Synthesis and aromatase inhibitory activity of some new 16E-arylidenosteroids

Article abstract of DOI:10.1016/j.bioorg.2012.08.005

A new series of 16E-arylidene androstene derivatives has been synthesized and evaluated for aromatase inhibitory activity. The impact of various aryl substituents at 16 position of the steroid skeleton on aromatase inhibitory activity has been observed. The 16E-arylidenosteroids 6, 10 and 11 exhibited significant inhibition of the aromatase enzyme. 16-(4-Pyridylmethylene)-4- androstene-3,17-dione (6, IC₅₀: 5.2 μM) and 16-(benzo-[1,3]dioxol- 5-ylmethylene)androsta-1,4-diene-3,17-dione (11, IC₅₀: 6.4 μM) were found to be approximately five times more potent in comparison to aminoglutethimide.

Full text of DOI:10.1016/j.bioorg.2012.08.005

Bioorganic Chemistry 45 (2012) 36–40
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and aromatase inhibitory activity of some new 16E-arylidenosteroids
a
a
a
b
b
Ranju Bansal a, , Sridhar Thota , Nalin Karkra , Maninder Minu , Christina Zimmer , Rolf W. Hartmann
⇑
a University Institute of Pharmaceutical Sciences, Sector-14, Panjab University, Chandigarh 160 014, India
b Pharmaceutical and Medicinal Chemistry, Saarland University, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C2.3, D-66123 Saarbrücken, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 January 2012
Available online 12 September 2012
A new series of 16E-arylidene androstene derivatives has been synthesized and evaluated for aromatase
inhibitory activity. The impact of various aryl substituents at 16 position of the steroid skeleton on aro-
matase inhibitory activity has been observed. The 16E-arylidenosteroids 6, 10 and 11 exhibited signifi-
cant inhibition of the aromatase enzyme. 16-(4-Pyridylmethylene)-4-androstene-3,17-dione (6, IC50
5.2 M) and 16-(benzo-[1,3]dioxol-5-ylmethylene)androsta-1,4-diene-3,17-dione (11, IC50 6.4 M)
were found to be approximately five times more potent in comparison to aminoglutethimide.
:
Keywords:
l
:
l
16E-arylidenosteroids
Aromatase inhibitory activity
Breast cancer
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
approach for reducing tumor growth [4]. Over the past two dec-
ades, substantial effort has been directed towards developing po-
tent inhibitors of aromatase. There are two classes of AIs,
steroidal and nonsteroidal compounds, which cause potent estro-
gen suppression [5]. The non-steroidal AIs are mostly azole type
compounds such as the clinically used anastrozole and letrozole,
which compete with the substrate for binding to the enzyme active
site [6]. Among steroidal agents, formestane (1) was widely used
during the early 1990s, but it is not used nowadays because of
the need to administer it by intramuscular injection. Therefore,
the orally active exemestane (2) (Fig. 2) is the main steroidal inhib-
itor of contemporary importance [7]. These steroidal inhibitors mi-
mic the natural substrate androstenedione and are converted by
the enzyme to reactive intermediates, which bind irreversibly to
the enzyme active site, resulting in inactivation of aromatase [8].
Despite the success of the third-generation steroidal and non-
steroidal AIs, they still have some major side effects, such as mus-
culoskeletal effects (arthritis, arthralgia, and/or myalgia) and bone
toxicity [9]. For this reason, it is important to search for other po-
tent and specific molecules with lower side effects.
Among the various nonsteroidal aromatase inhibitors, a series of
pyridyl-substituted indanones, indans, and tetralins have been re-
ported and developed as potent and selective agents. Of these, inda-
none derivatives 2-(4-pyridylmethylene)-1-indanone (3) (Fig. 2)
and its corresponding saturated pyridyl–methyl analogue have
been reported to possess good selectivity and increased potency to-
ward aromatase as compared to aminoglutethimide [10]. A large
number of potent steroidal derivatives with substitution at position
16 have been described in the literature as potent cytotoxic agents
[11,12]. Recent work from our laboratory has also demonstrated
the effectiveness of 16E-arylidenosteroids as potential cytotoxic
and aromatase inhibitors [13,14]. These observations prompted us
to prepare and study some more new 16E-arylidenosteroids as
Aromatase is a cytochrome P-450 dependent enzyme that cata-
lyzes the aromatization of androgens to estrogens and hence plays
a key role in endocrine physiology and estrogen-dependent breast
cancer. It is comprised of a polypeptide chain of 503 amino-acid
residues and a prosthetic heme group at its active site [1,2]. The
enzyme complex is bound in the endoplasmic reticulum of the cell.
The androgens are converted to estrogens by aromatase via three
sequential oxidation steps as depicted in Fig. 1. The process utilizes
three moles each of oxygen and NADPH for the overall conversion.
The first step consists of a typical cytochrome P-450 hydroxylation
of the angular C-19 methyl group which stereospecifically forms
the 19-hydroxyandrostenedione. The second step is another ste-
reospecific hydroxylation of the C-19 methyl, in which the 19-
pro-R hydrogen is displaced to give a C-19,19-gem diol. The gem
diol then can readily dehydrate to produce 19-oxoandrostenedione
[1,2]. The first two steps entails the hydroxylation of C19-methyl
group, which is modulated by three amino acid residues, compris-
ing A306 and T310, and two catalytic water molecules, which acti-
vates the ferrous dioxygen to the hydroxylating Fe(IV)@O form.
This is followed by a H2b abstraction of the 2,3-enolization process
in the aromatization step that essentially involves a nucleophilic
attack on 2b hydrogen by A306 and T310 along with concerted
electrophilic attack on C3-keto oxygen by D309 to drive H2b
abstraction and 2,3-enolization. The final step consists of another
oxidation that results in the elimination of the 1b hydrogen as
water and incorporation of the C-19 into formic acid [3].
In breast cancer, intratumoral aromatase is the source for local
estrogen production and inhibition of this enzyme is an important
⇑
Corresponding author. Fax: +91 172 2543101.
E-mail address: ranju29in@yahoo.co.in (R. Bansal).
0045-2068/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2012.08.005

R. Bansal et al. / Bioorganic Chemistry 45 (2012) 36–40
37
O
O
O
OH
HO
HO
NaDPH
O2
NaDPH
O2
O
O
O
- H2O
O
O
O
NaDPH
O
2
,
,
- H2O - CHO2H
O
HO
Fig. 1. Aromatization of androgens to estrogens through aromatase enzyme.
aromatase inhibitors, in which structural features of various nonste-
roidal aromatase inhibitors have been incorporated.
1080. 1H NMR (CDCl3): 0.99 (s, 3H, 18-CH3), 1.07 (s, 3H, 19-CH3),
3.54 (m, 1H, 3 -H), 5.38 (s, 1H, 6-CH), 7.32 (s, 1H, vinylic-H),
a
7.37 (d, 2H, J = 6 Hz, 3-CH and 5-CH, aromatic), 8.66 (d, 2H,
J = 6 Hz, 2-CH and 6-CH, aromatic). MS: m/z 378 [M+]. Anal. Calcd
for C25H31NO2: C, 79.54; H, 8.27; N, 3.71. Found: C, 79.77; H,
8.11; N, 3.55.
2. Materials and methods
Melting points were determined on a Veego melting point
apparatus and are uncorrected. UV (wavelengths in nm) was re-
corded on Lambda 15 and IR (wave numbers in cmÀ1) spectra on
Perkin–Elmer spectrum RX 1, FT-IR spectrophotometer models
using KBr pellets. 1H NMR spectra were recorded on BrukerAC-
300F, 300 MHz using deuterated chloroform (CDCl3) or deuterated
dimethylsulfoxide (DMSO-d6) containing tetramethylsilane as
internal standard (chemical shifts in ppm). Mass spectrum was re-
corded on a Vg-11-250J 70 S model (VG Analytical Ltd., Manches-
ter, England). Elemental analyses were carried out on a Perkin–
Elmer-2400 model CHN analyzer. Plates for TLC were prepared
according to Stahl (E. Merck) using EtOAc as solvent (activated at
110 °C for 30 min) and were visualized by exposure to iodine va-
pors. Anhydrous sodium sulfate was used as drying agent. All sol-
vents were distilled prior to use according to standard procedures.
2.1.1.2. 16-(3,4,5-Trimethoxybenzylidene)-17-oxo-5-androsten-3-ol
(2). Yield: 74.1%. m.p. 65–68 °C. IRmmax (KBr): 3400, 2971, 1638,
1442, 1224, 1132, 1023. 1H NMR (CDCl3): 0.99 (s, 3H, 18-CH3),
1.07 (s, 3H, 19-CH3), 3.5 (m, 1H, 3
a
-H), 3.9 (s, 9H, 3Â AOCH3),
5.39 (s, 1H, J = 4 Hz, 6-CH), 6.78 (s, 2H, CH, aromatic) and 7.37 (s,
1H, vinylic-H). MS: m/z 467 [M+]. Anal. Calcd for C29H38O5: C,
74.65; H, 8.21. Found: C, 74.97; H, 7.99.
2.1.1.3. 16-(4-Methoxy-2-nitrobenzylidene)-17-oxo-5-androsten-3-ol
(3). Yield: 63.8%. m.p. 158–160 °C. IR
1720, 1640, 1540, 1280, 1020. 1H NMR (CDCl3): 0.96 (s, 3H, 18-
CH3), 1.07 (s, 3H, 19-CH3), 3.53 (m, 1H, 3 -H), 4.01 (s, 3H, AOCH3),
mmax (KBr): 3400, 2880, 2820,
a
5.40 (d, 1H, 6-CH), 7.13 (d, 1H, 6-CH, Jo = 8.77 Hz, aromatic), 7.35 (s,
1H, vinylic-H), 7.67 (dd, 1H, 5-CH, Jo = 8.7 Hz and Jm = 1.47 Hz, aro-
matic), 8.04 (d, 1H, 3-CH, Jm = 1.68 Hz, aromatic). MS: m/z 452 [M+].
Anal. Calcd for C27H33NO5: C, 71.82; H, 7.37; N, 3.10. Found: C,
71.63; H, 7.48; N, 3.01.
2.1. Chemical synthesis
2.1.1. General method for the preparation of compounds 1–5
A mixture of dehydroepiandrosterone (2.60 mmol), the appro-
priate aldehyde (4-pyridine carboxaldehyde/3,4,5-trimethoxy-
benzaldehyde/4-methoxy-2-nitrobenzaldehyde/benzo-[1,3]diox-
ol-5-carboxaldehyde/imidazole-2-carboxaldehyde) and potassium
hydroxide (1 g) in methanol (20 ml) was stirred at room tempera-
ture for 2 h until the reaction was completed (monitored by TLC).
Cold water was added to the reaction mixture and the precipitate
obtained was filtered, washed with water, dried and crystallized
from methanol to give the corresponding 16-arylideno steroid 1–5.
2.1.1.4. 16-(Benzo-[1,3]dioxol-5-ylmethylene)-17-oxo-5-androsten-3-
ol (4). Yield: 73.5%. m.p. 238–240 °C. IRmmax (KBr): 3400, 2920,
1710, 1460, 1260, 1040. 1H NMR (CDCl3): 0.97 (s, 3H, 18-CH3),
1.07 (s, 3H, 19-CH3), 3.52 (m, 1H, 3a-H), 5.41 (d, 1H, 6-CH), 6.02
(s, 2H, AOACH2AOA), 6.86 (dd, 1H, 6-CH, Jm = 2.03 Hz, Jo = 6.62 -
Hz), 7.06 (d, 2H, 2-CH and 5-CH, aromatic, Jo = 6.59 Hz), 7.31 (s,
1H, vinylic-H). MS: m/z 421 [M+]. Anal. Calcd for C27H32O4: C,
77.11; H, 7.67. Found: C, 76.93; H, 7.48.
2.1.1.1. 16-(4-Pyridylmethylene)-17-oxo-5-androsten-3-ol (1). Yield:
61.5%. m.p. 220–224 °C. IRmmax (KBr): 3420, 2945, 1720, 1600,
2.1.1.5. 16-[(1H-imidazol-2-yl) methylene]-17-oxo-5-androsten-3-ol
(5). Yield: 73.2%. m.p. 213–215 °C. IRmmax (KBr): 3150, 3063, 2930,
O
O
O
O
O
CH2
(2)
OH
N
(3)
(1)
Fig. 2. Steroidal and nonsteroidal aromatase inhibitors.

38
R. Bansal et al. / Bioorganic Chemistry 45 (2012) 36–40
1712, 1636, 1548, 1441, 1059, 760. 1H NMR (CDCl3): 0.93 (s, 3H,
18-CH3), 1.05 (s, 3H, 19-CH3), 3.37 (m, 1H, NH), 3.40 (m, 1H, 3
2.1.3. 16-(Benzo-[1,3]dioxol-5-ylmethylene)androsta-1,4-diene-3,17-
dione (11)
a
-
H), 5.35 (s, 1H, 6-CH), 7.18 (s, 2H, CH, imidazole-H), 7.22 (s, 1H,
vinylic-H). MS: m/z 367 [M+]. Anal. Calcd for C23H30N2O2: C,
75.37; H, 8.25; N, 7.64. Found: C, 75.27; H, 8.48; N, 7.40.
A
solution of 16-(benzo-[1,3]dioxol-5-ylmethlene)-4-
androsten-3,17-dione (9, 0.66 g, 1.57 mmol) and 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone (DDQ, 1.2 g) was heated under reflux
in benzene (100 ml) for 50 h. The progress of the reaction was
monitored by TLC. The reaction mixture was cooled to room tem-
perature and filtered. The filtrate was washed several times with
an aqueous solution of KOH (1%) and then with water to neutrality.
The organic layer was dried over sodium sulfate and removed un-
der vacuum to yield a oily residue, which was crystallized from
ether to afford 11.
Yield: 9.1%. m.p. 232–234 °C. IRmmax (KBr): 2920, 2820, 1710,
1660, 1240, 1030. 1H NMR (CDCl3): 1.02 (s, 3H, 18-CH3), 1.29 (s,
3H, 19-CH3), 6.03 (s, 2H, AOACH2AO), 6.10 (s, 1H, 4-CH), 6.25
(dd, 1H, 1-CH, Jcis = 10.16 Hz and J = 1.93 Hz), 6.86 (d, 1H, 6-CH, aro-
matic, Jo = 8.29 Hz), 7.06 (m, 3H, 2-CH and 5-CH, aromatic and 2-
CH), 7.37 (s, 1H, vinylic-H). MS: m/z 417 [M+]. Anal. Calcd for
C27H28O4: C, 77.86; H, 6.78. Found: C, 77.68; H, 6.53.
2.1.2. General procedure for the synthesis of compounds 6–10
The aldol products 1–5 (2 mmol) were dissolved in a mixture of
cyclohexanone (10 ml) and dry toluene (150 ml). Traces of mois-
ture were removed by azeotropic distillation. The distillation was
continued at a slow rate while adding a solution of aluminum iso-
propoxide (1 g) in dry toluene (15 ml) dropwise. The reaction mix-
ture was refluxed for 5 h and allowed to stand at room
temperature overnight. The slurry formed was filtered and the res-
idue was washed thoroughly with dry toluene. The combined fil-
trate and the washings were steam distilled to remove the
organic solvent. The solid obtained was filtered, washed with
water, dried and treated with diethyl ether and n-hexane to furnish
the corresponding 4-ene-3-keto steroids 6–10, respectively.
2.1.4. 16-(4-Pyridylmethylene)-17-oximino-5-androsten-3-ol (12)
A solution of sodium acetate trihydrate (0.6 g) and hydroxyl-
amine hydrochloride (0.6 g) in water (20 ml) was added to a
refluxing solution of 16-(4-pyridylmethylene)-17-oxo-5-andros-
ten-3b-ol (1, 0.3 g) in aldehyde-free ethanol (30 ml). After refluxing
for 4 h, the solution was concentrated under reduced pressure,
diluted with water and allowed to stand. The precipitate was
isolated by filtration, washed with water, dried and crystallized
from acetone to afford 12.
2.1.2.1. 16-(4-Pyridylmethylene)-4-androstene-3,17-dione (6). Yield:
81.6%. m.p. 112–118 °C. IRmmax (KBr): 2925, 1720, 1675, 1550,
1210, 910. 1H NMR (CDCl3): 1.02 (s, 3H, 18-CH3), 1.25 (s, 3H, 19-
CH3), 5.76 (s, 1H, 4-CH), 7.34 (s, 1H, vinylic-H), 7.35 (d, 2H,
J = 6 Hz, 3-CH and 5-CH, aromatic), 8.67 (br, 2H, 2-CH and 6-CH,
aromatic). MS: m/z 376 [M+]. Anal. Calcd for C25H29NO2: C, 79.99;
H, 7.73; N, 3.75. Found: C, 79.80; H, 7.97; N, 3.57.
Yield: 35.5%. m.p. 224–230 °C. IRmmax (KBr): 3250, 2930, 1600,
1440, 1370, 1040. 1H NMR (CDCl3): 1.05 (s, 3H, 19-CH3), 1.10 (s,
2.1.2.2. 16-(3,4,5-Trimethoxybenzylidene)-4-androstene-3,17-dione
(7). Yield: 50.6%. m.p. 218–222 °C. IRmmax (KBr): 2910, 1722,
1674, 1631, 1135, 1017, 918. 1H NMR (CDCl3): 1.02 (s, 3H, 18-
CH3), 1.25 (s, 3H, 19-CH3), 3.90 (s, 9H, 3Â AOCH3), 5.76 (s, 1H, 4-
CH), 6.78 (s, 2H, CH, aromatic), 7.38 (s, 1H, vinylic-H). MS: m/z
465 [M+]. Anal. Calcd for C29H38O5: C, 74.97; H, 7.81. Found: C,
74.68; H, 7.45.
3H, 18-CH3), 3.45 (m, 1H, 3a-H), 5.33 (d, 1H, 6-CH), 7.09 (s), 8.19
(s){1.5:1 ratio,1H, vinylic-H}, 7.28 (m, 2H, 3-CH and 5-CH, Jo = 9 Hz,
aromatic), 8.55 (m, 2-CH and 6-CH, Jo = 9 Hz, aromatic), 9.62 ppm
(br, 1H, @NAOH; disappeared on D2O exchange). MS: m/z 393
[M+]. Anal. Calcd for C25H32N2O2: C, 76.50; H, 8.22; N, 7.14. Found:
C, 76.81; H, 8.0; N, 6.96.
2.1.2.3. 16-(4-Methoxy-2-nitrobenzylidene)-4-androstene-3,17-dione
2.1.5. 16-(4-Pyridylmethylene)-3,17-dioximino-4-androstene (13)
To a refluxing solution of 16-(4-pyridylmethylene)-4-andro-
stene-3,17-dione (6, 0.5 g) in aldehyde free ethanol (50 ml), a hot
solution of sodium acetate trihydrate (1.0 g) and hydroxylamine
hydrochloride (1.0 g) in water (20 ml) was added. After refluxing
for 4 h, the solution was concentrated, diluted with water
(150 ml) and allowed to stand. The precipitate was filtered, washed
with water, dried and crystallized from methanol to afford 13.
Yield: 29.9%. m.p. 194–196 °C. IRmmax (KBr): 3320, 2960, 1600,
1440, 960. 1H NMR (CDCl3): 1.03 (s, 3H, 19-CH3), 1.11 (s, 3H, 18-
CH3), 5.77 (s, 1H, 4-CH), 7.06 (s), 8.16 (s) {0.5:1 ratio, 1H, vinylic-
H}, 7.26 (m, 2H, J = 9 Hz, 3-CH and 5-CH, aromatic), 8.54 (m, 2H,
Jo = 9 Hz, 2-CH and 6-CH, aromatic), 8.66 (br, 1H, @NAOH; disap-
peared on D2O exchange), 8.92 (br, 1H, @NAOH; disappeared on
D2O exchange). MS: m/z 406 [M+]. Anal. Calcd for C25H30N3O2: C,
74.22; H, 7.47; N, 10.38. Found: C, 74.54; H, 7.75; N, 10.12.
(8). Yield: 58.6%. m.p. 220–222 °C. IRmmax (KBr): 2980, 2920, 1730,
1660, 1550, 1280, 1020. 1H NMR (CDCl3): 1.04 (s, 3H, 18-CH3), 1.25
(s, 3H, 19-CH3), 3.99 (s, 3H, AOCH3), 5.77 (s, 1H, 4-CH), 7.13 (d, 1H,
6-CH, Jo = 8.76 Hz, aromatic), 7.37 (s, 1H, vinylic-H), 7.68 (dd, 1H, 5-
CH, Jo = 8.76 Hz and Jm = 2.17 Hz, aromatic), 8.05 (d, 1H, 3-CH,
Jm = 2.15 Hz, aromatic). MS: m/z 450 [M+]. Anal. Calcd for
C27H31NO5: C, 72.13; H, 6.95; N, 3.11. Found: C, 72.23; H, 7.18;
N, 3.04.
2.1.2.4.
16-(Benzo-[1,3]dioxol-5-ylmethylene)-4-androstene-3,17-
dione (9). Yield: 58.2%. m.p. 248–250 °C. IRmmax (KBr): 2980,
1720, 1660, 1260, 1040, 1020. 1H NMR (CDCl3): 1.01 (s, 3H, 18-
CH3), 1.25 (s, 3H, 19-CH3), 5.76 (s, 1H, 4-CH), 6.03 (s, 2H, AOACH2-
AOA), 6.87 (d, 1H, 6-CH, Jo = 8.69 Hz, aromatic), 7.06 (d, 2H, 2-CH
and 5-CH, Jo = 7.37 Hz, aromatic), 7.36 (d, 1H, vinylic-H). MS: m/z
419 [M+]. Anal. Calcd for C27H30O45: C, 77.48; H, 7.23. Found: C,
77.29; H, 7.38.
2.2. Aromatase inhibitory activity
2.2.1. Preparation of aromatase
2.1.2.5.
16-[(1H-imidazol-2-yl)-methylene]-17-oxo-4-androstene-
The enzyme was obtained from the microsomal fraction of
freshly delivered human term placental tissue according to the
procedure of Thompson and Siiteri [15]. The isolated microsomes
were suspended in the minimum volume of phosphate buffer
(0.05 M, pH 7.4, 20% glycerol). Additionally DTT (dithiothreitol,
10 mM) and EDTA (1 mM) were added to protect the enzyme from
degradation. The enzyme preparation was stored at À70 °C.
3,17-dione (10). Yield: 56.7%. m.p. 189–190 °C. IRmmax (KBr):
3184, 2942, 1712, 1641, 1442, 1094, 1023, 745. 1H NMR (CDCl3):
0.99 (s, 3H, 18-CH3), 1.24 (s, 3H, 19-CH3), 3.33 (dd, 1H, 15-CH),
5.75 (s, 1H, 4-CH), 7.19 (S, 2H, imidazole-H), 7.22 (s, 1H, vinylic-
H). MS: m/z 365 [M+]. Anal. Calcd for C23H28N2O2: C, 75.79; H,
7.74; N, 7.69. Found: C, 75.65; H, 8.01; N, 7.53.

R. Bansal et al. / Bioorganic Chemistry 45 (2012) 36–40
39
O
2.2.2. Inhibition of aromatase in vitro
The assay was performed similar to the described methods
[16,17] monitoring the enzyme activity by measuring the 3H2O
formed from [1b-3H]androstenedione during aromatization. Each
incubation tube contained 15 nM [1b-3H]androstenedione
a
(9)
O
(0.08 lCi), 485 nM unlabeled androstenedione, 2 mM NADP,
O
20 mM glucose-6-phosphate, 0.4 U of glucose-6-phosphate-dehy-
drogenase and inhibitor (in three different concentrations for
determining the IC50 value) in phosphate buffer (0.05 M, pH 7.4).
The test compound had been dissolved in DMSO and diluted with
buffer. The final DMSO concentration in the control and inhibitor
incubation was 2%. Each tube was preincubated for 5 min at
30 °C in a shaking water bath. Microsomal protein (0.1 mg) was
added to start the reaction. The total volume for each incubation
was 0.2 ml. The reaction was terminated by the addition of
O
(11)
Scheme 2. Synthesis of the compound 11. Reagents and reaction conditions: (a)
2,3-dichloro-5,6-dicyanobenzoquinone (DDQ).
it with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) for 5 h to af-
ford
16-(benzo-[1,3]dioxol-5-ylmethylene)-androsta-1,4-diene-
3,17-dione (11), which showed 1H NMR singlet at d 6.1 for 4-CH
and a doublet at 6.25 ppm for 1-CH of A ring of steroid. Oxidation
of 1 was affected with hydroxylamine hydrochloride–sodium ace-
tate in aldehyde-free ethanol to get the 17-oximino derivative 12.
Vinyl-H of 16-(4-pyridylmethylene) derivative 12 appeared as split
singlets at 7.09 and 8.19 in 1.5:1 area ratio. Dioxime derivative 13
was prepared by treating 16E-arylidenosteroid 6 with hydroxyl-
amine hydrochloride and sodium acetate trihydrate in refluxing
aldehyde free ehanol. Work up of the reaction mixture gave com-
pound 13, which showed nuclear magnetic resonance signals at
5.77 (s, 1H, 4-CH) and split singlets for vinyl-H of 13 at 7.06 (s)
and 8.16 (s) in 0.5:1 area ratio.
200 ll of a cold 1 mM HgCl2 solution. After addition of 200 ll of
an aqueous dextran-coated charcoal (DCC) suspension (2%), the
vials were shaken for 20 min and centrifuged at 1500g for 5 min
to separate the charcoal-adsorbed steroids. Aliquots of the super-
natant were assayed for 3H2O by counting in a scintillation mixture
in a 1209 Rackbeta Wallac liquid scintillation spectrometer (Phar-
macia LKB, Freiburg, Germany).
3. Results and discussion
3.1. Chemistry
The synthesis of 16E-arylidenoandrostene derivatives 1–13 has
been carried out as depicted in Schemes 1–3. Aldol condensation of
dehydroepiandrosterone with various aromatic aldehydes at room
temperature in alkaline medium afforded the corresponding 16E-
benzylidene steroids 1–5. The methine-bridged proton at C16 ap-
peared at ꢀd 7.3 ppm in the 1H NMR spectra of all these aldol prod-
ucts. The configuration at C16 with respect to the carbonyl at C17
has been assigned E on the basis of earlier reports from our labora-
tory [12]. In view of the literature reports that 3,17-diketo steroids
with higher degree of unsaturation in A-ring exhibit potent aroma-
tase inhibitory activity [18], Oppenauer oxidation of aldol products
3.2. Aromatase inhibitory activity
Newly synthesized
a,b-unsaturated 3-keto-16-arylideno ste-
roids 6–13 were screened for aromatase inhibitory activity as
described earlier [16]. The data is presented in Table 1. A
comparative plot of concentration–response curves of the active
compounds 6, 10, 11 and standard drug aminoglutethimide for
aromatase inhibition has been presented in Fig. 3. Of these com-
pounds, 3-keto-4-ene steroids 6 (IC50: 5.2
lM) and 10 (IC50:
22.7 M) with a heteroaromatic ring at 16 position exhibited mod-
l
erate inhibition of the enzyme in comparison to 16-arylidene ste-
1–5 was carried out to afford the corresponding
a,b-unsaturated-3-
roids 7–9, which displayed very weak inhibition of the enzyme
keto steroids 6–10. A singlet for the 4-CH proton at about d
5.76 ppm was observed in the 1H NMR spectra of all these
products.
Insertion of a double bond between C-1 and C-2 allows the ste-
roid to bind irrereversibly to the aromatase enzyme active site,
resulting in inactivation of aromatase [18]. Therefore, we at-
tempted to explore such modification in compound 9 by treating
even at a higher concentration of 36 lM. It is anticipated that the
pyridyl and imidazolyl substituted steroids interfere with steroid
hydroxylations by the binding of the sterically available N atom
with the heme Fe (III) iron of cytochrome P-450. However 16-pyr-
idyl substituted steroidal oxime 12 with a 3-hydroxy group and
3,17-dioximino derivative 13 displayed reduced binding affinity
for aromatase enzyme in comparison to the pyridyl substituted
O
O
O
Ar
Ar
a
b
HO
HO
O
(1-5)
DHA
(6-10)
NO2
OCH3
(3,8) ;
OCH3
(2,7) ;
N
(1,6) ;
OCH3
OCH3
H
N
O
O
(4,9) ;
(5,10) ;
N
Scheme 1. Synthesis of the compounds 1–10. Reagents and reaction conditions: (a) ArACHO, MeOH, KOH, RT; (b) Al(t-BuO)3, cyclohexanone, reflux, 5 h.

40
R. Bansal et al. / Bioorganic Chemistry 45 (2012) 36–40
b
(1)
(6)
a
a
N-OH
N-OH
HO
N
HO-N
N
(12)
(13)
Scheme 3. Synthesis of the compounds 12 and 13. Reagents and reaction conditions: (a) hydroxylamine HCl, sodium acetate trihydrate; (b) Al(t-BuO)3, cyclohexanone, reflux,
5 h.
Table 1
45% inhibition at 36 lM. This is also in accordance with earlier lit-
Aromatase inhibitory data of various compounds.
erature reports [18]. From the above study it may be concluded
that the presence of a heteroaromatic ring so that the nitrogen of
the heteroaromatic ring is able to complex with the heme–iron
of the enzyme along with 3 and 17 carbonyl groups and enhanced
A ring unsaturation in the steroidal nucleus are important struc-
tural features for effective binding to the aromatase enzyme com-
plex. Given the significance of azole grouping of many potent
aromatase inhibitors [3], introduction of an imidazolyl or pyridyl
ring at C-16 position together with C-3 keto and C-1 and 4 double
bond of the steroid skeleton seem a rational approach to yield po-
tent and specific enzyme inhibitors. With this design it may be
possible to achieve dual inhibition by producing substrate like
compounds which not only interact with the steroid binding site
of the enzyme, thus introducing high specificity, but also provide
a ligand to enzyme heme iron resulting in tight binding.
S. no.
Compound no.
Inhibition on CYP 19a
IC50 M)
RPb
5.7
(
l
1
2
3
4
5
6
7
8
6
7
8
5.2 1.5
24% inhibition 0.8 at 36
36% inhibition 0.1 at 36
45% inhibition 3.6 at 36
22.7 2.9
6.4 1.8
7% inhibition 3.5 at 36
l
l
l
M
M
M
9
10
11
12
13
1.3
4.6
lM
32% inhibition 1.8 at 36
lM
a
Mean value of two independent experiments SDabsolute
;
substrate [1b-
3H]androstenedione, 500 nM.
b
Relative potency = relative to aminoglutethimide (RP = 1; IC50 = 28.5 lM).
Acknowledgments
The authors are thankful to Department of Science and Technol-
ogy, India for providing financial assistance. The generous supply
of steroids by Cipla Ltd., India is gratefully acknowledged.
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Fig. 3. Concentration–response-curves of compounds 6, 10, 11 and aminogluteth-
imide for aromatase enzyme inhibition. The log (inhibitor) vs. response model
(Prism 5.0) was applied for nonlinear regression.
3-keto-4-ene counterpart 6. This indicates the importance of 3 and
17-carbonyl groups for binding of the molecule with active site of
the aromatase in addition to the heteroaromatic ring. On the other
side, 16-(benzo-[1,3]dioxol-5-ylmethylene)androstane derivative
11 with increased unsaturation in ring A, although lacks a hetero-
aromatic ring produced better aromatase inhibitory activity (IC50
:
6.4 M) in contrast to less oxidized analogue 9, which showed only
l