[99mTc]oxotechnetium(V) complexes amine-amide-dithiol chelates with dialkylaminoalkyl substituents as potential diagnostic probes for malignant melanoma.

Article abstract of DOI:10.1021/jm0005407

[99mTc]oxotechnetium(V) complexes of amine-amide-dithiol (AADT) chelates containing tertiary amine substituents were synthesized and shown to have affinity for melanoma. For complexation the AADT-CH2[CH2]nNR2 (n = 1, 2; R = Et, n-Bu) ligand was mixed with a [99mTc]oxotechnetium(V)-glucoheptonate precursor to make the AADT-[99mTc]oxotechnetium(V) complexes in nearly quantitative yield. Structurally analogous nonradioactive oxorhenium(V) complexes were also synthesized and characterized. In vitro sigma-receptor affinity measurements indicate these complexes to possess sigma-affinity in the low micromolar range with K(i) values in the 7.8-26.1 and 0.18-2.3 microM range for the sigma1- and sigma2-receptors, respectively. In vitro cell uptake of the 99mTc complexes in intact B16 murine melanoma cells at 37 degrees C after a 60-min incubation ranged from 12% for complex 2 (n = 1, R = n-Bu) to 68% for complex 4 (n = 2, R = n-Bu). In vivo evaluation of complexes 1-Tc-4-Tc in the C57Bl/B16 mouse melanoma model demonstrated significant tumor localization. Complex 1-Tc (n = 1, R = Et) displayed an in vivo tumor uptake of 7.6% ID/g at 1 h after administration with initial melanoma/blood (M/B), melanoma/spleen (M/S), and melanoma/lung (M/L) ratios >4; these ratios increased to 10.8, 10.1, and 7.3, respectively, at 6 h. While complex 3-Tc (n = 3, R = Et) had an initial tumor uptake of 3.7% ID/g 1 h after administration with M/B, M/S, and M/L ratios >2, a greater tumor retention and slightly faster clearance from nontumor-containing organs resulted in M/B, M/S, and M/L ratios of 19.1, 19.1, and 12.7, respectively, at 6 h. The high tumor uptake and significant tumor/nontumor ratios indicate that such small technetium-99m-based molecular probes can be developed as in vivo diagnostic agents for melanoma and its metastases.

Full text of DOI:10.1021/jm0005407

3132
J . Med. Chem. 2001, 44, 3132-3140
[
99m Tc]Oxotech n etiu m (V) Com p lexes of Am in e-Am id e-Dith iol Ch ela tes w ith
Dia lk yla m in oa lk yl Su bstitu en ts a s P oten tia l Dia gn ostic P r obes for Ma lign a n t
Mela n om a
Matthias Friebe,† Ashfaq Mahmood,*,† Cristina Bolzati,†,‡ Antje Drews,§ Bernd J ohannsen,§ Michael Eisenhut,||
Daniel Kraemer,∧ Alan Davison,∧ and Alun G. J ones†
Department of Radiology, Harvard Medical School and Brigham and Women’s Hospital, Boston, Massachusetts 02115;
Laboratory of Nuclear Medicine, Department of Clinical and Experimental Medicine, University of Ferrara,
Ferrara 44100, Italy; Institute of Bioinorganic and Radiopharmaceutical Chemistry, Research-Center Rossendorf,
Dresden, Germany; Department of Nuclear Medicine, University of Heidelberg, Heidelberg, Germany; and Department of
Inorganic Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received December 18, 2000
[
99mTc]oxotechnetium(V) complexes of amine-amide-dithiol (AADT) chelates containing tertiary
amine substituents were synthesized and shown to have affinity for melanoma. For complex-
ation the AADT-CH2[CH2]nNR2 (n ) 1, 2; R ) Et, n-Bu) ligand was mixed with a [99mTc]-
oxotechnetium(V)-glucoheptonate precursor to make the AADT-[99mTc]oxotechnetium(V)
complexes in nearly quantitative yield. Structurally analogous nonradioactive oxorhenium(V)
complexes were also synthesized and characterized. In vitro σ-receptor affinity measurements
indicate these complexes to possess σ-affinity in the low micromolar range with Ki values in
the 7.8-26.1 and 0.18-2.3 µM range for the σ1- and σ2-receptors, respectively. In vitro cell
uptake of the 99mTc complexes in intact B16 murine melanoma cells at 37 °C after a 60-min
incubation ranged from 12% for complex 2 (n ) 1, R ) n-Bu) to 68% for complex 4 (n ) 2, R
) n-Bu). In vivo evaluation of complexes 1-Tc-4-Tc in the C57Bl/B16 mouse melanoma model
demonstrated significant tumor localization. Complex 1-Tc (n ) 1, R ) Et) displayed an in
vivo tumor uptake of 7.6% ID/g at 1 h after administration with initial melanoma/blood (M/B),
melanoma/spleen (M/S), and melanoma/lung (M/L) ratios >4; these ratios increased to 10.8,
10.1, and 7.3, respectively, at 6 h. While complex 3-Tc (n ) 3, R ) Et) had an initial tumor
uptake of 3.7% ID/g 1 h after administration with M/B, M/S, and M/L ratios >2, a greater
tumor retention and slightly faster clearance from nontumor-containing organs resulted in
M/B, M/S, and M/L ratios of 19.1, 19.1, and 12.7, respectively, at 6 h. The high tumor uptake
and significant tumor/nontumor ratios indicate that such small technetium-99m-based mo-
lecular probes can be developed as in vivo diagnostic agents for melanoma and its metastases.
In tr od u ction
Earlier attempts at imaging melanoma with radiola-
beled monoclonal antibodies8,9 or simpler radiolabeled
molecules, such as radioiodinated amino acids4,5 or
nucleic acids,6,7 as false precursors in the melanin
formation cycle were unsuccessful due to either insuf-
ficient tumor localization, low tumor/nontumor ratios,4-9
or poor pharmacokinetics.7,8 More recently, the use of
radiolabeled peptides, such as 188Re- and 99mTc-labeled
melanotropin,13,14 has generated encouraging results for
in vivo melanoma scintigraphy.
The search for small radiolabeled molecular probes
for in vivo scintigraphy has led to the identification of
several radioiodinated benzamide derivatives including
N -(2-diet h yla m in oet h yl)-3-iodo-4-m et h oxyben z-
amide15-19 (IMBA) (Figure 1). This radiolabeled benz-
amide displays an in vivo melanoma uptake of 6.7%
injected dose (ID)/g at 60 min after administration in
the C57Bl/B16 mouse melanoma model. It has also
entered phase II clinical trials, providing images in
patients of metastatic melanoma that are very encour-
aging.16,20 In efforts to elucidate the uptake mechanism
of these radiolabeled benzamides in melanoma, σ-recep-
tors21 have been explored, mainly because structurally
similar benzamide ligands have been known to possess
high affinity for σ-receptors, and σ-receptors are ex-
The incidence of malignant melanoma is rising faster
than that of any other cancer in the United States; it is
estimated that 47 700 new cases will be diagnosed
annually.1 Global estimates of 105 000 new cases per
year with 33 000 annual deaths are associated with
melanoma of the skin.1a Since the early detection of all
related metastases improves considerably the patient’s
prognosis,1-3 the search for in vivo diagnostic probes is
of special interest.4-9 While an 18F-labeled glucose
analogue, 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG), is
quite useful in positron-emission tomographic (PET)
imaging of tumors10 including melanoma,11,12 a single-
photon-emission computed tomographic (SPECT) radio-
pharmaceutical based on technetium-99m, a radioiso-
tope with widespread availability, would provide a cost-
effective means of early detection and diagnosis.
* Address for correspondence: Harvard Medical School, Department
of Radiology, Goldenson Building B-1/251, 220 Longwood Avenue,
Boston, Massachusetts, 02115. Tel: (617)-432-3995. Fax: (617)-432-
2419. E-mail: amahmood@hms.harvard.edu.
† Harvard Medical School and Brigham and Women’s Hospital.
‡ University of Ferrara.
§ Institute of Bioinorganic and Radiopharmaceutical Chemistry.
|| University of Heidelberg.
∧ Massachusetts Institute of Technology.
10.1021/jm0005407 CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/15/2001

[
99mTc]Oxotechnetium(V) Complexes of AADT
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3133
F igu r e 1. Iodine-123-labeled N-(2-diethylaminoethyl)-3-iodo-4-methoxybenzamide ([123I]IMBA), technetium-99m-labeled oxo-
technetium-bis-aminethiol-N-(2-diethylaminoethyl)benzamide ([99mTc]TcN-BAT-BZA), and two “3 + 1” oxotechnetium complexes
with pendent amine groups [99mTc]TcO(SNS)(S-C2-NEt2) (a) and [99mTc]TcO(SNS)(S-C2-NBu2) (b).
pressed on a number of tumor-cell types including
melanoma.22-25 However, recent investigations have
shown that the accumulation of these radiolabeled
benzamides by melanoma cells is not correlated with
their σ-affinity but rather with their melanin con-
tent.18,19
diagnostic probes with affinity for malignant melanoma.
Herein, we describe the synthesis and characterization
of these tertiary-amine-bearing 99mTc-AADT complexes
and their in vivo evaluation in a melanoma tumor
model.
While iodine-123 can be obtained commercially, tech-
netium-99m is available on demand through a 99Mo/
99mTc generator. The technetium-99m isotope emits
single γ-photons of 140 keV, has a high photon flux, and
decays with a 6-h half-life, making it very cost-effective
and the radiolabel of choice for routine clinical SPECT
imaging. Technetium-99m-labeled complexes based on
the complete N-(2-dialkylaminoalkyl)benzamide core,
such as nitridotechnetium-bis-aminethiol-benzamide
(TcN-BAT-BZA) (Figure 1) have been reported with
a maximum tumor uptake of 1.6% ID/g at 60 min after
administration in the mouse melanoma model.26,27 More
recently, we have reported “3 + 1” mixed-ligand 99mTc
complexes28,29 that integrate the radiometal within the
pharmacophore by replacing the aromatic ring with a
tridentate/monodentate chelated mono-oxometal. These
“3 + 1” complexes, TcO(SNS)(S-C2-NEt2) and TcO-
(SNS)(S-C2-NBu2) (Figure 1, a and b), display a
significantly higher tumor accumulation of 3.1% and 5%
ID/g, respectively, 60 min after administration. While
these “3 + 1” mixed-ligand complexes28 are easy to
synthesize, their known tendency to form transchelation
products with glutathione in vivo28,30 may hamper their
use as diagnostic probes.
Resu lts a n d Discu ssion
Ch em istr y. The N2S2-AADT chelate was synthesized
via multistep reactions (Scheme 1) as previously de-
scribed.31,32 Chelate derivatives containing the C2-linked
dialkylamino groups as substituents (1, 2) were syn-
thesized via N-alkylation of the amine nitrogen in the
AADT chelate using N-(2-dialkylamino)ethyl chloride
(alkyl ) Et, Bu), while the C3-linked dialkylamino
substituents (3, 4) were incorporated in the chelate via
alkylation of commercially available dialkylamines (alkyl
) Et, Bu) using an N-(3-chloropropyl)-AADT deriva-
tive.32
Technetium-99m-labeled complexes (1-Tc-4-Tc) were
synthesized by transmetalation of technetium-99m from
a prereduced 99mTc-glucoheptonate precursor (Scheme
1). Upon heating the reaction mixture at 70 °C, ligand
exchange of the N2S2-AADT ligand bearing the pendent
tertiary amines and the 99mTc(V)-glucoheptonate pre-
cursor yielded complexes 1-Tc-4-Tc in nearly quantita-
tive yields within 30 min. Typical mass amounts of the
99mTc complexes preclude their physical characteriza-
tion; however, since both technetium and rhenium form
structurally identical AADT complexes,32 analogous
nonradioactive rhenium complexes were synthesized
(vide infra) and used as surrogates for HPLC compari-
sons. Identical HPLC retention times established the
existence of the proposed technetium-99m species.
A tetradentate chelate that replaces the aromatic
moiety in the analogous benzamides would furnish the
required stability and, thus, might provide higher in
vivo tumor uptake. Using the tetradentate amine-
amide-dithiol (AADT) chelate,31,32 which lacks the gem-
dimethyl groups compared with the earlier bis-amine-
thiol chelates and forms stable mono-oxotechenetium
complexes with reduced lipophilicity, we synthesized
new 99mTc complexes that may serve as potential
Using a method similar to that for 99mTc complexes,
the mono-oxorhenium(V) complexes 1-Re-4-Re were
obtained by reduction of perrhenate(VII) with stannous
chloride in the presence of sodium glucoheptonate and
the deprotected N2S2-chelating ligand; heating the reac-

3134 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Friebe et al.
Sch em e 1. Synthesis of the Amine-Amide-Dithiol (AADT) Ligands 1-4 and Complexesa
a
(i) CH3CN, KI, K2CO3, reflux, 24 h; (ii) TFA, Et3SiH, 2 min; (iii) sodium glucoheptonate, SnCl2, 75 °C, 1 h.
tion mixture at 75 °C for 1 h afforded brownish-purple
solids of the rhenium complexes (1-Re-4-Re). These
complexes show distinct υRedO infrared vibrations in the
950-960 cm-1 region, typical for mono-oxorhenium
complexes. Upon chelation the N-substituent on the
chelate may adopt a syn or anti configuration with
respect to the asymmetric MdO core. The deshielding,
anisotropic environment of the MdO core and the
proximity of the N-substituent in the syn configuration
to the asymmetric oxometal core result in a downfield
shift of the proton resonances syn to the MdO core, thus
permitting differentiation of the syn and anti diaster-
1
eomers via NMR.33-36 For example, in the H NMR of
the 1-Re complex, the methylene protons of the N-
substituent (C7) appear as two separate multiplets
(doublets of doublets) downfield at 4.55 and 4.06 ppm,
indicating a syn configuration of the N-substituent. This
resonance pattern is also observed for the other com-
plexes synthesized.
F igu r e 2. Crystal structure of [ReOAADT]-C2-NEt2 (1).
Ta ble 1. Selected Bond Length and Angles of Complex 1-Re
bond length (Å)
bond angle (deg)
Re(1)-O(1)
1.691(8)
1.977(10)
2.172(9)
2.268(3)
2.275(3)
1.847(13)
1.835(12)
1.190(14)
1.50(2)
O(1)-Re(1)-N(1)
118.1(4)
101.6(4)
79.9(4)
116.7(3)
124.9(3)
83.8(2)
106.2(3)
82.6(3)
151.7(3)
88.17(11)
109.1(7)
Re(1)-N(1)
Re(1)-N(2)
Re(1)-S(2)
Re(1)-S(1)
S(1)-C(1)
S(2)-C(6)
O(2)-C(3)
N(2)-C(4)
N(2)-C(5)
N(2)-C(7)
O(1)-Re(1)-N(2)
N(1)-Re(1)-N(2)
O(1)-Re(1)-S(2)
N(1)-Re(1)-S(2)
N(2)-Re(1)-S(2)
O(1)-Re(1)-S(1)
N(1)-Re(1)-S(1)
N(2)-Re(1)-S(1)
S(2)-Re(1)-S(1)
C(4)-N(2)-Re(1)
Further confirmation of the syn configuration was
obtained by the crystal structure determination of 1-Re.
As expected, the structure displays a distorted square-
pyramidal geometry, with the amine-amide-dithiol do-
nor set forming the base-plane and the oxo group at the
apex of the pyramid (Figure 2). The rhenium atom lies
slightly above the AADT base-plane. The pendent
tertiary amine group connected by the C2 alkyl chain is
oriented syn to the MdO core. Selected bond lengths
and angles are listed in Table 1. While only the
geometric syn isomer is formed, due to the presence of
a stereogenic center at the substituted amine in the
chelate, these complexes exist as enantiomeric pairs of
two mirror images. Since most of the physiochemical
parameters (vide infra) are not expected to be signifi-
cantly different for the individual enantiomers, these
were not separated further and are used as such.
1.50(2)
1.55(2)
The physicochemical parameters of the rhenium
complexes, i.e., lipophilicity log P, log DpH7.4, and pKa
(Table 2) were determined using HPLC methods.37-39
As expected, the dibutylamine group in 2-Re (C2-linked)
displays a higher log P of 3.3 compared with the
diethylamine-containing complex 1-Re (C2-linked), which
has a log P of 1.6. Although the dibutyl groups in 2-Re
would normally lead to a more basic amine moiety

[
99mTc]Oxotechnetium(V) Complexes of AADT
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3135
Ta ble 2. pKa, Lipophilicity, RP-HPLC Retention Time, and σ1- and σ2-Receptor Affinity for the Oxorhenium(V) AADT Complexes
Ki (µM)
RP-HPLC
tR (min)
a
b
complex
n
R
pKa
DpH7.4
log DpH7.4
P
log P
σ1
σ2
1-Re
2-Re
3-Re
4-Re
1
1
2
2
ethyl
n-butyl
ethyl
7.7
7.7
9.2
9.5
14
80
0.3
5
1.1
1.9
-0.5
0.7
38.7
1.6
3.3
1.3
3.1
30.6
39.5
35.8
40.2
10.9 ( 2.4
ndc
26.1 ( 3.3
7.8 ( 7.0
2.3 ( 0.3
nd
0.18 ( 0.04
1.6 ( 0.25
2186
19.2
1349
n-butyl
a
b
Determined in guinea pig brain homogenate; radioligand, [3H]-(+)-pentazocine. Determined in rat liver homogenate; radioligand,
[3H]DTG in the presence of 1 µM dextrallorphan to mask σ1-receptors. c None detected.
compared with 1-Re, both complexes have a pKa ) 7.7.
Since log DpH7.4 is a composite measure of log P and pKa,
the log DpH7.4 of 2-Re (1.9) is also higher than that of
1-Re (1.1). A similar log P difference is found for the
C3-linked complexes 3-Re and 4-Re. However, unlike
1-Re and 2-Re, the dibutylamine complex 4-Re yields
a slightly higher pKa of 9.5 compared with 9.2 for the
diethylamine complex 3-Re. The log P of the C3-linked
complexes 3-Re and 4-Re is slightly lower than those
of the C2-linked analogues 1-Re and 2-Re. With pKa
values >9 for complexes 3-Re and 4-Re, the resulting
low log DpH7.4 values of -0.5 (3-Re) and 0.7 (4-Re) are
not surprising, since the complexes would exist in a
protonated form at pH 7.4.
In Vitr o Tu m or -Up ta k e Stu d ies. Tumor-cell up-
take studies in B16/F0 murine melanoma cells were
performed with complexes 1-Tc-4-Tc at 37 and 4 °C.
Additionally, tumor-cell uptake of complex 1-Tc and
3-Tc were investigated in another rapidly dividing
MCF-7 human breast cancer cell line.
All compounds display a rapid cell uptake within 10
min of incubation at 37 °C (Figure 3a,b). While the C2-
linked complex 1-Tc (R ) Et) has a maximal cell uptake
of 43%, its slightly less lipophilic C3 analogue 3-Tc (R
) Et) has a higher uptake of 62%. With the more
lipophilic dibutyl homologues, the C3-linked complex
4-Tc (R ) Bu) has the highest melanoma cell uptake of
68%, and its corresponding C2 analogue (R ) Bu) the
lowest of the entire test set (12%).
To distinguish an active uptake component from
passive diffusion, measurements were also carried out
at 4 °C. A decrease in the incubation temperature from
37 to 4 °C results in a lower cell uptake for complexes
F igu r e 3. In vitro cell uptake of complex 1-Tc (a) and
1-Tc, 3-Tc, and 4-Tc (Figure 3a,b), with the most
complexes 2-Tc-4-Tc (b) in B16 murine melanoma and MCF-7
lipophilic complex 4-Tc showing the least difference
(23% decrease) and the least lipophilic complex 3-Tc the
human breast cancer (5 × 106 cells/mL) at 37 and 4 °C.
greatest difference (77% decrease). To ensure that the
decreased uptake at 4 °C is due to decreased metabolism
and not cell death, the tumor cells were reincubated at
37 °C for 60 min following a 4 °C incubation; this
restored the tumor-cell uptake of the complexes to the
level observed at 37 °C (data not shown). These obser-
vations indicate a significant active accumulation pro-
cess occurring for these compounds in melanoma cells.
It is presumably the presence of this active component
in the cell-uptake process at 37 °C that makes it difficult
to deduce any correlations with either lipophilicity (log
P and log DpH7.4) or in vivo tumor uptake of these
complexes.
The in vitro uptake of complex 2-Tc is unexpectedly
low for reasons not yet understood. However, complex
2-Tc exhibits the highest log P (3.3) among the four
complexes and also a pKa of 7.7, making it very lipophilic
at pH 7.4, as indicated by its log DpH7.4 of 1.9, which
may contribute to this unusual behavior. The tumor-
cell uptake of complex 1-Tc in another rapidly dividing
tumor-cell line MCF-7 has a maximum cell uptake of
only 6% at 37 °C compared with 43% for the B16/F0
cell line (Figure 3a). Additionally, complex 3-Tc displays
only a 17% maximal uptake at 37 °C in the MCF-7 cell
line (data not shown) compared with the 62% maximal
uptake in the B16 melanoma cell line.

3136 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Friebe et al.
dextrallorphan41 (σ2) were used as high-affinity radio-
ligands. The apparent Kd values for the radioligands are
6.44 ( 0.53 nM (σ1) and 23.7 ( 2.0 nM (σ2), respectively.
All four Re complexes display only micromolar affinity
toward the σ1-receptor (Table 2). In general, their Ki (σ1)
values range from 7.8 to 26.1 µM. However, unlike the
iodobenzamides,19,22 all four complexes have a slightly
higher affinity for the σ2-receptor compared with the σ1-
receptor (Table 2). The Ki values for the σ2-receptor
range from 0.18 to 2.3 µM with 3-Re displaying 100-
fold greater affinity toward the σ2-receptor subtype.
The apparent low σ-receptor affinity of 1-Re-4-Re;
the low tumor-cell uptake of these compounds in MCF-7
cells, known to express σ2 receptors;22,24 and the high
micromolar concentrations of DTG required to inhibit
the tracer concentration of the 99mTc complex uptake
in intact B16 melanoma cells would seem to suggest
that, while binding to the σ-receptors cannot be excluded
from the accumulation process, the inhibitory effects
observed in the intact B16 cell-uptake assay may be due
to secondary effects induced by DTG on the growth and
proliferation of the B16 melanoma cells.42,43 Other
factors such as melanin production and content may
play a more significant role in the accumulation of these
complexes in melanomas, as recently shown with the
radioiodinated benzamides.18,19
F igu r e 4. In vitro cell uptake at 1 h of complexes 1-Tc-4-Tc
in B16 murine melanoma cells (5 × 106 cells/mL) incubated
at 37 and 4 °C, and 37 °C with DTG (90 µM).
In Vivo Tu m or Up ta k e. To study the tumor uptake
of 99mTc complexes 1-Tc-4-Tc in vivo, biodistribution
experiments at 1 and 6 h after their administration were
carried out in C57Bl6 mice with palpable B16 melanoma
nodules. The biodistribution data including melanoma/
nontumor (M/NT) ratios for selected organs are sum-
marized in Table 3 as percentage injected dose per gram
(% ID/g).
F igu r e 5. Dose-dependent uptake inhibition of 3-Tc with
DTG (90 µM) in B16 melanoma cells (5 × 106 cells/mL) at 37
Complex 1-Tc (C2-linked, R ) Et) displays the highest
tumor uptake of the entire test set with 7.6% ID/g and
high melanoma/blood (M/B) (7.6), melanoma/spleen (M/
S) (4.2), and melanoma/lung (M/L) (5.4) ratios at 1 h
after administration. Although the % ID/g in the tumor
decreases at 6 h after administration (3.5% ID/g), the
M/NT ratios increase for blood (10.8), spleen (10.1), and
lung (7.3) due to the faster clearance of the complex from
these tissues compared with the tumor. In comparison,
complex 2-Tc (C2-linked, R ) n-Bu), the more lipophilic
analogue (log DpH7.4 ) 1.9), has a lower melanoma
uptake of 3.0% ID/g 1 h after administration. While the
total tumor uptake is significantly lower than that for
1-Tc, the M/NT ratios are equivalent (except for a lower
M/L value of 2.1) at the 1-h time point. At 6 h after
administration, although the M/NT ratios increase, the
lower tumor content of complex 2-Tc (1.1% ID/g) may
be inadequate for in vivo imaging.
°C.
Since the radioiodinated benzamides have been re-
ported to possess high affinity for σ-receptors expressed
by various tumors,22-25 tumor-cell uptake studies of the
complexes were also performed in the presence of 1,3-
di-o-tolylguanidine (DTG), a known high-affinity σ-ligand.
Preincubation of the tumor cells with DTG (90 µM), 30
min prior to the uptake experiments with the 99mTc
complexes, yields a lower tumor-cell uptake for com-
plexes 1-Tc (29% less), 3-Tc (33% less), and 4-Tc (32%
less) at 37 °C (Figure 4). Additional dose-response
experiments conducted with intact B16/F0 cells at 37
°C with 3-Tc and DTG as the inhibitor (Figure 5) show
a DTG-concentration-dependent decrease in the uptake
of the complex. Complexes 1-Tc, 3-Tc, and 4-Tc exhibit
50% maximal inhibition at 21, 49, and 52 µΜ DTG,
respectively.
Recep tor -Bin d in g Stu d ies. To gain a better under-
standing of the involvement of σ-receptor binding in the
melanoma-cell uptake of these 99mTc complexes, we
further investigated the affinity of these complexes in
an established σ-receptor assay. Employing the struc-
turally similar nonradioactive rhenium complexes 1-Re,
3-Re, and 4-Re as surrogates for the 99mTc complexes,
competitive binding assays were carried out with guinea
pig brain membranes (σ1-receptors) and rat liver mem-
branes (σ2-receptors) to assess σ-receptor subtype se-
lectivity, while [3H]-(+)-pentazocine40 (σ1) and [3H]DTG/
Complex 3-Tc (C3-linked, R ) Et) has a melanoma
uptake of 3.7% ID/g at 1 h after administration and
M/NT ratios almost identical to those for complexes 1-Tc
and 2-Tc (except for M/L). However, a greater retention
in tumor tissue and a faster clearance from nontumor
tissues result in a significant increase in the M/NT
ratios for 3-Tc at the 6-h point, giving M/B, M/S, and
M/L ratios of 19, 19, and 12.7, respectively. The mela-
noma uptake of complex 4-Tc (1.3% ID/g 1 h after
administration) and the M/NT ratios are the lowest of
the test set and may be far from ideal for in vivo
diagnostic purposes.

[
99mTc]Oxotechnetium(V) Complexes of AADT
Ta ble 3. Biodistribution and Tumor/Nontumor Ratios of Complexes 1-Tc-4-Tc at 1 and 6 h Postinjectiona
1-Tc 2-Tc 3-Tc
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3137
4-Tc
organ
1 h
6 h
1 h
6 h
1 h
6 h
1 h
6 h
blood
heart
lung
spleen
liver
kidney
muscle
brain
melanoma
mel/blood
mel/spleen
mel/lung
mel/liver
1.00b ( 0.46
0.82 (0.37
1.40 ( 0.49
1.83 ( 0.55
12.7 ( 1.54
5.53 ( 0.81
0.35 ( 0.21
0.30 ( 0.10
7.62 ( 0.62
7.6 ( 0.54
4.2 ( 0.58
5.4 ( 0.60
0.6 ( 0.09
0.32 ( 0.19
0.11 ( 0.02
0.47 ( 0.21
0.34 ( 0.10
4.08 ( 0.92
1.98 ( 0.21
0.08 ( 0.01
0.05 ( 0.02
3.45 ( 1.2
10.8 ( 1.00
10.1 ( 1.11
7.3 ( 0.98
0.9 ( 0.35
0.39 ( 0.07
0.32 (0.20
1.39 ( 0.45
0.73 ( 0.16
10.9 ( 0.57
6.78 (1.12
0.98 ( 0.34
0.16 ( 0.08
2.95 ( 0.23
7.6 ( 0.33
4.0 ( 0.80
2.1 ( 0.81
0.3 ( 0.03
0.13 ( 0.01
0.07 ( 0.04
0.27 ( 0.12
0.17 ( 0.01
5.66 ( 0.53
3.30 ( 0.52
0.06 ( 0.03
0.01 ( 0.01
1.14 ( 0.26
8.8 ( 0.32
6.7 ( 0.24
4.2 ( 0.22
0.2 ( 0.18
0.48 ( 0.10
0.38 ( 0.29
1.55 ( 0.79
1.24 ( 0.33
14.6 ( 1.88
5.62 ( 0.41
0.38 ( 0.14
0.08 ( 0.01
3.70 ( 0.32
7.7 ( 0.43
3.0 ( 0.30
2.4 ( 1.00
0.3 ( 0.08
0.14 ( 0.05
0.02 ( 0.01
0.21 ( 0.04
0.14 ( 0.06
6.42 ( 1.12
2.97 ( 0.41
0.06 ( 0.02
0.01 ( 0.01
2.67 ( 0.29
19.1 ( 0.33
19.1 ( 0.28
12.7 ( 0.22
0.4 ( 0.2
0.31 ( 0.10
0.27 ( 0.15
0.86 ( 0.35
0.92 ( 0.26
6.83 ( 0.96
3.49 ( 0.60
0.13 ( 0.03
0.04 ( 0.01
1.31 ( 0.17
4.2 ( 0.21
1.4 ( 0.34
1.5 ( 0.41
0.2 ( 0.15
0.09 ( 0.02
0.04 ( 0.01
0.24 ( 0.13
0.34 ( 0.02
2.45 ( 1.12
2.00 ( 0.86
0.05 ( 0.02
0.01 ( 0.00
0.80 ( 0.29
8.9 ( 0.10
2.4 ( 0.09
3.3 ( 0.16
0.3 ( 0.19
a
b
n ) 4 animals per time point. Values represent % ID/g wet tissue.
with an ATR accessory. Mass spectra were obtained on a
MicroMass LCZ electrospray LC-MS instrument. HPLC pu-
rification was performed on a Waters Millennium Chroma-
tography System equipped with a 996 UV-Vis diode-array
detector attached in series to a γ-detector consisting of a
shielded photomultiplier powered by a Canberra voltage
amplifier and connected to a ratemeter. For the purification
of all complexes, a reversed-phase C8 column equipped with a
C18 guard was eluted with methanol (solvent A) and 0.005 M
phosphate-buffered saline, pH 7.4, (Sigma) (solvent B) using
a linear gradient from 15/85 (A/B) to 90/10 (A/B) at a 1.0 mL/
min flow rate.
The tetradentate chelating ligand N-[2-((2-(S-(triphenyl-
methyl)thio)ethyl)amino)acetyl]-S-(triphenylmethyl)-2-amino-
ethanethiol (AADT-(Trt)2) and the N-[(3-chloropropyl)-N-2-(2-((S-(tri-
phenylmethyl)thio)ethyl)amino)acetyl]-S-(triphenylmethyl)-
2-aminoethanethiol (AADT-(Trt)2-N-CH2CH2CH2Cl) derivative
were synthesized as described earlier.31,32
Similar to our earlier results with the “3 + 1” com-
plexes,29 a log DpH7.4 ) 1 seems to favor a higher
melanoma uptake of these 99mTc complexes. However,
unlike the “3 + 1” complexes, where pKa ) 9.9 gives the
highest melanoma uptake, the more rigid tetradentate
99mTc-AADT complexes, such as 1-Tc with pKa ) 7.7,
display a higher in vivo melanoma uptake.
The exact tumor-uptake mechanism of these com-
plexes still remains unclear. The higher in vivo tumor
accumulation of the relatively more stable tetradentate
AADT complexes (relative to the “3 + 1” complexes)
suggests a more specific involvement of intramolecular
processes than simple entrapment due to instability of
the complex or its transmetalation to intracellular
proteins.
Liga n d Syn th esis. N-[(2-Dieth yla m in oeth yl)-N-(2-(2-(S-
(tr ip h en ylm eth yl)th io)eth yl)a m in o)a cetyl]-S-(tr ip h en yl-
m et h yl)-2-a m in oet h a n et h iol [AADT-(Tr t )2-N-CH 2CH 2-
N(CH2CH3)2] (1). N-(2-Diethylamino)ethyl chloride (68.8 mg,
0.4 mmol), AADT-(Trt)2 (252 mg, 0.4 mmol), KI (199.2 mg, 1.2
mmol), and K2CO3 (276.4 mg, 2 mmol) were added to 50 mL
of CH3CN, and the solution was refluxed under argon atmo-
sphere for 24 h. After cooling to room temperature, the
inorganic salts were filtered, and the filtrate was evaporated
to dryness. The residue was redissolved in CH2Cl2 and
extracted with a basic aqueous solution (pH 11). The CH2Cl2
portion was evaporated to a minimum volume and chromato-
graphed on a silica gel column with the following sequence of
eluents: 100 mL of CH2Cl2, 200 mL of 1% CH3OH/CH2Cl2, and
200 mL of 2% CH3OH/CH2Cl2. TLC (SiO2): 7% NH3/CH3OH
(5% NH4OH in CH3OH)/93% CH2Cl2. The product was isolated
as a yellow viscous oil (42% yield): 1H NMR (CDCl3) δ 7.946
(t, 1H, -NH), 7.435-7.208 (m, 30H, Ar), 3.116-3.077 (q, 2H,
-CH2-), 2.94 (s, 2H, -CH2CO), 2.52-2.45 (m, 10H, -CH2-),
2.403 (t, 2H, -CH2-), 2.295 (t, 2H, -CH2-), 0.977 (t, 6H,
-CH3); MS (MW ) 777.4) observed 778 (M + H)+. Anal.
(C50H55N3OS2)‚1/2H2O calcd (found): C, 76.29 (76.10); H, 7.17
(7.04); N, 5.33 (5.34).
Con clu sion s
Integrating the radiometal within the pharmacophore
of melanoma-targeting dialkylaminoethyl benzamides,
such as IMBA, by replacing the aromatic ring with an
oxometal-AADT moiety leads to metal complexes that
display significant in vivo melanoma accumulation.
Similar to the earlier oxometal “3 + 1” complexes,29
these oxotechnetium(V)- and oxorhenium(V)-AADT
complexes also contain pendent tertiary amines, and
those with a log DpH7.4 ≈ 1 (complex 1-Tc) display
relatively high in vivo melanoma accumulation. While
the σ-receptor affinity for all the complexes is low to
moderate, in vitro cell-uptake measurements indicate
an active uptake component in B16 melanoma cells. The
relatively high in vivo melanoma uptake coupled with
the high melanoma/nontumor ratios displayed by these
technetium complexes indicates that technetium-based
small molecular probes that target melanoma may be
designed and could potentially be useful in the early
detection and diagnosis of melanoma and its metastases.
N-[(2-Dibu tylam in oeth yl)-N-(2-(2-(S-(tr iph en ylm eth yl)-
t h io)et h yl)a m in o)a cet yl]-S-(t r ip h en ylm et h yl)-2-a m in o-
eth a n eth iol [AADT-N-CH2CH2-N(C4H9)2] (2). This com-
pound was synthesized with N-(2-dibutylamino)ethyl chloride
and AADT-(Trt)2 using a procedure analogous to that described
above for 1. Purification was carried out on a silica gel TLC
plate that was developed in 7% methanolic NH3 (5% NH4OH
in CH3OH)/93% CH2Cl2. The product was obtained as a
yellowish viscous oil (38% yield): 1H NMR (CDCl3) δ 7.829-
7.745 (s, 1H, -NH), 7.440-7.350 (m, 12H, Ar), 7.295-7.242
(m, 12H, Ar), 7.230-7.100 (m, 6H, Ar), 3.090-3.035 (q, 2H,
-CH2-), 2.929 (s, 2H, -CH2CO), 2.540-2.300 (m, 12H,
-CH2-), 2.297-2.265 (m, 2H, -CH2-), 1.360 (brs, 4H, -CH2-),
1.285-1.220 (m, 4H, -CH2-), 0.894 (t, 6H, -CH3); MS (MW
Exp er im en ta l Section
All chemicals and reagents, purchased from commercial
sources (Aldrich Chemicals, Gibco Life Technologies), were of
analytical grade and were used without further purification.
Technetium-99m pertechnetate was obtained via a generator
(DuPont). Elemental analyses were performed on an elemental
analyzer LECO-CHNS-932. 1H NMR spectra were recorded
on a Varian XL500 MHz instrument. X-ray crystallography
was performed on a Siemens platform goniometer with a CCD
detector using a Mo KR radiation source. The structure was
solved by direct methods using SHELXTL version 5.0. FT-IR
spectra were recorded on a Bruker Vector 22 FTIR instrument

3138 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Friebe et al.
) 833.2) observed 834 (M + H)+. Anal. (C54H63N3OS2) calcd
(found): C, 77.75 (77.03); H, 7.61 (7.62); N, 5.04 (5.03).
CH3OH/95% CH2Cl2 to yield the desired product as a pale
purple solid.
N-[(3-Dieth yla m in op r op yl)-N-(2-(2-(S-(tr ip h en ylm eth -
yl)t h io)et h yl)a m in o)a cet yl]-S-(t r ip h en ylm et h yl)-2-a m i-
n oet h a n et h iol [AADT-N-CH 2CH 2CH 2-N(CH 2CH 3)2] (3).
AADT-N-CH2CH2CH2Cl32 (310 mg, 0.4 mmol), diethylamine
(59.9 mg, 0.4 mmol), KI (340.1 mg, 2.1 mmol), and K2CO3
(141.7 mg, 1.0 mmol) were added to 50 mL of CH3CN, and the
solution was refluxed for 24 h. The product was purified via
silica gel chromatography with 3% methanolic NH3 (5% NH4-
OH in CH3OH)/97% CH2Cl2, yielding a yellowish oil (72%
yield): 1H NMR (CDCl3) δ 7.535-7.500 (m, 1H, -NH), 7.465-
7.380 (m, 12H, Ar), 7.330-7.250 (m, 12H, Ar), 7.250-7.190
(m, 6H, Ar), 3.100-3.020 (q, 2H, -CH2-), 2.896 (s, 1H, -CH2-
CO), 2.600-2.520 (m, 4H, -CH2-), 2.500-2.400 (m, 6H,
-CH2-), 2.410-2.340 (m, 2H, -CH2-), 2.325-2.226 (m, 2H,
-CH2-), 1.620-1.540 (m, 2H, -CH2-), 1.025 (t, 6H, -CH3);
MS (MW ) 791.4) observed 792 (M + H)+. Anal. (C51H57N3-
OS2)‚1/2H2O calcd (found): C, 76.5 (76.65); H, 7.3 (7.24); N, 5.24
(5.30).
[ReOAADT]-C2-NEt2 (1-Re): yield 74.4%; 1H NMR (CDCl3)
δ 4.943 (d, 1H, -CH2CO), 4.554 (dd, 1H, -CH2-), 4.248 (d,
1H, -CH2CO), 4.065 (dd, 1H, -CH2-), 3.993 (m, 1H, -CH2-),
3.639 (m, 1H, -CH2-), 3.532 (dd, 1H, -CH2-), 3.419 (ddd,
1H, -CH2-), 3.212 (ddd, 1H, -CH2-), 3.160 (ddd, 1H,
-CH2-), 2.868 (dd, 2H, -CH2-), 2.795 (m, 1H, -CH2-), 2.570
(m, 4H, -CH2-), 1.579 (ddd, 1H, -CH2-), 1.060 (t, 6H, -CH3);
IR υRedO ) 952 cm-1; MS (MW ) 493.1) observed 494 (M +
H)+. Anal. (C12H24N3O2ReS2) calcd (found): C, 29.3 (29.5); H,
4.9 (5.1); N, 8.5 (8.4); S, 13.0 (12.6).
[ReOAADT]-C2-NBu 2 (2-Re): yield 15%; 1H NMR (CDCl3)
δ 4.963 (d, 1H, -CH2CO), 4.582 (dd, 1H, -CH2-), 4.209 (d,
1H, -CH2CO), 4.089 (dd, 1H, -CH2-), 3.975 (m, 1H, -CH2-),
3.646 (m, 1H, -CH2-), 3.492 (d, 1H, -CH2-), 3.426 (m, 1H,
-CH2-), 3.264 (m, 1H, -CH2-), 3.180 (m, 1H, -CH2-), 2.899
(m, 2H, -CH2-), 2.805 (m, 1H, -CH2-), 2.464 (m, 4H,
-CH2-), 1.606 (m, 1H, -CH2-), 1.450 (m, 4H, -CH2-), 1.336
(m, 4H, -CH2-), 0.943 (t, 6H, -CH3); IR υRedO ) 958 cm-1
;
MS (MW ) 549.1) observed 550 (M + H)+. Anal. (C16H32N3O2-
ReS2) calcd (found): C, 35.0 (34.9); H, 5.9 (5.7); N, 7.7 (7.7); S,
11.6 (11.9).
N-[(3-Dibu tyla m in op r op yl)-N-(2-(2-(S-(tr ip h en ylm eth -
yl)t h io)et h yl)a m in o)a cet yl]-S-(t r ip h en ylm et h yl)-2-a m i-
n oeth a n eth iol [AADT-N-CH2CH2CH2-N(C4H9)2] (4). Dibu-
tylamine (149.9 mg, 1.2 mmol), AADT-N-CH2CH2CH2Cl32 (584
mg, 0.8 mmol), KI (664 mg, 4 mmol), and K2CO3 (552.8 mg,
4.0 mmol) were dissolved in 50 mL of argon-saturated CH3-
CN and refluxed for 24 h. The product was purified via silica
gel chromatography using as eluent 4% CH3OH/96% CH2Cl2,
followed by 10% CH3OH/90% CH2Cl2, yielding a yellowish oil
(78% yield): 1H NMR (CDCl3) δ 7.460-7.440 (m, 1H, -NH),
7.410-7.360 (m, 12H, Ar), 7.300-7.180 (m, 18H, Ar), 3.040-
3.010 (q, 2H, -CH2-), 2.893 (s, 2H, -CH2-), 2.650-2.295 (m,
13H, -CH2-), 1.900-1.380 (m, 7H, -CH2-), 1.330-1.250 (m,
4H, -CH2-), 0.914 (t, 6H, -CH3); MS (MW ) 847.5) observed
848 (M + H)+. Anal. (C55H65N3OS2)‚1/2H2O calcd (found): C,
77.06 (77.08); H, 7.75 (7.81); N, 4.90 (5.11).
[ReOAADT]-C3-NEt2 (3-Re): yield 70.4%; 1H NMR (CDCl3)
δ 4.694 (d, 1H, -CH2CO), 4.567 (dd, 1H, -CH2-), 4.113 (d,
1H, -CH2CO), 4.081 (dd, 1H, -CH2-), 4.000 (ddd, 1H,
-CH2-), 3.610 (ddd, 1H, -CH2-), 3.399 (ddd, 1H, -CH2-),
3.239 (m, 2H, -CH2-), 3.184 (4d, 1H, -CH2-), 2.866 (dd, 1H,
-CH2-), 2.555 (m, 4H, -CH2-), 2.496 (m, 2H, -CH2-), 1.921
(m, 2H, -CH2-), 1.614 (ddd, 1H, -CH2-), 1.030 (t, 6H, -CH3);
IR υRedO ) 955 cm-1; MS (MW ) 507.1) observed 508 (M +
H)+. Anal. (C13H26N3O2ReS2) calcd (found): C, 30.8 (30.7); H,
5.2 (5.0); N, 8.3 (8.6); S, 12.6 (12.3).
[ReOAADT]-C3-NBu 2 (4-Re): yield 12.4%; 1H NMR (CDCl3)
δ 4.698 (d, 1H, -CH2CO), 4.564 (dd, 1H, -CH2-), 4.122 (d,
1H, -CH2CO), 4.071 (dd, 1H, -CH2-), 3.985 (ddd, 1H,
-CH2-), 3.624 (ddd, 1H, -CH2-), 3.351 (ddd, 1H, -CH2-),
3.268 (m, 2H, -CH2-), 3.202 (4d, 1H, -CH2-), 2.875 (dd, 1H,
-CH2-), 2.525 (m, 4H, -CH2-), 2.512 (m, 2H, -CH2-), 1.974
(m, 2H, -CH2-), 1.645 (ddd, 1H, -CH2-), 1.389 (m, 4H,
-CH2-), 0.94 (t, 6H, -CH3); IR υRedO ) 956 cm-1; MS (MW )
563.2) observed 564 (M + H)+. Anal. (C17H34N3O2ReS2) calcd
(found): C, 36.2 (36.3); H, 6.1 (6.2); N, 7.5 (7.8); S, 11.4 (11.5).
Gen er a l P r oced u r e for Dep r otection of Th iol Gr ou p s.
The bis-trityl-protected AADT ligand (6.0 mg) was dissolved
in 3 mL of trifluoroacetic acid (TFA) and stirred at room
temperature for 5 min. The solution was titrated with trieth-
ylsilyl hydride until the disappearance of the yellow color. The
TFA was evaporated completely and the dry residue was
placed under high vacuum overnight. The resulting white solid
was redissolved in 300 µL of argon-saturated methanol and
portioned into six vials using 50-µL aliquots. The contents of
the vials were then evaporated and the vials stored under
vacuum for subsequent labeling with technetium-99m.
X-r a y Str u ctu r e Deter m in a tion : formula, C12H24N3O2-
ReS2; formula weight, 493.1; unit cell dimensions, a ) 6.8929(8)
Å, b ) 9.8926(12) Å, c ) 12.2566(14) Å, R ) 93.074(2)°; â )
93.770(2)°; γ ) 103.706(2)°; density, 2.025 mg/m3 (calculated);
space group, P; wavelength, 0.71073 Å; reflections, 3246
(collected), 2265 (independent); absorption correction, semiem-
pirical from ψ-scans; refinement, full-matrix least-squares on
F2; final R indices [I > 2σ(I)], R1 ) 0.0550, wR2 ) 0.1361
(Supporting Information).
Tech n etiu m -99m La belin g (1-Tc-4-Tc). One milligram
of thiol-deprotected ligand (1-4) (vide supra) dissolved in 0.25
mL of phosphate buffer (0.005 M, pH 7.4) and the required
activity of 99mTc-glucoheptonate were placed in a vial, and
the reaction mixture was heated at 60-75 °C for 45 min.
HPLC evaluation of the 99mTc complexes typically showed 85-
98% radiochemical yield. Coinjection of the characterized
rhenium complexes with the analogous 99mTc complexes showed
coelution of the radioactive species with the corresponding UV-
active rhenium complex.
Deter m in a tion of Lip op h ilicity a n d p Ka Va lu es. The
lipophilicity and pKa values of all complexes were determined
using HPLC methods described previously.38,39 log P, log DpH7.4
,
and pKa values were determined on a Perkin-Elmer HPLC
system 1020 using a reversed phase PRP-1 column (250 × 4.1
mm; 10 µm; Hamilton) run under isocratic conditions with a
flow rate of 1.5 mL/min at room temperature. The mobile
phase was acetonitrile/phosphate buffer (0.01 M), 3/1, with the
aqueous buffer adjusted to the desired pH between 3 and
11.38,39 The capacity factor (k′) was calculated for each deter-
mination37,44 and the partition coefficient at a given pH (D or
log D) was calculated from the equation: log D ) a log k′ + b,
where the parameters a and b are predetermined using
Gen er a l P r oced u r e for Rh en iu m Com p lexa tion . The
bis-trityl-protected ligand (1-4) (100 mg, 0.1 mmol) was
dissolved in 0.25 mL of anisol and 10 mL of TFA. The resulting
yellow solution was stirred for 5 min and then titrated with
triethylsilyl hydride until colorless. The solution was evapo-
rated and the residue placed under high vacuum until com-
pletely dry. The residue was redissolved in 5 mL of argon-
saturated 20% CH3OH/water solution. To this was added an
aqueous solution of NaReO4 (30 mg, 0.1 mmol), sodium
glucoheptonate (55 mg, 0.2 mmol), and solid SnCl2 (21 mg, 0.1
mmol) with stirring. The solution began to turn brownish
purple. The pH of the reaction mixture was adjusted to 7, and
the reaction was heated at 75 °C for 1 h. The solution was
then cooled to room temperature, the pH adjusted to 8, and
the solution extracted with CH2Cl2. The extract was concen-
trated and chromatographed on silica gel, eluting with 4-5%
standard amines. The fitted points of inflection from the
38
sigmoidal DHPLC/pH profiles permit calculation of the pKHPLC
.
The aqueous ionization constants pKa were calculated from
the pKHPLC values after correction with a predetermined
correction factor obtained using standard amine compounds.
log P values of the neutral complexes were estimated from the
upper plateau of the respective sigmoidal log D/pH curves in
the alkaline range.

[
99mTc]Oxotechnetium(V) Complexes of AADT
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3139
In Vitr o Cell Stu d ies. Murine B16/F0 melanoma cells and
(0.15 mg of membrane protein) in Tris-HCl (50 mM), pH 8.0,
for 120 min at 25 °C in a 0.25-mL volume. All other manipula-
tions and data analysis were performed as described vide supra
for the σ1-receptor assay.
human MCF-7 breast cancer cells were obtained from Ameri-
can Type Culture Collection (ATCC, Manassas, VA) and were
grown in T-175 flasks in 14 mL of Dulbecco’s Modified Eagle
Medium (D-MEM; Gibco, Life Technology, Gaithersburg, MD)
containing 4500 mg/L D-glucose, L-glutamine, and pyridoxine
hydrochloride, 110 mg/L sodium pyruvate, 10% fetal bovine
serum (FBS), 0.2% gentamicin, and 0.5% penicillin-strepto-
mycin solution. All cells were harvested from cell culture flasks
by trypsinization with 1 mL of trypsin-EDTA solution (0.25%
trypsin, 1 mM EDTA · 4 Na) (Gibco). After being washed with
12 mL of Dulbecco’s phosphate-buffered saline (PBS), pH 7.2
(Ca2+- and Mg2+-free; g/L KCl, 0.20; KH2PO4, 0.20; NaCl, 8.00;
Na2HPO4, 1.15) (Gibco), the cells were counted and resus-
pended in 8 mL of S-MEM (Gibco) (Ca2+-free, with reduced
Mg2+content) and stored at 4 °C until use.
For in vitro tumor-cell accumulation studies, 5 × 106 cells
in polypropylene test tubes were incubated at 37 or 4 °C, with
intermittent agitation with 1-2 µCi (5 µL) 99mTc complex (1-
Tc-4-Tc) in a total volume of 350 µL of S-MEM. At appropri-
ate time intervals, the tubes were vortexed and 8-µL samples
were layered on 350 µL of cold FBS in a 400-µL Eppendorf
microcentrifuge tube. After centrifugation at 15 000 rpm for
2 min, the tubes were frozen in a dry ice-acetone bath. While
still frozen, the bottom tip of the microcentrifuge tube contain-
ing the cell pellet was cut and placed in a counting tube. The
remaining portion of the tube with the supernatant was placed
in a separate counting tube. Both fractions were counted for
radioactivity in a γ-counter (WALLAC, 1480 WIZARD 3′′). The
amount of supernatant in the cell pellet was determined to be
<1% in separate experiments. The percentage cell uptake of
the 99mTc complex was calculated as % uptake ) [cpm (pellet)]/
[cpm (pellet) + cpm (supernatant)] × 100.
An im a l Stu d ies. All animal experiments were performed
in compliance with the Principles of Laboratory Animal Care
(NIH publication #85-23, revised 1985). Biodistribution studies
and tumor-uptake measurements were performed in C57Bl6
mice (15-20 g) bearing the B16/F0 murine melanoma on the
hind limb.15,17,18,20 The tumor cells (B16/F0), obtained from
ATCC, were washed with PBS and transplanted subcutane-
ously on the left hind flank by an inoculation of 0.5 × 106 cells
(0.1 mL). Ten to 14 days later the animals developed palpable
tumor nodules 3-5 mm in diameter. The biodistribution
studies were carried out by tail-vein injection of 25-30 µCi
(0.05-0.1 mL) of the 99mTc-labeled complexes 1-Tc-4-Tc. At
the designated time after tail-vein administration, the animals
were weighed and sacrificed. The organs and tumors were
harvested and, when appropriate, blotted dry, weighed, and
counted in a γ-counter along with technetium-99m standards
of the injected dose. The results are expressed as % ID/g tissue
(Table 3).
Su p p or tin g In for m a tion Ava ila ble: ORTEP drawing of
1-Re, crystal data and refinement parameters, coordinates,
anisotropic temperature factors, bond lengths and angles,
hydrogen coordinates, and observed and calculated structure
factors. This material is available free of charge via the
Internet at http://pubs.acs.org.
Refer en ces
(1) (a) Parkin, D. M.; Pisani, P.; Ferlay, J . Global Cancer Statistics,
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