pharmaceutics-17-00100

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Academic Editor: Leonard I Wiebe
Received: 27 November 2024
Revised: 24 December 2024
Accepted: 4 January 2025
Published: 13 January 2025
Citation: de Araujo Fernandes, A.G.;
Lafratta, A.E.; Luz, C.P.; Levy, D.; de
Paula Faria, D.; Buchpiguel, C.A.;
Abram, U.; Deflon, V.M.; Navarro
Marques, F.L. [99mTc]Technetium and
Rhenium Dithiocarbazate Complexes:
Chemical Synthesis and Biological
Assessment. Pharmaceutics 2025, 17,
100. https://doi.org/10.3390/
pharmaceutics17010100
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Article
[99mTc]Technetium and Rhenium Dithiocarbazate Complexes:
Chemical Synthesis and Biological Assessment
André Gustavo de Araujo Fernandes 1,2,†, Alyne Eloise Lafratta 3,†, Carolina Portela Luz 3, Debora Levy 4 ,
Daniele de Paula Faria 3 , Carlos Alberto Buchpiguel 3 , Ulrich Abram 5 , Victor Marcelo Deflon 1,*
and Fabio Luiz Navarro Marques 3,*
1 Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos 13566-590, SP, Brazil;
agafernandes@uesc.br
2 Departamento de Ciências Exatas, Universidade Estadual de Santa Cruz, Ilhéus 45662-900, BA, Brazil
3 Laboratory of Nuclear Medicine (LIM-43), Hospital das Clinicas HCFMUSP, Faculdade de Medicina,
Universidade de Sao Paulo, Sao Paulo 05403-911, SP, Brazil; alyne@alumni.usp.br (A.E.L.);
carol.p.luz@gmail.com (C.P.L.); danielefaria1@gmail.com (D.d.P.F.); buch@usp.br (C.A.B.)
4 Lipids, Oxidation, and Cell Biology Team, Laboratory of Immunology (LIM19), Heart Institute (InCor),
Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo,
Sao Paulo 05403-900, SP, Brazil; d.levy@fm.usp.br
5 Institute of Chemistry and Biochemistry, Freie Universität Berlin, Fabeckstr. 34-36, D-14195 Berlin, Germany;
ulrich.abram@fu-berlin.de
* Correspondence: deflon@iqsc.usp.br (V.M.D.); fabio.marques@fm.usp.br (F.L.N.M.)
† These authors contributed equally to this work.
Abstract: Background/Objectives: Dithiocarbazates (DTCs) and their metal complexes
have been studied regarding their property as anticancer activities. In this work, using
S-benzyl-5-hydroxy-3-methyl-5-phenyl-4,5-dihydro-1H-pirazol-1-carbodithionate (H2bdtc),
we prepared [ReO(bdtc)(Hbdtc)] and [[99mTc]TcO(bdtc)(Hbdtc)] complexes for tumor up-
take and animal biodistribution studies. Methods: Re complex was prepared by a reaction
of H2bdtc and (NBu4)[ReOCl4], the final product was characterized by IR, 1H NMR, CHN,
and MS-ESI. 99mTc complex was prepared by the reaction of H2bdtc and [[99mTc]TcO4
−
and analyzed by planar and HPLC radiochromatography, and the stability was evalu-
ated against amino acids and plasma. Biodistribution was performed in C57B/6 mice
with B16F10 and TM1M implanted tumor. Results: Re is asymmetric coordinated by
two dithiocarbazate ligands, one with O,N,S chelation, and the other with N,S chelation;
[[99mTc]TcO(bdtc)(Hbdtc)] was prepared with a radiochemical yield of around 93%. The
radioactive complex is hydrophobic (LogP = 1.03), stable for 6 h in PBS and L-histidine
solution; stable for 1 h in plasma, but unstable in the presence of L-cysteine. Ex vivo biodis-
tribution demonstrated that the compound has a fast and persistent (until 2 h) uptake by the
spleen (55.46%), and tumor B16F10 and TM1M uptake is lower than 1%. In vivo SPECT/CT
imaging confirmed ex vivo biodistribution, except by heterogenous TM1M accumulation
but not in the B16-F10 lineage. Conclusions: H2bdtc proved to be an interesting chelator for
rhenium or [99mTc]technetium. The right spleen uptake opened the opportunity to deepen
the study of the molecule in this tissue and justifies future studies to identify the reason of
heterogenous uptake in TM1M tumor uptake.
Keywords: rhenium; technetium-99m; dithiocarbazates; tumor; TM1M; B16F10
1. Introduction
Dithiocarbazate (DTC) and thiosemicarbazone (TSC) molecules have nitrogen and
sulfur in their structure (Figure 1), which can be used to coordinate metals. DTCs or their
Pharmaceutics 2025, 17, 100 https://doi.org/10.3390/pharmaceutics17010100
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Pharmaceutics 2025, 17, 100 2 of 16
metal complexes have been evaluated and showed activity that is trypanocidal [1], antibac-
terial [2,3], and antifungal [4]. The same has happened with TSCs and their complexes [5,6].
Another important application is related to cancer treatment. A large number of molecules
have been evaluated as chemotherapeutics, mainly for TSCs [7–9] and in a minor number
for DTCs [10–12].
Pharmaceutics 2025, 17, x FOR PEER REVIEW 2 of 17 
 
 
1. Introduction 
Dithiocarbazate (DTC) and thiosemicarbazone (TSC) molecules have nitrogen and 
sulfur in their structure (Figure 1), which can be used to coordinate metals. DTCs or their 
metal complexes have been evaluated and showed activity that is trypanocidal [1], anti-
bacterial [2,3], and antifungal [4]. The same has happened with TSCs and their complexes 
[5,6]. Another important application is related to cancer treatment. A large number of 
molecules have been evaluated as chemotherapeutics, mainly for TSCs [7–9] and in a mi-
nor number for DTCs [10–12]. 
 
Figure 1. The backbone of a thiosemicarbazone (TSC) and a dithiocarbazate (DTC); R = alkyl or aryl 
groups. 
A less developed application for TSCs and DTCs is as a radiotracer or radiopharma-
ceutical. By the introduction of radioactive metal, it is possible to image metabolic path-
ways using [64Cu]Cu-CTS (CTS = 2,3-pentanedione bis(thiosemicarbazone)) for cardiac 
hypoxia [13–15] or [64Cu]Cu-ATSM (ATSM = 2,3-butanedione bis(N4-methylthiosemi-
carbazone)) for tumor hypoxia imaging [16–18], the blood pool for hepatic hemangioma 
diagnosis using [68Ga]Ga-TSC-dextran [19], or tumor localization by an unknown process 
as reported for [68Ga]Ga-AATS (AATS = acetylacetate bis(thiosemicarbazones)) for fibro-
sarcoma [20]. Also, the radioactive complex can be used to estimate the biodistribution of 
non-radioactive gallium and indium complexes [21]. [99mTc]technetium, one of the more 
exciting radiometals used in nuclear medicine, due to radioisotope characteristics such as 
the half-life of 6.02 h, gamma emission of 140 keV, and obtainment from a 99Mo/99mTc gen-
erator, has been investigated for TSC and DTC radiometal complexes, such as 
[99mTc][Tc(TSC)(CO)3]+ to be used as a precursor for bifunctional radiopharmaceuticals 
[22]. A nitrofuryl thiosemicarbazone complex was investigated for infection imaging [23], 
[99mTc]technetium TSC containing stilbene and other derivatives presented affinity to am-
yloid-β fibers but could not cross the blood-brain barrier [24] and 4-hydroxy-pyridine 
derivative presented potential as an estrogen receptor linker [25]. Other [99mTc]Tc-TSC 
complexes were prepared and evaluated for radiolabeling conditions, stability, and gen-
eral biodistribution [26,27]. Less common than TSCs is the preparation of the [99mTc]tech-
netium DTC complex, with just a few reports aiming the use of S-methyldithicarbazate 
(NH2NHCS2CH3) as an intermediate to obtain a nitride [[99mTc]TcN(NH2NHCS2CH3)] 
derivative [28] for further transchelation reactions [29]. 
Since our group has been working on the preparation of DTC metal complexes, in-
cluding rhenium [1,30], we decided to investigate the radiolabeling conditions of the S-
benzyl-5-hydroxy-3-methyl-5-phenyl-4,5-dihydro-1H-pyrazole-1-carbodithionate(H2bdtc) with [99mTc]technetium and evaluate the behavior of the radiotracer in vitro and 
in vivo using B16-F10 and TM1M murine melanoma cells. Furthermore, we prepared the 
non-radioactive Re complex for structure characterization using ESI-MS, IR, 1H NMR, X-
ray diffraction, and elemental analysis. This structurally characterized Re complex will be 
Figure 1. The backbone of a thiosemicarbazone (TSC) and a dithiocarbazate (DTC); R = alkyl or
aryl groups.
A less developed application for TSCs and DTCs is as a radiotracer or radiopharmaceuti-
cal. By the introduction of radioactive metal, it is possible to image metabolic pathways using
[64Cu]Cu-CTS (CTS = 2,3-pentanedione bis(thiosemicarbazone)) for cardiac hypoxia [13–15]
or [64Cu]Cu-ATSM (ATSM = 2,3-butanedione bis(N4-methylthiosemicarbazone)) for tumor
hypoxia imaging [16–18], the blood pool for hepatic hemangioma diagnosis using [68Ga]Ga-
TSC-dextran [19], or tumor localization by an unknown process as reported for [68Ga]Ga-AATS
(AATS = acetylacetate bis(thiosemicarbazones)) for fibrosarcoma [20]. Also, the radioactive
complex can be used to estimate the biodistribution of non-radioactive gallium and indium
complexes [21]. [99mTc]technetium, one of the more exciting radiometals used in nuclear
medicine, due to radioisotope characteristics such as the half-life of 6.02 h, gamma emis-
sion of 140 keV, and obtainment from a 99Mo/99mTc generator, has been investigated for
TSC and DTC radiometal complexes, such as [99mTc][Tc(TSC)(CO)3]+ to be used as a pre-
cursor for bifunctional radiopharmaceuticals [22]. A nitrofuryl thiosemicarbazone complex
was investigated for infection imaging [23], [99mTc]technetium TSC containing stilbene and
other derivatives presented affinity to amyloid-β fibers but could not cross the blood-brain
barrier [24] and 4-hydroxy-pyridine derivative presented potential as an estrogen receptor
linker [25]. Other [99mTc]Tc-TSC complexes were prepared and evaluated for radiolabel-
ing conditions, stability, and general biodistribution [26,27]. Less common than TSCs is
the preparation of the [99mTc]technetium DTC complex, with just a few reports aiming
the use of S-methyldithicarbazate (NH2NHCS2CH3) as an intermediate to obtain a nitride
[[99mTc]TcN(NH2NHCS2CH3)] derivative [28] for further transchelation reactions [29].
Since our group has been working on the preparation of DTC metal complexes,
including rhenium [1,30], we decided to investigate the radiolabeling conditions of the S-
benzyl-5-hydroxy-3-methyl-5-phenyl-4,5-dihydro-1H-pyrazole-1-carbodithionate (H2bdtc)
with [99mTc]technetium and evaluate the behavior of the radiotracer in vitro and in vivo
using B16-F10 and TM1M murine melanoma cells. Furthermore, we prepared the non-
radioactive Re complex for structure characterization using ESI-MS, IR, 1H NMR, X-ray
diffraction, and elemental analysis. This structurally characterized Re complex will be used
to confirm the 99mTc complex structure by comparing the HPLC retention time for both.
Pharmaceutics 2025, 17, 100 3 of 16
2. Materials and Methods
2.1. Materials
Chemical compounds were analytical grade (Sigma Aldrich Chemie GmbH, Schnell-
dorf, Germany or Merck KGaA, Darmstadt, Germany), and were used without additional
purification. B16-F10 murine melanoma cells were purchased from the American Type Cul-
ture Collection (ATCC CRL-6475TM), and TM1M cells were selected in our institution [31].
(NBu4)[ReOCl4] is routinely prepared in our laboratory, as described in the literature [32].
The ligand H2bdtc was prepared as published previously [1]. [99mTc]NaTcO4 solutions
were obtained from a [99Mo/99mTc] generator produced by the Instituto de Pesquisas
Energéticas e Nucleares (IPEN, Brazil).
2.2. Instruments
FTIR spectra were measured as KBr pellets on an IR Prestige-21 (Shimadzu Co.,
Kyoto, Japan) spectrophotometer between 400 and 4000 cm−1. Elemental analyses were
determined using the CHN EA 1108 equipment (Fisons Instruments, Rodano, Italy). 1H
NMR spectra were acquired in the NMR Avance III instrument—400 MHz (Bruker BioSpin
GMBH, Rheinstetten, Germany) at room temperature with deuterated chloroform (CDCl3)
as the solvent. HPLC analysis was performed on a Shimadzu VP series (Shimadzu Co.,
Kyoto, Japan), equipped with a UV detector and connected to a radiation detector, the
PerkinElmer Flow Scintillation Analyzer, Radiomatic 610RT. Planar chromatography was
analyzed using a Bioscan Scanner AR2000 (Eckert & Ziegler Radiopharma, Inc., Wilmington,
MA, USA) or a Perkin Elmer 1480 Automatic Gamma Counter Wallac Wizard 3” (Turku,
Finland). The same gamma counter was used to measure the radioactivity in animal organs.
SPECT/CT images were obtained on a Triumph SPECT/CT scanner (TriFoil Imaging, Los
Angeles, CA, USA).
2.3. Animal Handling
The C57B/6 male mice were acquired from the Faculdade de Medicina-USP vivarium
and kept in individually ventilated cages in a controlled room (20 ± 2 ◦C and 50–60%
humidity), under a 12/12 h light/dark cycle. Food and water were provided ad libitum,
except on the experiment day, when food was removed 3 h before the procedure. Animal
handling procedures were carried out under anesthesia with 3% isoflurane in 100% oxygen.
2.4. Synthesis of [ReO(bdtc)(Hbdtc)] and Chemical Characterization
For the preparation of the [ReO(bdtc)(Hbdtc)] complex, the ligand S-benzyl-5-hydroxy-
3-methyl-5-phenyl-4,5-dihydro-1H-pyrazole-1-carbodithionate (H2bdtc) was prepared by
the reaction of S-benzyldithiocarbazate (1) and 1-phenyl-1,3-butadione (2), as summarized
in Scheme 1 [1]. The complex was obtained from mixing a solution of (NBu4)[ReOCl4]
(0.058 g, 0.1 mmol) and H2bdtc (0.068 g, 0.2 mmol) in MeOH:CH2Cl2 (1:1, 10 mL), stirred
for 30 min, resulting in a rapid color change from light yellow to dark red. Red single
crystals, adequate for X-ray diffraction, were obtained by slow solvent evaporation for
2 days at room temperature. The single crystals were filtered off, washed with n-hexane,
and dried under reduced pressure. Yield: 0.082 g, 93%.
The crystal structure data were collected with Mo-Ka radiation (λ = 71.073 pm) at
a temperature of 200 K in an IPDS 2T diffractometer (STOE & Cie GmbH, Darmstadt,
Germany). Standard procedures were applied for data reduction and absorption correction.
The structures were solved by direct methods and refined with anisotropic displacement
parameters using the program SHELXS-97 [33]. Idealized hydrogen atom positions were
calculated with the riding model option of SHELXL-97. Crystal data and structure refine-
ment parameters are summarized in Table 1, and selected bond distances and angles are
Pharmaceutics 2025, 17, 100 4 of 16
listed in Table 2. CCDC 1549693 contains the supplementary crystallographic data for
this paper.
Pharmaceutics 2025, 17, x FOR PEER REVIEW 5 of 17 
 
 
C(6)-S(1) 175.9(7) N(1)-Re-S(1) 79.2(1) 
 N(1)-Re-S(12) 159.1(1) 
 N(12)-Re-S(1) 177.0(1) 
 N(12)-Re-S(12) 81.0(1) 
 S(1)-Re-S(12) 96.05(6) 
Elemental analysis for calculated mass for C36H33N4O3ReS4 (884.13 g·mol−1): C, 48.91; 
H, 3.76; N, 6.34; S, 14.50. Found: C, 48.79; H, 3.45; N, 6.26; S, 14.42. 
NMR spectra were obtained on a Varian Mercury plus 400 spectrometer (9.4 T field 
and frequency of 400 MHz for 1H), being internally referenced to TMS. 1H NMR (400 MHz, 
CDCl3-d1, δ ppm): 2.56 (CH3b, 3H, s), 2.98 (CH3a, 3H, s), 3.48 (CH2c″, 1H, dd, 2J = 13 Hz), 
3.68 (CH2a″, 1H, dd, 2J = 15 Hz), 3.81 (CH2c′, 1H, dd, 2J = 13 Hz), 4.49 (CH2b″, 1H, dd, 2J = 13 
Hz), 4.56 (CH2b′, 1H, dd, 2J = 13 Hz), 4.63 (CH2a′, 1H, dd, 2J = 15 Hz), 6.75 (CH, 1H, s), 7.61-
6.96 (Ar, 20H, m) (Figure S2). 
The mass spectra were obtained using the electrospray ionization (MS-ESI) method 
on an Agilent 6210 ESI-TOF spectrometer. Positive ESI-MS (m/z): 885.10 ([M + H]+), 907.08 
([M + Na]+), 923.06 ([M + K]+) (Figure S4). 
FTIR spectra were measured as KBr pellets on an IR Prestige-21—Shimadzu, with 
elected IR data (KBr, pallets, cm−1): νC=N 1601, νSCS 984, νC=O 1682, νRe=O 957. 
Spectroscopic and analyticaldata for full characterization of compounds are given in the 
Supplementary Materials (Figure S3, Table S1). 
 
Scheme 1. Synthesis of the ligand H2bdtc and [ReO(bdtc)(Hbdtc)]. Reagents and conditions. Step 1: 
(a) MeOH, stirring, room temperature, 8 h, and isolated; Step 2: (b) MeOH:CH2Cl2 (1:1), stirring, 
room temperature, 30 min. 
Scheme 1. Synthesis of the ligand H2bdtc and [ReO(bdtc)(Hbdtc)]. Reagents and conditions. Step 1:
(a) MeOH, stirring, room temperature, 8 h, and isolated; Step 2: (b) MeOH:CH2Cl2 (1:1), stirring,
room temperature, 30 min.
Table 1. Crystal data and details of structural determination for the [ReO(bdtc)(Hbdtc)] complex.
Parameter Evaluation Values
Empirical formula C36H33N4O3ReS4
Formula weight 884.10
Temperature (K) 200
Wavelength (Å) 0.71073
Crystal system Triclinic
Space group P ı̄
Unit cell dimensions
a (Å) 8.172(2)
b (Å) 15.097(4)
c (Å) 16.168(4)
α (◦) 110.804(2)
β (◦) 98.755(2)
γ (◦) 95.717(2)
Volume 1.81737(8)
Z 2
D calc (g/cm3) 1.616
Pharmaceutics 2025, 17, 100 5 of 16
Table 1. Cont.
Parameter Evaluation Values
Absorption coefficient (mm−1) 3.614
F(000) 880
2θ range for data collection (o) 2.65 to 24.77
Index ranges −9 ≤ h ≤ 9, −17 ≤ k ≤ 16, 0 ≤ l ≤ 19
Reflections collected 6120
Independent reflections 6120 [R(int) = 0.0000]
Completeness to 2θ = 50.00 98.3%
Refinement method Full-matrix least-squares on F2
Data/restraints/parameters 6120/0/433
Goodness-of-fit on F2 1.116
R indices (all data) R1 = 0.0573, wR2 = 0.0798
Final R indices [I > 2σ(I)] R1 = 0.0387, wR2 = 0.0748
Largest difference peak and hole (e Å−3) 0.688 and −0.635
Table 2. Selected bond distances and bond angles for the complex [ReO(bdtc)(Hbdtc)].
Distances (pm) Angles (◦)
Re-O(1) 168.04(4) O(1)-Re-O(2) 156.9(2)
Re-O(2) 206.1(4) O(1)-Re-N(1) 98.6(2)
Re-N(1) 207.1(5) O(1)-Re-N(12) 82.9(2)
Re-N(12) 225.5(5) O(1)-Re-S(1) 98.1(2)
Re-S(1) 230.8(2) O(1)-Re-S(12) 102.2(2)
Re-S(12) 233.5(2) O(2)-Re-N(1) 78.5(2)
C(4)-O(2) 130.4(7) O(2)-Re-N(12) 75.7(2)
C(42)-O(22) 123.3(8) O(2)-Re-S(1) 103.7(1)
C(2)-N(1) 133.0(7) O(2)-Re-S(12) 82.9(1)
C(6)-N(2) 128.2(8) N(1)-Re-N(12) 103.5(2)
C(6)-S(1) 175.9(7) N(1)-Re-S(1) 79.2(1)
N(1)-Re-S(12) 159.1(1)
N(12)-Re-S(1) 177.0(1)
N(12)-Re-S(12) 81.0(1)
S(1)-Re-S(12) 96.05(6)
Elemental analysis for calculated mass for C36H33N4O3ReS4 (884.13 g·mol−1): C, 48.91;
H, 3.76; N, 6.34; S, 14.50. Found: C, 48.79; H, 3.45; N, 6.26; S, 14.42.
NMR spectra were obtained on a Varian Mercury plus 400 spectrometer (9.4 T field
and frequency of 400 MHz for 1H), being internally referenced to TMS. 1H NMR (400 MHz,
CDCl3-d1, δ ppm): 2.56 (CH3b, 3H, s), 2.98 (CH3a, 3H, s), 3.48 (CH2c′′ , 1H, dd, 2J = 13 Hz),
3.68 (CH2a′′ , 1H, dd, 2J = 15 Hz), 3.81 (CH2c′ , 1H, dd, 2J = 13 Hz), 4.49 (CH2b′′ , 1H, dd,
2J = 13 Hz), 4.56 (CH2b′ , 1H, dd, 2J = 13 Hz), 4.63 (CH2a′ , 1H, dd, 2J = 15 Hz), 6.75 (CH, 1H,
s), 7.61-6.96 (Ar, 20H, m) (Figure S2).
The mass spectra were obtained using the electrospray ionization (MS-ESI) method on
an Agilent 6210 ESI-TOF spectrometer. Positive ESI-MS (m/z): 885.10 ([M + H]+), 907.08
([M + Na]+), 923.06 ([M + K]+) (Figure S4).
FTIR spectra were measured as KBr pellets on an IR Prestige-21—Shimadzu, with
elected IR data (KBr, pallets, cm−1): νC=N 1601, νSCS 984, νC=O 1682, νRe=O 957. Spec-
troscopic and analytical data for full characterization of compounds are given in the
Supplementary Materials (Figure S3, Table S1).
Pharmaceutics 2025, 17, 100 6 of 16
2.5. Radiolabeling and Quality Control
2.5.1. Preparation of [[99mTc]TcO(bdtc)(Hbdtc)]
The radiocomplex was prepared by adding, in a borosilicate vial, 300 µL of nitro-
genated solution of H2bdtc in DMSO (1 mg/mL), 300 µL of [99mTc]NaTcO4 (370–740 MBq)
solution, 20 µL of SnCl2 solution (2.5 mg of SnCl2.2H2O, 10 µL of HCl 37%, 10 mL nitro-
genated water), and 640 µL phosphate buffer (PB) solution (0.1 M, pH 7). The vial was
closed and the solution stood at room temperature for 15 min. The solution was analyzed
for pH using a universal paper indicator.
2.5.2. Radiochemical Purity
The radiochemical purity was assessed by planar chromatography using paper
W3MM/NaCl 0.9%, W3MM/acetone systems, and by HPLC using column RP-C18 Synergy-
Hydro-RP, 300 × 3.9 mm × 10 µm (Phenomenex, Torrance, CA, USA), mobile phase
acetone:H2O:MeOH (66:22:12), isocratic model at 1 mL/min. [ReO(bdtc)(Hbdtc)] standard
was co-injected with the radioactive samples in the HPLC, and the UV detector was set up
to 260 nm.
2.5.3. Partition Coefficient
Log Po/w for the 99mTc-complex was determined by adding 100 µL of the radioactive
solution to a mixture of 3 mL of n-octanol and 2.9 mL of phosphate buffer (0.1 M, pH
7). The mixture was vigorously vortexed for 1 min and stood for 5 min. Aliquots of
1 mL organic and inorganic layers were removed and activity was independently deter-
mined in a dose calibrator. The partition coefficient (P) was calculated using the equation:
Po/w = (radioactivity in n-octanol)/(radioactivity in water), and the result was given in a
logarithm value (Log Po/w). The results represent the mean of the three measurements.
2.5.4. Complex Stability
Complex stability was evaluated for saline, L-cysteine, L-histidine, and mice plasma.
In a borosilicate vial were added 100 µL of 99mTc complexes and 900 µL of: (a) saline (NaCl
0.9%); (b) 1 mM L-cysteine hydrochloride solution (PBS 0.1 M, pH 7.4); (c) 1 mM L-histidine
hydrochloride solution (PBS 0.1 M, pH 7.4); (d) mice plasma, isolated as described early [34].
The solutions were kept at 37 ◦C, sampling at times 0, 1, 3, and 6 h, and evaluated using the
HPLC conditions to analyze radiochemistry purity. For a, b, and c, sampling was carried
out directly from the test vials, and for d, proteins were precipitated using acetonitrile and
centrifuged; the liquid phase was filtered in a 0.45 µm filter, and the sample was removed
from the filtered [34]. In addition, a, b, c, and d were analyzed by planar chromatography
using the systems (1) W3MM/acetone and (2) W3MM/NaCl 0.9%; in this case, proteins
from plasma were not precipitated.
2.6. Biological Assay
2.6.1. In Vitro Cell Uptake
B16-F10 and TM1M murine melanoma cell lines were maintained as monolayer cul-
tures in RPMI-1640 medium containing 10% fetal bovine serum and penicillin (100 U/mL)-
streptomycin (100 µg/mL), under 37 ◦C, 5% CO2, and humidity of 95%. For uptake studies,
1 × 105 cells were plated in culture dishes (d = 16 mm) and left standing overnight for
adherence. For uptake experiments, the medium was changed by a fresh one, without
antibiotics, and 10 µL of a solution containing 37 KBq of the 99mTc complex was added to
each culture dish (n = 4/time/cell line). The incubation was maintained for 15, 60, and
120 min. At the end of each time, the medium was quickly removed, cells were washed
twice with cold PBS, and these solutions were transferred to counter tubes. The cells were
Pharmaceutics 2025, 17, 100 7 of 16
harvested using trypsin and transferred to counter tubes. The activity of the samples was
counted in a gamma counter, and uptake yield was calculated by dividing counts in the cell
tube by the total counts in the system. Cell viability was accessed by trypan blue staining.
2.6.2. Ex Vivo Biodistribution
Mice were anesthetized with isoflurane at 3% in O2, and B16-F10 and TM1M murine
melanoma cells suspended in saline were inoculated on the animal’s superior left and right
flanks, respectively. The TM1M grew for 13–14 days and B17F10 for 10–12 days, or until
it reached no more than 0.75 cm3. After tumor growth, animals were anesthetized, and
100 µL aliquot of 99mTc complex (20 MBq in saline) was injected into the penile vein of
male C57Bl/6 mice (8 weeks, 25–28 g). At 15, 60, and 120 min post-injection, the animals
(n = 3–4), under anesthesia effect, were euthanized by heart extirpation. The blood and urine
samples, tumor, muscle, bone, brain, thyroid, lungs, heart, kidneys, stomach, spleen, liver,
large intestine, small intestine, and bladder were dissected and weighed; their radioactivitywas measured in a gamma counter. The results expressed as a percentage of injected dose
per gram of tissue (%ID·g−1).
2.6.3. In Vivo Biodistribution
Another group of animals (n = 2/group), presenting the same tumor, were anes-
thetized, and 28.6–44.4 MBq of 99mTc complexes in 0.1 mL of saline:DMSO (5:1) was
injected into the penile vein. SPECT images (64 projections × 60 s) were acquired 60 min
after tracer injection using a dual-head camera with 1.0 mm five-pinhole collimators and
a CZT detector system. Images were reconstructed using the OSEM method. CT images
were acquired immediately before SPECT imaging to help with the best positioning of the
animal in the center of the field of view (FOV) and anatomical information for fusion with
SPECT images. Images were acquired using a flat-panel detector CT system with 45 kVp
and 390 µA. After reconstruction, images (SPECT and CT) were fused using PMOD™ 4.0
(PMOD Technologies Ltd., Zurich, Switzerland) and analyzed.
3. Results
3.1. Synthesis of [ReO(bdtc)(Hbdtc)]
The ligand H2bdtc was previously prepared [1] by the reaction of S-benzyldithiocarbazate
(1) and 1-phenyl-1,3-butadione (2), as summarized in Scheme 1. An equilibrium between
the open-chain form (3A), which is appropriate for chelation, and the cycle-chain of the 5-
hydroxypyrazolic form (3B) is possible. The [ReO(bdtc)(Hbdtc)] complex was obtained from
the ligand H2bdtc (3) and the precursor (NBu4)[ReOCl4] (4), and the reaction was followed by
a color change from light yellow to dark red.
Two dithiocarbazate ligands were coordinated to the metal center in different modes;
one with dianionic tridentate O,N,S chelation (bdtc2−) and the other with monoanionic
bidentate N,S chelation (Hbdtc1−). The ligand H2bdtc presents absorption bands at
3359 cm–1, ν(O–H), and no ν(N–H) band, in agreement with the cyclic structure found in
the solid state, as shown in Supplementary Data (Figure S3) [1]. The ν(O–H) signal was
lost in the rhenium complex, evidencing the deprotonation and thioenolization of the C=S
group (Table S1) upon coordination. The thiocarbonyl nature of the free ligand in the solid
state is supported by the absence of a ν(SH) 2600–2500 cm–1 [16]. The coordination through
the deprotonated thioenolic tautomeric form of the ligand [–N=C(SH)–functionality] is
commonly observed in their complexes, and it is further corroborated by the appearance of
a new band in the range of 710–779 cm–1, characteristic of ν(C–S) [35]. The strong ν(C=N)
1630 cm–1 (in H2bdtc) is shifted to a lower wavenumber, 1601 cm−1, and the intensity
in the corresponding complex indicates the coordination of the azomethine nitrogen to
Pharmaceutics 2025, 17, 100 8 of 16
the metal ion [36–38]. A νC=O 1682 cm−1 for the complex agrees with one DTC ligand
N,S coordinating in a monoanionic keto form. The bathochromic shift of νRe=O from
1002 cm−1 ([ReOCl4]− precursor) to 949 cm−1 confirms the complex formation.
1H NMR spectra of the free ligand H2dtc were measured in CDCl3 solution at
400 MHz. The spectra of the ligand were predominantly observed in the cyclic tautomeric
form, with an insignificant amount present in the open-ring form (Scheme 1). 1H NMR
(ppm): 2.10 (s, 3H, CH3), 3.05 (d, 1H, 2J = 19 Hz, ring CH2), 3.44 (d, 1H, 2J = 19 Hz, ring
CH2), 4.30 (d, 1H, 2J = 13 Hz, benzyl CH2), 4.41 (d, 1H, 2J = 13 Hz, benzyl CH2), 6.47
(s, 1H, OH), 7.24–7.41 (m, 10H, Ph). No NH resonance is observed, agreeing with the
cyclic pyrazoline ligand form (Figure S1) [39]. Most of the ligand signals are shifted for
the complex, except the OH and NH signals, which are absent for the complex (Figure S2),
according to the infrared and X-ray data.
The complex was analyzed by single crystal X-ray diffraction and the structure agrees
with the spectroscopic results. The molecular structure of the rhenium complex with
the atomic numbering scheme is shown in Figure 2 and the ortep plot is included as
Supplementary Data. In addition, selected bond lengths and angles are given in Table 2.
The general geometry around the metal center can be better described as a distorted
octahedral coordination sphere for the rhenium atom. The metal core is coordinated with
a dianionic O,N,S-tridentate facial TSC ligand, a monoanionic N,S-bidentate equatorial
TSC ligand, and an oxo group at trans position to the oxygen donor atom of the tridentate
TSC ligand.
Pharmaceutics 2025, 17, x FOR PEER REVIEW 8 of 17 
 
 
corresponding complex indicates the coordination of the azomethine nitrogen to the metal 
ion [36–38]. A νC=O 1682 cm−1 for the complex agrees with one DTC ligand N,S 
coordinating in a monoanionic keto form. The bathochromic shift of νRe=O from 1002 
cm−1 ([ReOCl4]− precursor) to 949 cm−1 confirms the complex formation. 
 1H NMR spectra of the free ligand H2dtc were measured in CDCl3 solution at 400 
MHz. The spectra of the ligand were predominantly observed in the cyclic tautomeric 
form, with an insignificant amount present in the open-ring form (Scheme 1). 1H NMR 
(ppm): 2.10 (s, 3H, CH3), 3.05 (d, 1H, 2J = 19 Hz, ring CH2), 3.44 (d, 1H, 2J = 19 Hz, ring 
CH2), 4.30 (d, 1H, 2J = 13 Hz, benzyl CH2), 4.41 (d, 1H, 2J = 13 Hz, benzyl CH2), 6.47 (s, 1H, 
OH), 7.24–7.41 (m, 10H, Ph). No NH resonance is observed, agreeing with the cyclic 
pyrazoline ligand form (Figure S1) [39]. Most of the ligand signals are shifted for the 
complex, except the OH and NH signals, which are absent for the complex (Figure S2), 
according to the infrared and X-ray data. 
The complex was analyzed by single crystal X-ray diffraction and the structure agrees 
with the spectroscopic results. The molecular structure of the rhenium complex with the 
atomic numbering scheme is shown in Figure 2 and the ortep plot is included as 
Supplementary Data. In addition, selected bond lengths and angles are given in Table 2. 
The general geometry around the metal center can be better described as a distorted 
octahedral coordination sphere for the rhenium atom. The metal core is coordinated with 
a dianionic O,N,S-tridentate facial TSC ligand, a monoanionic N,S-bidentate equatorial 
TSC ligand, and an oxo group at trans position to the oxygen donor atom of the tridentate 
TSC ligand. 
 
Figure 2. X-ray structure [ReO(bdtc)(Hbdtc)] complex. Hydrogen atoms are omitted for clarity. 
3.2. Complex Characteristics 
3.2.1. Radiochemical Purity and Partition Coefficient 
The [[99mTc]TcO(bdtc)(Hbdtc)] radiochemical purity was 93 ± 2%, as determined by 
reversed-phase radio-HPLC (n = 4), with a retention time (tR) of 3.2 min (Figure 3b), which 
matches the [ReO(bdtc)(Hbdtc)] standard, presenting a tR = 3.2–3.5 min, as observed in 
the UV detector (Figure 3c); unbonded [99mTc][TcO4]− reaching 5 ± 2% found at tR = 1.9–2.2 
min, within the value found in planar chromatography (Table 3). 
Planar chromatography (PC) was performed in parallel (n = 5) and found 3 ± 1% of 
[99mTc][TcO4]− (Figure 4a) and 4 ± 2% for [99mTc]TcO2 (Figure 4b). 
The partition coefficient (log p) value was 1.03 ± 0.06 (n = 3), demonstrating the com-
plex’s hydrophobic characteristic. 
 
Figure 2. X-ray structure [ReO(bdtc)(Hbdtc)] complex. Hydrogen atoms are omitted for clarity.
3.2. Complex Characteristics
3.2.1. Radiochemical Purity and Partition Coefficient
The [[99mTc]TcO(bdtc)(Hbdtc)] radiochemical purity was 93 ± 2%, as determined by
reversed-phase radio-HPLC (n = 4), with a retention time (tR) of 3.2 min (Figure 3b), which
matches the [ReO(bdtc)(Hbdtc)] standard, presenting a tR = 3.2–3.5 min, as observed in the
UV detector (Figure 3c); unbonded [99mTc][TcO4]− reaching 5 ± 2% found at tR = 1.9–2.2 min,
within the value found in planar chromatography (Table 3).
Planar chromatography (PC) was performed in parallel (n = 5) and found 3 ± 1% of
[99mTc][TcO4]− (Figure 4a) and 4 ± 2% for [99mTc]TcO2 (Figure 4b).
The partition coefficient (log p) value was 1.03 ± 0.06 (n = 3), demonstrating the
complex’s hydrophobic characteristic.
Pharmaceutics 2025, 17, 100 9 of 16
Pharmaceutics 2025, 17, x FOR PEER REVIEW9 of 17 
 
 
Table 3. Medium (n = 2) [[99mTc]TcO(bdtc)(Hbdtc)] stability evaluated by planar chromatography. 
Experimental 
Parameter Rf Values 
Radioactive Concentration (%) at Time 
0 1 h 3 h 6 h 
PBS 7.4 
0–0.3 
0.7–1 
2.8 
4.4 
3.1 
6 
3 
6.3 
3.2 
6.3 
L-His 
0–0.3 
0.7–1 
2.8 
4.4 
2.2 
3.8 
0.6 
5.9 
2.4 
7.2 
L-Cys 0–0.3 
0.7–1 
2.8 
4.4 
6.5 
5.5 
9 
9.7 
14.9 
15.4 
Plasma 0–0.3 
0.7–1 
2.8 
4.4 
17.7 
2.4 
38.6 
3.0 
74.5 
5.2 
Chromatographic systems: W3MM/acetone (Rf 0–0.3); W3MM/NaCl 0.9% (Rf 0.7–1). 
 
Figure 3. HPLC analysis for (a) [[99mTc]TcO4]−, (b) [[99mTc]TcO(bdtc)(Hbdtc)], and (c) 
[ReO(bdtc)(Hbdtc)]. 
 
Figure 3. HPLC analysis for (a) [[99mTc]TcO4]−, (b) [[99mTc]TcO(bdtc)(Hbdtc)], and (c) [ReO(bdtc)(Hbdtc)].
Table 3. Medium (n = 2) [[99mTc]TcO(bdtc)(Hbdtc)] stability evaluated by planar chromatography.
Experimental
Parameter Rf Values
Radioactive Concentration (%) at Time
0 1 h 3 h 6 h
PBS 7.4 0–0.3
0.7–1
2.8
4.4
3.1
6
3
6.3
3.2
6.3
L-His 0–0.3
0.7–1
2.8
4.4
2.2
3.8
0.6
5.9
2.4
7.2
L-Cys 0–0.3
0.7–1
2.8
4.4
6.5
5.5
9
9.7
14.9
15.4
Plasma 0–0.3
0.7–1
2.8
4.4
17.7
2.4
38.6
3.0
74.5
5.2
Chromatographic systems: W3MM/acetone (Rf 0–0.3); W3MM/NaCl 0.9% (Rf 0.7–1).
Pharmaceutics 2025, 17, x FOR PEER REVIEW 9 of 17 
 
 
Table 3. Medium (n = 2) [[99mTc]TcO(bdtc)(Hbdtc)] stability evaluated by planar chromatography. 
Experimental 
Parameter Rf Values 
Radioactive Concentration (%) at Time 
0 1 h 3 h 6 h 
PBS 7.4 
0–0.3 
0.7–1 
2.8 
4.4 
3.1 
6 
3 
6.3 
3.2 
6.3 
L-His 
0–0.3 
0.7–1 
2.8 
4.4 
2.2 
3.8 
0.6 
5.9 
2.4 
7.2 
L-Cys 0–0.3 
0.7–1 
2.8 
4.4 
6.5 
5.5 
9 
9.7 
14.9 
15.4 
Plasma 0–0.3 
0.7–1 
2.8 
4.4 
17.7 
2.4 
38.6 
3.0 
74.5 
5.2 
Chromatographic systems: W3MM/acetone (Rf 0–0.3); W3MM/NaCl 0.9% (Rf 0.7–1). 
 
Figure 3. HPLC analysis for (a) [[99mTc]TcO4]−, (b) [[99mTc]TcO(bdtc)(Hbdtc)], and (c) 
[ReO(bdtc)(Hbdtc)]. 
 
Figure 4. Representative planar chromatographic profile for [[99mTc]TcO(bdtc)(Hbdtc)]:
(a) W3MM/NaCl 0.9% solution and (b) W3MM/acetone.
Pharmaceutics 2025, 17, 100 10 of 16
3.2.2. Complex Stability
[[99mTc]TcO(bdtc)(Hbdtc)] was incubated in PBS (0.1 M, pH 7.4), in PBS containing
1 mM of L-cysteine (L-cys), 1 mM L-histidine (L-his), and mice plasma at 37 ◦C, for 1, 3,
and 6 h. HPLC analysis has demonstrated the complex stability in the presence of L-his
and plasma (mainly by 1 h), but in the presence of the L-cys, radiochemical purity is
diminished by the decrease of the radioactivity at tR = 2.3–4.1 min with an increase of this
one in the tR = 1.6–2.3 min, when compared to time 0 (Figure 5). Planar chromatographic
values at Rf = 0–0.3 in the W3MM/acetone, representing [[99mTc]O2 or [[99mTc]-amino
acid and plasma impurities, and at Rf = 0.7–1 in the W3MM/NaCl 0.9%, representing
[[99mTc]O4
− or [[99mTc]-amino acid impurities obtained at time zero, were considered to
compare stability. The results shown in Table 3, representing the radioactive amount in
the Rf listed, demonstrated good stability for the [[99mTc]TcO(bdtc)(Hbdtc)] in PBS and
in the presence of L-his, since substantial changes in the values of radioactivity on the Rf
analyzed during 6 h were not observed. On the other hand, the complex was unstable in
the presence of L-cys by increasing the radioactivity at both selected Rf, compatible with the
conversion of a hydrophobic compound to a hydrophilic one, but not [99mTc][TcO4]−, since
this molecule has migrated to Rf 0.7–1 in both mobile phases. In the plasma, at the system
W3MM/acetone, the radioactivity concentration at Rf 0–0.3 increased during the time
interval, suggesting compound protein binding. While HPLC and plana chromatography
gave similar results related to the action of amino acids over the complex, the phenomena
related to protein binding can not be observed in HPLC since, during the preparation of
plasma sampling for this analysis, the proteins were precipitated using acetonitrile. At
this point it is important to recommend the combined HPLC and planar chromatographic
analysis for full characterization of radiochemical species present in the samples.
Pharmaceutics 2025, 17, x FOR PEER REVIEW 10 of 17 
 
 
Figure 4. Representative planar chromatographic profile for [[99mTc]TcO(bdtc)(Hbdtc)]: (a) 
W3MM/NaCl 0.9% solution and (b) W3MM/acetone. 
3.2.2. Complex Stability 
[[99mTc]TcO(bdtc)(Hbdtc)] was incubated in PBS (0.1 M, pH 7.4), in PBS containing 1 
mM of L-cysteine (L-cys), 1 mM L-histidine (L-his), and mice plasma at 37 °C, for 1, 3, and 
6 h. HPLC analysis has demonstrated the complex stability in the presence of L-his and 
plasma (mainly by 1 h), but in the presence of the L-cys, radiochemical purity is 
diminished by the decrease of the radioactivity at tR = 2.3–4.1 min with an increase of this 
one in the tR = 1.6–2.3 min, when compared to time 0 (Figure 5). Planar chromatographic 
values at Rf = 0–0.3 in the W3MM/acetone, representing [[99mTc]O2 or [[99mTc]-amino acid 
and plasma impurities, and at Rf = 0.7–1 in the W3MM/NaCl 0.9%, representing [[99mTc]O4− 
or [[99mTc]-amino acid impurities obtained at time zero, were considered to compare 
stability. The results shown in Table 3, representing the radioactive amount in the Rf 
listed, demonstrated good stability for the [[99mTc]TcO(bdtc)(Hbdtc)] in PBS and in the 
presence of L-his, since substantial changes in the values of radioactivity on the Rf 
analyzed during 6 h were not observed. On the other hand, the complex was unstable in 
the presence of L-cys by increasing the radioactivity at both selected Rf, compatible with 
the conversion of a hydrophobic compound to a hydrophilic one, but not [99mTc][TcO4]−, 
since this molecule has migrated to Rf 0.7–1 in both mobile phases. In the plasma, at the 
system W3MM/acetone, the radioactivity concentration at Rf 0–0.3 increased during the 
time interval, suggesting compound protein binding. While HPLC and plana 
chromatography gave similar results related to the action of amino acids over the 
complex, the phenomena related to protein binding can not be observed in HPLC since, 
during the preparation of plasma sampling for this analysis, the proteins were 
precipitated using acetonitrile. At this point it is important to recommend the combined 
HPLC and planar chromatographic analysis for full characterization of radiochemical 
species present in the samples. 
 
Figure 5. HPLC stability studies of [[99mTc]TcO(bdtc)(Hbdtc)] against (a) L-histidine, (b) L-cysteine, 
and (c) plasma. tR = retention time. AUC = area under the curve. 
3.3. Biological Characteristics 
3.3.1. In Vitro Cellular Uptake 
[[99mTc]TcO(bdtc)(Hbdtc)] in vitro tumor uptake was evaluated for adhered B16-F10 
and TM1M cells. The uptake reached 1.03 ± 0.13% and 0.85 ± 0.24% at 15 min for TM1M 
Figure 5. HPLC stability studies of [[99mTc]TcO(bdtc)(Hbdtc)] against (a) L-histidine, (b) L-cysteine,
and (c) plasma. tR = retention time. AUC = area under the curve.
3.3. Biological Characteristics
3.3.1. In Vitro Cellular Uptake
[[99mTc]TcO(bdtc)(Hbdtc)] in vitro tumor uptake was evaluated for adhered B16-F10
and TM1M cells. The uptake reached 1.03 ± 0.13% and 0.85 ± 0.24% at 15 min for TM1M
and B16-F10, respectively, but decreased to 0.7 ± 0.08% and 0.63 ± 0.05% after 120 min
(Figure 6).
Pharmaceutics 2025, 17, 100 11 of 16
Pharmaceutics 2025, 17, x FOR PEER REVIEW 11 of 17 
 
 
and B16-F10, respectively, but decreased to 0.7 ± 0.08% and 0.63 ± 0.05% after 120 min 
(Figure 6). 
 
Figure 6. In vitro uptake of the [[99mTc]TcO(bdtc)(Hbdtc)] in murine melanoma B16-F10 and TM1M 
culture (n = 4). 
3.3.2. Ex Vivo Biodistribution 
The ex vivo biodistribution of the [[99mTc]TcO(bdtc)(Hbdtc)] was evaluated in 
C57Bl/6 mice with implanted murine melanoma tumor B16-F10 and TM1M cells. The 
uptake value was lower than 1% ID·g−1 and decreased by 15, 60, and 120 min after 
injection, as presented in Table 4. No statistical difference (ppoints for each cell type was observed. Two other characteristics to be observed for 
this compound are the fast blood washout, with concentrations as low as 0.41 ± 0.15% 
ID·g−1 at 15 min, while at the same time, the liver uptake was 26.47 ± 4.67 % ID·g−1 and 
spleen was 63.11 ± 2.93% ID·g−1, still high at 120 min, as shown in Table 4. No other organ 
registered a noticeable uptake. 
Table 4. Biodistribution of [[99mTc]TcO(bdtc)(Hbdtc)] in B16-F10 and TM1M tumor-expressing 
C57/Bl6 mice (n = 3), expressed as % ID·g−1. 
Samples 15 min 60 min 120 min 
Blood 0.41 ± 0.16 0.17 ± 0.04 0.17 ± 0.04 
Brain 0.02 ± 0.01 0.01 ± 0 0.02 ± 0.01 
Thyorid 0.51 ± 0.14 1.08 ± 0.46 0.45 ± 0.24 
Heart 0.21 ± 0.06 0.2 ± 0.09 0.06 ± 0.04 
Lungs 0.54 ± 0.19 0.82 ± 0.52 0.48 ± 0.24 
Kidneys 0.6 ± 0.04 1.3 ± 1.04 2.15 ± 0.25 
Stomach 0.7 ± 0.73 1.42 ± 0.94 1.42 ± 0.01 
Spleen 63.11 ± 2.93 60.11 ± 6.74 55.46 ± 2.9 
Liver 26.47 ± 4.67 29.07 ± 4.95 31.91 ± 2.73 
Large intestine 0.07 ± 0 0.1 ± 0.3 0.24 ± 0.02 
Small intestine 0.14 ± 0.04 0.32 ± 0.08 0.74 ± 0.1 
Bone 2.58 ± 3.9 0.71 ± 0.36 3.26 ± 0.16 
Muscle 0.05 ± 0.01 0.12 ± 0.15 0.04 ± 0.01 
B16-F10 0.34 ± 0.07 0.31 ± 0.09 0.25 ± 0.05 
TM1M 0.26 ± 0.08 0.22 ± 0.05 0.20 ± 0.06 
 
Figure 6. In vitro uptake of the [[99mTc]TcO(bdtc)(Hbdtc)] in murine melanoma B16-F10 and TM1M
culture (n = 4).
3.3.2. Ex Vivo Biodistribution
The ex vivo biodistribution of the [[99mTc]TcO(bdtc)(Hbdtc)] was evaluated in C57Bl/6
mice with implanted murine melanoma tumor B16-F10 and TM1M cells. The uptake
value was lower than 1% ID·g−1 and decreased by 15, 60, and 120 min after injection,
as presented in Table 4. No statistical difference (pthe paper chromatographic system, which is characteristic behavior for radiolabeled
proteins [41,42].
Tumor cell uptake was low, in vitro, and different from what occurs for several radio-
labeled molecules, mainly hydrophobic compounds, which are expected to internalize in
the cells by passive diffusion [43] and growth during the time. The same low uptake was
observed in vivo, in part due to the fast blood washout and high liver and spleen uptake
drastically decreasing the complex’s availability to be accumulated in other organs.
The ex vivo and in vivo biodistribution demonstrated a high and persistent liver
and spleen uptake, with no finding of a correlation with another hydrophobic compound
presented previously in the literature, such as 99mTc(V)-aminooxime derivatives, with LogP
= 1.35–1.43, having a maximum liver uptake of 23%; in spleen this was 1.45% ID·g−1 [44];
99mTc-daunorubicin, LogP = 1.7, with a liver uptake of 0.93% ID·g−1 and spleen 0.54%
ID·g−1, 1 h after injection [45]; in the [[99mTc]Tc(mibi)6]+, liver and spleen uptake was
about 2% ID·g−1, 60 min post-injection, but the spleen concentration can be increased
to 23.6% ID·g−1, when [[99mTc]Tc(mibi)6]+ was encapsulated in liposomes [46]. For col-
loidal compounds, such as 99mTc-sulfur colloidal, liver uptake is about 92.1% ID·g−1
between 15 min and 24 h after injections, whereas the spleen uptake was as low as 2.5
and 3.6% ID·g−1, during the same interval time [47]. Thus, [[99mTc]TcO(bdtc)(Hbdtc)] can
be a candidate for spleen imaging agents in the substitution of heat-denatured red blood
cells [48,49], which involves the manipulation of the patient’s blood and reinjection, with
potential risk to patient health.
Tumor uptake was relatively low as demonstrated by ex vivo and in vivo studies;
however, by imaging it was possible to visualize a heterogeneous uptake of the 99mTc
complex in the TM1M tumor (0.75 cm3), whereas in the B16-F10 tumor (0.5 cm3) it was
not observed; these findings are not uncommon for radiopharmaceuticals used in the
evaluation of different tumor types, usually associated with the differential expression
of some cell protein or receptor [50–52]. However, since ex vivo analysis did not show a
difference in the tumor uptake, as observed in the image, we can consider the possibility
that the tumor microenvironment components can play some role in this finding. These
results reinforce the importance of tomographic imaging methods in developing new drugs
or radiopharmaceuticals, especially over ex vivo biodistribution data.
5. Conclusions
The new [ReO(bdtc)(Hbdtc)] complex was prepared in high purity, allowing complete
structural characterization that was fundamental to confirming achieved the equal com-
pound radiolabeled with 99mTc, with the match in retention time in HPLC analysis. The
[[99mTc]TcO(bdtc)(Hbdtc)] was also obtained in proper purity and presented limited in vitro
stability that could compromise in vivo use as a diagnostic agent. However, the biodistri-
bution data showing a fast and high liver and spleen uptake could open opportunities for
developing new radiopharmaceuticals for the study of liver and spleen diseases. The lack
of difference in the ex vivo TM1M and B16F10 tumor uptake, but a clear difference by the
small animal SPECT imaging, can suggest that some components in microenvironments
can be associated with this behavior. To deepen the biological behavior findings in this
research, new projects focusing on gastroenterology and oncology need to be developed.
Supplementary Materials: The following supporting information can be downloaded at:
https://www.mdpi.com/article/10.3390/pharmaceutics17010100/s1. Figure S1: The 1H-NMR of
the ligand H2bdtc; Figure S2: The 1H-NMR of the complex [ReO(bdtc)(Hbdtc)], CDCl3, 400 MHz;
Figure S3. IR spectrum of the complex [ReO(bdtc)(Hbdtc)]; Figure S4: ESI-MS spectrum of the
complex [ReO(bdtc)(Hbdtc)] (full and expanded area); Table S1: Important IR spectral bands (cm–1)
and their assignments for ligand and metal complexes.
https://www.mdpi.com/article/10.3390/pharmaceutics17010100/s1
Pharmaceutics 2025, 17, 100 14 of 16
Author Contributions: Conceptualization, C.A.B., U.A., V.M.D. and F.L.N.M.; methodology, D.d.P.F.,
U.A., V.M.D. and F.L.N.M.; investigation, A.G.d.A.F., A.E.L., C.P.L., D.L. and D.d.P.F.; resources,
D.d.P.F., V.M.D., and F.L.N.M.; writing—original draft preparation, A.G.d.A.F., A.E.L. and D.d.P.F.;
writing—review and editing, A.G.d.A.F., A.E.L., V.M.D. and F.L.N.M.; visualization, A.G.d.A.F.,
A.E.L., D.d.P.F. and F.L.N.M.; supervision, V.M.D. and F.L.N.M.; project administration, V.M.D. and
F.L.N.M.; funding acquisition, V.M.D. and F.L.N.M. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo
(Grants: #2007/02595-0, #2007/03135-3, #2014/22250-1, and #2021/10265-8—Cancer Theranostics
Innovation Center (CancerThera)/CEPID—Centros de Pesquisa, Inovação e Difusão). Coordenadoria
de Aperfeiçoamento de Pessoal de Nível Superior (Alyne E. Lafratta master fellowship).
Institutional Review Board Statement: The experiments with animals were carried out in accordance
with the guidelines of the National Council for the Control of Animal Experimentation (CONCEA,
Brazil), a constituent body of the Ministry of Science, Technology, and Innovation (MCTI, Brazil). All
protocols were approved by the Ethics Committee on Animal Use of the Faculty of Medicine of the
University of São Paulo (CEUA-FMUSP 372/12, approved 7/11/2012, addendum 29/05/2014).
Data Availability Statement: Data are contained within the article or Supplementary Material.
Conflicts of Interest: The authors declare no conflicts of interest.
References
1. da Maia, P.I.S.; de Fernandes, A.G.A.; Silva, J.J.N.; Andricopulo, A.D.; Lemos, S.S.; Lang, E.S.; Abram, U.; Deflon, V.M.
Dithiocarbazate complexes with the [M(PPh3)]2+ (M=Pd or Pt) moiety: Synthesis, characterization and anti-Tripanosoma cruzi
activity. J. Inorg. Biochem. 2010, 104, 1276–1282. [CrossRef] [PubMed]
2. Bin Break, M.K.; Fung, T.Y.; Koh, M.Z.; Ho, W.Y.; Tahir, M.I.M.; Elfar, O.A.; Syed, R.U.; Khojali, W.M.A.; Alluhaibi, T.M.; Huwaimel,
B.; et al. Synthesis, Crystal Structure, Antibacterial and In Vitro Anticancer Activity of Novel Macroacyclic Schiff Bases and Their
Cu (II) Complexes Derived from S-Methyl and S-Benzyl Dithiocarbazate. Molecules 2023, 28, 5009. [CrossRef] [PubMed]
3. Latif, M.A.; Ahmed, T.; Hossain, M.S.; Chaki, B.M.; Abdou, A.; Zahan, K.E. Synthesis, Spectroscopic Characterization, DFT
Calculations, Antibacterial Activity, and Molecular Docking Analysis of Ni(II), Zn(II), Sb(III), and U(VI) Metal Complexes Derived
from a Nitrogen-Sulfur Schiff Base. Russ. J. Gen. Chem. 2023, 93, 389–397. [CrossRef]
4. Malik, M.A.; Lone, S.A.; Wani, M.Y.; Talukdar, M.I.A.; Dar, O.A.; Ahmad, A.; Hashmi, A.A. S-benzyldithiocarbazate imine
coordinated metal complexes kill Candida albicans by causing cellular apoptosis and necrosis. Bioorg. Chem. 2020, 98, 103771.
[CrossRef]
5. More, M.S.; Joshi, P.G.; Mishra, Y.K.; Khanna, P.K. Metal complexes driven from Schiff bases and semicarbazones for biomedical
and allied applications: A review. Mater. Today Chem. 2019, 14, 100195. [CrossRef]
6. Mir, I.A.; Ain, Q.U.; Qadir, T.; Malik, A.Q.; Jan, S.; Shahverdi, S.; Nabi, S.A. A review of semicarbazone-derived metal complexes
for application in biomedicine and related fields. J. Mol. Struct. 2024, 1295, 136216. [CrossRef]
7. Siqueira, L.R.P.; de Moraes Gomes, P.A.T.; Ferreira, L.P.L.; de Melo Rêgo, M.J.B.; Leite, A.C.L. Multi-target compounds acting in
cancer progression: Focus on thiosemicarbazone, thiazole and thiazolidinone analogues. Eur. J. Med. Chem. 2019, 170, 237–260.
[CrossRef]
8. Bai, X.-G.; Zheng, Y.; Qi, J. Advances in thiosemicarbazone metal complexes as anti-lung cancer agents. Front. Pharmacol. 2022, 13,
1018951. [CrossRef]
9. Jiang, X.; Fielding, L.A.; Davis, H.; Carroll, W.; Lisic, E.C.; Deweese,J.E. Inhibition of Topoisomerases by Metal Thiosemicarbazone
Complexes. Int. J. Mol. Sci. 2023, 24, 12010. [CrossRef]
10. Gou, Y.; Jia, X.; Hou, L.X.; Deng, J.G.; Huang, G.J.; Jiang, H.W.; Yang, F.J. Dithiocarbazate−FeIII, −CoIII, −NiII, and −ZnII
Complexes: Design, Synthesis, Structure, and Anticancer Evaluation. J. Med. Chem. 2022, 65, 6677–6689. [CrossRef]
11. Deng, J.G.; Peng, C.; Hou, L.X.; Wu, Y.R.; Liu, W.; Fang, G.H.; Jiang, H.W.; Qin, S.F.; Yang, F.; Huang, G.J.; et al. Dithiocarbazate-
Copper Complex Loaded Thermosensitive Hydrogel for Lung Cancer Therapy via Tumor in situ Sustained-Release. Inorg. Chem.
Front. 2022, 9, 6190–6201. [CrossRef]
12. Sahu, G.; Patra, S.A.; Lima, S.; Das, S.; Görls, H.; Plass, W.; Dinda, R. Ruthenium(II)-Dithiocarbazates as Anticancer Agents:
Synthesis, Solution Behavior, and Mitochondria-Targeted Apoptotic Cell Death. Chem. Eur. J. 2023, 29, e202202694. [CrossRef]
[PubMed]
https://doi.org/10.1016/j.jinorgbio.2010.08.009
https://www.ncbi.nlm.nih.gov/pubmed/20843554
https://doi.org/10.3390/molecules28135009
https://www.ncbi.nlm.nih.gov/pubmed/37446670
https://doi.org/10.1134/S1070363223020214
https://doi.org/10.1016/j.bioorg.2020.103771
https://doi.org/10.1016/j.mtchem.2019.100195
https://doi.org/10.1016/j.molstruc.2023.136216
https://doi.org/10.1016/j.ejmech.2019.03.024
https://doi.org/10.3389/fphar.2022.1018951
https://doi.org/10.3390/ijms241512010
https://doi.org/10.1021/acs.jmedchem.1c02186
https://doi.org/10.1039/D2QI01383E
https://doi.org/10.1002/chem.202202694
https://www.ncbi.nlm.nih.gov/pubmed/36598160
Pharmaceutics 2025, 17, 100 15 of 16
13. Medina, R.A.; Mariotti, E.; Pavlovic, D.; Shaw, K.P.; Eykyn, T.R.; Blower, P.J.; Southworth, R. 64Cu-CTS: A Promising Radio-
pharmaceutical for the Identification of Low-Grade Cardiac Hypoxia by PET. J. Nucl. Med. 2015, 56, 921–926. [CrossRef]
[PubMed]
14. Avila-Rodriguez, M.A.; Rios, C.; Carrasco-Hernandez, J.; Manrique-Arias, J.C.; Martinez-Hernandez, R.; García-Pérez, F.O.;
Jalilian, A.R.; Martinez-Rodriguez, E.; Romero-Piña, M.E.; Diaz-Ruiz, A. Biodistribution and radiation dosimetry of [64Cu]copper
dichloride: First-in-human study in healthy volunteers. EJNMMI Res. 2017, 7, 98. [CrossRef]
15. Pell, V.R.; Baark, F.; Mota, F.; Clark, J.E.; Southworth, R. PET Imaging of Cardiac Hypoxia: Hitting Hypoxia Where It Hurts. Curr.
Cardiovasc. Imaging Rep. 2018, 11, 7. [CrossRef]
16. Hueting, R.; Kersemans, V.; Cornelissen, B.; Tredwell, M.; Hussien, K.; Christlieb, M.; Gee, A.D.; Passchier, J.; Smart, S.C.;
Dilworth, J.R.; et al. A Comparison of the Behavior of 64Cu-Acetate and 64Cu-ATSM In Vitro and In Vivo. J. Nucl. Med. 2014,
55, 128–134. [CrossRef]
17. Pérès, E.A.; Toutain, J.; Paty, L.P.; Divoux, D.; Ibazizène, M.; Guillouet, S.; Barré, L.; Vidal, A.; Cherel, M.; Bourgeois, M.; et al.
64Cu-ATSM/64Cu-Cl2 and their relationship to hypoxia in glioblastoma: A preclinical study. EJNMMI Res. 2019, 9, 114. [CrossRef]
18. Kurihara, H.; Narita, Y.; Ito, K.; Sato, H.; Miyakita, Y.; Takahashi, M.; Yanagisawa, S.; Okita, N.T.; Sadachi, R.; Tateishi, K.; et al.
Phase I clinical trial of 64Cu-ATSM for malignant brain tumors. J. Clin. Oncol. 2024, 42, 2052. [CrossRef]
19. Almasi, T.; Jabbari, K.; Gholipour, N.; Kheirabadi, A.M.; Beiki, D.; Shahrokhi, P.; Akhlaghi, M. Synthesis, characterization, and
in vitro and in vivo 68Ga radiolabeling of thiosemicarbazone Schiff base derived from dialdehyde dextran as a promising pool
imaging agente. Int. J. Biol. Macromol. 2019, 125, 915–921. [CrossRef]
20. Jalilian, A.R.; Yousefnia, H.; Shafaii, K.; Novinrouz, A.; Rajamand, A.A. Preparation and Biodistribution Studies of a Radiogallium-
Acetylacetonate Bis(Thiosemicarbazone) Complex in Tumor-Bearing Rodents. Iran. J. Pharm. Res. 2012, 11, 523–531.
21. Prado, V.S.; Leitao, R.C.F.; Silva, F.; Gano, L.; Santos, I.C.; Marques, F.L.N.; Paulo, A.; Deflon, V.M. Gallium and indium complexes
with new hexadentate bis(semicarbazone) and bis(thiosemicarbazone) chelators. Dalton Trans. 2021, 50, 1631–1640. [CrossRef]
[PubMed]
22. Fuks, L.; Gniazdowska, E.; Kozminski, P.; Mieczkowski, J. Technetium(I) tricarbonyl complexed with the N-heterocyclic aldehyde
thiosemicarbazones: Potential precursors of the radiopharmaceuticals. J. Radioanal. Nucl. Chem. 2012, 292, 255–259. [CrossRef]
23. Halder, K.K.; Nayak, D.K.; Baishya, R.; Sarkar, B.R.; Sinha, S.; Ganguly, S.; Debnath, M.C. 99mTc-labeling of ciprofloxacin
and nitrofuryl thiosemicarbazone using fac-[99mTc(CO)3(H2O)3] core: Evaluation of their efficacy as infection imaging agents.
Metallomics 2011, 3, 1041–1048. [CrossRef]
24. Pino-Cuevas, A.; Raposinho, P.D.; Fernandes, C.; Paulo, A.; Abram, U.; Carballo, R.; Vazquez-L’opez, E.M. Thiosemicarbazonate
complexes with affinity for amyloid-β fibers: Synthesis, characterization and biological studies. Future Med. Chem. 2019,
11, 2527–2544. [CrossRef]
25. Argibay-Otero, S.; Núñez, A.; Muñoz, L.; Carballo, R.; Fernandes, C.; Paulo, A.; Vázquez-López, E.M. Study of Mixed
Re(I)/99mTc(I) Thiosemicarbazonate Complexes: Estrogen Receptor Affinity and Stability in Biological Media. Eur. J. Inorg. Chem.
2024, 27, e202400131. [CrossRef]
26. Argibay-Otero, S.; Gano, L.; Fernandes, C.; Paulo, A.; Carballo, R.; Vázquez-López, E.M. Chemical and biological studies of
Re(I)/Tc(I) thiosemicarbazonate complexes relevant for the design of radiopharmaceuticals. J. Inorg. Biochem. 2020, 203, 110917.
[CrossRef]
27. Kelderman, C.A.; Davey, P.R.; Ma, M.T.; de Veer, M.; Salimova, E.; Donnelly, P.S.; Paterson, B.M. Hexadentate technetium-99m
bis(thiosemicarbazonato) complexes: Synthesis, characterisation and biodistribution. Dalton Trans. 2022, 51, 14064. [CrossRef]
28. Liu, G. Direct interaction between S-methyl dithiocarbazate and pertechnetate-99m. Nucl. Med. Biol. 1997, 24, 119–122. [CrossRef]
29. Carta, D.; Jentschel, C.; Thieme, S.; Salvarese, N.; Morellato, N.; Refosco, F.; Ruzza, P.; Bergmann, R.; Pietzsch, H.-J.; Bolzati, C.
Assessment of the best N3− donors in preparation of [M(N)(PNP)]-based (M = 99mTc-; 188Re) target-specific radiopharmaceuticals:
Comparison among succinic dihydrazide (SDH), N-methyl-S-methyl dithiocarbazate (HDTCZ) and PEGylated N-methyl-S-
methyl dithiocarbazate (HO2C-PEG600-DTCZ). Nucl. Med. Biol. 2014, 41, 570–581. [CrossRef]
30. de Fernandes, A.G.A.; Viana, R.B.; Moreno-Fuquen, R.; Gatto, C.C.; Lang, E.S.; Modolo, M.Z.; Silva, A.K.; Lemos, S.S.; Hagenbach,
A.; Abram, U.; et al. Oxorhenium(V) complexes with a benzyldithiocarbazate ligand: Synthesis, crystal structure, spectroscopic
and DFT analyses. J. Mol. Struct. 2022, 1250, 131875. [CrossRef]
31. Oba-Shinjo, S.M.; Correay, M.; Riccay, T.I.; Molognoni, F.; Pinhal, M.A.; Neves, I.A.; Marie, S.K.; Sampaio, L.O.; Nader, H.B.;
Chammas, R.; et al. Melanocyte Transformation Associated with Substrate Adhesion Impediment. Neoplasia 2006, 8, 231–241.
[CrossRef] [PubMed]
32. Alberto, R.; Schibli, R.; Egli, A.; Schubiger, P.A.; Hermann, W.A.; Artus, G.; Abram, U.; Kaden, T.A. Metal carbonyl syntheses XXII.
Low pressure carbonylation of [MOCl4]− and [MO4]−: The technetium(I) and rhenium(I) complexes [NEt4]2[MCl3(CO)3]. J.
Organomet. Chem. 1995, 493, 119–127. [CrossRef]
33. Sheldrick, G.M. A short history of SHELX. Acta Cryst. 2008, A64, 112–122. [CrossRef] [PubMed]
https://doi.org/10.2967/jnumed.114.148353
https://www.ncbi.nlm.nih.gov/pubmed/25883129
https://doi.org/10.1186/s13550-017-0346-4
https://doi.org/10.1007/s12410-018-9447-3
https://doi.org/10.2967/jnumed.113.119917
https://doi.org/10.1186/s13550-019-0586-6
https://doi.org/10.1200/JCO.2024.42.16_suppl.2052
https://doi.org/10.1016/j.ijbiomac.2018.12.133
https://doi.org/10.1039/D0DT04028B
https://www.ncbi.nlm.nih.gov/pubmed/33480908
https://doi.org/10.1007/s10967-011-1404-4
https://doi.org/10.1039/c1mt00068c
https://doi.org/10.4155/fmc-2019-0013
https://doi.org/10.1002/ejic.202400131
https://doi.org/10.1016/j.jinorgbio.2019.110917
https://doi.org/10.1039/D2DT01264B
https://doi.org/10.1016/S0969-8051(96)00159-X
https://doi.org/10.1016/j.nucmedbio.2014.04.126https://doi.org/10.1016/j.molstruc.2021.131875
https://doi.org/10.1593/neo.05781
https://www.ncbi.nlm.nih.gov/pubmed/16611417
https://doi.org/10.1016/0022-328X(94)05373-J
https://doi.org/10.1107/S0108767307043930
https://www.ncbi.nlm.nih.gov/pubmed/18156677
Pharmaceutics 2025, 17, 100 16 of 16
34. Souza, A.M.; Pitombeira, M.S.; Souza, L.E.; Navarro Marques, F.L.; Buchpiguel, C.A.; Real, C.C.; Faria, D.P. 11C-PK11195 plasma
metabolization has the same rate in multiple sclerosis patients and healthy controls: A cross-sectional study. Neural Regen. Res.
2021, 16, 2494–2498.
35. Maurya, M.R.; Kumar, A.; Bhat, A.R.; Azam, A.; Bader, C.; Rehder, D. Dioxo- and Oxovanadium(V) Complexes of Thiohydrazone
ONS Donor Ligands: Synthesis, Characterization, Reactivity, and Antiamoebic Activity. Inorg. Chem. 2006, 45, 1260–1269.
[CrossRef]
36. Salem, N.M.; El-Sayed, L.; Iskander, M.F. Metal complexes derived from hydrazoneoxime ligands: IV. Molecular and supramolec-
ular structures of some nickel(II) complexes derived from diacetylmonoxime S-benzyldithiocarbazonate. Polyhedron 2008,
27, 3215–3226. [CrossRef]
37. Yazdanbakhsh, M.; Heravi, M.M.; Takjoo, R.; Frank, W. Synthesis and Spectroscopic Characterization of the Benzalde-
hyde Schiff Baseof S-Allyldithiocarbazate and its Copper(II) Complex and the X-ray CrystalStructure of Bis[S-allyl-β-N-
benzylidene)dithiocarbazato]Copper(II). Z. Anorg. Allg. Chem. 2008, 634, 972–976. [CrossRef]
38. Roy, S.; Mandal, T.N.; Barik, A.K.; Gupta, S.; Butcher, R.J.; Nethaji, M.; Kar, S.K. Syntheses, characterization and X-ray crystal
structures of Co(III) and Mn(II) complexes of pyrimidine derived Schiff base ligands. Polyhedron 2008, 27, 593–601. [CrossRef]
39. Pavan, F.R.; Maia, P.I.S.; Leite, S.R.A.; Deflon, V.M.; Batista, A.A.; Sato, D.N.; Franzblau, S.G.; Leite, C.Q.F. Thiosemicarbazones,
semicarbazones, dithiocarbazates and hydrazide/hydrazones: Anti—Mycobacterium tuberculosis activity and cytotoxicity. Eur.
J. Med. Chem. 2010, 45, 1898–1905. [CrossRef]
40. Kirsch, S.; Jankowsky, R.; Leibnitz, P.; Spies, H.; Johannsen, B. Crystal and solution structure of oxo rhenium(V) complexes with
cysteine and cysteine methyl ester. JBIC J. Biol. Inorg. Chem. 1999, 4, 48–55. [CrossRef]
41. Wang, Y.-F.; Chen, Y.-C.; Li, D.-K.; Chuang, M.-H. Technetium-99m-Labeled Autologous Serum Albumin: A Personal-Exclusive
Source of Serum Component. J. Biomed. Biotechnol. 2011, 413802. [CrossRef] [PubMed]
42. Lodhi, N.A.; Park, J.Y.; Kim, K.; Kim, Y.J.; Shin, J.H.; Lee, Y.-S.; Im, H.-J.; Jeong, J.M.; Khalid, M.; Cheon, G.J.; et al. Development
of 99m Tc-Labeled Human Serum Albumin with Prolonged Circulation by Chelate-then-Click Approach: A Potential Blood Pool
Imaging Agent. Mol. Pharm. 2019, 16, 1586–1595. [CrossRef] [PubMed]
43. Dearling, J.L.J.; Lewis, J.S.; Mullen, G.E.D.; Welch, M.J.; Blower, P.J. Copper bis(thiosemicarbazone) complex as hypoxia imaging
agents: Structure-activity relationships. J. Biol. Inorg. Chem. 2002, 7, 249–259. [CrossRef]
44. Mirković, M.; Janković, D.; Vranješ-Durić, S.; Radović, M.; Stanković, D.; Mijin, D.; Nikolić, N. Novel tetradentate diamine
dioxime ligands: Synthesis, characterization and in vivo behavior of their 99mTc-complexes. Appl. Organometal. Chem. 2012,
26, 347–355. [CrossRef]
45. Faheem, A.R.; Bokhari, T.H.; Roohi, S.; Mushtaq, A.; Sohaib, M. 99mTc-Daunorubicin a potential brain imaging and theranostic
agent: Synthesis, quality control, characterization, biodistribution and scintigraphy. Nucl. Med. Biol. 2013, 40, 148–152. [CrossRef]
46. Belhaj-Tayeb, H.; Briane, D.; Vergote, J.; Kothan, S.; Léger, G.; Bendada, S.-E.; Tofighi, M.; Tamgac, F.; Cao, A.; Moretti, J.-L. In vitro
and in vivo study of 99mTc-MIBI encapsulated in PEG-liposomes: A promising radiotracer for tumour imaging. Eur. J. Nucl. Med.
Mol. Imaging 2003, 30, 502–509. [CrossRef]
47. Noronha, O.P.D.; Sewatkar, A.B.; Ganatra, R.D. Factors Affecting the Preparation of 99mTc-Sulfur Colloid. Eur. J. Nucl. Med. 1978,
3, 47–54. [CrossRef]
48. Massey, M.D.; Steven, J.S. Residual Spleen Found on Denatured Red Blood Cell Scan Following Negative Colloid Scans. J. Nucl.
Med. 1991, 32, 2286–2287.
49. Barber, T.W.; Dixon, A.; Smith, M.; Yap, K.S.K.; Kalff, V. Ga-68 octreotate PET/CT and Tc-99m heat-denatured red blood cell
SPECT/CT imaging of an intrapancreatic accessory spleen. J. Med. Imaging Radiat. Oncol. 2016, 60, 227–229. [CrossRef]
50. Tanaka, T.; Furukawa, T.; Fujieda, S.; Kasamatsu, S.; Yonekura, Y.; Fujibayashi, Y. Double-tracer autoradiography with Cu-
ATSM/FDG and immunohistochemical interpretation in four different mouse implanted tumor models. Nucl. Med. Biol. 2006,
33, 743–750. [CrossRef]
51. Tzanopoulou, S.; Sagnou, M.; Paravatou-Petsotas, M.; Gourni, E.; Loudos, G.; Xanthopoulos, S.; Lafkas, D.; Kiaris, H.; Varvarigou,
A.; Pirmettis, I.C.; et al. Evaluation of Re and 99mTc Complexes of 2-(4′-Aminophenyl)benzothiazole as Potential Breast Cancer
Radiopharmaceuticals. J. Med. Chem. 2010, 53, 4633–4641. [CrossRef] [PubMed]
52. Rahmanian, N.; Hosseinimehr, S.J.; Khalaj, A.; Noaparast, Z.; Abedi, S.M.; Sabzevari, O. 99mTc-radiolabeled GE11-modified
peptide for ovarian tumor targeting. DARU J. Pharm. Sci. 2017, 25, 13. [CrossRef]
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https://doi.org/10.1021/ic050811+
https://doi.org/10.1016/j.poly.2008.07.009
https://doi.org/10.1002/zaac.200700521
https://doi.org/10.1016/j.poly.2007.10.012
https://doi.org/10.1016/j.ejmech.2010.01.028
https://doi.org/10.1007/s007750050288
https://doi.org/10.1155/2011/413802
https://www.ncbi.nlm.nih.gov/pubmed/21541233
https://doi.org/10.1021/acs.molpharmaceut.8b01258
https://www.ncbi.nlm.nih.gov/pubmed/30869911
https://doi.org/10.1007/s007750100291
https://doi.org/10.1002/aoc.2870
https://doi.org/10.1016/j.nucmedbio.2012.08.010
https://doi.org/10.1007/s00259-002-1038-4
https://doi.org/10.1007/BF00253477
https://doi.org/10.1111/1754-9485.12319
https://doi.org/10.1016/j.nucmedbio.2006.05.005
https://doi.org/10.1021/jm1001293
https://www.ncbi.nlm.nih.gov/pubmed/20518489
https://doi.org/10.1186/s40199-017-0179-8
	Introduction 
	Materials and Methods 
	Materials 
	Instruments 
	Animal Handling 
	Synthesis of [ReO(bdtc)(Hbdtc)] and Chemical Characterization 
	Radiolabeling and Quality Control 
	Preparation of [[99mTc]TcO(bdtc)(Hbdtc)] 
	Radiochemical Purity 
	Partition Coefficient 
	Complex Stability 
	Biological Assay 
	In Vitro Cell Uptake 
	Ex Vivo Biodistribution 
	In Vivo Biodistribution 
	Results 
	Synthesis of [ReO(bdtc)(Hbdtc)] 
	Complex Characteristics 
	Radiochemical Purity and Partition Coefficient 
	Complex Stability 
	Biological Characteristics 
	In Vitro Cellular Uptake 
	Ex Vivo Biodistribution 
	In Vivo Biodistribution 
	Discussion 
	Conclusions 
	References