A novel 99mTc-labeled testosterone derivative as a potential agent for targeting androgen receptors

Article abstract of DOI:10.1016/j.bmcl.2006.08.081

With an insight that ligands possessing a N₂S₂ tetradentate array of donor atoms serve as ideal bifunctional chelating agents (BFCA) in the radiolabeling of target-specific agents, 5-hydroxy-3,7-diazanonan-1,9-dithiol (DAHPES) with a derivatizable substituent in the form of a hydroxyl group in the backbone was synthesized. The preparation of a steroid conjugate via coupling of this BFCA with testosterone-3-(O-carboxymethyl) oxime and the subsequent radiolabeling of the conjugate under optimized conditions with ^99mTc, the ideal diagnostic radionuclide in nuclear medicine procedures, are reported. The immunoreactivity of the radiolabeled conjugate was demonstrated in a study using anti-testosterone antibodies, wherein the radiolabeled conjugate exhibited significant binding with antiserum to testosterone. Cell-uptake studies in DU145 prostate carcinoma cell line bearing androgen receptors (ARs) and comparison with AR non-bearing breast carcinoma cell line revealed the specific binding of the steroidal moiety with the testosterone receptor.

Full text of DOI:10.1016/j.bmcl.2006.08.081

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5788–5792
99m
A novel Tc-labeled testosterone derivative as a potential agent
for targeting androgen receptors
*
Tapas Das, Sharmila Banerjee, Grace Samuel, Ketaki Bapat,
ꢀ
Suresh Subramanian, Maroor R. A. Pillai and Meera Venkatesh
Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai 400085, India
Received 23 May 2006; revised 4 August 2006; accepted 17 August 2006
Available online 1 September 2006
2 2
Abstract—With an insight that ligands possessing a N S tetradentate array of donor atoms serve as ideal bifunctional chelating
agents (BFCA) in the radiolabeling of target-specific agents, 5-hydroxy-3,7-diazanonan-1,9-dithiol (DAHPES) with a derivatizable
substituent in the form of a hydroxyl group in the backbone was synthesized. The preparation of a steroid conjugate via coupling of
this BFCA with testosterone-3-(O-carboxymethyl) oxime and the subsequent radiolabeling of the conjugate under optimized con-
99m
ditions with
Tc, the ideal diagnostic radionuclide in nuclear medicine procedures, are reported. The immunoreactivity of the radi-
olabeled conjugate was demonstrated in a study using anti-testosterone antibodies, wherein the radiolabeled conjugate exhibited
significant binding with antiserum to testosterone. Cell-uptake studies in DU145 prostate carcinoma cell line bearing androgen
receptors (ARs) and comparison with AR non-bearing breast carcinoma cell line revealed the specific binding of the steroidal moiety
with the testosterone receptor.
ꢀ
2006 Elsevier Ltd. All rights reserved.
Several methods of radiolabeling steroidal substrates
with radioisotopes of choice for targeting specific recep-
tors over-expressed in target tissues in diseased states
have been reported. Testosterone is a steroidal hor-
mone which binds to androgenic receptors which are
envisage a strategy whereby testosterone could be radio-
9
9m
labeled with
non-invasive imaging of androgen receptors. The desir-
Tc in designing a potential agent for
1
–3
99m
able features of
Tc as an isotope of choice in diagnos-
tic nuclear medicine emerge from its manifold
advantages attributable to its suitable nuclear decay
characteristics (half-life 6 h, decay by isomeric transition
with the emission of 140 keV gamma photon suitable for
1
over-expressed in cancerous regions of the prostate.
Agents to image the prostate or tumors therein, based
on the androgen content, serve as a useful tool in staging
4
of the disease and monitoring the course of therapy.
Various derivatives of androgens have been labeled with
imaging procedures), easy availability from
Mo– Tc generator, and amenable chemistry. The
a
9
9
99m
7
7
82
125
99m
several c-emitting radionuclides, such as, Br, Br, I,
present work reports an attempt to prepare a
Tc-
7
5
5–8
18 4,9
Se
as well as with the positron emitter F, in
labeled testosterone conjugate for targeting androgenic
receptors.
search of an ideal androgen receptor-based prostatic
imaging agent. However, rapid metabolic cleavage,
low receptor binding affinity, and inadequate specific
activity are the major impediments toward use of these
radiolabeled conjugates. The aforementioned inadequa-
cies of currently available diagnostic methods for in vivo
imaging of prostatic carcinoma have prompted us to
In our search for novel bifunctional chelating agents, a
tetradentate ligand possessing an array of donor atoms
identical with those in ethylene dicysteine (ECD) or
1
0–14
diaminodithiols (DADT) was envisaged.
5-Hy-
droxy-3,7-diazanonan-1,9-dithiol (DAHPES) with a
N S donor atom set and a derivatizable substituent in
the form of a hydroxyl group in the backbone was syn-
2
2
Keywords: Testosterone; BFCA–testosterone conjugate; Receptor-spe-
cific agent; Androgen receptor imaging.
thesized by applying a single-step synthetic strategy
15–18
(
Fig. 1).
Successful radiolabeling of DAHPES with
Tc at room temperature with high yield and a desir-
*
Corresponding author. Tel.: +91 22 2559 0616; fax: +91 22 2550
151; e-mail: sharmila@apsara.barc.gov.in
9
9m
5
ꢀ
able biodistribution pattern of the labeled ligand with
no major uptake in any organ and fast clearance via
Present address: Industrial Application and Chemistry Section,
International Atomic Energy Agency, Wagramerstrasse, A-1400
Vienna, Austria.
1
6
the renal route provided impetus for its use as a BFCA
0
960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.081

T. Das et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5788–5792
5789
OH
OH
Toluene
Reflux, N
HN
NH
S
+
2
NH
H N
2
40 h
2
SH HS
Ethylene sulphide
1
,2-diamino-3-
5
-hydroxy-3,7-diaza-
hydroxy propane
nonan-1,9-dithiol
(DAHPES)
OH
OH
NH -O-CH -COOH
2
2
Pyridine:Methanol:Water (1:4:1)
2
h, Reflux
O
HOOCH CON
2
Testosterone
Testosterone-3-(O-carboxymethyl) oxime
OH
HN
NH
SH HS
DAHPES
Isobutylchloroformate
Tributylamine
Dioxane
RT, 4 h
OH
O
C-CH -O-N
2
O
HN
NH
SH HS
DAHPES-testosterone conjugate
Figure 1. Synthesis of DAHPES–testosterone conjugate.
2
0
for the present study. It is well reported that testosterone
when functionalized at the C position retains its immu-
which in turn was synthesized from testosterone. The
coupling of the testosterone derivative with the BFCA
involved the activation of testosterone-3-(O-carboxym-
ethyl)-oxime using isobutyl chloroformate and tributyl
amine. The mixed anhydride thus obtained was allowed
3
noreactivity, and hence a strategy toward preparation of
a radiolabeled agent for androgen receptor targeting
1
9
was envisaged using a C functionalized testosterone.
3
2
1
In the present paper, we report the synthesis, purifica-
tion, and
to react with the DAHPES in dry dioxane. The prod-
22
uct was characterized by H NMR and mass spectra.
The peak integrations and multiplicities in the
NMR spectrum observed were as expected and provided
evidence in favor of the formation of the desired conju-
gate. The desired derivatization is indicated by the
appearance of an 8-H multiplet between 3.20 and 3.36
corresponding to the CH protons of the DAHPES moi-
ety. A shift in the d values of the CH(O) proton from
9
9m
1
Tc labeling of a testosterone–BFCA conju-
1
gate, and the preliminary biological evaluation of the
developed radiolabeled conjugate in suitable in vitro
systems.
H
The BFCA–testosterone conjugate was prepared via a
two-step reaction (Fig. 1) involving the testosterone-3-
(
O-carboxymethyl)-oxime derivative as the precursor,

5790
T. Das et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5788–5792
3
.50–3.52 to 3.72–3.78 at the site of derivatization pro-
favor of the extent of complexation determined by using
the combination of PC and TLC systems described
above. A dual pump HPLC unit with a C18 reversed-
phase column (25 cm · 0.46 cm) was used for the purifi-
cation of the radiolabeled conjugate. The elution was
monitored both by UV absorbance at 240 nm as well
as by radioactivity signals. The flow rate was maintained
at 1 mL/min. Acetonitrile (A) and water (B) mixtures
containing 0.1% trifluoro acetic acid were used as the
mobile phase and the following gradient elution tech-
nique was adopted for the separation (0–3 min 10% A
to 90% A, 3–10 min 90% A to 10% A, and 10–20 min
10% A to 90% A). Typical HPLC patterns representing
the radioactive chromatogram and UV profile are
depicted in Figures 2a and b, respectively. It is evident
vided confirmatory evidence toward formation of the
steroid–BFCA conjugate. The appearance of two-pro-
ton singlet at 4.43 and 4.46 corresponding to the
1
@
NOCH COO– proton in the H NMR spectrum of
2
the conjugate is indicative of the formation of geometri-
cal isomers (60:40). The observation of the molecular
ion peak at m/z 553 in the mass spectrum conclusively
showed the formation of the DAHPES–testosterone
conjugate.
For the labeling of DAHPES–testosterone conjugate,
99m
À
0
.2 mL of freshly eluted
TcO4 (500 lCi) from a
Mo– Tc generator was added to 1 mg (1.8 mM) of
the conjugate in 0.4 mL phosphate buffer (0.05 M, pH
2) and 0.2 mL of normal saline. 0.2 mL of
SnCl Æ 2H O (0.2 mg) solution in dilute HCl was added
9
9
99m
9
9m
1
from the figure that while
Tc–DAHPES–testosterone
À
99m
complex exhibited a retention time of ꢀ210 s,
TcO4
Tc–DAH-
2
2
9
9m
to the reaction mixture. The mixture was purged with
nitrogen for 2 min and then incubated for 30 min to
1
eluted out from the column at ꢀ280 s.
PES–testosterone complex thus purified was subse-
quently used for all biological evaluations.
h at room temperature.
9
9m
The extent of
Tc labeling achieved was determined by
Stability studies carried out using quality control tech-
niques mentioned above showed that the radiolabeled
conjugate retained its radiochemical purity when stored
at room temperature up to 4 h after its preparation.
paper chromatography (PC), thin-layer chromatogra-
phy (TLC), and paper electrophoresis techniques follow-
ing standard procedures reported elsewhere.
2
3,24
In PC,
using saline as the solvent, it was observed that a consid-
erable portion of the activity remained at the point of
spotting (R = 0), which could be attributed to the pres-
f
ence of either the radiolabeled conjugate or reduced
technetium. The presence of minor amount of activity
at the solvent front indicated the presence of either per-
a
350
99m
Tc-DAHPES-testosterone
9
9m
technetate or
impurity), both of which appeared at R = 0.8–1 under
Tc-labeled DAHPES (as radiochemical
3
2
2
1
1
00
50
00
50
00
f
identical conditions. However, on TLC/saline, the major
activity moved toward the solvent front, thereby differ-
entiating the radiolabeled conjugate from the reduced
technetium. Therefore, a combination of these two chro-
matographic methods enabled the preliminary determi-
nation of the extent of complexation which was
further verified by paper electrophoresis studies. This
was carried out using 0.025 M phosphate buffer of pH
99m
TcO4
-
7
7
1
.5 at a potential gradient of 10 V/cm for a period of
À
5
0
0
99m
5 min. It was observed that, while
0 cm toward the anode,
TcO4 moved
Tc-labeled conjugate
9
9m
remained at the point of spotting under identical
conditions.
0
200
400
600
800
1000
1200
Retention time (s)
In optimization studies of the complexation parameters
for achieving maximum yield, it was observed that 1 mg
of the radiolabeled steroidal–BFCA conjugate at pH 12
yielded ꢀ60% complexation when incubated at room
temperature for 30 min to 1 h. Further increase in the
ligand concentration did not show any appreciable effect
on the complexation yield. Stannous chloride was used
as the reducing agent and 0.2 mg of this compound
was required to obtain maximum complexation. PC
and TLC studies revealed that a maximum of 60% com-
plexation was achieved when the complex was prepared
under optimized conditions.
b
9
9m
9
9m
Radiochemical purification of the
Tc-labeled DAH-
Figure 2. (a) HPLC pattern (radioactive chromatogram) of
labeled DAHPES–testosterone conjugate. (b) HPLC pattern (UV
Tc-
PES–testosterone conjugate was achieved by HPLC.
This technique also provided an additional support in
9
9m
profile) of
Tc-labeled DAHPES–testosterone conjugate.

T. Das et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5788–5792
9m 99m
5791
9
Biological activity of the resultant
Tc–DAHPES–tes-
beling with
Tc. Retention of immunological activity
tosterone complex was studied by carrying out prelimin-
ary in vitro cell-uptake studies with DU145, a human
prostate carcinoma cell line known to express testoster-
one receptors, as well as by binding studies with anti-tes-
tosterone antibodies.
and thereby a gross indication of structural integrity of
testosterone in the radiolabeled conjugate was demon-
strated in serum antibody binding studies. While in vitro
cell binding studies showed significant uptake in the
human prostatic carcinoma cell line, negligible uptake
observed in the androgen receptor negative breast
carcinoma cell line is indicative of the androgen receptor
specificity of the developed agent. These studies showed
2
5
Antibody binding studies were carried out to prelimi-
narily ascertain the retention of immunological activity
which could provide a clue toward structural integrity
of the steroid after introduction of the BFCA. It was ob-
served that the radiolabeled conjugate exhibited a bind-
ing of ꢀ50% with 1:5 diluted antiserum, while binding
was ꢀ43% with a 1:10 diluted antiserum. The decrease
in binding with increased dilution of the testosterone-
specific antiserum indicated that the radiolabeled conju-
gate retained its binding capacity to the antiserum. The
binding was comparable to the observation made in ear-
lier studies carried out in our laboratory wherein 100 pg
of a radioiodinated (1000 lCi/lg) testosterone derivative
showed a binding of 35–45% with a 1:150,000 diluted
9
9m
that the radiolabeled agent
Tc–DAHPES–testoster-
one possesses considerable promise toward use as a
potential targeting agent for androgen receptors.
Acknowledgments
The authors thankfully acknowledge Dr. V. Venugopal,
Director, Radiochemistry and Isotope Group, Bhabha
Atomic Research Centre, for his keen interest and con-
stant encouragement. The authors also thank the
National Centre for Cell Science, Pune for supplying
the cancer cell lines used in the study.
2
6
antiserum. It was also observed in the present studies
9
9m
with
Tc-labeled DAHPES–testosterone conjugate
that, as the amount of conjugate was increased from 3
to 6 lg, the tracer showed a decrease in binding from
References and notes
ꢀ
50% to ꢀ40%. Blank studies where the antiserum
À
4
99m
was incubated with either only TcO or with
Tc–
1
2
. Katzenellenbogen, J. In Radiopharmaceuticals: Chemistry
and Pharmacology; Nunn, A. D., Ed.; Marcel Dekker Inc.:
New York, 1992; p 297.
. Hom, H. K.; Katzenellenbogen, J. A. Nucl. Med. Biol.
1
DAHPES gave a binding of 2–5%. These results indicat-
ed that the binding of testosterone toward the specific
antiserum was retained after radiolabeling.
997, 24, 485.
Prostate cancer is one of the most common forms of
malignant growth in human males. Prostatic carcinoma
cells have been reported to bear androgen receptors not
only intracellularly, but also on the membrane surface.
The latter are hypothesized to be responsible for ra-
3. Katzenellenbogen, J. A. J. Nucl. Med. 1995, 36, 8S.
4. Liu, A.; Dence, C. S.; Welch, M. J.; Katzenellenbogen, J.
A. J. Nucl. Med. 1992, 33, 724.
5
. Ghanadian, R.; Walters, S. L.; Chisholm, G. D. Eur. J.
Nucl. Med. 1977, 2, 155.
2
7
6. Tarle, M.; Padovan, R.; Spaventi, S. Eur. J. Nucl. Med.
981, 6, 79.
pid-acting non-genomic effects. The DU145 prostatic
carcinoma cell line is androgen-independent, but pos-
1
7
. Hoyte, R. M.; Rosner, W.; Hochberg, R. B. J. Steroid
Biochem. 1982, 16, 621.
2
8
sesses androgen receptors and therefore could serve
as a target for radiolabeled steroidal substrates possess-
ing affinity for androgenic receptors. Blank experiments
were also performed by incubating the DU145 cells with
8
. Skinner, R. W. S.; Pozerac, R. V.; Counsell, R. E.; Hsu, C.
F.; Weinhold, P. A. Steroids 1977, 30, 15.
9. Liu, A.; Katzenellenbogen, J. A.; Van Brocklin, H. F.;
Mathias, C. J.; Welch, M. J. J. Nucl. Med. 1991, 32, 81.
0. Baidoo, K. E.; Lever, S. Z. Bioconjugate Chem. 1990, 1,
9
9m
À
TcO4 and a negative control was set up by incubat-
1
1
1
ing the radiolabeled BFCA–testosterone conjugate with
an androgen receptor negative cell line, namely, MDA-
MB-468, a human breast carcinoma cell line, keeping
1
32.
1. Davison, A.; Jones, A. G.; Orgiv, C.; Sohn, M. Inorg.
Chem. 1981, 20, 1629.
2. Vallbhajosula, S.; Zimmerman, R. E.; Picard, M.; Stritke,
P.; Mena, I.; Hellman, R. S.; Tikofsky, R. S.; Stabin, M.
G.; Morgan, R. A.; Goldsmith, S. J. J. Nucl. Med. 1989,
2
9
other experimental conditions identical. About ꢀ24–
3
1% cell uptake was observed when 0.5–1 lg of the
Tc-labeled DAHPES–testosterone conjugate was
9
9m
4
incubated with 4 · 10 cells. When the amount of tracer
was increased 10-fold (10 lg), the cell uptake decreased
from an average of 31% to 22%, whereas with a 100-fold
3
0, 599.
13. Liang, F. H.; Virzi, F.; Hnatowich, D. J. Nucl. Med. Biol.
1987, 14, 63.
(
decreased from 31% to <5%. Blank experiments carried
out using TcO4 in place of the radiolabeled conjugate
and negative controls with MDA-MB-468 breast carci-
noma cell line in place of DU145 showed <5% cell
uptake. These results appear to indicate that the
radiolabeled conjugate has specific affinity toward
testosterone receptors on the prostate cancer cell line.
100 lg) increase in the amount of tracer, the uptake
14. Oya, S.; Plossl, K.; Kung, M. P.; Stevenson, D. A.; Kung,
H. F. Nucl. Med. Biol. 1998, 25, 135.
15. Corbin, J. L.; Miller, K. F.; Pariyadath, N.; Wherland, S.;
Bruce, A. E.; Stiefel, E. I. Inorg. Chem. Acta 1984, 90, 41.
À
1
6. Banerjee, S.; Das, T.; Samuel, G.; Sarma, H. D.;
Venkatesh, M.; Pillai, M. R. A. Ind. J. Nucl. Med. 2003,
1
8, 25.
1
7. Synthesis of 5-hydroxy-3,7-diazanonan-1,9-dithiol (DAH-
PES). A mixture of ethylene sulfide (5.87 g, 97 mM) in
2
0 mL of dry toluene was added to a stirred solution of
The present study describes the preparation of a novel
BFCA–testosterone conjugate and its successful radiola-
1,3-diamino-2-hydroxypropane (4 g, 44 mM) in 30 mL dry
toluene under reflux and with nitrogen flushing over a

5792
T. Das et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5788–5792
period of 3 h. The refluxing was continued for 40 h after
23. Das, T.; Banerjee, S.; Samuel, G.; Sarma, H. D.; Rama-
moorthy, N.; Pillai, M. R. A. Nucl. Med. Commun. 2000,
21, 939.
24. Das, T.; Chakraborty, S.; Banerjee, S.; Samuel, G.; Sarma,
H. D.; Venkatesh, M.; Pillai, M. R. A. J. Labelled Compd.
Radiopharm. 2003, 46, 197.
which toluene was removed under vacuum distillation,
whereby a white residue was obtained. The residue was
extracted several times with a mixture of methanol and
dichloromethane (1: 1 v/v, 5· 10 mL). The pooled organic
extracts were concentrated to yield a viscous liquid, which
subsequently solidified to a pale white solid (5.2 g, yield
25. Protocol for antibody binding studies. Antiserum was raised
in rabbit against testosterone-3-(O-carboxymethyl)-ox-
ime–bovine serum albumin (BSA) conjugate in our labo-
ratory. When the specific binding of the antiserum with
testosterone was taken as 100%, <10% cross-reactivity
with dihydrotestosterone and <1% cross-reactivity with all
other structurally related compounds were observed.
0.1 mL of the purified diluted radiolabeled conjugate
(ꢀ3 lg) was incubated with diluted antiserum in 0.1 M
Tris buffer of pH 8.5 containing 0.1% gelatin. 0.1 mL of
3% c-globulin was added to facilitate precipitation. At the
end of the 1 h incubation at room temperature, 1 mL of
22% polyethylene glycol in normal saline was added to
57%) on storage. The crude product thus obtained was
purified by column chromatography over silica gel using
10% ammonium hydroxide in methanol as the eluant.
1
8. Characterization of 5-hydroxy-3,7-diazanonan-1,9-dithiol
À1
(
DAHPES). FT-IR (KBr, m cm ): 3364 (–NH), 2960
1
–SH). H NMR, CD
( OD (d ppm): 2.55–2.58 (2H, m)
CH H NH, 2.64–2.71 (2H, dd) NHCH H (CHOH),
3
A
B
A
B
2
–
.67–2.69 (2H, m) CH
NHCH (CHOH) and –NHCH
SH, 3.50–3.52 (1H, m) –CHOH. Mass
A
H
B
NH, 2.79–2.93 (6H, m)
H
A B
2
CH SH, 3.16 (4H, t,
2
J = 5.4 Hz) –CH
2
+
spectra (EI) m/z: 210 (M ).
9. Autenrieth, D.; Kan, G.; Van Lier, J. E. Eur. J. Med.
Chem. 1981, 16, 525.
1
2
9
9m
precipitate the
Tc-labeled DAHPES–testosterone con-
0. Synthesis of testosterone-3-(O-carboxymethyl)-oxime. A
mixture of 25 mg (0.087 mM) of testosterone and 45 mg
jugate bound to antibody. The precipitate, which carried
the radioactivity bound to antibody, was separated by
centrifugation and the radioactivity in the precipitate was
measured. Blank studies were also performed simulta-
(
2
0.413 mM) of aminoxy acetic acid was dissolved in
.1 mL of pyridine/methanol/water (1:4:1 v/v) and the
neously with 9 TcO4 as well as with the
9m
À
99m
Tc–DAH-
resulting solution was refluxed for 2 h and then, stirred
overnight at room temperature. Removal of the solvent
produced a semi-solid mass of testosterone-3-(O-carb-
oxymethyl)-oxime, which was subsequently utilized with-
out any further purification for the coupling with
DAHPES.
PES complex in place of the radiolabeled conjugate.
26. Grace, S.; Meera, V.; Balakrishnan, S. A.; Sarma, H. D. J.
Radioanal. Nucl. Chem. 1997, 223, 83.
27. Hatzoglou, A.; Kampa, M.; Kogia, C.; Charalampopou-
los, I.; Theodoropoulos, P. A.; Anezinis, P.; Dambaki, C.;
Papakonstanti, E. A.; Stathopoulos, E. N.; Stournaras, C.;
Gravanis, A.; Castanas, E. J. Clin. Endocrinol. Metab.
2005, 90, 893.
2
1. Coupling of DAHPES with testosterone-3-(O-carboxym-
ethyl)-oxime. In a typical reaction, 20 mg of testosterone-
3-(O-carboxymethyl)-oxime (0.055 mM) in 1 mL of dry
dioxane was incubated at 4 ꢁC for 30 min with 10 lL of
isobutyl chloroformate and 20 lL of tributyl amine. A
solution of 12 mg DAHPES (0.057 mM) in 2 mL dry
dioxane was added to the mixture and stirred at room
temperature for 4 h at pH 8. The desired coupled product
28. Brolin, J.; Skoog, L.; Ekman, P. Prostate 1992, 20,
281.
29. Protocol for cell binding studies. Cell lines were cultured
at 37 ꢁC in humidified atmosphere with 5% CO
2
.
RPMI-1640 medium was used for DU145 and Dul-
becco’s modified Eagle’s medium (DMEM) was used
for MDA-MB-468, both supplemented with 10% fetal
bovine serum. Once sufficient cellular mass was
obtained, the cells were harvested by trypsinization,
washed repeatedly with assay buffer (phosphate-buffered
saline, pH 7.5), and finally dispensed into reaction
(
DAHPES–testosterone conjugate) thus obtained was
purified by preparative thin-layer chromatography
TLC) on pre-coated silica gel plates using 15% methanol
(
in ethyl acetate as the eluting solvent.
2. Spectral data for DAHPES–testosterone conjugate.
1
2
H
NMR, CD OD (d ppm): 0.78 (3H, s) C –H , 1.11 (3H, s)
3
18
3
4
–
C
C
19–H
3
, 2.22–2.38 (4H, m) –CH
A
H
B
NH, 3.0–3.1 (2H, m)
of testosterone, 3.20–3.25 and 3.34–3.36 (8H,
tubes at a concentration of 4 · 10 cells/tube in assay
6
–H
A
H
B
buffer. The radiolabeled conjugate was diluted in assay
buffer and added in varying concentrations to the cells.
Total reaction volume was maintained at 0.3 mL/tube.
After an incubation for 2 h at 37 ꢁC, the cells were
washed twice with cold assay buffer and counted in a
NaI(Tl) scintillation counter.
m) C –H C –H C –H C –H of DAHPES, 3.57 (1H, t,
9
1
2
8
J = 9 Hz) C –H, 3.72–3.78 (1H, m), DAHPES–CH(O)-
17
COCH
metrical isomers), 5.71 (1H, s) C
2
–, 4.43 and 4.46 (2H, s) @N–O–CH
2
COO (geo-
–H of testosterone. Mass
4
+
spectra (EI) m/z: 553 (M ).