Synthesis, characterization, and biological evaluation of M(I)(CO) 3(NNO) complexes (M = Re, 99mTc) conjugated to 2-(4-aminophenyl)benzothiazole as potential breast cancer radiopharmaceuticals

Article abstract of DOI:10.1021/jm0606387

The synthesis and biological evaluation of new M(I)(CO)₃(NNO) (M = Re, ^99mTc) complexes attached to the antitumor agent 2-(4-aminophenyl)benzothiazole are reported. The fluorescent rhenium complex enters MCF-7 breast cancer cells but

Full text of DOI:10.1021/jm0606387

5408
J. Med. Chem. 2006, 49, 5408-5410
Chart 1
Synthesis, Characterization, and Biological
Evaluation of M(I)(CO)3(NNO) Complexes (M )
Re, 99mTc) Conjugated to
2-(4-Aminophenyl)benzothiazole as Potential
Breast Cancer Radiopharmaceuticals
Stamatia Tzanopoulou,† Ioannis C. Pirmettis,‡
George Patsis,‡ Maria Paravatou-Petsotas,‡
Evangelia Livaniou,‡ Minas Papadopoulos,‡ and
Maria Pelecanou*,†
Institute of Biology and Institute of Radioisotopes and
Radiodiagnostic Products, National Centre for Scientific Research
“Demokritos”, 15310 Athens, Greece
ReceiVed May 30, 2006
Emax ) 1.07 MeV) and 188Re (t1/2 ) 0.7 day, Emax ) 2.12 MeV),
have been introduced in the field of nuclear medicine for
radiotherapy applications aiming at the delivery of therapeuti-
cally significant radiation doses to malignant lesions without
adversely affecting normal tissue.11 Attempts toward the de-
velopment of analogous complexes of 99mTc and 186Re, 188Re
as “matched pairs” for cancer imaging and targeted radiotherapy
have been reported in the literature.12
Abstract: The synthesis and biological evaluation of new M(I)(CO)3-
(NNO) (M ) Re, 99mTc) complexes attached to the antitumor agent
2-(4-aminophenyl)benzothiazole are reported. The fluorescent rhenium
complex enters MCF-7 breast cancer cells but does not enter normal
HFFF-2 and MRC-5 cells. The analogous radioactive 99mTc complex
produces fast blood and soft tissue clearance when administered to
healthy mice. These complexes are promising candidates for developing
radiopharmaceuticals for imaging (99mTc) and targeted radiotherapy
(
186Re, 188Re) of breast cancer.
Within this context and aiming at the development of
analogous technetium and rhenium complexes with affinity for
breast tumors as potential target-specific breast cancer radio-
pharmaceuticals, we report herein the synthesis of new M(I)-
(CO)3(NNO) complexes 1a (M ) Re) and 1b (M ) 99mTc)
conjugated to the antitumor agent 2-(4-aminophenyl)benzothia-
zole (2, Chart 1).13 Compound 2 elicits pronounced inhibitory
activity in vitro on the nanomolar scale against a panel of human
breast cancer cell lines with a biphasic dose response and is
the lead compound in the production of a series of potent and
selective antitumor benzothiazole agents that culminated in the
identification of a clinical candidate, Phortress.14
The active molecule 2 was joined (through an acetyl group)
to the ethyl ester of the metal chelator (N-(2-pyridylmethyl)-
aminoacetic acid (5) to generate the appropriate ligand 3, as
outlined in Scheme 1.15 Standard literature procedures were used
in order to obtain 2 and 5.16 All synthesized products were
characterized by NMR and IR spectroscopies and elemental
analyses.
Breast cancer is the most frequent malignancy among women
(31% in the U.S.1 and 27% in Europe2) and one of the leading
causes of cancer-related death. Early diagnosis and treatment
remain the key to surviving breast cancer. While mammography
is the most effective tool for screening and early detection of
breast cancer in women,3 its sensitivity is limited by dense
breasts, the presence of implants and scars, and the use of
estrogen replacement therapy. As a result, other noninvasive
imaging techniques such as single-photon-emission computed
tomography (SPECT), positron-emission tomography (PET), and
magnetic resonance imaging (MRI) complement mammography
in the characterization and staging of breast tumors, the
evaluation of high-risk patients, the detection of axillary lymph
node metastatic involvement and the assessment of tumor
response to chemotherapy.4,5
The most widely used radionuclide for diagnostic imaging
with SPECT is the metastable isotope of technetium, 99mTc,
because of its favorable physical properties (t1/2 ) 6 h, Eγ )
140 keV), low cost, and widespread availability.6 Three 99mTc
complexes, 99mTc-sestamibi,7 99mTc-tetrofosmin,8 and 99mTc-
methylene diphosphonate,9 are already employed in the clinical
practice of breast cancer diagnosis as adjuncts to mammography,
and research in this area is active aiming at improving sensitivity
and specificity in the detection of breast tumors. The develop-
ment of technetium complexes as potential radiopharmaceuticals
is facilitated by the use of rhenium, the group VIIB congener
of technetium. Rhenium, a mixture of the 185Re and 187Re
isotopes, generally produces complexes with similar physical
and biodistribution properties to those of technetium and is often
used as a nonradioactive alternative to technetium for large-
scale synthesis and structural characterization.10 Furthermore,
the â-emitting radioisotopes of rhenium,186Re (t1/2 ) 3.8 days,
The synthesis of complex 1a15 was effected by ligand
exchange reaction using the organometallic Re tricarbonyl
precursor [NEt4]2[fac-Re(CO)3(Br)3].17 Equimolar quantities of
the precursor and ligand 3 were stirred under reflux for 3 h in
acetonitrile to afford 1a as a white precipitate in 80% yield.
Basic hydrolysis of the ethyl ester group of 3 with aqueous
NaOH preceded its reaction with the precursor. Complex 1a
was fully characterized by IR, NMR, elemental analysis, and
1
mass spectrometry. Detailed H and 13C NMR chemical shift
data in DMSO-d6 at 25 °C are in Supporting Information.
Biological evaluation of complex 1a was accomplished by
cell uptake experiments monitored by fluorescence micro-
scopy.15 Complex 1a can be used as a fluorescent probe because
it absorbs light in the UV region with a peak absorbance at
332 nm and emits fluorescence with maximum intensity at 407
nm. Cellular entry of 1a was tested in the MCF-7 human breast
cancer cell line, which is particularly sensitive in 2,13 and in
two human normal fibroblast cell lines, the fetal lung fibroblast,
* To whom correspondence should be addressed. Phone: +30210
6503555. Fax: +30210 6511767. E-mail: pelmar@bio.demokritos.gr.
† Institute of Biology.
‡ Institute of Radioisotopes and Radiodiagnostic Products.
10.1021/jm0606387 CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/10/2006

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5409
Scheme 1a
Figure 2. Summary of the biodistribution data (% ID/g) of the 99mTc
complex 1b in healthy mice.
a Reagents and conditions: (a) NEt3 (1.5 equiv), dry CH2Cl2, 0 °C,
chloroacetyl chloride (1.1 equiv), under nitrogen, 0 °C, 30 min, followed
by room temp, 60 min, yield 40%; (b) BrCH2COOEt (1 equiv), dry THF,
0 °C, 30 min, followed by room temp, 48 h, yield 75%; (c) 4 (1 equiv), 5
(1.1 equiv), Hunig’s base (1.5 equiv), CH3CN, 80 °C, 24 h, yield 56%.
fac-[99mTc(CO)3(H2O)3]+.18 The concentration of ligand 3 was
approximately 10-4 M. The mixture was analyzed by reversed-
phase HPLC, and the analysis demonstrated that a single
complex was formed (radiochemical yield of >90%) that was
stable for at least 2 half-lives of the isotope (12 h). The identity
of the complex was established by comparative HPLC studies
using a sample of the well-characterized complex 1a as
reference. When samples of the complexes 1a and 1b were co-
injected on a reversed-phase HPLC column, they eluted with
practically identical retention times as witnessed by simultaneous
UV (1a) and radioactivity (1b) detection, revealing their
structural analogy.15 Complex 1b was stable against the histidine
and cysteine challenge.19,15
In vivo biological evaluation of complex 1b was effected by
biodistribution studies in Swiss albino mice following intrave-
nous administration of the HPLC purified complex (1-2 µCi).15
The tissue biodistribution data shown in Figure 2 demonstrate
rapid blood and soft tissue clearance. The low stomach and
spleen values are indicative of the lack of in vivo oxidation of
technetium to pertechnetate or Tc colloid. The radioactivity was
mainly excreted via the hepatobiliary system while tissues in
the thoracic cavity remained practically free of radioactivity,
leaving a clear background suitable for a possible application
in breast cancer imaging.
In conclusion, the isostructural Re and 99mTc complexes 1a
and 1b are amenable to fluorescent and radioimaging studies
and offer the opportunity of directly correlating their in vitro
and in vivo biological behavior.20 Their preliminary evaluation
data show selective uptake of 1a by the MCF-7 breast cancer
cells and no specific tissue accumulation of 1b in healthy mice.
These results are encouraging for further exploration of their
properties toward the development of 99mTc, 186Re, and 188Re
complexes for breast imaging and therapy. Determination of
the cytotoxicity of 1a, quantitative assessment of the uptake of
1b into MCF-7 cells, and biodistribution study of 1b in tumor
bearing mice are currently in progress.
Figure 1. Fluorescence microscopy photos showing the selective
cellular entrance of complex 1a into human breast cancer cells (MCF-
7) and not into healthy human foreskin (HFFF-2) and healthy human
lung (MRC-5) fibroblast cell lines along with the control experiment
for each cell line: (A) MCF-7 cells after 24 h of exposure to 100 µM
1a; (B) MCF-7 cells cultivated in growth medium free of 1a; (C)
HFFF-2 cells after 24 h of exposure to 100 µM 1a; (D) HFFF-2 cells
cultivated in growth medium free of 1a; (E) MRC-5 cells after 24 h of
exposure to 100 µM 1a; (F) MRC-5 cells cultivated in growth medium
free of 1a.
Acknowledgment. M. Pelecanou gratefully acknowledges
financial support by the National Bank of Greece. The authors
thank Dr. D. Kletsas of the Institute of Biology of NCSR
“Demokritos” for kindly providing the MCF-7, HFFF-2, and
MRC-5 cell lines.
MRC-5, and fetal foreskin fibroblast, HFFF-2. After a 24 h
incubation, satisfactory cellular entry of 1a was evident (for
concentrations of 1a ranging from 100 to 20 µM)15 in the MCF-7
breast cancer cells but not in the HFFF-2 or MRC-5 normal
cells (Figure 1).
Supporting Information Available: Synthetic procedures for
1
1a,b and 2-5, H and 13C NMR assignments for 1a and 2-5,
procedures for stability tests of 1a and 1b, cell uptake experiments
of 1a, and biodistribution of 1b in healthy mice. This material is
available free of charge via the Internet at http://pubs.acs.org.
The stability of 1a in cell culture supernatant at 37 °C was
checked by NMR to ensure that the fluorescent signal is due to
complex 1a and not to a product of its potential decomposition.15
No peaks were present in the NMR spectra that could be
attributed to any decomposition products.
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JM0606387