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acetic acid. The reaction mixture was stirred at room
temperature for 28 h. Subsequently, it was diluted with
150 mL of distilled water and extracted with diethyl
ether (6 · 75 mL). The organic layers were pooled to-
gether and thoroughly washed with saturated aqueous
sodium bicarbonate solution until the organic layer ap-
peared light pink in colour. The organic layer was fur-
ther washed with 1 N aqueous sodium carbonate
solution followed by distilled water and dried over anhy-
drous sodium sulfate. Evaporation under vacuum affor-
ded 2.03 g (78.14%) of crude 6-keto-17b-estradiol
diacetate. The crude product was purified by silica-gel
column chromatography using ethyl acetate–petroleum
ether (1:9, v/v) as eluting solvent to give a white crystal-
line solid (II).
At the end of the reaction, the reaction mixture was
acidified at 0 ꢁC with 1 N HCl to pH 6. Concentration
of the reaction mixture in vacuo afforded 6a-amino-
17b-estradiol as a solid.
Melting point: 159–161 ꢁC. FT-IR (KBr, m cmÀ1): 3394
(–OH), 2927, 2861, 1670, 1617, 1459, 1222 and 1021
(–NH2). 1H NMR (CD3OD, d ppm): 0.77 (3H s,
C18-H3), 1.20–1.78 (11H m, C8, C9, C14-H and C11,
C12, C15, C16-H2), 2.2–2.4 (2H m, C7-HAHB), 3.63–
3.71 (1H dd, C6-Hb, J = 4.0, 8.1 Hz), 4.67–4.72 (1H m,
C17-H), 6.55–6.65 (1H m, Ar-H), 6.97–7.15 (1H m,
Ar-H), 7.32–7.36 (1H m, Ar-H).
The stereoselective amination of the 6-keto derivative to
the less sterically hindered 6a-amine via reductive ami-
nation was achieved using ammonium acetate and so-
Melting point: 173 ꢁC. FT-IR (Nujol, m cmÀ1): 1769
(C17-OCOCH3), 1730 (Ar-OCOCH3) 1677 (C6-CO).
1H NMR (CDCl3, d ppm): 0.83 (3H s, C18-H3), 1.20–
1.92 (11H m, C8, C9, C14-H and C11, C12, C15, C16-
H2), 2.07 (3H s, C17-OCOCH3), 2.31 (3H s, Ar-
OCOCH3), 2.53–2.55 (1H m, C7-HAHB), 2.73–2.79
(1H dd, C7-HAHB, J = 16.8, 3.5 Hz), 4.72 (1H q,
C17-H, J = 9.2, 7.8 Hz), 7.24–7.55 (3H m, Ar-H).
1
dium borohydride in methanol. The high resolution H
NMR of the unhydrolyzed as well as the hydrolyzed
6-amino derivative shows that the Hb proton exhibits
a one-proton dd with J values of the order of 4.4 Hz
and 10.7 Hz suggestive of an axial equatorial and diaxial
coupling respectively with the adjacent methylene pro-
tons at C7. Owing to the puckering of the B-ring in pres-
ence of the aromatic A-ring, the Hb proton at C6
acquires a pseudo-axial-orientation resulting in a slight
deviation from true axial equatorial and diaxial cou-
pling constants.
4.3.3. 6a-Amino-17b-estradiol diacetate (III). The con-
version of 6-keto-17b-estradiol diacetate (II) to 6a-ami-
no-17b-estradiol diacetate was achieved by reductive
amination using sodium cyanoborohydride as the reduc-
ing agent.18 The reaction was carried out by refluxing a
mixture of 6-keto-17b-estradiol diacetate (0.75 g,
0.002 M) and ammonium acetate (2.7 g, 0.035 M) in
60 mL of dry methanol under nitrogen atmosphere. So-
dium cyanoborohydride (0.66 g, 0.01 M) was added to
the refluxing mixture in five equal portions at an interval
of 30 min between two successive additions. The reflux-
ing was continued for 35 h. The solvent was evaporated
to obtain the crude product. The crude product was dis-
solved in 60 mL of 1 N HCl and extracted with ethyl
acetate to remove the unreacted 6-keto-17b-estradiol
diacetate. The aqueous layer was then neutralized with
1 N aqueous NaOH solution to yield the free amine
from the hydrochloride salt and re-extracted with ethyl
acetate. The organic layers were pooled together, dried
over anhydrous sodium sulfate and concentrated under
vacuum to obtain pure 6a-amino-17b-estradiol diacetate
(0.5 g, 66.49%).
4.3.5. p-NCS-benzyl-DOTA-6a-amino-17b-estradiol con-
jugate (V). 6a-Amino-17b-estradiol (0.01 g, 0.035 mM)
and p-NCS-benzyl-DOTA, 4HCl (24 mg, 0.035 mM)
were dissolved in 5 mL of distilled N,N-dimethyl form-
amide and the mixture was stirred at room temperature
for 3 h after adjusting the pH to 9 using 1 N NaOH
solution. The pH was maintained at 9 throughout the
reaction. The progress of the reaction was monitored
by TLC in 30% ammonia in methanol. On completion
of the reaction, the solvent was removed to yield the
crude product. The purification of the steroidal–BFCA
conjugate (V) was carried out by preparative TLC on sil-
ica gel plates using 10% ammonia in methanol. The
compound exhibited a Rf value of 0.2–0.3 in this solvent
system.
FT-IR (KBr, m cmÀ1): 3382 (–OH), 2932, 2867, 1670,
1619, 1463, 1233 and 1021 (–NH2). H NMR (CD3OD,
1
d ppm): 0.90 (3H s, C18-H3), 1.25–1.59 (11H m, C8, C9,
C14-H and C11, C12, C15, C16-H2), 2.0–2.05 (1H m, C7-
HAHB), 2.1–2.2 (1H m, C7-HAHB), 3.31 (8H s,
–CH2COOH), 3.52–3.58 (15H m, DOTA-CH2), 3.65–
3.68, (1H m, C6-H), 4.0 (2H br t, Ar-CH2-DOTA),
7.1–7.37 (6H m, Ar-H), 7.75–7.77 (1H m, Ar-H).
Melting point: 220 ꢁC. FT-IR (Nujol, m cmÀ1): 1769
(C17-OCOCH3), 1730 (Ar-OCOCH3), 2923, 2852,
1
1611, 1462, 1246 and 1021 (–NH2). H NMR (CD3OD,
d ppm): 0.77 (3H s, C18-H3), 1.20–1.85 (11H m, C8, C9,
C14-H and C11, C12, C15, C16-H2), 2.01 (3H s, C17-
OCOCH3), 2.2–2.3 (2H m, C7-HAHB), 2.50 (3H s, Ar-
OCOCH3), 3.64–3.7 (1H dd, C6-Hb, J = 4.4, 10.7 Hz),
4.65–4.70 (1H br t, C17-H), 6.69–6.85 (2H m, Ar-H),
7.17–7.21 (1H m, Ar-H).
The appearance of the 1H NMR signals in the conjugate
(V) corresponding to the p-NCS-benzyl-DOTA moiety
is indicative of the desired derivatization.
4.3.4. 6a-Amino-17b-estradiol (IV). The deacetylation of
6a-amino-17b-estradiol diacetate (III) was effected by
stirring a mixture of 6a-amino-17b-estradiol diacetate
(0.5 g, 0.0013 M) dissolved in 20 mL ethanol and 4 mL
of 40% aqueous NaOH for 24 h at room temperature.
4.4. Radiolabelling of p-NCS-benzyl-DOTA with 177Lu
For 177Lu labelling of p-NCS-benzyl-DOTA, a stock
solution of the ligand was prepared in 0.1 M ammonium
acetate buffer of pH ꢀ 5.5 with a concentration of