Insight into technetium amidoxime complex: Oxo technetium(V) complex of N-substituted benzamidoxime as new basic structure for molecular imaging

Article abstract of DOI:10.1021/ic101714q

In search of benzamidoxime (BHam) derivatives that provide a single 99mTc-labeled compound of high in vivo stability, we synthesized three N-alkyl compounds of benzamidoxime (BHam) ligand. They provided a single 99mTc-labeled compound by ligand exchange reaction of 99mTc-glucoheptonate in high radiochemical yields (over 95% at MBHam concentration of 1 × 10-5 M). 99mTc-N-methyl benzamidoxime (99mTc-MBHam) showed higher stability than the parental 99mTc-BHam. The complex of this compound with 99gTcO+ ion was prepared, isolated, and characterized by FT-IR and NMR spectra as well as X-ray diffraction.99gTc-MBHam crystallized in an orthorhombic space group Pna2 ₁ with a = 13.4823(5), b = 15.5410(7), c = 7.7907(3) A, V = 1632.39(11) A3, and Z =4. The 99gTc complex possessed square base pyramid coordination geometry. The equatorial plane was formed by two-amine nitrogen and two-oxime oxygen atoms in trans position, while the oxo core of the technetium(V) occupied the apical position. The 99gTc-MBHam proved to be identical with the 99mTc-MBHam prepared at the no-carrier-added level by comparison of their HPLC profiles.

Full text of DOI:10.1021/ic101714q

992 Inorg. Chem. 2011, 50, 992–998
DOI: 10.1021/ic101714q
Insight into Technetium Amidoxime Complex: Oxo Technetium(V) Complex of
N-Substituted Benzamidoxime as New Basic Structure for Molecular Imaging
Khajadpai Thipyapong,†,‡ Tomoya Uehara,‡ Yuji Tooyama,§ Henrik Braband,§ Roger Alberto,§ and
Yasushi Arano*,‡
†Department of Chemistry, Faculty of Science, Burapha University, Chonburi 20131, Thailand,
‡Graduate School of Pharmaceutical Sciences, Chiba University,1-8-1 Inohana, Chuo, Chiba 260 8675, Japan,
and §Institute of Inorganic Chemistry, University of Zu€rich, Winterthurerstrasse 190, CH-8057 Zu€rich,
Switzerland
Received August 24, 2010
In search of benzamidoxime (BHam) derivatives that provide a single 99mTc-labeled compound of high in vivo stability,
we synthesized three N-alkyl compounds of benzamidoxime (BHam) ligand. They provided a single 99mTc-labeled
compound by ligand exchange reaction of 99mTc-glucoheptonate in high radiochemical yields (over 95% at MBHam
concentration of 1 ꢀ 10-5 M). 99mTc-N-methyl benzamidoxime (99mTc-MBHam) showed higher stability than the
parental 99mTc-BHam. The complex of this compound with 99gTcO3þ ion was prepared, isolated, and characterized by
FT-IR and NMR spectra as well as X-ray diffraction. 99gTc-MBHam crystallized in an orthorhombic space group Pna21
3
with a = 13.4823(5), b = 15.5410(7), c = 7.7907(3) A, V = 1632.39(11) A , and Z = 4. The 99gTc complex possessed square
base pyramid coordination geometry. The equatorial plane was formed by two-amine nitrogen and two-oxime oxygen atoms
in trans position, while the oxo core of the technetium(V) occupied the apical position. The 99gTc-MBHam proved to be
identical with the 99mTc-MBHam prepared at the no-carrier-added level by comparison of their HPLC profiles.
˚
˚
Introduction
of 99mTc-labeled probes for molecular imaging. However,
BHam always provides two 99mTc-labeled compounds, which
hinders the application of BHam to 99mTc-coordination mole-
cules. On the other hand, tetradentate BHam compounds, N,
N0-ethylene bis(benzohydroxamamide) [(C2(BHam)2)] and
N,N0-propylene bis(benzohydroxamamide) [(C3(BHam)2)],
generated a single 99mTc-labeled compound of high stability
with over 95% radiochemical yields over a wide pH range at
ligand concentration as low as 2.5 ꢀ 10-6 M.3 The C3(BHam)2-
based bifunctional chelating agent provided 99mTc-labeled
antibodies of high stability and high specific activity under
mild conditions.4 However, characterization of 99mTc-labeled
compounds with either bidentate or tetradentate BHam
derivatives has been limited so far. Our earlier attempts to
prepare rhenium-labeled counterparts were abortive.
Molecular imaging is an emerging field that aims to integrate
patient-specific and disease-specific molecular information
derived from diagnostic imaging. This technique is also
expected to have a major economic impact on the development
of new drugs at both preclinical and clinical stages. Among a
variety of modalities, single photon emission computed
tomography (SPECT) has the advantages of high intrinsic
sensitivity, unlimited depth penetration, and wide availabil-
ity. 99mTc constitutes one of the most useful radionuclides for
the purpose because of its suitable nuclear properties and
availability from the 99Mo-99mTc generator system. Efforts
have been and are being made to develop new 99mTc-labeled
probes to promote the application.
Benzamidoxime or benzohydroxamamide (BHam) is a
thiol-free bidentate ligand that provides 99mTc-labeled com-
pounds of high stability with high radiochemical yields even
at low BHam concentrations.1,2 Such properties render
BHam attractive as a chelating molecule to prepare a variety
Previously, we used Hartree-Fock and density functional
theory (DFT) calculations to evaluate the structures of
BHam ligand and their oxo-technetium(V) complexes.5
(3) Xu, L.-C.; Nakayama, M.; Harada, K.; Nakayama, H.; Tomiguchi,
S.; Kojima, A.; Takahashi, M.; Arano, Y. Nucl. Med. Biol. 1998, 25, 295–
303.
(4) Xu, L.-C.; Nakayama, M.; Harada, K.; Kuniyasu, A.; Nakayama, H.;
Tomiguchi, S.; Kojima, A.; Takahashi, M.; Ono, M.; Arano, Y.; Saji, H.;
Yao, Z.; Sakahara, H.; Konishi, J.; Imagawa, Y. Bioconjugate Chem. 1999,
10, 9–17.
*To whom correspondence should be addressed. Phone: þ81 43 226 2896.
Fax: þ81 43 226 2897. E-mail: arano@p.chiba-u.ac.jp.
(1) Nakayama, M.; Saigo, H.; Kai, E.; Koda, A.; Ozeki, H.; Harada, K.;
Sugii, A.; Tomiguchi, S.; Kojima, A.; Hara, M.; Kinoshita, R.; Takahashi,
M. Nucl. Med. Commun. 1992, 13, 445–449.
(2) Nakayama, M.; Xu, L. C.; Koga, Y.; Harada, K.; Sugii, A.; Nakayama,
H.; Tomiguchi, S.; Kojima, A.; Ohyama, Y.; Takahashi, M.; Okabayashi, I.
Appl. Radiat. Isot. 1997, 48, 571–577.
(5) Thipyapong, K.; Arano, Y.; Ruangpornvisuti, V. J. Mol. Struct.
(THEOCHEM) 2004, 676, 65–71.
r
pubs.acs.org/IC
Published on Web 01/12/2011
2011 American Chemical Society

Article
Inorganic Chemistry, Vol. 50, No. 3, 2011 993
ligands coordinated to one TcO3þ core at the trans-position
without impairing the unique coordinating ability of BHam.
Experimental Section
General Procedures for Ligand Synthesis and Characteriza-
tion. All reactions were performed under nitrogen atmosphere
unless otherwise noted. Solvents and chemicals obtained from
commercial sources were of analytical grade or higher and were
used without further purification. BHam was synthesized accord-
ing to the procedure of Bedford et al. with slight modification.15
The melting points were measured using a Thomas-Hoover
capillary melting point apparatus and were uncorrected. The
1H and 13C NMR spectra were recorded using a JEOL ECP-500
spectrometer (JEOL, Tokyo, Japan). The chemical shifts (δ)
were reported in ppm downfield by reference to proton reso-
nances resulting from incomplete deuteration of the NMR
solvent. The elemental analyses were performed at the Chemical
Analysis Center, Chiba University (Chiba, Japan). The infrared
(IR) spectra were recorded using a Perkin-Elmer 2000 FT-IR
spectrometer (Perkin-Elmer, MA, U.S.A.). Fast atom bom-
bardment mass spectrometry (FAB-MS) was performed using
a JEOL JMS-HX-110A mass spectrometer (JEOL Ltd., Tokyo,
Japan). The mass spectrum of 99m/99gTc measurements were carried
out using an Agilent 6130 Series Quadrupole LC/MS electrospray
system equipped with diode array detector (Agilent technolo-
gies, Tokyo).
Figure 1. Structures of (A) BHam (Mw. 136, formula C7H8N2O), (B)
MBHam (Mw. 150, formula C8H10N2O), (C) EBHam (Mw. 164, formula
C9H12N2O), and (D) PBHam (Mw. 178, formula C10H14N2O).
Two hydroxyimino and ten hydroxyamino conformers of
BHam were found as tautomers. The calculation also sug-
gested that [L2TcO]þ of C2 conformer would be the most
stable. Recent study on the crystal structure of BHam showed
that BHam molecules are connected via intermolecular N-
H
O and O-H N hydrogen bonds to form a two-
3 3 3
3 3 3
dimensional supramolecular structure6 as a result of tauto-
merization.7-9 The tautomerization through migration of an
amine proton to imine leads to a formation of an imino
isomer that has lower acidity than the oximino form.10-12 In
the complex formation with technetium, this isomerization
of amidoxime may have caused a mixture of 99mTc-labeled
Synthesis of N-methyl-benzamidoxime (MBHam). The solu-
tion of 2.0 g (13.0 mmol) of benzaldehyde oximinoyl chloride16
in 20 mL of diethyl ether was cooled to 0 °C in an ice bath. Three
milliliters of 40% w/v methylamine (16.3 mmol) dissolved in a
mixture of 10 mL of diethyl ether and 2 mL of triethylamine
(∼13.0 mmol) were added slowly to the benzaldehyde oximinoyl
chloride solution at 0 °C.17 The temperature of the mixture was
maintained at 0 °C during the addition. To complete the reaction,
the mixture was stirred at room temperature for 3 h and then
diluted with 50 mL of H2O. The aqueous layer was separated
and extracted three times with 10 mL of chloroform. The combined
organic extracts were washed three times with 10 mL of water
and dried over anhydrous magnesium sulfate. After removing
the solvent in vacuo, the residue was dissolved in 20 mL of
chloroform and triturated with hexane. The solution began to
deposit white needle crystals. After standing overnight, the
crystal were collected, washed with hexane and then dried under
vacuum to yield 1.71 g (79.5%) of N-methyl-benzamidoxime. Mp:
162-163 °C (Lit.18 163 °C). FAB-MS: m/z 151 [MH]þ. 1H NMR
(CDCl3, 300 MHz): δ 2.73 (s, 3H, -CH3), 5.28 (br s, 1H, OH),
7.36-7.50 (m, 5H, Ar). 13C NMR (CDCl3, 100 MHz): δ 157.1
(CdN), 131.2, 129.4, 128.6(2C), 128.3(2C) (Ar), 30.5 (-CH3) IR
(KBr, cm-1) 3390(s), 3300-2500(br), 1650(s), 1492(m), 1350(m),
928 and 908(m) Anal. Calcd for C8H10N2O: C, 63.98; H, 6.71; N,
18.65 found C, 63.62; H, 6.63; N, 18.49.
compounds, as was also observed with 99mTc-racemic
D,L
penicillamine.13 The N,N-disubstituted aminoxime provides
only one oximino isomer.12,14 In addition, when the fractio-
nated two components of 99mTc-BHam were reanalyzed by
RP-HPLC, each component remained unchanged at pH 7.
However, two components reemerged from each component
at pH 5.2 These findings suggested that a decrease in the
number of tautomers by decreasing the number of protons in
the amino group of BHam would be favorable to provide a
single technetium complex. Thus, N-substituted amidoxime
would constitute an interesting compound to reduce tauto-
mers and generate new class of technetium complexes for
molecular imaging.
In the present study, we synthesized three N-substituted
BHam ligands, N-methyl-benzamidoxime (MBHam), N-ethyl-
benzamidoxime (EBHam), and N-propyl-benzamidoxime
(PBHam), as shown in Figure 1. The reaction of 99mTc-
glucoheptonate with the three N-substituted BHam deriva-
tives was compared to that with BHam. MBHam was selected
as ligand for further studies, and the reaction product of
[nBuN][99gTcOCl4] with MBHam was characterized by X-ray
diffraction (XRD), nuclear magnetic resonance, and infrared
spectroscopy. The gathered studies indicated that MBHam
provided a single 99mTc-labeled compound with two MBHam
Synthesis of N-ethyl-benzamidoxime (EBHam). EBHam was
synthesized following the procedure described above except that
ethylamine was used in place of methylamine. A pale yellow
viscous oil of EBHam (1.82 g) was obtained after silica gel
chromatography using 10% MeOH/CHCl3 as eluent (84.9%).
FAB-MS: m/z 165 [MH]þ. 1H NMR (CDCl3, 300 MHz): δ 1.06
(t, 3H, J=7.2 Hz, -CH3), 3.03 (d, 2H, J=6.4 Hz, -CH2-N),
5.22 (br s, 1H, OH), 7.24-7.46 (m, 5H, Ar). 13C NMR (CDCl3,
100 MHz): δ 156.3 (CdN), 131.5, 129.2, 128.3(2C), 128.1(2C)
(6) Xu, S.-Q.; Li, J.-M. Acta Crystallogr., Sect. E 2008, 64, o1469.
(7) NazIr, H.; YildIz, M.; Yilmaz, H.; Tahir, M. N.; lk, D. J. Mol. Struct.
2000, 524, 241–250.
(8) Voegel, J. J.; von Krosigk, U.; Benner, S. A. J. Org. Chem. 1993, 58,
7542–7547.
(15) Bedford, C. D.; Howd, R. A.; Dailey, O. D.; Miller, A.; Nolen,
H. W.; Kenley, R. A.; Kern, J. R.; Winterle, J. S. J. Med. Chem. 1986, 29,
2174–2183.
(16) Gennet, D.; Zard, S. Z.; Zhang, H. Chem. Commun. 2003, 1870–1871.
(17) Cho, S. Y.; Kang, S. K.; Ahn, J. H.; Ha, J. D.; Choi, J.-K.
Tetrahedron Lett. 2006, 47, 9029–9033.
(9) Alkorta, I.; Elguero, J. J. Org. Chem. 2002, 67, 1515–1519.
(10) Bushey, D. F.; Hoover, F. C. J. Org. Chem. 1980, 45, 4198–4206.
(11) Boyer, J. H.; Frints, P. J. A. J. Org. Chem. 1968, 33, 4554–4556.
(12) Ungnade, H.; Kissinger, L. J. Org. Chem. 1958, 23, 1794–1796.
(13) Johnson, D. L.; Fritzberg, A. R.; Hawkins, B. L.; Kasina, S.; Eshima,
D. Inorg. Chem. 1984, 23, 4204–4207.
(14) Katritzky, A. R.; Khashab, N. M.; Kirichenko, N.; Singh, A. J. Org.
Chem. 2006, 71, 9051–9056.
(18) Buscemi, S.; Vivona, N.; Caronna, T. J. Org. Chem. 1995, 60, 4096–
4101.

994 Inorganic Chemistry, Vol. 50, No. 3, 2011
Thipyapong et al.
(Ar), 38.2 (-CH2-), 16.4 (-CH3) IR (Thin film KBr, cm-1
)
and BHam solution of different concentrations (10-7-10-2 M).
The radiochemical yields were determined by RP- HPLC under
the isocratic conditions.
3340(s), 3300-2500(br), 1641(s), 1481(m), 1382(m), 933 and
900(m).
Synthesis of N-propyl-benzamidoxime (PBHam). PBHam was
also synthesized following the procedure described above using
propylamine. The product was isolated as 2.19 g of a pale yellow
Electrical Properties of 99mTc-Labeled Compound. Cellulose
acetate electrophoresis was performed to determine the net
charge of the 99mTc-labeled compound. Cellulose acetate strips
(Separax SP, Johko Co., Tokyo) were soaked in phosphate
buffer (pH=7.4, I=0.05) for 30 min. The strips were placed in an
electrophoresis chamber containing the same buffer. Each
sample was spotted on the strip. Each strip was run at a constant
current of 1 mA/cm for 25 min. After drying, the strips were cut
into 0.5 cm sections, and the radioactivity level of each section
was determined by a well-type gamma counter.
1
viscous liquid (91.6%). FAB-MS: m/z 179 [MH]þ. H NMR
(CDCl3, 300 MHz): δ 0.83 (t, 3H, J=7.4 Hz, -CH3), 1.42 (m,
2H, C-CH2-C), 2.95 (s, 2H, -CH2-N), 5.29 (br s, 1H, OH),
7.34-7.46 (m, 5H, Ar). 13C NMR (CDCl3, 100 MHz): δ 156.2
(CdN), 131.4, 129.0, 128.3(2C), 128.0(2C) (Ar), 45.1 (-CH2-),
24.2 (-CH2-), 10.7(-CH3) IR (Thin film KBr, cm-1) 3362(s),
3300-2500(br), 1632(s), 1459(m), 1343(m), 940 and 914(m).
General Procedures for Radioactive Material. Caution! 99gTc
is a weak β- emitter (E = 0.292 MeV; t1/2=2.12 ꢀ 105 years) and
may only be handled in laboratories approved for use of low-level
radioactivity. Sodium [99mTc]pertechnetate was daily eluted
from a commercial 99Mo/99mTc generator (Nihon Medi-Physics
Co. Ltd., Nishinomiya, Japan or Mallinckrodt-Tyco Inc., Petten,
Netherlands). Ammonium [99gTc]pertechnetate was purchased
from Oak Ridge National Laboratory (TN, U.S.A.). Tetrabu-
tylammonium tetrachlorooxotechnetate (TBA[99gTcOCl4]) was
synthesized following the published procedure.19 The in-house
lyophilized kit containing 4 mg of glucoheptonate (GH) and 1.2
Preparation of 99m/99gTc-Carrier for Mass Analysis. A lyoph-
ilized GH kit containing 0.015 mg of SnCl2 2H2O and 10 mg of
3
sodium glucoheptonate was reacted with 200 μL (concentrated
by MEK extraction of a solution of 99m/99gTcO4- elute from a
100 mCi 99Mo-99mTc generator that had not been eluted for a
week). The 99m/99gTc-GH (100 μL) was then reacted with a same
volume of MBHam (1 ꢀ 10-2 M), and 99m/99gTc-MBHam
(20 μL) was purified by RP-HPLC under the isocratic systems.
The 99m/99gTc-MBHam fractions (4.0 to 5.0 min; 1 mL) were
collected. After removing methanol at room temperature under
N2 flow, the 20 μL solution was subjected to LC/MS analysis.
The same procedure was conducted to determine 99m/99gTc-BHam.
The fractions of the two species were collected at 3.5-4.5 and
4.5-5.5 min.
μg of SnCl2 2H2O was prepared using ultrapure water (Milli Q,
3
Millipore Japan, Tokyo) at pH 9 and used throughout this
experiment. The labeling reactions were performed for all solu-
tions under nitrogen atmosphere. The reversed-phase HPLC
(RP-HPLC) analysis was carried out with a Cosmosil 5C18-AR-
300 column (4.6 ꢀ 150 mm2, Nacalai Tesque, Kyoto, Japan)
under isocratic conditions using a mixture of 50% solvent A
(5 mM phosphate buffer pH 7) and 50% solvent B (methanol) at a
flow rate of 1 mL/min. The eluent was monitored with an online
UV/visible single beam spectroscopy (Model 100-10, Hitachi
Co. Ltd., Tokyo) and a radiodetector (Gibi Star, Raytest,
Straubenhardt, Germany). The RP-HPLC was also conducted
with a Nucleosil 100-5 C18 column (3 ꢀ 250 mm2, Macherey-
Nagel AGC, Oensingen, Switzerland) at a flow rate of 0.5 mL/min,
with a gradient mobile phase starting from 100% A (50 mM
triethylammonium phosphate buffer pH 2.5) and 0% B (aceto-
nitrile) to 0% A and 100% B at 30 min. The eluent was monitored
with an online ultraviolet detector (250 nm) and a radiodetector
(Model LB 508, EG&G Berthold LB, Regensdorf, Switzerland).
The 1H NMR spectra were recorded using a Varian Gemini 300
(Varian, CA, U.S.A.). The infrared (IR) spectra were recorded
using a Perkin-Elmer BX II IR spectrometer (Perkin-Elmer,
MA, U.S.A.).
Synthesis of 99gTc-MBHam. Method A. To a 2 mL solution
of TBA[99gTcOCl4] (42.8 mg; 0.086 mmol) dissolved in acetoni-
trile was dropwise added a 4 mL solution of MBHam (64.4 mg;
0.43 mmol; 5 equiv) in acetonitrile. The reaction mixture was
kept stirring for 1.5 h under N2 atmosphere. The yellow powder
of 99gTc-MBHam (24.8 mg; 69.9%) was collected and washed
with water. IR (KBr, cm-1): 3300-2500 (br), 1656 (s), 1348 (m),
1286 (w), 1196 (w), and 957 (m). 1H NMR (DMSO: ppm) 3.36 (s,
6H, CH3), 7.59 (m, 10H, Ar).
Method B. TBA[99gTcOCl4] (17.2 mg; 0.034 mmol) and
MBHam (26 mg; 0.17 mmol; 5 equiv) were dissolved separately
in 2 mL of methanol. The MBHam solution was slowly added to
the (TBA)[99gTcOCl4] solution without stirring. Afterward, the
mixture was left for a few days, allowing slow diffusion of the
two components. This produced the orange crystals suitable for
X-ray crystallography.
Synthesis of 99gTc-BHam. The precursor, 99gTc-ethylenegly-
col was prepared by in situ. Ten drops of triethylamine were
added to a mixture of TBA[99gTcOCl4] (160 mg, 0.32 mmol) and
ethyleneglycol (10 drops) in THF (7 mL). 99gTc-ethyleneglycol
was obtained as a violet solution. After removing the chloride
salt by filtration, a solution of BHam (82 mg, 0.64 mmol,
2 equiv) in THF (15 mL) was added dropwise to the 99gTc-
ethyleneglycol solution. The reaction mixture was continuously
stirred for 3 h under N2 atmosphere. During these times, the
color of the solution changed from violet to deep orange. After
removal of the solvent in vacuo, the residue was dissolved in
methanol (2 mL). Brown solid (65.1 mg, 52.9%) was precipi-
tated by H2O (2 mL) treatment. IR (KB, cm-1): 3300-2500 (br),
1635 (w), 1480 (m), 1366 (s), and 948 (m). 1H NMR (DMSO: ppm)
7.43-8.98 (br, 10H, Ar).
Preparation of 99mTc-Labeled Compound. The lyophilized Sn-
GH kit (4 mg) was reconstituted with 100 μL of oxygen-free
0.9% normal saline, sonicated and N2-purged for 15 min before
use. Twenty microliters (0.8 mg Sn-GH) of the solution were
mixed with a solution of 1 mL of Na[99mTcO4]. After incubation
at 25 °C for 30 min, 99mTc-glucoheptonate (99mTc-GH) was
obtained. Radiochemical yields were determined by TLC (Silica
gel 60 F254, Merck Ltd.) with 100% acetone or 100% saline as
the mobile phases. The chemical purity of 99mTc-GH higher
than 99% was use for the subsequent reaction. The 99mTc-GH
solution (100 μL) was mixed with a same volume of oxygen-free
MBHam (1 ꢀ 10-2 M) in 0.02 M phosphate buffer solution (pH 7),
and the reaction mixture was kept for 30 min at room tempera-
ture. The radiochemical yields were determined by RP-HPLC
under the isocratic conditions mentioned above. To estimate the
stability, the eluent of the major radioactive peak was collected
and reinjected after 30 min. The similar labeling procedure was
employed for 99mTc-BHam, 99mTc-EBHam, and 99mTc-PBHam.
Effects of Ligand Concentration. Similar to the procedure
described above, the 99mTc-GH was added to a series of MBHam
X-ray Diffraction (XRD). The XRD data of the complexes
were collected at 183(2) K using an Oxford Diffraction Xcalibur
system (Oxfordshire, U.K.) equipped with a Ruby detector and
˚
graphite-monochromated Mo-KR radiation (λ = 0.7107 A).
The suitable crystals were covered with oil (Infineum V8512,
formerly known as Paratone N), mounted on the top of the glass
fiber and immediately transferred to the diffractometer. The
CrysalisPro program was used for the data collection, semiem-
pirical absorption correction, and data reduction.20 The crystal
(19) Preetz, W.; Peters, G. Z. Naturforsch. 1980, 35B, 1355–1358.
(20) CrysAlis PRO, 171.32; Oxford Diffraction Ltd.: Oxford, U.K., 2009.

Article
Inorganic Chemistry, Vol. 50, No. 3, 2011 995
Figure 2. Radiochromatograms of 99mTc-labeled BHam derivatives:
99mTc-BHam (retention times = 3.88 and 4.96 min), 99mTc-MBHam
(retention time = 4.67 min), 99mTc- EBHam (retention times = 7.01 min)
and 99mTc-PBHam (retention times = 16.15 min).
Scheme 1
Figure 3. Percent radiochemical yields of 99mTc-BHam (9) and 99mTc-
MBHam (b) as a function of the logarithmic ligand concentration.
conditions. Contrary to 99mTc-BHam, 99mTc-MBHam,
99mTc-EBHam, and 99mTc-PBHam generated a single peak
at the retention times of 4.67 min, 7.01 min, and 16.15,
respectively. The incorporation of two methyl groups at the
amine group of BHam failed to produce 99mTc-labeled
compounds of high stability (data not shown). These results
suggested that N-monoalkylation of BHam would be useful
to produce a single 99mTc-labeled compound. To further
estimate the effect of N-alkylation on the chelating ability of
BHam and to elucidate the role played by the chemical
modification on the formation of a single 99mTc-labeled
compound, further studies were conducted with BHam and
MBHam.
structures were solved by direct methods using Sir9721 and refined
by full-matrix least-squares methods on F2 using Shelxl-97.22
The structures were checked for higher symmetry using Platon23
Results and Discussion
Synthesis of N-alkyl substituted BHam Ligand. MBHam
were prepared by reacting methylamine with phenyl chloro-
oxime that was obtained from the chlorination of benzal-
doxime with N-chlorosuccinimide (NCS) in dimethylfor-
mamide (DMF).24 The synthetic route for the MBHam
ligand is illustrated in Scheme 1. By replacing the methyla-
mine with ethylamine or n-propylamine, EBHam or PBHam
was obtained in high yields. MBHam was a white solid,
whereas EBHam and PBHam were yellow viscous oils.
The 1H and 13C NMR spectra showed that all compounds
were in one isomer (Z). The IR spectra for all compounds
registered a number of characteristic absorption bands. The
Figure 3 shows the radiochemical yields of 99mTc-
BHam and 99mTc-MBHam as a function of ligand con-
centration. For 99mTc-BHam, the radiochemical yields of
the two radiolabeled species were combined and pre-
sented as radiochemical yields. MBHam generated a
99mTc-labeled compound in higher radiochemical yields
than BHam at low ligand concentration. Moreover, the
radiochemical yields of 99mTc-MBHam at MBHam con-
centration below 1 ꢀ 10-5 M were similar to those of the
tetradentate ligand, C3(BHam)2.1-3 Figure 4 compares
the % intact 99mTc-labeled species remaining at room
temperature as a function of post-labeling time when both
99mTc-MBHam and 99mTc-BHam were prepared at ligand
concentrations of 10-5 M. This indicated that 99mTc-
MBHam possesses higher stability than 99mTc-BHam.
Furthermore, no changes in RP-HPLC radiochromato-
grams were observed after removing the excess of MBHam
ligand from 99mTc-MBHam by RP-HPLC and reana-
lyzed under the isocratic conditions (the retention times
of 99mTc-MBHam and MBHam were 4.68 and 2.8 min,
respectively under the isocratic conditions). The gathered
findings confirmed that N-methylation provided BHam
an ability to form a single 99mTc-labeled compound while
enhancing the unique chelating properties. The electro-
phoresis analysis showed that 99mTc-MBHam possessed
net neutral charge, as also observed with 99mTc-BHam,
99mTc-C3(BHam)2, and 99mTc-C2(BHam)2.2,3 The mass
spectroscopies of 99m/99gTc-MBHam registered the most
oxime group showed a broad band at about 3350 cm-1
,
corresponding to O-H vibrations, and the amine showed
a sharp absorption band of N-H stretching at ∼3300-
3400 cm-1. The ν CdN energy of 1630 ( 20 cm-1 is typical
for aryl amidoximes.25 In FAB-MS analyses, the molecular
ion peaks at m/z=151, 165, and 179 were due to molecular
mass of MBHam, EBHam, and PBHam, respectively. The
peaks at m/z=135, 149, and 163 were attributable to loss of
the OH moiety from the molecular ion. The other fragment
at m/z=104 and 77 arose from the loss of azomethane and
alkyl group (R=-CH3, -CH2CH3, and -CH2CH2CH3).
For all compounds, the molecular ion peak was also the
base ion peak.
99mTc-Labeled Compounds. Figure 2 shows the RP-HPLC
radiochromatograms of 99mTc-BHam, 99mTc-MBHam,
99mTc-EBHam, and 99mTc-PBHam under the isocratic
(21) Altomare, A.; Burla, M. C.; Camalli, M.; Cascarano, G. L.; Giacovazzo,
C.; Guagliardi, A.; Moliterni, A. G. G.; Polidori, G.; Spagna, R. J. Appl.
Crystallogr. 1999, 32, 115–119.
(22) Sheldrick, G. M. Acta Crystallogr. 2008, 64, 112–122.
(23) Spek, A. J. Appl. Crystallogr. 2003, 36, 7–13.
(24) Kumar, V.; Kaushik, M. P. Tetrahedron Lett. 2006, 47, 1457–1460.
(25) Dondoni, A.; Barbaro, G.; Battaglia, A. J. Org. Chem. 1977, 42,
3372–3377.

996 Inorganic Chemistry, Vol. 50, No. 3, 2011
Thipyapong et al.
Table 1. IR Absorption of Free Ligand and Its 99gTc-MBHam in the Range of
4000-400 cm-1 a
IR absorption (ν, cm-1
)
MBHam
99gTc-MBHam
bond
3300-2500 (br)
3390 (s)
1650 (s)
1350 (m)
N.D.
3300-2500 (br)
N.D.
1656 (s)
1348 (m)
957 (m)
O-H stretch
N-H stretch
CdN stretch
C-N stretch
TcdO stretch
a Abbreviations: s = strong, m = medium, w = weak, br = broad.
Figure 4. Percent radiochemical purity of 99mTc-BHam (9) and 99mTc-
MBHam (b) as a function of time when the ligand concentration was
fixed at 10-5 M.
Figure 5. Molecular structure of 99gTc-MBHam with atom-labeling
scheme. Hydrogen atoms are omitted for clarity.
intensive signal at m/z = 413 while the two /
99m 99gTc-
BHam showed the most intensive signal at m/z=385. This
suggested a formulation of [TcO(L-2)(LH-1)] for both
99mTc-MBHam[C16H17N4O3Tc] and 99mTc-BHam [C14H13-
N4O3Tc], which supported the earlier speculation for the
two species of 99mTc-labeled BHam compounds.1,2
ligands.26,27 Owing to functional groups in MBHam,
the other bands were also observed. The broad and intense
band observed in 3300-2500 cm-1 as the stretching
vibrations of the O-H groups suggested the deposition
of solvent (H2O) on complex to form hydrogen bonds.
The strong bands were also observed in the range 1656
and 1348 cm-1 resulting from the CdN and C-N groups,
respectively. The new bands appearing at 1286 and
1196 cm-1 were assigned to Tc-N and Tc-O vibration of
a chelate ring.
Structural Elucidation of 99gTc-MBHam and 99gTc-BHam
Complexes. All complexes were soluble in dimethyl sulf-
oxide (DMSO) and 0.1% trifluoroacetic acid (TFA)/
methanol, but almost insoluble in other organic solvents
such as methanol, ethanol, acetonitrile, THF, ether, and
dichloromethane. Therefore, 99gTc-complexes in 0.1%
TFA/methanol solution were prepared and analyzed by
the RP-HPLC under the gradient conditions. A mixture
of an acidic eluting solvent (50 mM triethylammonium
phosphate buffer pH 2.5) and acetonitrile was used to
enhance the solubility of the carrier 99gTc-complexes. The
retention time for 99gTc-MBHam probed by the UV detec-
tor was at 19.73 min, and 99gTc-BHam was at 19.45 and
25.82 min. They were similar to those of 99mTc-MBHam
(20.12 min) and 99mTc-BHam (21.03 and 26.92 min)
monitored by a radioactive detector. This suggested that
the structures of 99gTc complexes were equivalent to those
of 99mTc-labeled compounds prepared at very low tech-
netium concentration. The relative peak areas of the two
99gTc-BHam were found to be 32.2% for the earlier complex
(21.03 min) and 67.8% for the latter complex (26.92 min).
Crystal Structures. Method B was found to be useful to
produce single crystals of 99gTc-MBHam of XRD quality.
The perspective XRD crystal structure of 99gTc-MBHam
is shown in Figure 5. The crystal data and structure
refinement parameters for 99gTc-MBHam are presented
in Table 2. The selected bond distances and bond angles
are listed in Table 3. 99gTc-MBHam crystallized in the
orthorhombic system in space group Pna21. The metal
atom is coordinated by three oxygen and two nitrogen
atoms in a square pyramid, with an oxygen atom at the
apex and the other atoms forming a basal plane. The Tc
atom is displaced from the basal plane defined by the
N(9), N(36), O(3), and O(9) atoms toward O(12) by 0.706
A. 99gTc is coordinated by two MBHam ligands with the
˚
N-hydroxyl oxygen atoms being at the trans position. The
˚
TcdO (oxygen core) bonds (1.610 A) were shorter than
1
The H NMR spectrum of 99gTc-MBHam prepared
the Tc-O (N-hydroxyl oxygen atoms) bonds (1.932-
from Method A showed a singlet peak at δ=3.36 due to
the N-methyl group, suggesting that the two N-methyl
groups of MBHam were away at the trans-position. Table 1
shows representative IR absorptions of MBHam and its
Tc-complex. The IR spectrum of 99gTc-MBHam showed
a vital band at 957 cm-1 attributable to TcdO double
bond. This value was comparable to those observed for
other oxo complexes of Tc containing σ- and π-donating
˚
1.940 A). The C-N single and double bond distances in
˚
the coordinated ligands ranged 1.37-1.39 A and 1.26-
1.29, respectively. They were similar to those of CdN (C
sp2-N sp2) in non chelate BHam6 and its derivatives.28,29
˚
The short distance of Tc-N bond on N(36) 1.968 A and
N(9) 2.001 A indicated deprotonation of the amine
donor, as also observed with TcO-amine oxime and
˚
(26) Cattabriga, M.; Marchi, A.; Marvelli, L.; Rossi, R.; Vertuani, G.;
Pecoraro, R.; Scatturin, A.; Bertolasi, V.; Ferretti, V. J. Chem. Soc., Dalton
Trans. 1998, 1453–1460.
(27) Jurisson, S.; Schlemper, E. O.; Troutner, D. E.; Canning, L. R.;
Nowotnik, D. P.; Neirinckx, R. D. Inorg. Chem. 1986, 25, 543–549.
(28) Srivastava, R. M.; Brinn, I. M.; Machuca-Herrera, J. O.; Faria,
H. B.; Carpenter, G. B.; Andrade, D.; Venkatesh, C. G.; F. de Morais,
L. c. P. J. Mol. Struct. 1997, 406, 159–167.
(29) Buzykin, B. I.; Dokuchaev, A. S.; Kharitonova, O. A. Russ. Chem.
Bull. 1995, 44, 1456–1459.

Article
Inorganic Chemistry, Vol. 50, No. 3, 2011 997
Table 2. Crystal Data and Structure Refinement for 99gTc-MBHam
C16H15FN4O3Tc
428.32
orthorhombic
Pna21
13.4823(5)
15.5410(7)
7.7907(3)
90.00
90.00
90.00
formula mass
crystal system
space group
˚
a, A
˚
b, A
˚
c, A
R, deg
β, deg
γ, deg
unit cell volume, A
Z
3
˚
1632.39(11)
4
Figure 6. One-dimensional intermolecular hydrogen bond network of
99gTc-MBHam.
temperature, K
radiation type
183(2)
MoKR
0.917
8075
3173
0.0467
0.0694
0.1667
0.0992
0.1764
0.968
absorption coefficient, μ/mm-1
no. of reflections measured
no. of independent reflections
Rint
Scheme 2
final R1 values (I > 2σ(I))
final wR(F2) values (I > 2σ(I))
final R1 values (all data)
final wR(F2) values (all data)
goodness of fit on F2
Table 3. Selected Bond Distances (A) and Angles (deg) for Compound 99gTc-
˚
MBHam
˚
Bond Lengths (A)
Tc (1)-N (9)
Tc (1)-O (12)
Tc (1)-O (3)
Tc (1)-O (9)
Tc (1)-N (36)
2.001
1.610
1.932
1.940
1.967
C(34)-N(11)
C (34)- N (36)
C (34)-C (13)
C (32)-N (10)
C (32)-N (9)
C (32)-C (20)
1.261
1.395
1.488
1.295
1.371
1.449
Bond Angles (deg)
O (12)-Tc (1)-O (3)
O (12)-Tc (1)-O (9)
O (12)-Tc (1)-N (36)
O (12)-Tc (1)-N (9)
O (3)-Tc (1)-O (9)
111.24
109.31
111.75
110.71
139.44
O (3)-Tc (1)-N (36)
78.57
86.90
85.80
79.84
137.53
O (3)-Tc (1)-N (9)
O (9)-Tc (1)-N (36)
O (9)-Tc (1)-N (9)
N (36)-Tc (1)-N (9)
TcO-mercaptoacetyltriglycine.27,30,31 Angles found for
the N(36) and N(9) center 111.91°, 120.68°, 126.60° and
126.93°, 113.33°, 119.55, respectively, were typical of
a sp2 hybridized (N-center). The smaller Tc-N-C,
113.33°, 111.91° would be attributable to 5 member ring
strain, not to the geometry of a neutral NH (sp3) donor.
These findings suggested a small degree of delocalization
of the amidoxime group in 99gTc-MBHam. This was
supported by the delocalization of the π electron over
the amidoximato (ONCN) functional as observed in
chromium, aluminum and iron amidoxime complex.32,33
Figure 6 shows intermolecular hydrogen bonds from the
C-N-H on oxime group to CdN on neighboring complex.
This N-H-N hydrogen bonding in the coordination
system was supported by tautomerism of hydrogen on the
amidoxime group of LH-1. The formula [TcO(L-2)(LH-1)]
suggested different coordination modes of the two ligands
in the coordination sphere. The X-ray studies indicated
that only a LH- provides the migration of a proton from
the amine group to the oxime group. On the other side,
the unprotonated oxime nitrogen in L-2 was a pair to
generate the hydrogen bond. The alteration between
single and adjacent double bonds accompanied by the
migration of a proton would cause the deviation of ligand
structure in the coordinated ligand.34-36 The formation
of a single Tc-MBHam complex would be attributable to
a proton migration on the coordinated ligand. The iso-
merization of MBHam by a proton migration is not
possible in the L-2 form (Scheme 2). The N-methyl
substitution of BHam not only provided a single 99mTc-
MBHam but also improved stability and radiochemical
(30) Cyr, J. E.; Nowotnik, D. P.; Pan, Y.; Gougoutas, J. Z.; Malley, M. F.;
Di Marco, J.; Nunn, A. D.; Linder, K. E. Inorg. Chem. 2001, 40, 3555–3561.
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Chimi. Acta 1991, 180, 63–67.
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A. T.; Marzilli, L. G. Inorg. Chem. 1999, 38, 5351–5358.
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998 Inorganic Chemistry, Vol. 50, No. 3, 2011
Thipyapong et al.
peptide is attached via each amine group in BHam,
the two RGD peptides in the resulting 99mTc-labeled
compound would point to each direction of the two
heavy chains of an IgG antibody, which might increase
the drive toward binding to a target molecule (e.g., RvβIII
integrin).
Conclusions
Three N-substituted BHam were found to provide a single
99mTc-labeled compound. MBHam generated a 99mTc-
labeled compound in high radiochemical yields under mild
conditions even at low ligand concentration. 99m/99gTc-
MBHam is the first Tc-amidoxime complex characterized
by IR, NMR spectroscopy, and XRD analysis. The complex
structure supports the formation of metal to ligand ratio of
1:2. The conformation of the two ligands is at the trans-
position upon forming a square-pyramid. In addition, amine
function on the bidentate ligands may provide a variety of
molecular designs for new 99mTc-labeled probes.
Figure 7. 99mTc-labeled N-substituted BHam as an IgG antibody mi-
mic. The Y-like shaped structure of 99gTc-MBHam suggests that when a
targetingmolecule is incorporatedviaeachaminegroup in BHam, the two
targeting molecules would point to each direction of the two heavy chains
of an IgG antibody.
yield of the Tc-MBHam complex. This could be attribu-
table in part to an electron-donating effect of the methyl
groups,37,38 or a delocalization degree in the amidoxime
group. The steric interference by N-alkyl group may also
be involved. Unfortunately, owing to low solubility,
99mTc-BHam mixtures could not be separated to prepare
crystals suitable for X-ray analysis. According to RP-
HPLC and spectroscopic data of 99gTc-BHam, one of the
99mTc-BHam species would be closely related to the
structure of 99mTc-MBHam. In addition, the methyl
groups in Tc-MBHam pointed to each direction of the
two heavy chains of an IgG antibody (Figure 7). This
suggests that when a targeting molecule such as RGD
Acknowledgment. We gratefully acknowledge support
of this work by Japan Society for the Promotion of
Science (JSPS) Grant NRCT-10928. This work was sup-
ported in part by a Grant-in-Aid for Scientific Research
(B) and for Exploratory Research, and Special Funds for
Education and Research (Development of SPECT Probes
for Pharmaceutical Innovation) from the Ministry of Edu-
cation, Culture, Sports, Science and Technology, Japan.
Supporting Information Available: Crystallographic data in
CIF format. Further details are given in Figures S1-S3. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(37) Percy, G. C.; Thornton, D. A. J. Inorg. Nucl. Chem. 1972, 34, 3369–
3376.
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255, 97–99.