Journal of Nuclear and Radiochemical Sciences, Vol. 6, No. 3, pp. 187-188, 2005

Synthesis and In Vitro Evaluation of Tc(CO) 3 + Complexes with Dithiocarbamate Ligands
Containing Morpholine Moiety
G. E. Kodina,*, a A. O. Malysheva,a O. E. Klement’eva,a N. I. Gorshkov,b A. A. Lumpov,band D. N. Suglobovb
aISSC Institute of Biophysics, 123182, Moscow, Russia, Zhivopisnaya st., 46
RPA Khlopin Radium Institute, 194021, St.-Petersburg, Russia, 2nd Murinskii, 28bReceived: July 31, 2005; In Final Form: September 30, 2005The aim of this work was to synthesize and study complexes of M(CO) 3 + with dithiocarbamate ligands included
morpholine moiety which have high affinity to melanoma. The corresponding complexes of rhenium (A and B)
were obtained by simultaneous mixing of bidentate (dithiocarbamate) and monodentate (isonitrile) ligands with[Re(CO)3Br3][NEt4] 2. For the comparative evaluation of these technetium-99m complexes for diagnostics of melanoma the study of affinity of these complexes to the cells of melanoma B 16 in vitro was carried out.

1. Introduction
During the last ten years a lot of technetium and rhenium
complexes with mixed ligands included M(CO) 3 + group wasstudied and described in the literature. It is due to the development of new radiopharmaceuticals and first of all with radiopharmaceuticals for tumor investigations.
It was found earlier1, 2 that dithiocarbamates react withM(CO)3 (H2O)3+ to form complexes extremely stable for a long
period in both basic and acidic medium.
The aim of this work was to synthesize and study complexesof Tc(CO)3+ (99mTc-TC) with dithiocarbamate ligands included
morpholine moiety which have high affinity to melanoma.3
2. Material and Methods
The technetium complexes (99mTc-TC-I, 99mTc-TC-II, 99mTcTC-III, 99mTc-TC-IV, 99mTc-TC-V, 99mTc-TC-VI) were obtainedin two steps. Firstly the solution of 99mTc(CO) 3 (H 2O) 3 + withpH 7 was obtained from “IsoLink” kit. Then different ligands[propyl-morpholin-dithiocarbamate (L 1), morpholin-dithiocarbamate (L 2), isocyanoacetic acid (L3), tret-butyl-isonitryl (L 4),
nitrozyl-sulfuric acid (L 5), diethyl-dithiocarbamate (L 6), ethylisocyanoacetate (L 7) and propyl-morpholin-isonitryl (L 8)] wereadded to the solution. The ligand concentration in the mixture
was 1.5×10−3 M. The reaction mixture was heated in the waterboiling bath during 20 min. After cooling the solution to room
temperature pH was adjusted to 7 with 0.1 M HCl. The reaction mixture was analyzed using TLC on silica gel in MeOH/HCl conc. (99/1).
Ligand in position A

Ligand in position B

Tc-TC-I

L4L199m

Tc-TC-II

L5L699m

Tc-TC-IIIL3L299m

Tc-TC-IV

L7L199m

Tc-TC-V

L3L199m

Tc-TC-VI

L8L6complex
99m

For the cell culture and cell uptake study the breast adeno*Corresponding author. E-mail: vkorsuns@rcibph.dol.ru. FAX:
+7-495-190-3590.

carcinoma MCF-7 and melanoma B 16 cell lines were grown inmonolayers in RPMI 1640 medium, supplemented with 10%
fetal bovine serum, amino acids, vitamins and penicillin-streptomycin, in humidified 5% CO2 /air atmosphere at 37 ˚C. Cellgrowth status was monitored by inverted microscope withphase contrast, and cell viability was assessed by trypan blueexclusion. For all experiments, the cells were seeded at densityof 3 million cells/ flask 25 cm 2 . Before the experiments themedium was changed to RPMI 1640 without serum. Then theneeded amount of radiopharmaceuticals was added to the cell
culture and the cells were incubated at 37 ˚C. After various
incubation times (30, 60, 90, 120, 180 min) cellular uptake wasstopped by removing medium from cells and washing twicewith 5 mL phosphate-buffered saline (PBS). The cell uptake
was calculated as percentage of the activity counted in theflasks containing cells (after subtracting the absorbed activity)
relative to the total activity (in the flasks containing cells andmedium). The cell uptake was presented in percentage per 1
million of cells.
The efflux of radioactivity from the cells was determined inexperiment with 99mTc(CO)3Cit and TC-99mTc-VI (containingL 2 and L 8) in both cell lines. The cells were incubated for 90
min to allow cell uptake of tracers to reach a maximal level.
The radioactive medium was then removed, the cells werewashed twice with 5 mL of PBS and then 5 mL of fresh RPMI
1640 was added to the cells for re-incubation. The cell uptake
was determined once again at the end of re-incubation. Thecell efflux was calculated as difference of the total activity (inthe flasks containing cells and medium) and activity in theflasks containing cells (after subtracting the absorbed activity).
3. Results and Discussion
For the comparative evaluation of synthesized technetium99m complexes for diagnostics of melanoma the study of affinity of these complexes to the cells of melanoma B 16 and breast
adenocarcinoma MCF 7 in vitro was carried out. 99mTc(CO)3Cit,
which was earlier described4, was used as a comparative radiopharmaceutical.
The uptake of 99mTcO 4 − (0.2%/million) was significantly
lower than that of 99mTc(CO) 3 Cit, 99mTc-TC-I, 99mTc-TC-II,99m
Tc-TC-III, 99mTc-TC-IV, 99mTc-TC-V, 99mTc-TC-VI in allcells. So we can conclude that technetium-99m in the form ofpertechnetate-ions does not bind with cells.
The kinetics of cellular uptake of 99mTc-TC-I, 99mTc-TC-II,© 2005 The Japan Society of Nuclear and Radiochemical Sciences
Published on Web 12/28/2005

188 J. Nucl. Radiochem. Sci., Vol. 6, No. 3, 2005

KodinaCell efflux of 99mTc-TC-Cit and 99mTc-TC-VI

50

99mTc-TC-Cit

40

99mTc-TC-I
99mTc-TC-V

30

99mTc-TC-IV

20

99mTc-TC-VI

10
00

30

60

90

120

150

180

100

Cell-associated activity, %uptake (%/106 cells)

Melanoma B 16 cell line

MCF-7 99mTc-TC-Cit

80

MCF-7 99mTc-TC-VI

70

B 16 99mTc-TC-Cit

60

B16 99mTc-TC-VI

50
40
30
20time of incubation (minutes)

Figure 1. Uptake of90

10099m

Tc-complexes in cell lines.

030

60

90

re-incubation time, min

Figure 3. Cell efflux.
Brest adenocarcinoma MCF-7 cell line
Ouptake (%/106 cells)

O

99mTc-TC-I

50

99mTc-TC-II

40

99mTc-TC-Cit

30

CS

OC

Tc

OCS

99m

OCO

N

Tc

N

N

S

C

S

OC

COTc
CO

99m

Tc-TC- I

Tc-TC- II

99m

Tc-TC-

III

O

10OO

N

N

30

60

90

time of incubation (minutes)

Figure 2. Uptake of 99mTc-complexes in cell lines.

OCSTc

OCN

S

Tc-TC- IV

STc

OCN

CO99m

Tc-TC-VI and 99mTc(CO)3Cit in breast adenocarcinoma MCF
7 cell line was shown in Figure 1. The kinetics of cellular
uptake of 99mTc-TC-I, 99mTc-TC-IV, 99mTc-TC-V, 99mTc-TC-VI
and 99mTc(CO)3Cit in melanoma B 16 cell line was shown inFigure 2.
The maximal accumulation in the cells was observed after
90–120 min from the begging of incubation. The accumulationof 99mTc(CO) 3 Cit does not exceed 2.3%/million in both celllines. The highest uptake in both cell lines was for 99mTc-TC-VI(about 50%/million). The affinity of the other complexes werein several times lower and decreases in the following sequence
99mTc-TC-VI > 99mTc-TC-I >> 99mTc-TC-II > 99mTc-TC-Cit(breast adenocarcinoma MCF 7 cell line) and 99mTc-TC-VI >99m
Tc-TC-I >> 99mTc-TC-V > 99mTc-TC-IV > 99mTc-TC-Cit (melanoma B 16 cell line). It is very difficult to explain why the complex 99mTc-TC-VI posses the highest affinity to melanoma cells,
because the date of its biological behavior in vivo does not coincide with date in vitro. The accumulation in tumor in melanoma
bearing-mice is about 1.8%/g after 1 h post injection. This fact
we can explain, if we assume, that this complex has a very highlipophilicity in comparison with other complexes because its
accumulation in the intestine is about 68% after 1 h post injection. It is the highest accumulation in the intestine in comparison with other complexes. So the mechanism of cell uptake canbe the following-more lipophilic complex easier penetrates
through the cell membrane and accumulates inside the cell.
Due to the highest cell uptake 99mTc-TC-VI was used for cellefflux study. It is shown that cell efflux does not exceed 30%
after 1.5 h re-incubation for both complexes(99mTc(CO)3Cit and 99mTc-TC-VI) in both cell lines. Figure
3 shows the percentage of cell activity after 1.5 h re-incubation.

NC

C

OCO

N

O

0O

N

S

OC

20099m

S

OC

N

CO

99mTc-TC-VI

O

NNO

C

N

S

N

COO

OCS

Tc

OCN

S

CO

99m

Tc-TC- V

99m

Tc-TC- VI

Figure 4. Structures of the 99mTc-complexes.

4. Conclusions
1. Technetium-99m in the form of pertechnetate-ions does notbind with all cells.
2. Affinity of the 99mTc-complexes to melanoma B 16 cells
decreases in the following order:
99m
Tc-TC-VI >> 99mTc-TC-I >> 99mTc-TC-V > 99mTc-TC-IV
99m> Tc-TC-Cit
3. Affinity of the 99mTc-complexes to breast adenocarcinoma
MCF 7 cells decreases in the following order:
99m
Tc-TC-VI > 99mTc-TC-I >> 99mTc-TC-V > 99mTc-TC-II >99m
Tc-TC-Cit
4. The cell efflux does not exceed 30% after 1.5 h re-incubation for 99mTc-TC-Cit and 99mTc-TC-VI in both cell lines.
Acknowledgement. This work was financially supported
by ISTC (Grant 1723).
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(2) N. I. Gorshkov, R. Scbibly, and A. P. Schubiger, et al. J.
Organomet. Chem. 689/25, 4757 (2004).
(3) M. Friebe, A. Mahmood, and H. Spies, et al., J. Med. Chem.
43, 2745.
(4) G. E. Kodina, A. O. Malysheva, and O. E. Klementieva, etal. Eur. J. Nucl. Med. 30, P27/384, S319 (2003).