Benzimidazole derivatives as NSO ligands for the fac-[M(CO) 3]+ (M = Re, 99mTc)

Article abstract of DOI:10.1016/j.ica.2011.07.035

Two rhenium(I) tricarbonyl complexes with the tridentate monoanionic NSO ligands, 4-(benzimidazol-2-yl)-3-thiabutanoic acid (complex 3) and [1-(11-carboxyundecanyl)-4-(benzimidazol-2-yl)]-3-thiabutanoic acid (complex 4) were synthesized and characterized by spectroscopic methods and elemental analysis. X-ray crystallographic analysis of complex 3 revealed a distorted octahedral geometry around rhenium defined by the three facially bound CO groups and the NSO donor atom set of the tridentate ligand. The analogous technetium-99m complexes (complexes 5 and 6) were also prepared quantitatively by reaction of the NSO ligands with the fac-[^99mTc(H ₂O)₃(CO)₃]⁺ synthon and their identity was established by chromatographic comparison to their rhenium congeners. Biodistribution in mice of complex 6 bearing the fatty acid chain showed significant heart uptake (6.26 ± 0.79% ID/g p.i.) at 1 min accompanied, however, with a heart:blood ratio below 1.

Full text of DOI:10.1016/j.ica.2011.07.035

Inorganica Chimica Acta 377 (2011) 62–68
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Benzimidazole derivatives as NSO ligands for the fac-[M(CO)3]+ (M = Re, 99mTc)
a
a
a
b
c
c
c
´
´
´
T. Tsotakos , C. Tsoukalas , G. Patsis , A. Panagiotopoulou , N. Nikolic , D. Jankovic , D. Djokic ,
C.P. Raptopoulou d, A. Terzis d, D. Papagiannopoulou e, M. Pelecanou b, M. Papadopoulos a, I. Pirmettis a,
⇑
a Institute of Radioisotopes – Radiodiagnostic Products, National Center for Scientific Research ‘‘Demokritos’’, 15310 Ag. Paraskevi, Athens, Greece
b Institute of Biology, National Center for Scientific Research ‘‘Demokritos’’, 15310 Ag. Paraskevi, Athens, Greece
c University of Belgrade, Vinca Institute of Nuclear Sciences, Laboratory for Radioisotopes, P.O.B. 522, 11001 Belgrade, Serbia
d Institute of Materials Science, National Center for Scientific Research ‘‘Demokritos’’, 15310 Ag. Paraskevi, Athens, Greece
e Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Two rhenium(I) tricarbonyl complexes with the tridentate monoanionic NSO ligands, 4-(benzimidazol-
2-yl)-3-thiabutanoic acid (complex 3) and [1-(11-carboxyundecanyl)-4-(benzimidazol-2-yl)]-3-thiabuta-
noic acid (complex 4) were synthesized and characterized by spectroscopic methods and elemental
analysis. X-ray crystallographic analysis of complex 3 revealed a distorted octahedral geometry around
rhenium defined by the three facially bound CO groups and the NSO donor atom set of the tridentate
ligand. The analogous technetium-99m complexes (complexes 5 and 6) were also prepared quantitatively
by reaction of the NSO ligands with the fac-[99mTc(H2O)3(CO)3]+ synthon and their identity was estab-
lished by chromatographic comparison to their rhenium congeners. Biodistribution in mice of complex
6 bearing the fatty acid chain showed significant heart uptake (6.26 0.79% ID/g p.i.) at 1 min accompa-
nied, however, with a heart:blood ratio below 1.
Received 27 May 2011
Accepted 26 July 2011
Available online 5 August 2011
Keywords:
Technetium-99m
Rhenium
Tricarbonyl
Fatty acid
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
chelating agent. This compound contains a pyridine nitrogen (N-3),
a thioether and a carboxylate group and can therefore act as a tri-
The gamma emitting nuclide technetium-99m is the preferred
radionuclide in diagnostic nuclear medicine for scintigraphy and
dentate NSO ligand with the fac-[M(CO)3]+ core (M = Re, 99mTc) to
generate neutral [M(NSO)(CO)3] complexes. Moreover, in addition
to the NSO donor atom system, ligand 1 contains a NH group (N-1)
that can function as the anchoring point for the attachment of
receptor-targeting molecule of interest. As an example, the
undecanoic acid has been tethered to 1, generating ligand 2. The
development of 99mTc-labelled fatty acid analogs for heart imaging
purposes is of broad scientific interest because fatty acids are sub-
strates for beta-oxidation, the main source of energy production in
the myocardium [20–23]. Herein we report the synthesis and char-
acterization of the Re (3 and 4) and 99mTc (5 and 6) complexes of
both ligands. The biodistribution of the 99mTc fatty acid analog 6
in mice is also reported.
SPECT imaging because of its physical decay characteristics (t1/2
=
6 h, E = 140 keV), its availability through commercially-available
c
generator systems and its low cost per dose [1–4]. The initial devel-
opment of a low pressure method and the later development of a sin-
gle vial freeze dried kit ([5,6], IsolinkÒ) for the preparation of the
[
99mTc(H2O)3(CO)3]+ synthon thrusted the organometallic chemis-
try of technetium and rhenium in the last decade. The three aqua li-
gands of the fac-[99mTc(H2O)3(CO)3]+ synthon are labile and readily
substituted by a variety of functional groups including amines, thio-
ethers, imines, thiols, carboxylates and phosphines to give stable
hexacoordinated complexes [7–14]. Furthermore, the small size
and kinetic inertness of the [99mTc(I)(CO)3]+ core make it suitable
for the labelling of receptor targeting molecules [15–19]. The label-
ling of such molecules usually requires the use of a bifunctional che-
lating agent, which on one hand binds to the metal and on the other
hand holds the receptor-targeting organic molecules that will ren-
der specificity to the complex.
2. Experimental
2.1. Materials and methods
In this work, we have focused on the study of 4-(benzimidazol-
2-yl)-3-thiabutanoic acid (1, Scheme 1) as a potential bifunctional
All laboratory chemicals were reagent grade and were used
without further purification. Solvents for high-performance liquid
chromatography (HPLC) were HPLC-grade. They were filtered
⇑
through membrane filters (0.22
degassed by a helium flux before and during use.
lm, Millipore, Milford, MA) and
Corresponding author. Tel.: +30 210 6503921; fax: +30 210 6524480.
E-mail address: ipirme@rrp.demokritos.gr (I. Pirmettis).
0020-1693/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2011.07.035

T. Tsotakos et al. / Inorganica Chimica Acta 377 (2011) 62–68
63
14-18
12
20
(CH2)5
OH
13
21
19
11
H
2
3
O
N
NH2
NH2
6
N
1
5
4
Br(CH2)10COOH
S(CH2COOH)2
6M HCl
COOH
S
S
COOH
7
N
H2O/NaOH
N
10
8
2
9
1
R
N
X
O
X
3
R = H, M = Re
X
1 or 2
S
M
4
5
6
R = (CH2)10COOH, M = Re
R = H, M =Tc-99m
N
O
i or ii
OC
CO
M
CO
R = (CH2)10COOH, M =Tc-99m
CO
OC
CO
i. for 3 and 4: M=Re, X=Br, MeOH/H2O, reflux, 5 hrs. ii. for 5 and 6: M = 99mTc, X = H2O, 80 oC, 30 min
Scheme 1. Synthetic scheme for ligands 1, 2 and complexes 3–6.
IR spectra were recorded as KBr pellets on a Perkin–Elmer 1600
FT-IR spectrophotometer in the region 4000–500 cmꢀ1. The NMR
spectra were recorded in DMSO-d6, at 25 °C on a Bruker 500 MHz
Avance DRX spectrometer using TMS as internal standard.
are given in Table 1. Anal. Calc. for C21H30N2O4SꢂH2O: C, 59.41; H,
7.60; N, 6.60. Found: C, 59.77; H, 7.29; N, 6.99%.
2.4. Synthesis of fac-[Re(L1)(CO)3] (3)
Elemental analyses for C, H and N were conducted on a Perkin–
Elmer 2400 automatic elemental analyzer. HPLC analysis was per-
formed on a Waters 600 chromatography system coupled to both a
Waters 2487 Dual k Absorbance detector and a Gabi gamma detec-
tor from Raytest. Separations were achieved on a Nucleosil C18
A mixture of 1 (48 mg 0.2 mmol), [NEt4]2[ReBr3(CO)3] (154 mg,
0.2 mmol) in acetonitrile (5 mL) and 2 mL 0.1 N NaOH (0.2 mmol)
was refluxed for 4 h. Upon standing at room temperature a pale
yellowish crystalline product precipitated and was collected by fil-
tration. Crystals suitable for X-ray structure analysis were isolated
by recrystallization from a mixture of acetonitrile/water. Yield
80 mg (81%). tR: 17.5 min. IR (KBr, cmꢀ1): 2028, 1904 (br), 1653
(br). 1H and 13C NMR data are given in Table 1. Anal. Calc. for
(10
l
m, 250 mm ꢁ 4 mm) column eluted with a binary gradient
system at a 1 mL minꢀ1 flow rate. Mobile phase A was methanol
containing 0.1% trifluoroacetic acid, while mobile phase B was
water containing 0.1% trifluoroacetic acid. The elution gradient
was 0–1 min 5% A (95% B), followed by a linear gradient to 95% A
(5% B) in 15 min; this composition was held for another 15 min.
The precursor [Et4N]2[ReBr3(CO)3] was synthesized according to
literature method [24]. Na99mTcO4 was obtained in physiological
saline as a commercial 99Mo/99mTc generator eluate (Mallinckrodt
Medical B.V., The Netherlands). The radioactive precursor fac-
C13H9N2O5ReS: C, 31.77; H, 1.85; N, 5.70. Found: C, 31.41; H,
1.79; N, 5.59%.
2.5. Synthesis of fac-[Re(L2)(CO)3] (4)
The compound was prepared as above using 2 (81.6 mg,
0.2 mmol). Recrystallization from acetonitrile afforded 4 as yellow-
ish crystals. Yield: 100 mg (74%). tR: 19.9 min. IR (KBr, cmꢀ1): 2928,
2853, 2029, 1896(br), 1717, 1632. 1H and 13C NMR data are given
in Table 1. Anal. Calc. for C24H29N2O7ReS: C, 42.66; H, 4.33; N,
4.15. Found: C, 42.75; H, 4.56; N, 4.04%.
[
99mTc(H2O)3(CO)3]+ was prepared using a commercially available
IsoLinkÒ kit (Malinckrodt), according to the manufacturers’
instructions.
2.2. Synthesis of L1H (1)
The product was synthesized according to literature method
[25,26]. Yield 31%. tR: 11.3 min. Mp: 193–196 °C (lit. 186 °C). IR
(KBr, cmꢀ1): 2459(br), 1633, 1541. 1H and 13C NMR data are given
in Table 1. Anal. Calc. for C10H10N2O2SꢂH2O: C, 49.99; H, 5.03; N,
11.66. Found: C, 49.62; H, 5.16; N, 12.03%.
2.6. X-ray crystal structure determination of Re complex 3
A colorless crystal of 3 (0.05 ꢁ 0.15 ꢁ 0.65 mm) was mounted in
air on a Crystal Logic Dual Goniometer diffractometer using graph-
ite monochromated Mo K
a radiation. Unit cell dimensions were
determined by using the angular settings of 25 automatically cen-
tered reflections in the range 11 < 2h < 23°. Intensity data were re-
corded using a h–2h scan. Three standard reflections monitored
every 97 reflections showed less than 3% variation and no decay.
Lorentz, polarization and psi-scan absorption corrections were ap-
plied using Crystal Logic software. The structures were solved by
direct methods using SHELXS-97 [27] and refined by full-matrix least
squares techniques on F2 using SHELXL-97 [28]. Important crystallo-
graphic data are listed in Table 2. Further crystallographic details
for 3: 2hmax = 50°; reflections collected/unique/used 5780/5666
2.3. Synthesis of L2H (2)
In a 100 mL round bottom flask 2.22 g (10 mmol) of 1 and 2.45 g
(10 mmol) of 11-bromoundecanoic acid were dissolved in 40 mL
1 N NaOH. The reaction mixture was refluxed for 72 h and then it
was left to cool down at room temperature. The pH of the solution
was gradually adjusted to 6 with 1 N HCl and the solid formed was
discarded. Subsequently the pH was adjusted to 5 and the solid
formed was collected by filtration and washed with cold aqueous
methanol (20%). The final product 2 was further purified by succes-
sive recrystallizations with methanol. Yield 38% Mp: 136 °C. tR:
17.3 min. IR (KBr, v/cmꢀ1): 2928, 2852, 1724. 1H and 13C NMR data
[Rint = 0.0261]/5666; 497 parameters refined; [
D
q
]max/[D
q
]
min
=
0.961/ꢀ1.341 e Å3;
[D/r]max = 0.001, R1/wR2 (for all data) =
0.0527/0.0960. All hydrogen atoms were located by difference

64
T. Tsotakos et al. / Inorganica Chimica Acta 377 (2011) 62–68
Table 1
1H and 13C chemical shifts (ppm) for ligands 1 and 2 and complexes 3 and 4 in DMSO-d6 at 25 °C. The atom numbering used is shown in Scheme 1.
Ligand 1
Complex 3
Ligand 2
Complex 4
Ligand 1
Complex 3
Ligand 2
Complex 4
H-2
H-3
H-6
H-7
H-8
H-9
H-11
H-12
H-13
H-14–H-18
H-19
H-20
3.39
4.00
7.49
7.15
7.15
7.49
3.66
4.79, 4.74
7.63
7.40
7.46
3.39
4.11
7.53
7.23
7.19
7.59
4.23
1.75
1.29
1.22
1.45
2.16
3.70, 3.59
4.96, 4.83
7.81
7.48
7.53
7.83
4.35
1.82, 1.71
1.28
1.22
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
171.06
33.38
28.59
151.42
138.08
114.66
121.66
121.66
114.66
138.08
178.05
35.94
35.13
160.04
134.40
113.37
125.00
124.47
118.05
141.18
171.07
33.06
26.94
150.72
135.18
110.35
122.16
121.53
118.74
141.91
43.21
29.32
178.86
34.20
36.28
159.74
134.76
112.54
125.32
125.16
118.43
140.54
46.31
7.79
C-9
C-10
C-11
C-12
C-13
C-14-C-18
1.46
2.17
29.11
25.95
29.18, 29.03
26.17
OH
NH
not visible
11.94
28.85, 28.78, 28.68, 28.63, 28.52
not visible
13.9 broad
C-19
C-20
C-21
C„O
24.47
33.65
174.50
24.91
34.20
175.61
195.43, 195.09
195.69, 195.35
Table 2
Tc-99m complexes 5 and 6 was established by comparative HPLC
studies using as references the well characterized analogous rhe-
nium complexes 3 and 4, respectively. In order to evaluate the sta-
bility of the 99mTc complexes, samples were periodically analyzed
by HPLC for a period up to 24 h.
Crystallographic data for complex 3.
3ꢂ0.5MeCN
C14H10.5N2.5O5ReS
Formula
Fw
512.01
Space group
a (Å)
b (Å)
C2/c
23.195(13)
8.933(5)
32.850(16)
90.00
109.025(16)
90.00
6435(6)
16
293
2.8. Cysteine and histidine challenge of the 99mTc complexes 5 and 6
c (Å)
a
(°)
Aliquots of 100 l
L of the 99mTc complexes 5 and 6 (final concen-
tration of ligands 10ꢀ6 M) were added to 900 L of a 10ꢀ2 M histi-
l
b (°)
c
(°)
dine and 10ꢀ2 M cysteine solution in PBS, pH 7.4, respectively. The
samples were incubated at 37 °C and periodically aliquots were re-
moved and analyzed by HPLC.
V (Å3)
Z
T (°C)
Radiation
0.71073 (Mo K
2.114
7.711
a)
qcalc (g cmꢀ3
)
2.9. Determination of partition coefficient (log Do/w
)
l
(mmꢀ1
)
Reflections with I > 2
r(I)
4215
0.0332
0.0814
a
R1
For the evaluation of the lipophilicity of the compounds, the
a
partition coefficient of the 99mTc complexes 5 and 6 was deter-
mined by the shake-flask method. 10 lL of the HPLC-purified
wR2
2(F2o ) + (
a
P)2 + bP] and P = (max Fo2, 0)+2Fc2)/3, R1
=
R
(|Fo| ꢀ |Fc|)/
R(|Fo|)
a
w = 1/[
r
R
99mTc complexes were mixed with 1.5 mL of 1-octanol and
1.5 mL phosphate buffer (0.125 M, pH 7.4) in a centrifuge tube.
The mixture was vortexed at room temperature for 1 min and fi-
nally centrifuged at 5000 rpm for 5 min. Three weighed samples
from the octanol and aqueous layers were then counted in a gam-
ma counter and the cpm/mL was calculated. Partition coefficient
(log Do/w) was calculated by dividing the cpm/mL(oct) to cpm/
mL(buffer), and the log log Do/w was found to be 1.5 and 1.7 for 5
and 6, respectively.
and wR2 = {
[w(F2o ꢀ F2c )2]/
R .
[w(F2o )2]}1/2
maps and were refined isotropically; all non-H atoms were refined
anisotropically. Plots of all structures were drawn using the DIAMOND
3.1 program package [29].
2.7. Synthesis of fac-[99mTc(L1)(CO)3] (5) and fac-[99mTc(L2)(CO)3] (6)
[
99mTc]-sodium pertechnetate (370–740 MBq, 1 mL) was added
2.10. Animal distribution studies
to the Isolink kit formulation (Mallinckrodt Medical B.V.) contain-
ing sodium tetraborate (2.85 mg), sodium carbonate (7.15 mg), so-
dium boranocarbonate decahydrate (2.85 mg) and sodium tartrate
dihydrate (8.5 mg) and the mixture was incubated at 95 °C for
20 min. After cooling, the pH was adjusted to 8 by addition of
0.1 N HCl and formation of fac-[99mTc(H2O)3(CO)3]+ was checked
by HPLC, tR = 4.5 min. An amount of the fac-[99mTc(H2O)3(CO)3]+
Three groups of four Swiss Albino mice each (male, 25 3 g)
were injected with the HPLC purified 99mTc complex 6 (0.1 mL,
1–2 lCi) through the tail vein. The animals were sacrificed by car-
diectomy under slight ether anesthesia at predetermined times
(1, 15 and 60 min). The organs of interest were excised, weighed
and counted in an automatic gamma counter. Bladder and excreted
urine were not weighed. The stomach and intestines were not
emptied of food contents prior to radioactivity measurements.
The percentage of injected dose per organ (%ID/organ) was calcu-
lated by comparison of sample radioactivity to standard solutions
containing 10% of the injected dose. The calculation for blood
and muscle were based upon measured activity, sample weight
and body composition data (considering that blood and muscle
precursor (450
or 2 (0.5–200
l
L) was added to a vial containing a solution of 1
lg, 50 L DMSO). The vial was sealed with a rubber
l
stopper and aluminum crimp cap and the solution was incubated
at 80 °C for 30 min. After cooling, a sample was analyzed by HPLC
and in each case the formation of a single radioactive complex, in
more than 95% yield, was observed. The retention time of 5 and
6 is 17.8 min and 20.2 min, respectively. The identity of the

T. Tsotakos et al. / Inorganica Chimica Acta 377 (2011) 62–68
65
comprise 7% and 43% of body weight). The percentage of injected
dose per gram (%ID/g) was calculated by dividing the %ID/organ
by the weight of the organ or tissue. All the animal experiments
were carried out in compliance with the relevant national laws
relating to the conduct of animal experimentation.
coordinating NSO atoms. The chemical shift differentiation of the
diastereotopic geminal protons is quite small for H-3 and non exis-
tent for H-2, a fact that has been observed also in other 4-(imidaz-
olin-2-yl)-3-thiabutanoic acid NSO complexes with the [M(CO)3]+
core [10]. As can be seen in Table 1, tautomeric proton exchange
in the benzimidazole moiety averages the proton and carbon
chemical shifts of ligand 1. In complex 3 the symmetry of the benz-
imidazole moiety is lost and all protons and carbons appear at dis-
tinct chemical shifts. Compared to ligand 1 all atoms are shifted
downfield with the exception of carbons C-5 and C-6 that are
shifted upfield by 3.7 and 1.3 ppm, respectively.
3. Results and discussion
3.1. Chemistry
The synthesis of the 99mTc complex 5 was carried out by ligand
substitution reaction employing the precursor fac-[99mTc(H2O)3(-
CO)3]+. The precursor was prepared by the addition of pertechne-
tate to a commercially available IsoLink kit that contains sodium
boranocarbonate which acts simultaneously as reductant and
‘‘in situ’’ source of CO [6]. The reaction was monitored by HPLC
(tR of pertechnetate 3.0 min, tR of precursor 4.5 min under the em-
ployed conditions) and was quantitative (>95%). Reaction of ligand
1 (10ꢀ5 M) with the precursor fac-[99mTc(OH2)3(CO)3]+ at pH 8.0 for
30 min at 80 °C resulted in the formation of 5. HPLC analysis dem-
onstrated the formation of a single complex with yield greater than
95%, eluting at 17.8 min. Even at the lower ligand concentration of
10ꢀ6 M, quantitative labelling (>90%) was achieved by increasing
the pH to 10.0. The structure of the Tc-99m complex 5 was effected
by comparative HPLC analysis using as reference the analogous
non radioactive rhenium complex 3, eluting at 17.5 (Fig. 4). Stabil-
ity studies performed by HPLC, demonstrate that the 99mTc com-
plex 5, remained stable for a period of at least 24 h. Its stability
in the presence of excess histidine and cysteine was also monitored
by HPLC and no changes were observed in the radiochromatogram
indicating the high stability of the 99mTc complex.
4-(Benzimidazol-2-yl)-3-thiabutanoic acid (L1H, 1) was synthe-
sized in good yield according to the literature [25,26], employing
the conventional Philips condensation reaction by using o-phenyl-
enediamine and thiodiglycolic acid in refluxing 6 N HCl (Scheme
1). It was designed to act as a monoanionic NSO tridentate ligand
with the fac-[M(CO)3]+ core (M = Re and 99mTc).
The synthesis of non-radioactive Re(I)-complex, 3 (Scheme 1)
was effected by ligand exchange reaction using the organometallic
Re tricarbonyl precursor [NEt4]2[ReBr3(CO)3] and equimolar quan-
tities of 1. HPLC analysis of the reaction mixture showed that only
one product is formed with a retention time 17.5 min. The complex
was isolated as yellowish crystals in high yield (81%) and was fully
characterized by IR and NMR spectroscopies, elemental analysis
and by X-ray crystallography. It is stable in the solid state as well
as in solution as shown by HPLC and NMR.
The characteristic finding in the infrared spectra of the rhenium
complex 3 is the presence of two very strong bands at 2028 and
1904 cmꢀ1. These bands are attributed to the symmetric and asym-
metric stretching vibration of the C„O bond and they are charac-
teristic for the fac-[Re(CO)3]+ core [9–14]. In addition, 3 exhibits a
strong broad absorption at 1653 cmꢀ1 which corresponds to the
asymmetrical C@O stretching vibration of the coordinated
carboxylate.
Due to the high affinity of 1 for the [99mTc(CO)3]+ core and high
stability of the complex formed we further explored its use as a
bifunctional chelator. Thus a fatty acid was tethered at the pyrrole
nitrogen (N-1) of the benzimidazole ring by alkylation with 11-
bromomoundecanoic acid resulting in ligand 2, L2H (Scheme 1).
Reaction of 2 with the [NEt4]2[ReBr3(CO)3] precursor afforded com-
plex 4 in high yield. All elemental and spectroscopic analyses are in
agreement with the expected structure shown in Scheme 1. By
1H and 13C chemical shift assignments for complex 3 were
based on the combined analysis of a series of 1H–1H and 1H–13
C
correlation spectra (Fig. 1A) and are reported in Table 1 along with
those of the corresponding ligand 1 for comparison purposes. Upon
coordination downfield shifts are noted for the H-2 (by appr.
0.3 ppm) and H-3 (by approx. 0.7 ppm) protons close to the
Fig. 1. 1H–13C long-range correlation (HMBC) spectra of A. complex 3 (range dH 8.06–3.44, range dC 183.00–109.15) and B. complex 4 (range dH 8.38–1.08, range dC 185.44–
105.25) in DMSO-d6 at 25 °C. The numbering of the atoms is shown in Scheme 1.

66
T. Tsotakos et al. / Inorganica Chimica Acta 377 (2011) 62–68
comparison of the 1H and 13C NMR data of the NSO chelate part in
complexes 3 and 4 (Table 1 and Fig. 1), it is clearly seen that the
fatty acid derivatized ligand 2 coordinates around the [Re(CO)3]+
core in the same mode as ligand 1. Further confirmation for the fact
that in complex 4 coordination is as expected through the pyridine
nitrogen and not through the fatty acid substituted nitrogen (N-1)
is provided by the presence in the NOESY spectrum of a correlation
peak between the H-3 and H-11 protons that is only possible if
coordination takes place through the pyridine nitrogen (N-3).
The synthesis of the radioactive 99mTc complex 6 was carried
out as for 5, using ligand 2 (10ꢀ5 M). HPLC analysis demonstrated
the formation of a single complex eluting at 20.2 min that co-
elutes with the corresponding rhenium complex (4) 19.9 min.
Complex 6 was found to be resistant to transchelation in the pres-
ence of excess histidine or cysteine. These results indicate that the
derivatization with a bulky substituent did not affect the coordina-
tion properties of the NSO donor atom system.
Table 3
0
Selected bond distances (AÅ) and angles (°) in 3.
3A
3B
Distances
Re(1)–C(11)
Re(1)–C(12)
Re(1)–C(13)
Re(1)–O(1)
Re(1)–N(1)
Re(1)–S(1)
1.896(9)
1.896(10)
1.930(9)
2.142(5)
2.177(6)
2.483(2)
Re(2)–C(31)
Re(2)–C(33)
Re(2)–C(32)
Re(2)–O(21)
Re(2)–N(11)
Re(2)–S(2)
1.910(9)
1.920(8)
1.923(9)
2.147(5)
2.178(6)
2.474(2)
Angles
C(11)–Re(1)–C(12)
C(11)–Re(1)–C(13)
C(12)–Re(1)–C(13)
C(11)–Re(1)–O(1)
C(12)–Re(1)–O(1)
C(13)–Re(1)–O(1)
C(11)–Re(1)–N(1)
C(12)–Re(1)–N(1)
C(13)–Re(1)–N(1)
O(1)–Re(1)–N(1)
C(11)–Re(1)–S(1)
C(12)–Re(1)–S(1)
C(13)–Re(1)–S(1)
O(1)–Re(1)–S(1)
N(1)–Re(1)-S(1)
88.7(4)
87.4(4)
89.5(4)
97.7(3)
173.4(3)
91.9(3)
171.2(3)
94.4(3)
100.4(3)
79.0(2)
92.0(3)
98.2(3)
172.3(3)
80.5(1)
79.4(2)
C(31)–Re(2)–C(32)
C(31)–Re(2)–C(33)
C(32)–Re(2)–C(33)
C(31)–Re(2)–O(21)
C(32)–Re(2)–O(21)
C(33)–Re(2)–O(21)
C(31)–Re(2)–N(11)
C(32)–Re(2)–N(11)
C(33)–Re(2)–N(11)
O(21)–Re(2)–N(11)
C(31)–Re(2)–S(2)
C(32)–Re(2)–S(2)
C(33)–Re(2)–S(2)
O(21)–Re(2)–S(2)
N(11)–Re(2)–S(2)
86.9(3)
88.4(3)
91.0(4)
99.2(3)
173.7(3)
90.5(3)
171.7(3)
94.5(3)
99.7(3)
79.3(2)
91.7(2)
98.2(2)
170.8(3)
80.4(1)
80.0(2)
3.2. Description of the structure
The molecular structure of 3 is given in Fig. 2, selected bond dis-
tances and angles are listed in Table 3. The asymmetric unit of 3
contains two crystallographically independent molecules, thereaf-
ter denoted as A and B, and one acetonitrile solvate molecule. The
distorted octahedral environment of the Re atom in the structure
of 3 is defined by the three facially bound CO groups and the
0
H2NꢂꢂꢂO22 = 2.387 ÅA, 0 N2–H2NꢂꢂꢂO22 = 157.6°; N12ꢂꢂꢂO2 (0.5 + x,
0
ꢀ0.5 + y, z) = 3.000 ÅA, H12NꢂꢂꢂO2 = 2.387 ÅA, N12–H12NꢂꢂꢂO2 =
NSO donor atom set of the tridentate ligand. The Re-carbonyl bond
0
154.0°] (Fig. 3).
distances (3A: 1.896–1.930 Å, 3B: 1.910–1.923 ÅA) are consistent
which those found in other Re-tricarbonyl complexes [9–14]. The
Re–S, Re–N and Re–O bond distances fall well in the ranges ob-
served in analogous complexes. In the coordination sphere of 3A
and 3B, the Re–N–C–C–S and Re–S–C–C–O five-membered rings
adopt the envelope configuration. Atoms C3 and C23 are displaced
by 0.37 and 0.28 Å, respectively, out of the best mean plane of their
respective remaining four atoms for the former rings, and atoms C2
and C22 are displaced by 0.31 and 0.42 Å, respectively, out of the
best mean plane of their respective remaining four atoms for the
latter rings. In the lattice structure of 3, there are intermolecular
N–HꢂꢂꢂO hydrogen bonding interactions between 3A and 3B in an
3.3. Biodistribution studies of 6
The 99mTc complex 6, purified by HPLC from excess ligand, was
evaluated in Swiss albino mice and the data are presented in Table
4. The complex showed maximum heart uptake of 6.26 0.79 ID/g
at 1 min p.i. that decreases to 0.16 0.07 and 0.11 0.04 ID/g at 15
and 60 min, respectively. The initial heart uptake is comparable
with the values obtained in mice with 99mTc-labeled penta- or
hexadecanoic acid analogs [Tc–CpTT-hexadecanoic acid (9.03%
ID/g at 1 min p.i.) [30], 99mTc(CO)3–15-[N-(acetyloxy)-2-picolyla-
mino]-pentadecanoic acid (6.38 %ID/g at 1 min p.i.) [31], 99mTc–
MAMA-hexadecanoic acid (11.22 %ID/g and 5.46 %ID/g at 0.5 and
2 min p.i. respectively) [32]] and higher than those of other
99mTc-labeled undecanoic or dodecanoic acid analogs [99mTc–
MAMA-dodecanoic acid (5.68 %ID/g at 0.5 min p.i.) [14],
alternating fashion (i.e. 3Aꢂꢂꢂ3Bꢂꢂꢂ3A); the so-formed chains extend
0
parallel to the b axis [N2ꢂꢂꢂO21 (ꢀ0.5 + x, ꢀ0.5 + y, z) = 3.065 ÅA,
Fig. 3. Partially labeled plot of the chains between 3A and 3B due to hydrogen
bonding interactions (dashed lines). Color code: Re, black; S, dark gray; O, medium
gray; N, large open circles; C, small open circle.
Fig. 2. Partially labeled plot of 3A with ellipsoids drawn at the 30% probability level.
Only the imidazolato H atom is shown, the rest have been omitted for clarity.

T. Tsotakos et al. / Inorganica Chimica Acta 377 (2011) 62–68
67
that the complex remains stable in vivo regarding its re-oxidation
to pertechnetate.
(a)
A
0.7
17.5
4. Conclusions
0.6
0.5
0.4
0.3
0.2
0.1
0.0
-0.1
In conclusion, the tridentate NSO ligands 4-(benzimidazol-2-
yl)-3-thiabutanoic acid (1) and its fatty acid derivative (2) coordi-
nate with the fac-[M(CO)3]+ (M = Re, 99mTc) core through the pyri-
dine nitrogen (N-3) of benzimidazole, the sulfur of the thioether
and the carboxylate oxygen forming neutral and lipophilic com-
plexes. The techentium-99m complexes are formed at significantly
low ligand concentration of 10ꢀ6 M and are stable against transch-
elation reactions. The biodistribution study of the fatty acid deriv-
ative show interesting heart uptake, however the heart to blood
ratio is low. Overall, the high affinity of this benzimidazole NSO li-
gand for the fac-[M(CO)3]+ core together with the high stability of
the complexes formed indicate that this system merits further
investigation towards the improvement of the biological properties
of the complexes. Furthermore, given that in principle the pyrrole
nitrogen (N-1) of benzimidazole can be derivatized by a variety of
pharmacophores, this ligand system may be suitable for the design
of a wide range of target-specific radiopharmaceuticals.
0
5
10
15
20
25
30
time (min)
(b)
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
mV
17.85
Acknowledgment
This work was supported by the GSRT, Hellenic Ministry of
Development, Measure 3.3., Excellence in Research II, ‘‘Functional
and Functionalized Biomolecules in Biodiagnosis and Radiophar-
macy’’ and Greek-Serbian Bilateral Co-operation Research Project,
No. 709. We thank Mallinckrodt Medical B.V., The Netherlands,
for providing the Isolink boranocarbonate kits.
Appendix A. Supplementary material
CCDC 826886 contains the supplementary crystallographic data
for complex 3. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif. Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.ica.2011.07.035.
0
5
10
15
20
25
30
time (min)
Fig. 4. HPLC chromatographs for complex 3 (a) and complex 5 (b).
Table 4
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