6) δ (ppm): 168.87 (C=O), 167.61
(C=O), 158.39 (7-C), 156.59 (4-C), 153.92 (2-C), 152.91 (6-C), 148.41 (9-C), 143.99 (3Ar-C), 129.03
(6Ar-C), 128.04 (6Ar-C), 126.77 (3Ar-C), 108.26 (10-C), 106.21 (8-C), 102.02 (5-C), 65.92 (-CH),
55.94 (-OCH3), 55.71 (-OCH3), 47.46 (-CH2), 42.35 (-CH2), 37.96 (-CH2), 35.89 (-CH2); MS (ESI):
M = 621.2 (Found: 622.2); Anal. Calcd. for C35H35N5O4S: C, 67.61; H, 5.67; N, 11.26%. Found: C,
67.58; H, 5.72; N, 11.39%.
Conjugation of Tr-MAG3 and ADMQ (Tr-MAG3-ADMQ,14). Compound 14 was using the same
procedure as for the synthesis of 13 except using Tr-MAG3 instead of Tr-MAG (yield: 60.1%). m.p.:
1
210 °C; IR (cm−1): ν 3428, 3290, 1632, 1506, 1247, 1221, 704; H-NMR (DMSO-d6) δ (ppm): 8.42
(1H, s, 2-H), 8.34 (1H, m, -NH), 8.19 (1H, m, -NH), 8.17 (1H, m, -NH), 8.11 (1H, m, -NH), 7.99 (1H,
m, -NH), 7.62 (1H, s, 5-H), 7.24–7.26 (15H, m, Ar), 7.08 (1H, s, 8-H), 3.89 (6H, s, -OCH3), 3.58-3.88
13
(10H, m, -CH2), 2.85 (2H, s, -CH2); C-NMR (DMSO-d6) δ (ppm): 169.13 (C=O), 169.09 (C=O),
167.77 (2C=O), 158.34 (4, 7-C), 153.85 (2-C), 153.10 (6-C), 148.36 (9-C), 143.95 (3Ar-C), 129.01
(6Ar-C), 128.03 (6Ar-C), 126.75 (3Ar-C), 108.35 (10-C), 106.47 (8-C), 102.01 (5-C), 65.92 (-CH),
55.92 (-OCH3), 55.64 (-OCH3), 42.31 (2CH2), 42.05 (2CH2), 38.02 (-CH2), 35.85 (-CH2); MS (ESI):

Molecules 2014, 19
5517
M = 735.3 (Found: 736.3); Anal. Calcd. for C39H41N7O6S: C, 63.66; H, 5.62; N, 13.32%. Found: C,
63.58; H, 5.71; N, 13.39%.
Synthesis of 99mTcN-MAG-ADMQ ([99mTcN]-1)
Compound 13 (5 mg) was treated with trifluoroacetic acid under cation trapping conditions (5%
triethylsilane) at room temperature for 5 min and then the solvent was removed under a stream of
nitrogen to give a residue which was neutralized with 0.1 M NaOH. The solution was extracted with
dichloromethane for three times, and then the aqueous phase was placed under nitrogen protection.
Saline (1 mL) containing [99mTcO4]− (15 MBq) 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 and then the MAG-ADMQ
solution (1 mL) was added. The reaction mixture was allowed to stand for 15 min at 100 °C to give the
final complex 99mTcN-MAG-ADMQ ([99mTcN]-1).
Synthesis of 99mTcN-MAG3- ADMQ ([99mTcN]-2)
[
99mTcN]-2 was prepared using the same method as [99mTcN]-1 except using compound 14 instead
of compound 13.
3.3. In Vitro Stability Study
Stability of the two complexes was studied using radio-HPLC analysis at different time intervals as
soon as they were prepared. After the complexes were added to test tubes containing PBS solution
(0.025 M, pH 7.4), the mixtures were incubated at 37 °C by shaking in a thermomixer. The
radiochemical purity was measured at 30 min, 60 min and 120 min.
3.4. Radio-HPLC Analysis
The formation of the two complexes was routinely determined by Radio-HPLC. Water (solvent A)
and acetonitrile (solvent B) were used for elution. For analysis of the product, the HPLC gradient
system 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. Sample (5 μL) was used for analysis. Recovery was determined by summing the
total counts in all fractions and comparing them to the total injected activity.
3.5. Paper Electrophoresis
A 1 µL sample was spotted on a piece of Whatman 1 chromatography paper (length 15 cm) which
was saturated with 0.05 M pH 7.4 phosphate buffer in an electrophoresis bath in advance. 150 V was
applied for 2 h. The strip was then dried and divided into three segments. The distribution of
radioactivity on each strip was determined.

Molecules 2014, 19
5518
3.6. Measurement of Partition Coefficients
The two complexes were each mixed with an equal volume of 1-octanol and phosphate buffer
(0.025 M, pH 7.4) in a centrifuge tube. After vortexing at room temperature for 1 min, the mixture was
centrifuged at 5,000 r/min for 5 min. The aliquot of each phase (0.1 mL) was pipetted and counted in a
well-type NaI(Tl) detector and the process was repeated three times. The partition coefficient
value was usually 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)
3.7. Biodistribution Study
Biodistribution studies of the two complexes were performed in Kunming female mice (18–22 g)
bearing S 180 tumors, which grew to a leg diameter of 10–15 mm. 99mTcN-MAG-ADMQ, and
99mTcN-MAG3-ADMQ (0.1 mL, 7.4 × 105 Bq) were injected via the tail vein and the injected
radioactivity was measured with a well-type NaI(Tl) detector, respectively. The mice were sacrificed
at 30 min, 60 min and 120 min post-injection and 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. Conclusions
In summary, 4-(2-aminoethylamino)-6,7-dimethoxyquinazoline (ADMQ) was successfully
synthesized and conjugated with MAG and MAG3, respectively. The two new compounds were
labeled with technetium-99m in high yields through a ligand exchange reaction, which was easily used
for the preparation of a radiopharmaceutical through a freeze-dried kit formulation. Moreover,
[
99mTcN]-2 demonstrates tumor accumulation, high tumor-to-muscle (T/M) ratios and rapid washout
from blood. Thus, it appears of high interest to explore the [99mTcN]-2 as a potential single photon
emission computed tomography (SPECT) tumor imaging agent.
Acknowledgments
This work is supported by the National Natural Science Foundation of China (No. 21071022) and
the Fundamental Research funds for the Central Universities. We especially thank Ms. Man Feng for
her excellent work and kindly help for what we have done in the paper.
Author Contributions
M.X.Z. synthesized all ligands and complexes and participated in all steps of the research; H.Y.N.
helped a lot in the complexes characterization; M.F. participated in all steps of the research; S.L.L. and
J.C. participated on the writing of manuscript. C.M.Q. gave the concepts of work, interpreted the
results and prepared the manuscript.

Molecules 2014, 19
5519
Conflicts of Interest
The authors declare no conflict of interest.
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