99mTc-labeling of colchicine using [99mTc(CO) 3(H2O)3]+ and [ 99mTc≡N]2+ core for the preparation of potential tumor-targeting agents

Article abstract of DOI:10.1016/j.bmc.2005.09.006

Multidrug resistance (MDR) mediated by over-expression of P-glycoprotein (Pgp) is one of the major causes of failure of chemotherapy in cancer treatment. Colchicine, a naturally occurring alkaloid, is a Pgp substrate and acts as an antimitotic agent by binding to microtubules. Hence, Colchicine and its analogues radiolabeled with ^99mTc may have potential for visualization of MDR in tumors. Here we report ^99mTc-labeling of colchicine derivatives using [^99mTc(CO)₃(H ₂O)₃]⁺ and [^99mTc≡N] ²⁺ cores. Trimethylcolchicinic acid synthesized from colchicine was used as the precursor to prepare iminodiacetic acid and dithiocarbamate derivatives which were then radiolabeled with [^99mTc(CO) ₃(H₂O)₃]⁺ and [ ^99mTc≡N]²⁺ cores, respectively. Radiolabeling yield for both the complexes was >98% as observed by HPLC and TLC patterns. In vitro studies in tumor cell lines showed significant uptake for ^99mTc-carbonyl as well as for ^99mTc-nitrido colchicine complexes. Biodistribution studies in Swiss mice bearing fibrosarcoma tumor showed 4.1 ± 1.2% ID/g of uptake at 30 min pi for ^99mTc(CO) ₃-complex as against 0.42 ± 0.24% ID/g for the ^99mTcN-complex. ^99mTc(CO)₃-colchicine complex exhibited better pharmacokinetics with lower liver accumulation as compared to the ^99mTcN-complex. Thus, colchicine radiolabeled with [ ^99mTc(CO)₃(H₂O)₃]⁺ core is more promising with respect to in vivo distribution characteristics in tumor model.

Full text of DOI:10.1016/j.bmc.2005.09.006

Bioorganic & Medicinal Chemistry 14 (2006) 793–799
99mTc-labeling of colchicine using [99mTc(CO)3(H2O)3]+
and [99mTc„N]2+ core for the preparation of potential
tumor-targeting agents
Aruna Korde,a Drishty Satpati,a Anupam Mathur,a Madhava Mallia,a
Sharmila Banerjee,a,* Kanchan Kothari,a H. D. Sarma,b
Pradeep Choudharic and Meera Venkatesha
aRadiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai 400085, India
bRadiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400085, India
cLaboratory and Nuclear Medicine Section, Bhabha Atomic Research Centre, Mumbai 400085, India
Received 9 June 2005; revised 31 August 2005; accepted 1 September 2005
Available online 11 November 2005
Abstract—Multidrug resistance (MDR) mediated by over-expression of P-glycoprotein (Pgp) is one of the major causes of failure of
chemotherapy in cancer treatment. Colchicine, a naturally occurring alkaloid, is a Pgp substrate and acts as an antimitotic agent by
binding to microtubules. Hence, Colchicine and its analogues radiolabeled with 99mTc may have potential for visualization of MDR
in tumors. Here we report 99mTc-labeling of colchicine derivatives using [99mTc(CO)3(H2O)3]+ and [99mTc„N]2+ cores. Trim-
ethylcolchicinic acid synthesized from colchicine was used as the precursor to prepare iminodiacetic acid and dithiocarbamate deriv-
atives which were then radiolabeled with [99mTc(CO)3(H2O)3]+ and [99mTc„N]2+ cores, respectively. Radiolabeling yield for both
the complexes was >98% as observed by HPLC and TLC patterns. In vitro studies in tumor cell lines showed significant uptake for
99mTc-carbonyl as well as for 99mTc-nitrido colchicine complexes. Biodistribution studies in Swiss mice bearing fibrosarcoma tumor
showed 4.1 1.2% ID/g of uptake at 30 min pi for 99mTc(CO)3-complex as against 0.42 0.24% ID/g for the 99mTcN-complex.
99mTc(CO)3–colchicine complex exhibited better pharmacokinetics with lower liver accumulation as compared to the 99mTcN-com-
plex. Thus, colchicine radiolabeled with [99mTc(CO)3(H2O)3]+ core is more promising with respect to in vivo distribution character-
istics in tumor model.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
[11C]Colchicine has been reported to be a feasible sub-
strate for imaging Pgp functions in tumors.2,3 Tri-
methylcolchicinic acid, a colchicine derivative, has also
been used for preparation of 99mTc–EC(ethylenedicys-
teine)–colchicine in targeting tumors and reported to
exhibit good tumor uptake.4 Bearing in mind the poten-
tial of trimethylcolchicinic acid to exhibit tumor affinity,
we report herein labeling of trimethylcolchicinic acid
derivatives with two different novel cores of 99mTc viz.
Colchicine is the main bioactive alkaloid of Colchicum
autmnale and is used in diagnosis and treatment of gout.
It acts as a potent inhibitor of cellular mitosis by binding
to microtubules. Colchicine, like many other cytotoxic
drugs, enters the cell through the lipid bilayer by passive
diffusion and binds reversibly to P-glycoprotein (Pgp).1
Pgp is one of the mechanisms involved in multidrug resis-
tance (MDR) for chemotherapeutic drugs. Pgp-mediated
transport of chemotherapeutic drugs has been studied
using single photon emission computed tomography
(SPECT) and positron emission tomography (PET).
[
evaluation of the resultant complexes for tumor imaging.
99mTc(CO)3(H2O)3]+ and [99mTc„N]2+ cores and the
Commonly used technetium complexes based on
99mTc(V) oxo core suffer from low specific activity due
to high concentration of chelating agents required for
stabilization of +5 oxidation state. Advent of novel
cores of technetium has opened fresh avenues for prep-
aration of stable complexes with high specific activity.5,6
Keywords: Colchicine; [99mTc(CO)3(H2O)3]+; [99mTc„N]2+; Iminodi-
acetic acid; Dithiocarbamate; Tumor.
*
Corresponding author. Tel.: +91 22 25595371; fax: +91 22
25505345; e-mail: sharmila@apsara.barc.ernet.in
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.006

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A. Korde et al. / Bioorg. Med. Chem. 14 (2006) 793–799
Alberto et al. have developed and demonstrated the
merits of using the new organometallic 99mTechnetium
carbonyl precursor [99mTc(CO)3(H2O)3]+ containing
technetium in +1 oxidation state which is less sensitive
to oxidation and hence does not require high concentra-
tion of ligands for stabilization.7 Low ligand concentra-
tion is a specific feature aimed at the designing of 99mTc-
labeled targeted radiopharmaceuticals. The tricarbonyl
core [99mTc(CO)3(H2O)3]+ possesses a low spin d6
Tc(I) center which is kinetically inert and hence offers
more flexibility toward choosing ligands for designing
complexes of desirable size, charge, and lipophilicity
suitable for the specific study. The three substitutionally
labile aqua ligands facilitate the formation of stable
complexes with a tridentate ligand. The N-containing li-
gands such as histidine, histamine, imidazole, iminodi-
acetic acid, diethylenetriamine, and SchiffÕs bases
which readily form stable complexes with the tricarbon-
yl precursor have been functionalized for the develop-
ment of site-specific radiopharmaceuticals using varied
biologically avid molecules.8,9 Another novel nitrido
core [99mTc„N]2+ isoelectronic with the [99mTcO]3+ core
used earlier was synthesized by Baldas and Bonnyman,
and found to exhibit a very high stability over a wide
range of experimental conditions. It shows high affinity
toward chelating ligands containing sulfur and phos-
phorus atoms. Dithiocarbamate ligand forms complexes
of high radiochemical purity with the [99mTc„N]2+ syn-
thon.10,11 Colchicine has been functionalized to iminodi-
acetic acid (IDA) and dithiocarbamate (DTC) capable
of complexation with the respective cores [99mTc(CO)3]+
and [99mTc„N]2+ in near-quantitative yields. In vitro
binding studies with tumor cell lines as well as in vivo
studies in tumor-bearing animals were carried out to
explore the potential of these radiolabeled species to
localize in tumors.
carbamate 4 could be characterized with the help of ele-
mental analysis and mass spectra.
To prepare the technetium-99m complexes of 3 and 4,
99mTc-carbonyl and 99mTc-nitrido cores were first pre-
pared. The 99mTc-tricarbonyl synthon was prepared
in situ according to the reported procedure and then
used for complexation.7 It could be prepared in >98%
yield as determined by C-18 reverse-phase HPLC sys-
tem. The 99mTc(CO)3–colchicine–IDA complex could
be obtained by incubation of IDA
[
3 with the
99mTc(CO)3(H2O)3]+ synthon. While the retention time
of the synthon was 13.7 min, that of the radiolabeled
complex, eluted as a single species in HPLC, was ob-
served to be 18 min. The percentage complexation as ob-
tained by HPLC analyses was >98% (Fig. 2).
Complexation yields were studied at different pH by
adjusting the pH of the [99mTc(CO)3(H2O)3]+ synthon
3ꢁ
prepared at pH ꢀ 10 using PO4
buffer (pH 7.5,
0.5 M): HCl (1.0 M) (1:3, v/v), prior to addition of the
ligand colchicine–IDA. Standardization and optimiza-
tion studies for obtaining maximum complexation
showed that the radiolabeling yield depends on the reac-
tion pH and concentration of the ligand. The reaction
was favorable at acidic pH (4.0) leading to 98% com-
plexation. Increasing the pH led to a fall in the complex-
ation yield which decreased to 70% and 40% at pH 7 and
9, respectively. In paper electrophoresis studies, it was
observed that the 99mTc(CO)3–colchicine–IDA complex
5 does not show any movement. Lipophilicity of the
radiolabeled complex was determined by distribution
in octanol and water, and the partition coefficient (logP)
was found to be 1.3. In vitro serum stability of the com-
plex was studied by 1 h incubation of 5 at 37 ꢁC. Obser-
vation of a single peak at 18 min, on analysis by HPLC,
indicated serum stability of the complex.
99mTc-niꢁtrido core was prepared by addition of
99mTcO4 to the kit vial followed by preparation of
the complex by incubation of 4 with [99mTc„N]2+ pre-
cursor. Formation of 99mTc-nitrido complex in good
yields (>98%) was confirmed by TLC and electrophore-
sis. TLC was carried out in ethanol/chloroform/toluene:
0.5 M ammonium acetate mixture (6:3:3:0.5 v/v) as well
as in saline. In the former solvent system, 99mTcN inter-
mediate as well as reduced technetium remained at the
point of spotting (Rf = 0–0.1), whereas 99mTcO4ꢁ moved
2. Results and discussion
2.1. Chemistry
Derivatization of colchicine to the suitable precursor
was necessary for subsequent complexation with the
99mTc-cores. Hence, introduction of amino group to
yield the precursor, trimethylcolchicinic acid, was
attempted by acid hydrolysis of the parent colchicine 1
using 30% concentrated sulfuric acid.12 The amino
group of trimethylcolchicinic acid 2 was further deriva-
tized to the iminodiacetic acid 3 and dithiocarbamate 4
for radiolabeling with [99mTc(CO)3]+ and [99mTc„N]2+
cores, respectively. The IDA of trimethylcolchicinic acid
was prepared by reaction with 1.6 equiv of bromoacetic
acid under alkaline conditions. The DTC of colchicine
was prepared by reaction with carbon disulfide in the
presence of KOH (Fig. 1). Hydrolysis of the –NHC-
OCH3 group as well as the concomitant demethylation
at Rf 0.4–0.6. However, in saline, both 99mTcO4 and
ꢁ
99mTcN intermediate moved with the solvent front
(Rf = 0.8–1.0) with reduced technetium at the point of
spotting. Radiolabeling yield of the complex (>98%)
could be determined by TLC in saline wherein the com-
plex remained at the point of spotting (Rf = 0). In paper
electrophoresis, while the 99mTcN intermediate showed a
movement of 5 cm toward the anode, the complex did
not show any movement indicating neutrality of the
complex.
1
of –OCH3 group of 1 could be ascertained by the H
NMR of 2 wherein the two 3H singlets corresponding
to –OCH3 and –NHCOCH3 observed in 1 were found
to be absent in 2. The formation of the IDA 3 was con-
firmed by the appearance of a 4H singlet at d 3.36
corresponding to the N–CH2–COOH group. The dithio-
2.2. In vitro cell binding studies
99mTc(CO)3–colchicine complex as well as [99mTcN]–col-
chicine complex corresponding to 1 nmol of colchicine
were studied for in vitro binding with fibrosarcoma

A. Korde et al. / Bioorg. Med. Chem. 14 (2006) 793–799
795
CH3O
CH3O
CH3O
NHCOCH3
NH2
100oC, 5h
CH3O
30% H2SO4
CH3O
CH3O
O
O
OCH3
OH
BrCH2COOH
K2CO3
Colchicine
RT, 5 days
(1)
Trimethyl colchicinic acid
(2)
S
CH3O
CH3O
CH3O
COOH
COOH
NH
C
N
CH3O
-
+
S
Na
CH3O
CH3O
O
O
OH
OH
Colchicine-DTC
Colchicine-IDA
(4)
(3)
[
99mTc(CO)3]+
[
99mTcN]+2
O
-
C
O
N
OCH3
OCH3
H3CO
S
S
S
S
H
H
CO
CH3O
CH3O
N
N
Tc
H3CO
Tc
N
CO
OCH3
OCH3
O
O
CO
C
O
CH3O
HO
OH
O
O
99mTcN-colchicine-DTC
OH
99mTc(CO)3-colchicine-IDA
(6)
(5)
Figure 1. Synthesis of 99mTc(CO)3–colchicine–IDA and 99mTcN–colchicine–DTC complexes.
and melanoma tumor cells. Compound 5 exhibited sig-
nificant binding of 50.6 2.2% and 54.2 2.3% with
fibrosarcoma and melanoma cells, respectively. Similar-
ly 6 also showed binding of 54.3 1.7% and 57.4 2.9%
with respective tumor cells. Since tumor cells have more
percentage of dividing cells as compared to normal
spleen, normal murine spleen cells showed comparatively
much less binding (15%) with both the tracers. Blank
experiments were carried out using [99mTc(H2O)3(CO)3]+
synthon and [99mTc„N]2+ intermediate under similar
experimental conditions. In this case, activity associated
with the cell pellet was only around 10% and 6%, respec-
tively. The in vitro results (Table 1) clearly indicate the
affinity of both the products for tumor cells.
1 h pi) (Table 2). On the contrary, 99mTc(CO)3–colchi-
cine–IDA complex exhibited greater tumor accumula-
tion (4.1 1.2% ID/g at 30 min pi) and better
pharmacokinetics. The compound partly excreted via
the hepatobiliary route and around 25% ID/g was re-
tained in the liver at 4 h pi. Figure 3 shows the detailed
results of the biodistribution study with 99mTc(CO)3–
colchicine–IDA complex.
2.4. Imaging studies
99mTc(CO)3–colchicine–IDA complex was injected in
nude mice bearing MCF-7 tumors and scintigraphic
images (Fig. 4) were obtained at 1 h pi and 3 h pi. The
uptake in tumor is observable, more predominantly at
3 h pi, albeit with significant amount of radioactivity
associated with the liver. The activity observed in the
bladder was found to be cleared at 3 h pi in concurrence
with the results obtained from biodistribution studies as
well.
2.3. Biodistribution studies
In biodistribution studies in mice bearing fibrosarcoma
tumor, [99mTcN]–colchicine complex showed significant
liver accumulation (ꢀ60% ID/organ) within 30 min with
slow hepatic clearance. There was also very high uptake
in lungs and spleen. Although tumor/blood and tumor/
muscle ratios were found to increase from 0.52 to 2.4
and 0.68 to 1.16 between 30 min and 4 h, the actual
tumor uptake was not very significant (0.42% ID/g at
The complexation of tropolone with a number of metal-
lic ions has been reported indicating a possibility of the
tropolone moiety complexing with 99mTc.13 However, in
the present case since the colchicine molecule has been

796
A. Korde et al. / Bioorg. Med. Chem. 14 (2006) 793–799
Table 2. Biodistribution studies of 99mTcN–colchicine–DTC complex
1200
800
400
0
Organ
% injected dose per organ (n = 3)
30 min
1 h
0.84 (0.23)
4 h
0.29 (0.06)
Blood
Liver
1.2 (0.16)
61.7 (1.28)
2.57 (0.27)
13.5 (0.69)
2.68 (0.33)
0.3 (0.08)
0.52
63.4 (0.85)
5.29 (0.46)
11.4 (1.21)
3.21 (0.12)
0.42 (0.24)
0.95
62.6 (4.08)
11.25 (0.7)
10.69 (3.3)
2.65 (0.65)
0.36 (0.1)
2.4
Int + GB
Lungs
Spleen
Tumor/gm
Tumor/blood
Tumor/muscle
0.68
0.97
1.16
0
500
1000
1500
2000
RT [sec]
99m
HPLC pattern of
Tc(CO) -colIDA at pH4.0
3
1200
800
400
0
30
25
20
15
10
5
30 min.pi
1 h.p.i
4 h.p.i
0
500
1000
1500
2000
RT [sec]
99m
HPLC pattern of
Tc(CO) -colIDA at pH 7
3
1200
1000
800
600
400
200
0
0
Figure 3. Biodistribution studies of 99mTc(CO)3–colchicine–IDA in a
murine fibrosarcoma tumor model (n = 3 for each time point).
chelator) at a low concentration, the complexation with
the iminodiacetate derivative is expected to be by far a
greater possibility in comparison with that of the chelat-
ing groups of tropolone. This has been confirmed by
carrying out a blank complexation of trimethylcolchici-
nic acid 2 with [99mTc(CO)3(H2O)3]+ wherein no com-
plexation was observed.
0
500
1000
RT [sec]
99m
1500
2000
HPLC pattern of
Tc(CO) -colIDA at pH 9
3
Since the [99mTcN]–colchicine complex was found to be
neutral, a possible structure which can be proposed indi-
cates the probable involvement of two DTC molecules.
Such a speculation could be further corroborated by
the observation of Abram et al.14 wherein formation
of an analogous complex structure has been confirmed.
However, 99mTc(CO)3–colchicine–IDA complex with
nitrogen and carboxyl as coordinating groups of the
Figure 2. HPLC pattern of 99mTc(CO)3–colchicine–IDA at different
pH.
derivatized to the iminodiacetate substituent and the
but
ꢁ
radiolabeling precursor is not 99mTcO4
[
99mTc(CO)3(H2O)3]+ which is known to form stable
complexes with the iminodiacetate group (a tridentate
Table 1. In vitro studies in tumor cells
Tracer
Fibrosarcoma
Melanoma
Normal spleen cells
99mTc(CO)3–colchicine—IDA
50.6 2.2
54.3 1.7
1.9 0.8
6.0 1.2
10.4 0.7
54.2 2.3
57.4 2.9
2.2 1.1
5.8 2.5
9.5 1.6
12 1.6
15 2.1
—
[
99mTc„ꢁN]–colchicine– dithiocarbamate
99mTcO4
[
[
99mTc„N]2+ intermediate
—
—
99mTc(H2O)3(CO)3]+

A. Korde et al. / Bioorg. Med. Chem. 14 (2006) 793–799
797
Figure 4. Scintigraphic images of 99mTc(CO)3–colchicine–IDA images in nude mice bearing MCF-7 tumor.
IDA moiety is expected to be uni-negative. 99mTc-car-
bonyl complex bearing polar IDA group has shown low-
er liver accumulation compared to the 99mTc-nitrido
complex bearing two lipophilic DTC molecules.
Although both the radiolabeled complexes exhibited sig-
nificant binding in vitro with tumor cells, the difference
in tumor uptake and pharmacokinetic patterns in vivo
clearly indicates the influence of lipophilicity of ligands
used for complexation. Scintigraphic images also sup-
port the results obtained by biodistribution studies.
Jasco PU 1580 system with a Jasco 1575 tunable absorp-
tion detector and a radiometric detector system. A C-18
reversed-phase HiQ Sil (5 lM, 250 · 4 mm) column has
been used. About 25 lL of the test solution was injected
into the column and the elution was monitored by
observing the radioactivity profile. The flow rate was
maintained at 1 mL/min. The gradient system consisting
of eluting solvents H2O (solvent A) and acetonitrile (sol-
vent B) with 0.1% trifluoroacetic acid was used (0–
28 min, 90% A–10% A; 28–30 min, 10% A; 30–32 min,
10% A–90% A). Proton NMR spectra were recorded
on a 300 MHz Varian VXR 300 S spectrophotometer.
Mass spectra were recorded on a QTOF Micromass
Instrument using electron spray ionization (ESI) in posi-
tive mode. Elemental analyses were performed on C, H,
N, and S elemental analyzer, Thermofinnigan, Flash EA
1112 series. Melanoma and Fibrosarcoma cell lines were
procured from ÔNational Centre of Cell Sciences,Õ Pune,
India. Fibrosarcoma tumors were raised in Swiss mice
by subcutaneous injection of ꢀ106 cells/animal. MCF-
7 (human breast cancer) tumor-bearing animals were
maintained at the nude mice housing facility of the
ÔLaboratory & Nuclear Medicine Section.Õ All the
animal experiments were carried out in compliance with
the relevant national laws as approved by the Local
Committee on the Conduct and Ethics of Animal
Experimentation.
3. Conclusion
Colchicine derivatives could be successfully radiolabeled
with [99mTc(H2O)3(CO)3]+ and [99mTc„N]2+ in high
yield and purity. Both the products showed good in vitro
tumor cell uptake. However, 99mTc(CO)3–colchicine
complex showed more promising results in vivo with re-
spect to reduced liver accumulation and higher uptake in
tumor. The tumor-specificity of this agent provides
further insight toward its possible utility for imaging
multi-drug resistant states.
4. Experimental
4.1. General
4.2. Chemical synthesis
All reagents were of commercial grade. Carbon monox-
ide in 0.5 L refillable canisters was obtained from M/s
4.2.1. Trimethylcolchicinic acid 2. A mixture of 1
(150 mg, 0.37 mmol) and 4.5 mL of 30% concd. sulfu-
ric acid was heated for 5 h at 100 ꢁC in an oil bath.
After 5 h, while hot, neutralization of the reaction
mixture to pH 7–7.5 was effected with solid sodium
carbonate. The resulting frothy mass was filtered
and washed with cold water. The filtrate was extract-
ed in chloroform (3· 10 mL), the combined extracts
Alchemie Gases
&
Chemicals, Mumbai, India.
99mTcO4 was eluted from an in-house 99Mo/99mTc col-
umn generator using normal saline. Commercial kit for
preparation of 99mTc-nitrido species was obtained from
CIS Bio International. Electrophoresis experiments were
carried out using 0.025 M phosphate buffer (pH 7.5) at
300 V/cm for 1 h. HPLC analyses were performed on a
ꢁ

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A. Korde et al. / Bioorg. Med. Chem. 14 (2006) 793–799
were washed with water and dried over anhydrous
sodium sulfate. TLC (silica): 5% glacial acetic acid/
acetonitrile. Rf (colchicine) = 0.4, Rf (product) = 0.
MS (ESI): mass calcd for C19H21O5N, 343; m/z
eluted 99mTcO4ꢁ (1 mCi, 37 MBq) was added to the vial,
vortexed, and allowed to stand at room temperature for
20 min. The 99mTc-nitrido intermediate thus prepared
was characterized by TLC.
1
(obsd): 344 [M+H]+. H NMR (CD3OD, d ppm) 3.6
(s, 3H, –OCH3); 3.86 (s, 3H, –OCH3); 3.9 (s, 3H, –
OCH3) 6.7 (s, 1H, 8H); 7.16 (d, 11H); 7.42 (d,
12H); 7.9 (s, 1H, Ar).
4.2.7. 99mTcN–colchicine–dithiocarbamate. To 0.5 mL
solution
of
colchicine–dithiocarbamate
(1 mg,
4.34 mM), 0.5 mL of freshly prepared [99mTc„N]2+ pre-
cursor was added, vortexed, and incubated for 30 min at
room temperature. The final 99mTcN-complex formed
herein was characterized by TLC and paper
electrophoresis.
4.2.2. Colchicine–IDA derivative (3). Compound 2
(15 mg, 43.4 lmol) was dissolved in methanol and
bromoacetic acid (10 mg, 71.96 lmol) was added to it
along with potassium carbonate (3 mg, 21.7 lmol) and
stirred at room temperature for 5 days. The solvent
was removed under vacuum. The product was charac-
terized by TLC in 30% glacial acetic acid/acetonitrile.
Rf (trimethylcolchicinic acid) = 0.1, Rf (product) = 0.4.
MS (ESI): mass calcd for C23H25O9N, 459; m/z found,
413, 414 (MꢁCOOH). 1H NMR (CD3OD, d ppm)
3.36 (s, 4H, NCH2); 3.55 (s, 3H, –OCH3); 3.84 (s, 6H,
–OCH3).
4.3. In vitro studies in tumor cells
Melanoma and fibrosarcoma cells were isolated under
aseptic conditions from tumor-bearing mice. The cells
were washed and cell suspension of 5 · 106 cells/mL
was prepared in plain DulbeccoÕs modified Eagle medi-
um (DMEM). The radiolabeled products namely
99mTc(CO)3–colchicine–IDA as well as 99mTcN–colchi-
cine–dithiocarbamate corresponding to 1 nmol colchi-
cine were incubated at 37 ꢁC for 1 h with 5 · 105 cells.
After incubation, the cells were separated by centrifuga-
tion. The cell pellet was washed twice and was counted
to determine the percentage of activity associated with
cells. Nonspecific binding of [99mTc(H2O)3(CO)3]+ syn-
thon as well as of [99mTc„N]2+ intermediate was stud-
ied similarly following identical experimental
conditions. Uptake in normal spleen cells was also stud-
ied for comparison to determine specific binding to tu-
mor cells.
4.2.3. Colchicine–dithiocarbamate derivative (4). Com-
pound 2 (20 mg, 58 lmol) was dissolved in diethyl ether
and 1:1 equivalent of carbon disulfide (3.5 lL) was add-
ed to it along with crushed NaOH (20 mg). The reaction
mixture was stirred at room temperature for 2 h when
the formation of a yellow precipitate was observed.
After removal of the solvent, the precipitate was repeat-
edly washed with diethyl ether to remove any unreacted
trimethylcolchicinic acid. The product was characterized
by elemental analyses. C, H, N, S: obsd (calcd) 53.95
(54.16), 5.45 (5.00), 3.12 (3.16), 14.35 (14.46). MS
(ESI): mass calcd for C20H20O5NS2Na, 441; m/z found,
419 [(M+H)ꢁNa].
4.4. Biodistribution studies
Biodistribution studies for both the products were
carried out in Swiss mice bearing fibrosarcoma tumor.
0.1 mL of the product containing 3–7 MBq of activity
was injected via the tail vein. The studies were carried
out in triplicate at 30 min, 1 h and 4 h post injection.
All major organs as well as tumor were excised,
rinsed, weighed, and counted in a NaI(Tl) flat geome-
try detector in order to estimate the percent of inject-
ed dose per gm of the tissue. Blood, muscle, and bone
were taken as 7%, 40%, and 10% of total body
weight.
4.2.4. [99mTc(H2O)3(CO)3]+ synthon. The synthon was
prepared by a modified procedure reported by Alberto
et al.7 A typical procedure consists of preparation of a
mixture of 0.5 mL aqueous solution of NaBH4
(5.5 mg), Na2CO3 (4 mg), and Na/K tartrate (15 mg),
and purging of carbon monoxide through it for 5 min.
After the addition of 1 mL 99mTcO4 (37 MBq) to the
ꢁ
solution, it was heated at 70 ꢁC for 20 min. The reaction
mixture was cooled and the pH was adjusted to 8 with
300 lL of 1:3 mixture of 0.5 M phosphate buffer (pH
7.5): 1 M HCl.
4.5. Imaging studies
4.2.5. 99mTc(CO)3–colchicine IDA. 0.1 mL of methano-
lic solution of the colchicine IDA derivative (800 lg,
3.4 mM) was added to 0.4 mL [99mTc(H2O)3(CO)3]+
synthon, adjusted to pH 4.0, and heated at 70 ꢁC
for 20 min. The complex was characterized by
HPLC.
A Single Head Digital SPECT Gamma Camera (MPS
GE, USA) with a parallel hole LEAP collimator was
used for imaging studies. 99mTc(CO)3–colchicine–IDA
complex (ꢀ15 MBq) was injected intravenously through
tail vein in nude mice bearing MCF-7 tumors. Static
images were acquired using 256 · 256 matrix with 125
KCounts at 1 and 3 h post injection.
4.2.6. [99mTc„N]2+ core. The commercially available kit
vial, containing succinic dihydrazide (5.0 mg), stannous
chloride dihydrate (100 lg), 1,2-diaminopropane–
N,N,N0,N0-tetraacetic acid (5 mg), sodium dihydrogen
phosphate (0.5 mg), and disodium hydrogen phosphate
(5.8 mg) in freeze-dried form, was used for preparing
the precursor. The kit vial stored at 4 ꢁC was allowed
to attain ambient temperature. One milliliter of freshly
Acknowledgments
The authors are grateful to Dr. V. Venugopal, Director,
Radiochemistry and Isotope Group, for his constant
support and encouragement.

A. Korde et al. / Bioorg. Med. Chem. 14 (2006) 793–799
799
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6. Banerjee, S.; Pillai, M. R. A.; Ramamoorthy, N. Semin.
Nucl. Med. 2001, 32, 260.
7. Alberto, R.; Schibli, R.; Egli, A.; Schubiger, A. P.; Abram,
U.; Kaden, T. A. J. Am. Chem. Soc. 1998, 120, 7987.
8. Egli, A.; Alberto, R.; Tannahill, L.; Schibli, R.; Abram,
U.; Schaffland, A. J. Nucl. Med. 1999, 40, 1913.
9. Waibel, R.; Alberto, R.; Willuda, J.; Finnern, R.;
Schibli, R.; Stichelberger, A. Nat. Biotechnol. 1999, 17,
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2005.09.006.
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