Synthesis of imidazole-derived steroidal hybrids as potent aromatase inhibitors

Article abstract of DOI:10.1007/s00044-012-0059-1

Imidazolyl substituted 16E-arylidenosteroidal derivatives have been synthesized and evaluated for aromatase inhibitory activity. The steroidal hybrids displayed moderate inhibition of the aromatase enzyme. The enzyme activity was monitored by measuring th

Full text of DOI:10.1007/s00044-012-0059-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:692–698
DOI 10.1007/s00044-012-0059-1
ORIGINAL RESEARCH
Synthesis of imidazole-derived steroidal hybrids as potent
aromatase inhibitors
•
Ranju Bansal Sheetal Guleria Sridhar Thota
•
•
•
Rolf W. Hartmann Christina Zimmer
Received: 18 August 2011 / Accepted: 5 April 2012 / Published online: 25 April 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Imidazolyl substituted 16E-arylidenosteroidal
derivatives have been synthesized and evaluated for aro-
matase inhibitory activity. The steroidal hybrids displayed
moderate inhibition of the aromatase enzyme. The enzyme
activity was monitored by measuring the tritiated H2O
released from [1b-3H] androstenedione during aromatiza-
tion. 16-[3-{3-(Imidazol-1-yl)propoxy}benzylidene]-4-andro-
stene-3,17-dione (10, IC50: 4.4 lM) was found to be seven
times more potent in comparison to standard drug
aminoglutethimide.
and prostate cancer (Gasi et al., 2001; Miller and Jackson,
2003). Over the past two decades, substantial efforts have
been directed toward developing potent inhibitors of aro-
matase. Clinical studies initially demonstrated that the
administration of the first generation inhibitor, aminoglu-
tethimide, caused regression of hormone-dependent breast
cancer in women. This served as the impetus for devel-
oping the second and third generation inhibitors, which
resulted in the availability of compounds that are much
more potent than aminoglutethimide (Brueggemeier et al.,
2005; Saberi et al., 2006). Among steroidal agents, for-
mestane (1) (Fig. 1) was used widely during the early
1990 s, but it is not used nowadays because of the need to
administer it by intramuscular injection. Therefore, the
orally active steroid exemestane (2) and non-steroidal
anastrozole (3) (Fig. 1) are the main aromatase inhibitors
of contemporary importance in these days (Lombardi 2002;
Brueggemeier et al., 2005).
Keywords 16E-Arylidenosteroids Á
Aromatase inhibitory activity Á Breast cancer
Introduction
Aromatase is a cytochrome P450 enzyme, which catalyzes
the conversion of androgens into estrogens in the last step
of estrogen biosynthesis (Chumsria et al., 2011). Com-
pounds that inhibit aromatase have potential applications in
the treatment of advanced estrogen-dependent tumors; such
as breast cancer, endometrial cancer, prostatic hyperplasia,
Taking into consideration the significance of azole
groupings of many specific and potent cytochrome P450
inhibitors including aromatase (Leze et al., 2006), we
thought it worthwhile to condense imidazole group with
androstane nucleus. 16-Substituted steroids have shown
diversified pharmacological activities and are of interest for
a medicinal chemist to develop new molecules (Numazawa
and Osawa 1981). Many medicinally active steroidal
derivatives with substitution at position 16 have already
been described in the literature (Vicker et al., 2006; Bansal
and Guleria, 2008). Recent study from our laboratory has
also demonstrated the effectiveness of 16E-arylidenoster-
oids as potential antitumor agents (Bansal and Guleria,
2008; Chattopadhaya et al., 2004). These observations
encouraged us to prepare and study some more new 16E-
arylidenosteroids possessing an imidazole group to obtain
potent aromatase inhibitors.
R. Bansal (&) Á S. Guleria Á S. Thota
University Institute of Pharmaceutical Sciences,
Panjab University, Sector-14, Chandigarh 160014, India
e-mail: ranju29in@yahoo.co.in
R. W. Hartmann Á C. Zimmer
Pharmaceutical and Medicinal Chemistry, Saarland University,
66041 Saarbru
¨cken, Germany
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Med Chem Res (2013) 22:692–698
693
Fig. 1 Structures of clinically
used steroidal and non-steroidal
aromatase inhibitors:
O
O
N
N
N
Formestane (1), Exemestane
(2), and Anastrozole (3)
O
O
OH
NC
CN
CH2
(1)
(2)
(3)
O
Materials and methods
CHO
Chemistry
O(CH2)3Cl
+
Melting points were determined on a Veego melting point
apparatus and are uncorrected. UV (wavelengths in nm)
HO
(4)
DHA
was recorded on Lambda 15, IR (wavenumbers in cm-1
)
a
spectra on Perkin-Elmer spectrum RX 1 FT-IR spectro-
photometer models using KBr pellets. 1H NMR spectra
were recorded on Bruker AC-300F, 300 MHz using deu-
terated chloroform (CDCl3) or deuterated dimethylsulfox-
ide (DMSO-d6) containing tetramethylsilane as internal
standard (chemical shifts in ppm). 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 vapors.
Anhydrous sodium sulfate was used as drying agent. All
the solvents were distilled before use according to standard
procedures.
O
O(CH2)3Cl
HO
(5)
b
O
N
O(CH2)3
N
The synthetic routes to the preparation of various new
steroidal derivatives have been outlined in Schemes 1, 2, 3
in accordance with the literature method (Bansal et al.,
2011b). Aldol condensation of DHA with substituted
benzaldehydes 4 and 11 afforded 16-benzylidene steroidal
derivatives 5 and 12, respectively. 16-Arylideno steroids 5
and 12 were then thermally fused with powdered imidazole
to afford the corresponding imidazolyl substituted products
6 and 13, which on Oppenauer oxidation in aluminum
isopropoxide–cyclohexanone–toluene system yielded the
corresponding 4-ene-3,17-dione derivatives 10 and 14.
Reduction of compound 6 afforded 3b,17b-diol derivative
7. Acetylation of the 7 and 6 using acetic anhydride
afforded 8 and 9, respectively.
HO
(6)
c
OH
N
O(CH2)3
N
(7)
HO
d
OCOCH3
The synthesis of substituted aldehydes 4 and 11 was
performed according to reported method (Bansal et al.,
2011a).
N
O(CH2)3
N
H3COCO
(8)
General method for the preparation of compounds 5, 12
Scheme 1 Synthesis of the compounds 4–8. Reagents and reaction
conditions: a MeOH, KOH, RT; b Imidazole, fusion 110–120 °C,
5 h; c NaBH4; d (CH3CO)2O/dry pyridine, steam bath, 2 h.
A mixture of dehydroepiandrosterone (0.75 g, 2.60 mmol),
appropriate substituted aldehyde (4, 11; oily residues
123

694
Med Chem Res (2013) 22:692–698
Scheme 2 Synthesis of
compounds 9 and 10. Reagents
and reaction conditions:
O
a (CH3CO)2O/dry pyridine,
steam bath, 2 h; b Al(t-BuO)3,
cyclohexanone, reflux, 5 h.
N
O(CH2)3
N
H3COCO
a
b
(9)
(6)
O
N
O(CH2)3
N
O
(10)
obtained from 1 g of respective aldehyde), and sodium
hydroxide (1 g) in methanol (20 ml) was stirred at room
temperature for 8 h, the reaction being 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 yield corresponding
16-arylideno steroids 5 and 12.
CHO
+
DHA
O(CH2)3Cl
(11)
a
16-[3-(3-Chloropropoxy)benzylidene]-17-oxo-5-
androsten-3b-ol (5)
O
Yield: 40.98 %; m.p. 145–147 °C; UVmax (MeOH):
292.0 nm (Log e 4.24); IR: 3419, 2932, 1715, 1628, 1580,
1445, 1375, 1257, 1215, 1163, 1054, 1008, 918, 873 cm-1
;
1H NMR (CDCl3): d 0.90 (s, 3H, 18-CH3), 1.00 (s, 3H,
19-CH3), 2.19 (m, 2H, –OCH2CH2CH2Cl), 3.40 (m, 1H,
3a-H), 3.67 (t, 2H, –CH2Cl), 4.06 (t, 2H, –OCH2–), 5.27 (d,
1H, 6-CH), 6.79 (d, 1H, Jo = 8.01 Hz, 4-CH, aromatic),
6.93 (s, 1H, 2-CH, aromatic), 7.02 (d, 1H, Jo = 7.55 Hz,
6-CH, aromatic), and 7.18–7.24 ppm (m, 2H, 5-CH, aro-
matic and vinylic-H, 16-arylidene). Anal. Calc. for
C29H37O3Cl: C: 74.26, H: 7.95; found; C: 74.64, H: 8.12.
HO
(12)
O(CH2)3Cl
b
O
(13)
HO
N
16-[4-(3-Chloropropoxy)benzylidene]-17-oxo-5-
androsten-3b-ol (12)
O(CH2)3
N
c
Yield: 55.37 %; m.p.111–113 °C; UVmax (MeOH):
317.2 nm (log e 5.45); IR: 3419, 2934, 1710, 1604, 1459,
O
1373, 1253, 1174, 1047, 830, 717 cm-1
;
1H NMR
(CDCl3): d 0.99 (s, 3H, 18-CH3), 1.10 (s, 3H, 19-CH3),
2.28 (m, 2H, –OCH2CH2CH2Cl), 3.60 (m, 1H, 3a-H), 3.78
(t, 2H, –CH2Cl), 4.19 (t, 2H, –OCH2–), 5.42 (d, 1H, 6-CH),
6.97 (d, 2H, Jo = 8.8 Hz, 3-CH and 5-CH, aromatic), 7.42
(s, 1H, vinylic-H, 16-arylidene), and 7.54 ppm (d, 2H,
Jo = 8.8 Hz, 2-CH and 6-CH, aromatic). Anal. Calc. for
C29H37O3 Cl: C: 74.26, H: 7.95; found: C: 74.35, H:
8.08 %.
O
N
(14)
O(CH2)3
N
Scheme 3 Synthesis of compounds 11–14. Reagents and reaction
conditions: a MeOH, KOH, RT; b Imidazole, fusion 110–120 °C,
5 h; c Al(t-BuO)3, cyclohexanone, reflux, 5 h.
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Med Chem Res (2013) 22:692–698
695
General method for the preparation of compounds 6, 13
and the washings were steam distilled until the removal of
organic solvent was affected. The solid obtained was fil-
tered, washed with water, dried, and treated with diethyl
ether and n-hexane to furnish the corresponding 4-ene-3-
keto steroids 10 and 14, respectively.
A mixture of benzylidene 5 or 12 (0.47 g, 1 mmol) and
powdered imidazole (0.68 g, 10 mmol, in excess) was
fused at 110–120 °C for 5 h. The completion of reaction
was monitored by TLC. The reaction was quenched with
cold water, and the solid obtained was filtered, washed with
water, dried, and crystallized from ethyl acetate to yield 6
and 13, respectively.
16-[3-{3-(Imidazol-1-yl)propoxy}benzylidene]-4-
androstene-3,17-dione (10)
Yield: 50.25 %; m.p. 81–83 °C; UVmax (MeOH):
239.6 nm (log e 4.34), 291.0 nm (log e 4.25); IR: 2937,
1717, 1663, 1622, 1576, 1448, 1256, 1226, 1179, 1162,
16-[3-{3-(Imidazol-1-yl)propoxy}benzylidene]-17-oxo-5-
androsten-3b-ol (6)
1
1080, 1026, 915, 864 cm-1; H NMR (CDCl3): d 1.02 (s,
Yield: 45.87 %; m.p. 211–212 °C. UVmax (MeOH):
290.4 nm (log e 4.25); IR: 3253, 2932, 2837, 1713, 1629,
1576, 1448, 1385, 1269, 1160, 1067, 919, 872 and
779 cm-1; 1H NMR (CDCl3): d 0.98 (s, 3H, 18-CH3), 1.07
(s, 3H, 19-CH3), 2.22 (m, 2H, OCH2CH2CH2N\), 3.49 (m,
1H, 3a-H), 3.92 (t, 2H, –CH2N\), 4.21 (t, 2H, OCH2–),
5.35 (s, 1H, 6-CH), 6.84 (dd, 1H, Jm = 2.17 Hz,
Jo = 8.17 Hz, 4-CH, aromatic) 6.88 (s, 1H, 5-CH, imid-
azole), 7.02 (m, 2H, 2-CH, aromatic and 4-CH, imidazole),
7.13 (d, 1H, Jo = 7.64 Hz, 6-CH, aromatic), 7.26–7.34 (m,
2H, 5-CH, aromatic and vinylic-H, 16-arylidene), and
7.44 ppm (s, 1H, 2-CH, imidazole). Anal. Calc. for
C32H40N2O3: C: 76.77, H: 8.05, N: 5.59; found; C: 76.85,
H: 8.19, N: 5.65.
3H, 18-CH3), 1.25 (s, 3H, 19-CH3), 2.26 (m, 2H,
–OCH2CH2CH2N\), 3.94 (t, 2H, –CH2N\), 4.23 (t, 2H,
–OCH2–), 5.76 (d, 1H, 4-CH), 6.90 (dd, 1H, Jm = 1.90 Hz,
Jo = 8.12 Hz, 4-CH, aromatic) 6.95 (s, 1H, 5-CH, imid-
azole), 7.02 (s, 1H, 2-CH, aromatic), 7.08 (s, 1H, 4-CH,
imidazole), 7.16 (d, 1H, Jo = 7.68 Hz, 6-CH, aromatic),
7.33 (t, 1H, Jo = 7.91 Hz, 5-CH, aromatic), 7.40 (s, 1H,
vinylic-H, 16-arylidene), and 7.57 ppm (s, 1H, 2-CH,
imidazole). Anal. Calc. for C32H38N2O3: C: 77.08, H: 7.68,
N: 5.62; found; C: 77.22, H: 7.49, N: 5.88.
16-[4-{3-(Imidazol-1-yl)propoxy}benzylidene]-4-
androstene-3,17-dione (14)
Yield: 23.2 %; m.p. 171–173 °C; UVmax (MeOH):
316.4 nm (log e 5.66); IR: 2933, 1713, 1665, 1606, 1508,
16-[4-{3-(Imidazol-1-yl)propoxy}benzylidene]-17-oxo-5-
androsten-3b-ol (13)
1
1458, 1251, 1179, 1091, 1027, 923, 828, 748 cm-1; H
NMR (CDCl3): d 1.01 (s, 3H, 18-CH3), 1.24 (s, 3H,
19-CH3), 2.25 (p, 2H, –OCH2CH2CH2N\), 3.94 (t, 2H,
–CH2N\), 4.21 (t, 2H, –OCH2–), 5.76 (s, 1H, 4-CH), 6.90
(d, 3H, Jo = 8.76 Hz, 3-CH, 5-CH, aromatic and 5-CH,
imidazole), 7.07 (s, 1H, 4-CH, imidazole), 7.41 (s, 1H,
vinylic-H, 16-arylidene), and 7.49 ppm (m, 3H, 2-CH,
6-CH, aromatic and 2-CH, imidazole). Anal. Calc. for
C32H38N2O3: C: 77.08, H: 7.68, N: 5.62; found; C: 77.24,
H: 7.66, N: 5.82 %.
Yield: 35.85 %; m.p. 201–203 °C. UVmax (MeOH):
317.6 nm (log e 4.47); IR: 3219, 2932, 1710, 1601, 1510,
1464, 1371, 1248, 1176, 1060, 830, and 742 cm-1 1H
.
NMR (CDCl3): d 0.98 (s, 3H, 18-CH3), 1.08 (s, 3H,
19-CH3), 2.24 (p, 2H, –OCH2CH2CH2N\), 3.51 (m, 1H,
3a-H), 3.94 (t, 2H, –CH2N\), 4.21 (t, 2H,–OCH2–), 5.40
(s, 1H, 6-CH), 6.92 (m, 3H, 5-CH, imidazole, 3-CH and
5-CH, aromatic), 7.06 (s, 1H, 2-CH, imidazole), 7.40 (s,
1H, vinylic-H, 16-arylidene), and 7.49 ppm (m, 3H, 4-CH,
imidazole, 2-CH and 6-CH, aromatic).
16-[3-{3-(Imidazol-1-yl)propoxy}benzylidene]-5-
androstene-3b,17b-diol (7)
General procedure for the synthesis of compounds 10, 14
To a stirred suspension of 6 (1 g, 2 mmol) in methanol
(100 ml) at room temperature, sodium borohydride (1.5 g)
was added in small fractions over a period of 2 h. The
reaction mixture was further stirred for 6 h. Solvent was
removed under reduced pressure, and cold water was
added. The precipitate obtained was filtered, washed with
water, dried, and crystallized from acetone to afford 7.
Yield: 50.02 %; m.p. 133–135 °C; UVmax (MeOH):
256.6 nm (log e 4.02); IR: 3373, 2936, 1601, 1438, 1366,
1252, 1161, 1050, 955 cm-1; 1H NMR (CDCl3): d 0.72 (s,
Compounds 6 or 13 (1 g, 2 mmol) was dissolved in a
mixture of cyclohexanone (10 ml) and dry toluene
(150 ml). Traces of moisture were removed by azeotropic
distillation. The distillation was continued at a slow rate
while adding a solution of aluminum isopropoxide (1 g) in
dry toluene (15 ml) dropwise. The reaction mixture was
refluxed for 5 h, and allowed to stand at room temperature
overnight. The slurry was filtered, and the residue was
washed thoroughly with dry toluene. The combined filtrate
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Med Chem Res (2013) 22:692–698
3H, 18-CH3), 1.04 (s, 3H, 19-CH3), 2.25 (m, 2H,
–OCH2CH2CH2N\), 3.54 (m, 1H, 3a-H), 3.91 (t, 2H,
–CH2N\), 4.06 (s, 1H, 17a-H), 4.21 (t, 2H, –OCH2–), 5.38
(d, 1H, 6-CH), 6.48 (d, 1H, vinylic-H, 16-arylidene), 6.73
(dd, 1H, Jm = 2.29 Hz, Jo = 8.03 Hz, 4-CH, aromatic),
6.87 (m, 4H, 2-CH, 6-CH, aromatic and 4-CH, 5-CH,
imidazole), 7.26 (m, 1H, 5-CH, aromatic) and 7.48 ppm (s,
1H, 2-CH, imidazole). Anal. Calc. for C32H42N2O3: C:
76.46, H: 8.42, N: 5.57; found; C: 76.42, H: 8.59, N: 5.41.
7.30 (m, 2H, 5-CH, aromatic and vinylic-H, 16-arylidene),
and 7.43 ppm (s, 1H, 2-CH, imidazole). Anal, Calc. for
C33H42N2O4: C: 74.69, H: 7.98, N: 5.28; found; C: 74.82,
H: 7.49, N: 5.31.
Biological activity
Preparation of aromatase
16-[3-{3-(Imidazol-1-yl)propoxy}benzylidene]-5-
androstene-3b,17b-diol diacetate (8)
The enzyme was obtained from the microsomal fraction of
freshly delivered human termplacental tissue according to
the procedure of Thompson and Siiteri (Thompson and
Siiteri 1974). The isolated microsomes were suspended in
the minimum volume of phosphate buffer (0.05 M, pH 7.4)
and stored at -30 °C as described. No loss of activity was
observed within 4 months.
A mixture of 7 (0.52 g, 1 mmol), acetic anhydride (1 ml),
and dry pyridine (2 ml) was heated in a steam bath for 2 h.
The reaction mixture was then poured into cold water and
basified with liquid ammonia. The precipitate formed was
collected by filtration, washed with water, dried, and
crystallized from n-hexane to get the compound 8.
Inhibition of aromatase in vitro
Yield: 34.48 %; m.p. 97–99 °C; UVmax (MeOH):
255.8 nm (log e 4.47); IR: 2939, 1732, 1600, 1440, 1372,
The assay was performed similar to the described methods
(Foster et al., 1983; Graves and Salhanick 1979; Hartmann
and Batzl 1986), monitoring enzyme activity by measuring
the 3H2O formed from [1b-3H]androstenedione during
aromatization. Each incubation tube contained 15 nM
[1b-3H]androstenedione (0.08 lCi), 485 nM unlabeled
androstenedione, 2 mM NADP, 20 mM glucose-6-phos-
phate, 0.4 units of glucose-6-phosphate-dehydrogenase,
and inhibitor (in at least three different concentrations for
determining the IC50 value) in phosphate buffer (0.05 M,
pH 7.4). The test compounds were 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 water bath. Micro-
somal protein was added to start the reaction (0.1 mg). The
total volume for each incubation was 0.2 ml. The reaction
was terminated by the addition of 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 1,5009g for
5 min to separate the charcoal-absorbed steroids. The
1
1241, 1162, 1034, 907.6 cm-1; H NMR (CDCl3): d 0.79
(s, 3H, 18-CH3), 1.05 (s, 3H, 19-CH3), 2.04 (s, 3H,
3b-OCOCH3), 2.21 (s, 3H, 17b-OCOCH3), 2.32 (m, 2H,
–OCH2CH2CH2N\), 3.91 (t, 2H, –CH2N\), 4.20 (t, 2H,
–OCH2–), 4.59 (m, 1H, 3a-H), 5.37 (s, 1H, 17a-H), 5.41 (d,
1H, 6-CH), 6.17 (s, 1H, vinylic-H, 16-arylidene), 6.73 (d,
1H, Jo = 7.96 Hz, 4-CH, aromatic), 6.84 (s, 1H, 2-CH,
aromatic), 6.93–7.06 (m, 3H, 6-CH, aromatic and 4-CH,
5-CH, imidazole), 7.26 (m, 1H, 5-CH, aromatic), and
7.49 ppm (s, 1H, 2-CH, imidazole); Anal. Calc. for
C36H46N2O5: C: 73.69, H: 7.90, N: 4.77; found; C: 73.51,
H: 7.99, N: 4.94.
16-[3-{3-(Imidazol-1-yl)propoxy}benzylidene]-17-oxo-5-
androsten-3b-yl-acetate (9)
A mixture of 6 (0.5 g, 1 mmol), acetic anhydride (1 ml),
and dry pyridine (2 ml) was heated in a steam bath for 2 h.
The reaction contents were then poured into iced water and
basified with liquid ammonia. The precipitate obtained was
filtered, washed with water, dried, and crystallized from
diethyl ether to afford 9.
3
supernatant was assayed for H2O by counting in a scin-
tillation mixture using a LKB-Wallac b-counter.
Yield: 47.16 %; m.p. 148–150 °C; UVmax (MeOH):
291.0 nm (log e 4.38); IR: 2940, 1728, 1631, 1575, 1508,
1
1443, 1374, 1254, 1163, 1030, 908, 873 cm-1; H NMR
Results and discussion
(CDCl3): d 0.98 (s, 3H, 18-CH3), 1.09 (s, 3H, 19-CH3),
2.03 (s, 3H, –OCOCH3), 2.23 (m, 2H, –OCH2CH2CH2N\),
3.91 (t, 2H, –CH2N\), 4.21 (t, 2H, –OCH2–), 4.58 (m, 1H,
3a-H), 5.41 (d, 1H, 6-CH), 6.83 (dd, 1H, Jm = 2.15 Hz,
Jo = 8.11 Hz, 4-CH, aromatic) 6.87 (s, 1H, 5-CH, imid-
azole), 6.97 (s, 1H, 2-CH, aromatic), 7.02 (s, 1H, 4-CH,
imidazole), 7.13 (d, 1H, Jo = 7.73 Hz, 6-CH, aromatic),
Chemistry
Base catalyzed aldol condensation of DHA with substituted
benzaldehydes 4 and 11 afforded 16-benzylidene steroidal
derivatives 5 and 12, respectively. The methine-bridged
proton of 16-arylidene resonated downfield at d 7.3 ppm in
123

Med Chem Res (2013) 22:692–698
697
RPb
1
the H NMR spectra. The configuration at C16 is assigned
Table 1 Aromatase inhibitory data of various compounds
E with respect to the keto group at C17 in analogy with
earlier reports (Thamotharan et al., 2004). 16-Arylideno
steroids 5 and 12 were then thermally fused with powdered
imidazole to afford the corresponding imidazolyl substi-
tuted products 6 and 13, which on Oppenauer oxidation in
aluminum isopropoxide-cyclohexanone-toluene system
yielded the corresponding 4-ene-3,17-dione derivatives 10
and 14. The appearance of a downfield 4-CH proton at d
5.76 ppm (in the proton NMR spectra), and the vibrational
bands at 1,713 and 1,665 cm-1 confirmed the formation of
10 and 14.
S. no.
Compound no.
Inhibition on CYP 19a
IC50 (lM)
1
2
3
4
5
6
7
8
a
6
34 % Inhibition at 36 lM
9.1
7
3.3
6.8
2.6
8
34 % Inhibition at 36 lM
38 % Inhibition at 36 lM
4.4
9
10
12
13
14
0.2 % Inhibition at 5 lM
4.1 % Inhibition at 5 lM
11.9
To study structure activity relationship in this particular
type of compounds, further modifications of steroidal
nucleus substituted with 3-hydroxybenzaldehyde deriva-
tives were carried out. Reduction of compound 6 using
sodium borohydride in methanol at room temperature
afforded 3b,17b-diol derivative 7. The broad vibrational
band for O–H stretching absorption at 3373.7 cm-1, and
1H NMR signals for 3a-H and 17a-H were observed at d
3.54 and 4.06 ppm, respectively. The vinylic proton at C16
was found at an upfield position (d 6.48 ppm), as compared
with its parent compound 6 (d 7.3 ppm). Acetylation of the
dihydroxy derivative 7 using acetic anhydride afforded
diacetoxy compound, 16-[3-{3-(imidazol-1-yl)propoxy}-
benzylidene]-5-androsten-3b,17b-diol diacetate (8). The
methine-bridged proton was found further at an upfield
value at d 6.17 ppm, as compared with its 3,17-diol
counterpart. Acetylation of 6 with acetic anhydride yielded
monoacetylated 3b-acetoxy derivative 9.
[1b-3H] androstenedione
b
Relative potency = relative to aminoglutethimide (RP = 1;
IC50 = 28.5 lM)
substituted analogue 14. Although it is expected that the
presence of imidazole group possessing a sterically avail-
able N will be able to enhance the interaction of steroid
with the active site of aromatase by complexing the Fe(III)
iron of cytochrome P450, it is observed from the biological
data that structural modifications of steroids might lead to
changes in 3D attachments of the compounds with the
enzyme site. This may result in significant loss in binding
affinity. Although shift in position of phenyl substituent
from meta to para position brought marginal changes in
aromatase inhibitory properties of the steroidal derivatives,
meta seems more promising. The ‘not so potent’ aromatase
inhibitory activity of the synthesized compounds discour-
aged us to further explore the structural modifications in
steroidal series with para phenyl substituents.
Aromatase inhibitory activity
Acknowledgments The authors are thankful to the Department of
Science and Technology and Council for Scientific and Industrial
Research, India for providing financial assistance. The generous
supply of steroids by Cipla Ltd., India is gratefully acknowledged.
The newly synthesized imidazolyl substituted 16-arylideno
steroids were screened for aromatase inhibitory activity by
measuring the tritiated water released during aromatiza-
tion. Table 1 displays the IC50 values for aromatase inhi-
bition and relative potency of these compounds in
comparison to standard drug aminoglutethimide. Despite
the presence of an imidazole group, the 3-hydroxy-17-keto
aldol products 6 and 13 displayed insignificant inhibition of
the enzyme irrespective of the meta or para substitution of
the side chain. Although hydroxylation at 3 and 17 posi-
tions in case of derivative 7 resulted in moderate inhibition
of the enzyme with IC50 = 9.1 lM, the presence of acet-
oxy groups in compounds 8 and 9 resulted in significant
loss of aromatase inhibitory activity. Increase in oxidation
of A ring as in case of 4-ene-3-keto steroids 10 (IC50:
4.4 lM) and 14 (IC50 = 11.9 lM) improved upon the
binding affinity of steroids with aromatase enzyme. Imid-
azolyl substituted benzylidene derivative 10 with side
chain at meta position exhibited *2.5 times more binding
affinity for aromatase enzyme, as compared to its para
References
Bansal R, Guleria S (2008) Synthesis of 16E-[3-methoxy-4-(2-
aminoethoxy) benzylidene androstene derivatives as potent
cytotoxic agents. Steroids 73:1391–1399
Bansal R, Narang G, Zimmer C, Hartmann RW (2011a) Synthesis of
some imidazolyl-substituted 2-benzylidene indanone derivatives
as potent aromatase inhibitors for breast cancer therapy. Med
Chem Res 20:661–669
Bansal R, Guleria S, Thota S, Hartmann RW, Zimmer C (2011b)
Synthesis and biologicalevaluation of 16E-arylidenosteroids as
cytotoxic and anti-aromatase agents. Chem Pharm Bull 59:
327–331
Brueggemeier RW, Hackett JC, Diaz-Cruz ES (2005) Aromatase
inhibitors in the treatment of breast cancer. Endo Rev 26:
331–345
Chattopadhaya R, Jindal DP, Maninder M, Gupta R (2004) Synthesis
and cytotoxic studies of hydroximino derivatives of some 16E-
arylidenosteroids. Arzneim Forsch/Drug Res 54:551–556
123

698
Med Chem Res (2013) 22:692–698
Chumsria S, Howesb T, Baoa T, Sabnisc G, Brodiec A (2011)
Aromatase, aromatase inhibitors, and breast cancer. J Steroid
Biochem Mol Biol 125:13–22
Numazawa M, Osawa Y (1981) Synthesis of 16a-bromoacetoxy
androgens
potential affinity labels of human placental aromatase. Steroids
and
17b-bromoacetylamino-4-androsten-3-one:
Foster AB, Jarman M, Leung S, Rowlands MG, Taylor GN (1983)
Analogues of aminoglutethimide: selective inhibition of choles-
terol side-chain cleavage. J Med Chem 26:50–54
Gasi KMP, Stankovic SM, Csanadi JJ, Djurendic EA, Sakac MN,
Mijacevic LM, Arcson ON, Stojanovic SZ, Andric S, Gabor DM,
Kovacevic R (2001) New D -modified androstane derivatives as
aromatase inhibitors. Steroids 66:645–653
Graves PE, Salhanick HA (1979) Stereoselective inhibition of
aromatase by enantiomers of aminoglutethimide. Endocrinology
105:52–57
38:149–159
Saberi MR, Vinh TK, Yee SW, Griffiths BJN, Evans PJ, Simons C
(2006) Potent CYP19 (aromatase) 1-[(benzofuran-2-yl)(phenyl-
methyl)pyridine, -imidazole, and -triazole inhibitors: synthesis
and biological evaluation. J Med Chem 49:1016–1022
Thamotharan S, Parthasarathi V, Gupta R, Guleria S, Jindal DP, Linden
A (2004) Two androst-5-ene derivatives: 16-[4-(3-chloropropoxy)-
3-methoxybenzylidene]-17-oxoandrost-5-en-3b-ol and 16-[3-met-
hoxy-4-(2-pyrrolidin-1-ylethoxy)benzylidene]-3b-pyrrolidinoandrost-
5-en-17b-ol monohydrate. Acta Cryst C 60:o75–o784
Hartmann RW, Batzl C (1986) Aromatase inhibitors. Synthesis and
evaluation of mammary tumor inhibiting activity of 3-alkylated
Thompson EA, Siiteri PK (1974) Utilization of oxygen and reduced
nicotinamide adenine dinucleotide phosphate by human placen-
tal microsomes during aromatization of androstenedione. J Biol
Chem 249:5364–5372
3-(4-aminophenyl)piperidine-2,6- diones.
1362–1369
J Med Chem 29:
Leze MP, Borgne ML, Pinson P, Palusczak A, Duflos M, Baut GL,
Hartmann RW (2006) Synthesis and biological evaluation of
5-[(aryl)(1H-imidazol-1-yl)methyl]-1H- indoles: potent and
selective aromatase inhibitors. Bioorg Med Chem Lett 16:
1134–1137
Vicker N, Lawrence HR, Allan GM, Bubert C, Smith A, Tutill HJ,
Purohit A, Day JM, Mahon MF, Reed MJ, Potter BV (2006)
Focused libraries of 16-substituted estrone derivatives and
modified e-ring steroids: inhibitors of 17-beta-hydroxysteroid
dehydrogenase type 1. Chem Med Chem 4:464–481
Lombardi P (2002) Exemestane, a new steroidal aromatase inhibitors
of clinical relevance. Biochim Biophys Acta 1587:326–337
Miller WR, Jackson J (2003) The therapeutic potential of aromatase
inhibitors. Expert Opin Investig Drugs 12:337–352
123