Design, synthesis and evaluation of novel 16-imidazolyl substituted steroidal derivatives possessing potent diversified pharmacological properties

Article abstract of DOI:10.1016/j.steroids.2012.02.005

As a part of our investigations into the structural-activity relationship studies of a novel class of medicinally active 16-substituted steroids, several new 16-imidazolyl substituted steroidal derivatives have been synthesized and pharmacologically evaluated in the current study. The new steroidal analogues 5, 6, 8, 9, 11 and 12 exhibited moderate cytotoxic effects in sixty cancer cell lines derived from nine cancers types. The imidazolyl substituted steroidal derivatives 6 (DPJ-RG-1241) and 7 (RB-401) were obtained as the powerful inhibitors of aromatase with IC₅₀ = 0.18 μM and IC₅₀ = 0.168 μM, respectively, approximately 1.2 and 1.4 times more potent in comparison to standard drug exemestane. The bis-quaternary steroids 13 and 14 displayed potent skeletal muscle relaxant properties. An affinity constant of 0.007 μM was observed for compound 14 on frog rectus abdominis muscle preparation and 13 displayed a very high anticholinesterase activity K _i = 25 nM, approximately 115-fold higher in comparison to standard drug galanthamine (K_i = 2.9 μM).

Full text of DOI:10.1016/j.steroids.2012.02.005

Steroids 77 (2012) 621–629
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Design, synthesis and evaluation of novel 16-imidazolyl substituted steroidal
derivatives possessing potent diversified pharmacological properties
Ranju Bansal a, , Sheetal Guleria , Sridhar Thota , Subhash L. Bodhankar , Moreshwar R. Patwardhan ,
⇑
a
a
b
b
c
c
d
Christina Zimmer , Rolf W. Hartmann , Alan L. Harvey
a
University Institute of Pharmaceutical Sciences, Sector-14, Panjab University, Chandigarh 160014, India
b
c
Department of Pharmacology, Bharti Vidyapeeth Deemed University, Poona College of Pharmacy, Erandawane, Pune 411038, India
Pharmaceutical and Medicinal Chemistry, Saarland University and Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C2.3, D-66123 Saarbrücken, Germany
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0NR, United Kingdom
d
a r t i c l e i n f o
a b s t r a c t
Article history:
As a part of our investigations into the structural–activity relationship studies of a novel class of medic-
inally active 16-substituted steroids, several new 16-imidazolyl substituted steroidal derivatives have
been synthesized and pharmacologically evaluated in the current study. The new steroidal analogues
Received 20 November 2011
Received in revised form 20 January 2012
Accepted 6 February 2012
5
, 6, 8, 9, 11 and 12 exhibited moderate cytotoxic effects in sixty cancer cell lines derived from nine can-
cers types. The imidazolyl substituted steroidal derivatives 6 (DPJ-RG-1241) and 7 (RB-401) were
obtained as the powerful inhibitors of aromatase with IC50 = 0.18 M and IC50 = 0.168 M, respectively,
Available online 15 February 2012
l
l
Keywords:
approximately 1.2 and 1.4 times more potent in comparison to standard drug exemestane. The bis-
quaternary steroids 13 and 14 displayed potent skeletal muscle relaxant properties. An affinity constant
1
6-Imidazolyl steroids
Aromatase inhibitors
Bis-quaternary steroids
Skeletal muscle relaxants
Anticholinesterases
of 0.007
played a very high anticholinesterase activity K
to standard drug galanthamine (K = 2.9 M).
lM was observed for compound 14 on frog rectus abdominis muscle preparation and 13 dis-
i
= 25 nM, approximately 115-fold higher in comparison
i
l
Ó 2012 Elsevier Inc. All rights reserved.
1
. Introduction
receptor antagonists, e.g., RU-5135 [2], aromatase inhibitors such
as formestane and exemestane [3], anticancer agents like 2-
methoxyestradiol [4] and neuromuscular junction blocking agents
like pipecuronium [5].
Heterosteroids have been accredited with a great amount of
attention over the years by medicinal chemists for drug discovery.
The interesting structural and stereochemical features of the ste-
roid nucleus provide additional fascination to the researchers and
thereby alterations in the steroidal skeleton have been envisaged
to discover new chemical entities with a potential to afford some
promising drugs of the future. The incorporation of a heterocyclic
ring or a heteroatom in the steroid backbone affects the chemical
properties of a steroid and often results in useful alterations in
its biological activities [1,2]. The products obtained by introduction
of heteroatom in the steroid nucleus are called nuclear heteroster-
oids. When heteroatom(s) forms a part of fused ring system, at-
tached group or a side chain of the steroid nucleus then the
products are known as extranuclear heterosteroids. Heterosteroids
encompass a wide range of compounds with varied biological
16-Substituted steroids have shown diversified pharmacological
activities and are of interest for a medicinal chemist to develop new
molecules. Many potent steroidal derivatives with substitution at
position 16 have been described in the literature [6,7]. Some inter-
esting 16E-arylidenosteroids have recently been reported from our
laboratory as strong in vitro inhibitors of the growth of many types
of human tumor cells [8–10]. These findings motivated us to con-
tinue the exploration of biological properties of 16-substituted ste-
roidal derivatives by designing and synthesizing new analogues
with suitable structural modifications. This paper embodies the
synthesis, study and biological evaluation of newer 16-imidazolyl
substituted steroids for their cytotoxic, aromatase inhibitory, NMJ
blocking and acetylcholinesterase inhibitory activities.
activities, e.g., 5
a
-reductase inhibitors like finasteride [2], GABA
Development of hybrid structures, in which pharmacologically
crucial structural elements from two molecules are combined to
produce a non-identical twin drug, is a rational approach to obtain
therapeutically useful molecules. The main focus of the current
study is to design and develop synthetic strategies to produce new
chemical hybrids of steroidal aromatase inhibitors such as formes-
tane, exemestane and non-steroidal aromatase inhibitors, e.g.,
Abbreviations: DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; SRB, sulforho-
damine B; DCC, dextran-coated charcoal; AChE, acetylcholinesterase; AD, Alzhei-
mer’s disease.
⇑
Corresponding author. Tel.: +91 172 2541142; fax: +91 172 2543101.
E-mail address: ranju29in@yahoo.co.in (R. Bansal).
0
039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2012.02.005

6
22
R. Bansal et al. / Steroids 77 (2012) 621–629
fadrozole (Fig. 1). These compounds may show high specificity and
increased potency as aromatase inhibitors. In view of the signifi-
cance of azole moiety to inhibit P450 enzyme inhibitors including
aromatase [11,12], we hypothesized that introducing imidazole
moiety to the androstane nucleus might yield specific and potent
P450 inhibitors. With this design, it may be possible to produce sub-
strate like chemical entities, which not only interact with the steroid
binding site of the enzyme, thus introducing high specificity, but
also provide a ligand to the enzyme heme iron resulting in tight
binding [13]. Literature survey further indicates that structural
modifications in steroidal A and D ring brings out noticeable changes
in aromatase inhibitory potential of steroidal molecules and pro-
vides potent, easily obtainable and structurally simple aromatase
inhibitors [7,14,15]. Therefore, as performed with non-steroidal
inhibitors [16–18], imidazolyl-substituted D-ring modified steroi-
dal derivatives containing a suitably positioned heteroatom capable
of binding to cytochrome P450 enzymes have been synthesized.
Neuromuscular junction blocking agents act on cholinergic
receptors on the skeletal muscle endplate to produce muscle paral-
ysis. These agents are clinically used to provide adequate skeletal
neuromuscular relaxation during surgery, controlled respiration
and orthopedic manipulation. These agents are used to induce
safer anesthesia, decrease the severity of muscle contraction dur-
ing electroconvulsive treatment and may also be used in the man-
agement of tetanus, in various spastic disorders and in diagnosis of
myasthenia gravis [2,5].
Quaternary ammonium steroids represent an important class of
skeletal muscle relaxants [5]. These are non-depolarising type NMJ
blockers and act as competitive antagonists at the acetylcholine
receptors. The block can be reversed by acetylcholinesterase inhib-
itors due to increase in concentration of acetylcholine at the junc-
tion by inhibiting the enzyme acetylcholinesterase. Most of the
acetylcholinesterase inhibitors are also quaternary ammonium
compounds [19]. Interonium distance between the two quaternary
centres in steroids is crucial for NMJ blocking activity. It has also
been observed from our previous studies that a variation in the
interonium distance, although increased, resulting from the built
in flexibility about the single bond on the moieties linked to ring
D of a bisquaternary steroid is not desirable for favourable NMJ
blocking effects [19]. Pipecuronium bromide with an interonium
distance of 16.07 Å produces less cardiovascular side effects in
comparison to pancuronium bromide (11.00 Å) (Fig. 2) [20,21],
however an increase in onset of action was observed. A value of
about 10–14 Å is considered as optimal interonium distance for
skeletal muscle relaxant effects. Taking a note of this observation,
some biquaternary steroids with a little extended, but fixed intero-
nium distance has also been prepared and evaluated for NMJ
blocking and anticholinesterase activity in the current study.
2. Experimental section
2.1. General
Melting points were determined on a Veego melting point
À1
apparatus and are uncorrected. IR (wave numbers in cm ) spectra
on Perkin-Elmer spectrum RX 1, FT-IR spectrophotometer models
1
using KBr pellets. H NMR spectra were recorded on Bruker AC-
3
300F, 300 MHz using deuterated-chloroform (CDCl ) or deuterated
dimethylsulfoxide (DMSO-d containing tetramethylsilane as
6
)
internal standard (chemical shifts in d, 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 visual-
ized by exposure to iodine vapors. Anhydrous sodium sulphate was
used as a drying agent. All solvents were distilled prior to use
according to standard procedures. All target compounds possessed
a purity of P95% as verified by elemental analyses by comparison
with the theoretical values.
16a-Bromo-17-oxo-5-androsten-3b-ol (2) was synthesized
according to reported procedure [22].
2
.1.1. Preparation of 16
-Bromo-17-oxo-5-androsten-3b-ol (2, 1 g, 2.72 mmol) was
dissolved in a mixture of cyclohexanone (10 mL) and dry toluene
100 mL). Traces of moisture were removed by azeotropic distilla-
a/b-bromo-4-androstene-3,17-dione (3)
16a
(
tion of toluene. The distillation was continued at a slow rate while
adding a solution of aluminium isopropoxide (1 g) in dry toluene
O
O
(
20 mL) dropwise. The reaction mixture was refluxed for 1 h and
N
N
allowed to stand at room temperature overnight. The slurry was
filtered and the residue was washed with dry toluene. The com-
bined filtrate and the washings were steam distilled until the com-
plete removal of organic solvents was affected. The solid obtained
was filtered, washed with water, dried and crystallized from a mix-
ture of acetone and n-hexane to obtain 3. Yield 60%, mp 128–
130 °C, IR (KBr): 2930.0, 1748.5, 1667.2, 1612.1, 1453.5, 1228.6,
O
O
OH
CH2
CN
Formestane
Exemestane
Fadrozole
À1
1
1
017.9 cm
.
3
H NMR (CDCl ): d 5.76 ppm (s, 1H, 4-CH), 4.56 (d)
Fig. 1. Structures of steroidal and non-steroidal aromatase inhibitors used in the
and 4.12 (t) (4:6 area ratio, 1H, 16b-H and 16a-H), 1.22 (s, 3H,
clinical practice.
1
9-CH
3 3
), 1.13 (s, 3H, 18-CH ).
H3C
O
H C
3
O
O
O
H3C
+
N
+
CH3
CH3
_
+
H3C
N
N
+
N
N
O
N
H3C
H3C
_
2Br
O
H
2Br
H
H3C
O
H3C
O
Pipecuronium bromide
Pancuronium bromide
Fig. 2. Structures of neuromuscular junction blockers used in the clinical practice.

R. Bansal et al. / Steroids 77 (2012) 621–629
623
O
O
Br
a
HO
HO
1
2
d
e
b
O
Br
O
O
Br
N
c
N
O
O
3
4
HO
5
d
b
d
O
O
N
f
N
N
N
c
O
O
7
OH
6
N
OCOCH3
N
N
N
g
HO
H COCO
3
8
9
Scheme 1. Synthesis of compounds 2–9. Reagents and conditions: (a) Cupric bromide, Dry methanol and Dry benzene; (b) Cyclohexanone, Toluene, Aluminum isopropoxide;
c) DDQ, Dioxane; 50 h; (d) Thermal fusion, imidazole; (e) DDQ, Dioxane; 4 days; (f) NaBH /Methanol; and (g) Acetic anhydride in dry pyridine.
(
4
2
.1.2. 16
A mixture of 16
.68 mmol), 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ, 0.5 g)
a
/b-Bromoandrosta-1,4-diene-3,17-dione (4)
J
cis = 10.23 Hz, 1-CH), 6.09 (s, 1H, 4-CH), 4.10 (t, 1H, 16
a-H), 1.26
a
/b-bromo-4-androstene-3,17-dione (3, 0.25 g,
(s, 3H, 19-CH ), 1.17 (s, 3H, 18-CH ).
3
3
0
was heated under reflux in dry dioxane (50 mL) for 50 h. The pro-
gress of the reaction was monitored by TLC. The reaction mixture
was cooled at room temperature and filtered. Chloroform (50 mL)
was added to the filtrate and washed with 1% aqueous solution
of potassium hydroxide (2 Â 25 mL) and then with water to neu-
trality. The organic layer was dried and removed under vacuum
to yield an oily residue, which revealed two spots on TLC and were
separated by fractional crystallization. The oily residue was re-
fluxed with diethyl ether, filtered and filtrate was concentrated
2.1.3. General procedure for the synthesis of compounds 5–7
A finely triturated mixture of requisite bromo steroid 2–4 (1 g,
2.74 mmol) and powdered imidazole (1.5 g, 10 mmol) was heated
at 130–135 °C for 1 h. The reaction mixture was cooled to room
temperature and cold water was added to it. The solid obtained
was filtered, washed with water, dried and crystallized from a mix-
ture of acetone and n-hexane to afford 5–7, respectively.
2
1
.1.3.1. 16b-(Imidazol-1-yl)-17-oxo-5-androsten-3b-ol (5) (DPJ-RG-
240). Yield 51%, mp 249–251 °C, IR (KBr): 3303.8, 2943.0, 1744.8,
to afford crystals of 16a-isomer 4a (30%). The residue left was crys-
tallized from a mixture of petroleum ether and diethyl ether to ob-
tain 16b-isomer 4b (70%).
1
9
7
499.5, 1456.1, 1374.7, 1318.3, 1243.9, 1065.8, 1041.4 and
À1 1
14.2 cm . H NMR (CDCl
.12 (s, 1H, 4-CH, imidazole), 6.95 (s, 1H, 5-CH, imidazole), 5.39
-H), 3.55 (m, 1H, 3 -H),
1.07 (s, 3H, 19-CH ), 1.02 (s, 3H, 18-CH ). Calcd for C22H30N O :
3
): d 7.70 ppm (s, 1H, 2-CH, imidazole),
2
(
J
(
.1.2.1. 16
CDCl ): d 7.04 ppm (d, 1H, Jcis = 10.15 Hz, 2-CH), 6.25 (d, 1H,
cis = 10.14 Hz, 1-CH), 6.10 (s, 1H, 4-CH), 4.54 (t, 1H, 16b-H), 1.26
s, 3H, 19-CH ), 0.99 (s, 3H, 18-CH ).
a
-Bromoandrosta-1,4-diene-3,17-dione (4a). 1
H
NMR
(d, 1H, J = 4.9 Hz, 6-CH), 4.47 (m, 1H, 16
a
a
3
3
3
2 2
C, 74.54; H, 8.53; N, 7.90. Found: C, 74.29; H, 8.84; N, 7.73.
3
3
2
.1.3.2. 16b-(Imidazol-1-yl)-4-androstene-3,17-dione (6) (DPJ-RG-
2
(
.1.2.2. 16b-Bromoandrosta-1,4-diene-3,17-dione (4b). 1
CDCl ): d 7.04 ppm (d, 1H, Jcis = 10.15 Hz, 2-CH), 6.23 (d, 1H,
H
NMR
1241). Yield 41%, mp 179–181 °C, IR (KBr): 2947.6, 1748.7,
1667.5, 1497.7, 1456.0, 1229.8, 1108.9, and 1045.0 cm . H NMR
À1
1
3

6
24
R. Bansal et al. / Steroids 77 (2012) 621–629
(
CDCl
imidazole), 6.95 (s, 1H, 5-CH, imidazole), 5.76 (s, 1H, 4-CH), 4.46
m, 1H, 16 -H), 1.24 (s, 3H, 19-CH ), 1.06 (s, 3H, 18-CH ). Calcd
for C22 : C, 74.97; H, 8.01; N, 7.95. Found: C, 74.71; H,
.28; N, 8.12.
3
): d 7.66 ppm (s, 1H, 2-CH, imidazole), 7.09 (s, 1H, 4-CH,
(KBr): 2937.5, 1732.9, 1494.1, 1435.5, 1371.8, 1250.1, 1072.9,
À1
1
1037.7 and 906.0 cm
CH, imidazole), 7.02 (s, 1H, 4-CH, imidazole), 6.89 (s, 1H, 5-CH,
imidazole), 5.39 (d, 1H, J = 4.89 Hz, 6-CH), 4.79 (m, 2H, 16 -H
and 17 -H), 4.61 (m, 1H, 3 -H), 2.04 (s, 3H, 3b-OCOCH ), 1.74 (s,
H, 17b-OCOCH ), 1.07 (s, 3H, 19-CH ),1.02 (s, 3H, 18-CH ). Calcd
for C26 : C, 70.88; H, 8.24; N, 6.36. Found: C, 70.59; H,
8.45; N, 6.49.
3
. H NMR (CDCl ): d 7.48 ppm (s, 1H, 2-
(
a
3
3
H
28
N
2
O
2
a
8
a
a
3
3
3
3
3
2
4
1
.1.3.3. 16b-(Imidazol-1-yl)-androsta-1,4-diene-3,17-dione (7). Yield
36 2 4
H N O
5%, mp 209–211 °C, IR (KBr): 2928.4, 1750.0, 1657.3, 1495.0,
À1
1
457.0, 1241.9, 1110.2, and 1046.7 cm
): d 7.54 ppm (s, 1H, 2-CH, imidazole), 7.03 (d, 1H,
cis = 12 Hz, 2-CH), 6.97 (s, IH, 4-CH, imidazole), 6.92 (s, 1H, 5-CH,
imidazole), 6.14 (d, 1H, Jcis = 12.1 Hz, 1-CH), 5.99 (s, 1H, 4-CH), 4.58
t, 1H, J = 8.36 Hz, 16 -H), 1.23 (s, 3H, 19-CH ), 1.03 (s, 3H, 18-
CH ). Calcd for C22 : C, 75.40; H, 7.48; N, 7.99. Found: C,
5.21; H, 7.22; N, 8.02.
.
3
H NMR (CDCl +
DMSO-d
J
6
2.1.5.2. 16b-(Imidazol-1-yl)-3b-pyrrolidino-5-androsten-17b-yl ace-
tate (12) (DPJ-RG-1320). Yield 45%, mp 220–222 °C, IR (KBr):
2937.9, 2791.6, 1735.9, 1491.9, 1455.5, 1372.9, 1239.3, 1139.4,
À1
1
(
a
3
1073.8, 1037.9 and 897.1 cm
1H, 2-CH, imidazole), 7.00 (s, 1H, 4-CH, imidazole), 6.88 (s, 1H, 5-
CH, imidazole), 5.34 (d, 1H, J = 4.42 Hz, 6-CH), 4.78 (m, 2H, 17 -H
and 16 -H), 2.63 (brs, 4H, –N–(CH , pyrrolidine), 1.73 (s, 3H, –
OCOCH ), 1.05 (s, 3H, 19-CH ), 1.02 (s, 3H, 18-CH ). Calcd for
3
. H NMR (CDCl ): 7.42 ppm (s,
3
26 2 2
H N O
7
a
a
3
2 2
)
2
1
.1.3.4. 16b-(Imidazol-1-yl)-5-androstene-3b,17b-diol (8) (DPJ-RG-
317). To a stirred suspension of 16b-(imidazol-1-yl)-17-oxo-5-
3
3
C28H41N O : C, 74.46; H, 9.15; N, 9.30. Found: C, 74.71; H, 9.39;
3
2
androsten-3b-ol (5, 1 g, 2.82 mmol) in methanol (100 mL) at room
temperature, sodium borohydride (1.5 g) was added in small frac-
tions over a period of 2 h. The reaction mixture was further stirred
for 4 h. Solvent was removed under reduced pressure and cold
water was added. The precipitate obtained was filtered, washed
with water, dried and crystallized from methanol to afford 8. Yield
N, 9.11.
2.1.6. General procedure for the synthesis of compounds 13 and 14
Methyl iodide (2.0 ml) was added to a solution of compound 11
or 12 (0.15 g, mmol) in dichloromethane (20.0 ml). The reaction
mixture was left at room temperature for 7 days and stirred inter-
mittently. The solvent was removed under reduced pressure and
the residue was treated with dry solvent ether to remove impurities.
The solid obtained was dried and crystallized from dry diethylether
in case of 13 and from a mixture of dry methanol–acetone for 14.
7
1
4%, mp 285–287 °C, IR (KBr): 3259.0, 2930.9, 1501.4, 1459.5,
À1
1
365.1, 1235.8, 1152.7, 1083.2 and 955.1 cm
3
. H NMR (CDCl ):
d 7.59 ppm (s, 1H, 2-CH, imidazole), 7.04 (s, 1H, 4-CH, imidazole),
.97 (s, 1H, 5-CH, imidazole), 5.32 (d, 1H, J = 4.9 Hz, 6-CH), 4.59
t, 1H, 16 -H), 3.79 (d, 1H, 17 -H), 3.43 (m, 1H, 3 -H), 1.03 (s,
), 0.89 (s, 3H, 18-CH ), Calcd for C22 : C, 74.12;
6
(
a
a
a
3
H, 19-CH
3
3
H
32
N
2
O
2
2.1.6.1. 16b-(1-Imidazolyl)-3b-pyrrolidino-5-androsten-17b-ol dim-
ethiodide (13). Yield 90%, mp 270–272 °C, IR (KBr): 3485, 2938,
H, 9.05; N, 7.86. Found: C, 74.39; H, 9.32; N, 8.02.
À1.
1
3 6
H NMR (CDCl -DMSO-d ):
1
605, 1447, 1267 and 1085 cm
2.1.4. 16b-(Imidazol-1-yl)-3b-pyrrolidino-5-androsten-17b-ol (11)
9
.19 ppm (s, 1H, 2-CH, imidazole), 7.58 (s, 1H, 5-CH imidazole),
(
DPJ-RG-1319)
7.66 (s, 1H, 4-CH imidazole), 5.52 (d, 1H, J = 4 Hz, 6-CH), 5.24 (d,
È
Pyrrolidine (1 mL) was added to a refluxing solution of 16b-
imidazol-1-yl)-4-androstene-3,17-dione (6, 1 g, 2.84 mmol) in
1
H, J = 5 Hz, 17
a
-H), 4.83 (t, 1H, 16
a
-H, J = 9 Hz), 3.96 (s, 3H, N -
È
(
CH3, imidazole), 3.55 (m, 4H, N -(CH
2
)
2
, pyrrolidine), 2.98 (s, 3H,
), 0.81 (s, 3H, 18-CH ).
: C, 48.49; H, 6.54; N, 6.06. Found: C, 48.84;
methanol (20 mL). The reaction mixture was further refluxed for
h and chilled on ice. The crystalline material 10 obtained was fil-
È
N -CH
3
, pyrrolidine), 1.08 (s, 3H, 19-CH
OI
3
3
1
Calcd for C28
H
45
N
3
2
tered, washed with methanol and immediately used for further
reaction.
H, 6.40; N, 6.15.
To a stirred suspension of above obtained 16b-(imidazol-1-yl)-
2
.1.6.2. 16b-(1-Imidazolyl)-3b-pyrrolidino-5-androsten-17b-yl ace-
3
(
-pyrrolidino-3,5-androstadiene-17-one
(10)
in
methanol
tate dimethiodide (14). Yield 78%, mp 220–222 °C, IR (KBr): 3442,
100 mL) at room temperature, sodium borohydride (1 g) was
À1.
1
3
H NMR (CDCl -DMSO-
2
942, 1732, 1624, 1241 and 1032 cm
): 9.57 ppm (s, 1H, 2-CH, imidazole), 7.62 (s, 1H, 4-CH, imidaz-
ole), 7.47 (s, 1H, 5-CH, imidazole), 5.52 (d, 1H, 6-CH), 5.15 (t, 1H,
added in small amounts over a period of 2 h at room temperature
and stirring was further continued for 4 h. Solvent was removed
under reduced pressure and iced water was added. The precipitate
obtained was filtered, washed with water, dried and crystallized
from acetone to furnish 11. Yield 41%, mp 265–267 °C, IR (KBr):
d
6
È
1
6
a
-H), 5.08 (d, 1H, 17
a
-H), 4.04 (s, 3H, N -CH3, imidazole), 3.54
È
È
(s, 4H, N -(CH
1.89 (s, 3H, 17b-OCOCH
CH ). Calcd for C30
2
)
2
, pyrrolidine), 3.02 (s, 3H, N -CH
3
, pyrrolidine),
), 0.98 (s, 3H, 18-
: C, 48.99; H, 6.44; N, 5.71. Found: C,
À1
1
3
(
379.7, 2939.1, 1661.7, 1453.1, 1234.0 and 1085.6 cm
CDCl + DMSO-d ): d 7.52 ppm (s, 1H, 2-CH, imidazole), 7.06 (s,
H, 4-CH, imidazole), 7.00 (s, 1H, 5-CH, imidazole), 5.37 (m, 1H,
-CH), 4.62 (t, 1H, J = 9.1 Hz, 16 -H), 3.81 (d, 1H, J = 9.26 Hz,
-H), 2.77 (brs, 4H, -N-(CH , pyrrolidine), 1.05 (s, 3H, 19-
), 0.89 (s, 3H, 18-CH ). Calcd for C26 O: C, 76.24; H, 9.60;
N, 10.26. Found: C, 76.59; H, 10.01; N, 10.51.
.
H NMR
3 3
), 1.09 (s, 3H, 19-CH
3
H
47
N
3
O
2
I
2
3
6
1
6
1
49.25; H, 6.48; N, 5.63.
a
7
a
2
)
2
2.2. Biological activity
CH
3
3
39 3
H N
2.2.1. Antineoplastic activity
The synthesized compounds were screened at National Cancer
2.1.5. General procedure for the synthesis of compounds 9 and 12
Institute, Bethesda, USA for in vitro antineoplastic activity.
A mixture of compound 8 or 11 (2.80 mmol), acetic anhydride
(
2
2 ml) and dry pyridine (2 ml) was heated in a steam bath for
h. The reaction contents were then poured into cold water and
2.2.2. 60-Cell line assay
The compounds 5, 6, 8, 9, 11 and 12 were selected by Drug Syn-
thesis and Chemistry Branch, National Cancer Institute, based in
general, on the basis of degree of novelty of the structure and com-
puter modeling techniques for anticancer screening. These com-
pounds were assayed in vitro against a panel consisting of 60
human tumor cell lines, derived from nine cancer types (leukemia,
lung, colon, CNS, melanoma, ovarian, renal, prostate and breast
basified with liquid ammonia. The precipitate obtained was fil-
tered, washed with water, dried and crystallized from acetone to
afford 9 and 12, respectively.
2.1.5.1. 16b-(Imidazol-1-yl)-5-androstene-3b,17b-diol diacetate (9)
(
DPJ-RG-1318). Sixty-four percentage yield, mp 197–199 °C, IR

R. Bansal et al. / Steroids 77 (2012) 621–629
625
cancers), using five concentrations at 10-fold dilutions, the highest
being 10 M. A 48 h continuous drug protocol was used and a sul-
(2%) (Serva, Heidelberg, Germany), the vials were shaken for
À4
20 min and centrifuged at 1500g for 5 min to separate the char-
3
forhodamine B (SRB) protein assay was used to estimate the cell
viability or growth [23,24]. Mean log dose response parameters
such as GI50 (drug concentration resulting in a 50% reduction in
the net protein increase), TGI (drug concentration for total growth
inhibition) and LC50 (concentration of drug resulting in a 50%
reduction in the measured protein at the end of the drug treatment
as compared to that at the beginning) were calculated. Two stan-
dard drugs, meaning that their activities against the cell lines are
well documented, were tested against each cell line: NSC 19893
coal-absorbed steroids. The supernatant was assayed for
H
2
O by
counting in a scintillation mixture using a b-counter. Exemestane
was used as a positive control that irreversibly binds to aromatase,
as a negative control (not binding irreversibly) aminoglutethimide
was used. The inhibition values after the three different incubation
times were related to the DMSO control. The compounds well as
the two reference compounds aminoglutethimide and exemestane
were tested for irreversible inhibition of aromatase in a concentra-
tion around their IC50 values.
(
5-Fluorouracil) and NSC 123127 (Adriamycin).
2
2
.2.3. Aromatase inhibitory activity
.2.3.1. Preparation of aromatase. The enzyme was obtained from
2.2.3.4. Neuromuscular blocking activity. Quaternary ammonium
steroids 13 and 14 were screened for in vitro neuromuscular block-
ing activity using paralysis in chicks [29], Rabbit head drop method
U.S.P.1975 [30] and isolated frog rectus abdominis muscle prepara-
tion [31].
the microsomal fraction of freshly delivered human term placental
tissue according to the procedure of Thompson and Siiteri [25]. 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 as described. No loss of activity was observed within four
months.
2.2.3.5. Paralysis in chicks. Paralysis in chicks was carried out as per
the procedure described by Buttle and Zaimis [29]. Female layer
chicks weighing between 20–25 g were divided into eight groups.
Each group consisted of six chicks. Quaternary ammonium steroids
13 and 14 and d-tubocurarine were administrated at various doses
intravenously in neck vein. The onset and duration of action was
recorded. The end point was considered when the chick was un-
able to stand on its legs and showed a typical contracture with flac-
cid paralysis. The mean onset and duration of action against dose
of drug with mean standard deviation was calculated.
2.2.3.2. Inhibition of aromatase in vitro. The assay was performed
similar to the described methods [26–28] monitoring enzyme
3
3
activity by measuring the H
2
O formed from [1b- H]androstenedi-
one during aromatization. Each incubation tube contained 15 nM
3
[
1b- H]androstenedione (0.08 lCi), 485 nM unlabeled andro-
stenedione, 2 mM NADP, 20 mM glucose-6-phosphate, 0.4 U 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 concen-
tration in the control and inhibitor incubation was 2%. Each tube
was preincubated for 5 min at 30 °C in a water bath. Microsomal
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
2.2.3.6. Rabbit head drop method. Rabbit head drop method was
carried out as per USP-1975 [30]. New Zealand white rabbits
weighing between 1.5 and 3 kg and of either sex were used. The
rabbits were divided in two equal groups of at least four rabbits
each. They were suitably restraint in prone position with the head
free, taking precautions to prevent struggling. The standard drug
was filled in a small bore burette which was joined with flexible
polythene tube to a hypodermic needle (21 gauge) inserted in
the marginal ear vein. The drug solution was administered at the
rate of 0.1 ml every 15 s into each rabbit until head drop occurred.
The end point was complete when rabbit was unable to raise and
hold its head in response to a light tap on the back. The number
of doses injected was recorded. The procedure was repeated in rab-
bits of both the groups. On the following day crossover was com-
pleted. Potency and confidence interval were calculated
according to the method described in USP-1975.
the addition of 200
of 200 l of an aqueous dextran-coated charcoal (DCC) suspension
2%), the vials were shaken for 20 min and centrifuged at 1500g for
2
ll of a cold 1 mM HgCl solution. After addition
l
(
5
min to separate the charcoal-absorbed steroids. The supernatant
3
was assayed for
2
H O by counting in a scintillation mixture using
a LKB-Wallac b-counter. The calculation of the IC50 values was per-
formed by plotting the percent inhibition versus the concentration
of inhibitor on a semi-log plot. From this the molar concentration
causing 50% inhibition was calculated.
2.2.3.3. Irreversible inhibition of aromatase. The assay was per-
formed similar to the test procedure described above. A preincuba-
tion of the aromatase containing microsomes was performed along
with a regenerating system (2 mM NADP, 20 mM glucose-6-phos-
phate, 0.4 U of glucose-6-phosphate-dehydrogenase) and inhibitor
in phosphate buffer (0.05 M, pH 7.4) for 30 min at 30 °C. The test
compounds were dissolved in DMSO and diluted with buffer. The
final DMSO concentration in the control and inhibitor incubation
was 2%. After preincubation an aqueous dextran coated charcoal
2.2.3.7. Effect on isolated frog rectus abdominis muscle prepara-
tion. Frog rectus abdominis muscle preparation was carried out as
described [31]. Standard procedure for setting up isolated tissue
was used. Responses to various concentrations of acetylcholine
were recorded. Frog ringer solution present in the reservoir was re-
placed by modified frog ringer solution containing test drug and
responses to various concentrations to acetylcholine were re-
corded. Concentrations of acetylcholine were increased four times
to study the nature of antagonism. Mean height of contractions
was measured and a log dose response curve was plotted.
(
DCC) suspension (2%) (Sigma, St. Louis, MO) was added followed
by a shaking step for 20 min at 4 °C. After full-speed centrifugation
50 l of the supernatant were supplemented with 50 l of regen-
erating system and 50 l substrate (30 nM [1b- H]androstenedi-
one (0.2 Ci), 185 nM unlabeled androstenedione) to start the
enzymatic reaction at 30 °C. After several time points (8, 16,
2
l
l
3
l
l
2.2.3.8. Anticholinesterase activity. The compound 13 was also
tested for in vitro AChE inhibitory activity for its ability to inhibit
the activity of electric eel acetylcholinesterase by modified Ellman
method [32].
2
1
4 min) 100
00 l of a cold 1 mM HgCl
solution. After addition of 100 l
l
l of the sample were stopped by the addition of
solution. After addition of 100 l of
l of Norit A
l
2
l
a cold 1 mM HgCl
2

6
26
R. Bansal et al. / Steroids 77 (2012) 621–629
3
. Results and discussion
resonated characteristically downfield at d 9.91 ppm indicating
the quaternization of imidazolyl nitrogen. H-NMR signals for pro-
1
3.1. Chemistry
tons of quaternary pyrrolidino methyl at d ꢀ2.98 and quaternary
imidazolyl methyl at 4 ppm were observed.
The synthetic routes to the preparation of various new steroidal
The interonium distances of the quaternary compounds were
calculated using drieding models, which indicated a value of
12.5 Å.
derivatives have been outlined in Schemes 1 and 2. Bromination of
was carried out using cupric bromide in dry methanol and dry
benzene to afford 16 -bromo-17-oxo-androsten-3b-ol (2) [22].
The configuration at position 16 has been assigned alpha in accor-
dance with the earlier reports, which states that 16b- and 16 -pro-
1
a
3.2. Biological activity
a
tons in 16-bromosteroids resonates at d 4.46 and 4.14 ppm,
respectively [22,33]. The proton NMR spectrum of compound 2
exhibited a triplet at d 4.55 ppm (16b-H). Oppenauer oxidation of
Newly synthesized 16-imidazolyl substituted steroidal deriva-
tives 5, 6, 8, 9, 11 and 12, selected for screening by NCI, exhibited
antiproliferative effects in sixty cancer cell lines. Mean inhibitory
2
using aluminium isopropoxide–cyclohexanone–toluene system
resulted into formation of a mixture of 16 -bromo (16b-H at
.56, 40%) and 16b-bromo (16 -H at 4.12, 60%) isomers of ,b-
unsaturated ketosteroid compound 3. Such epimerization of 16
concentrations ranging from 10 to 100 lM were observed for var-
a
ious response parameters such as GI50, TGI and LC50 in the 60-cell
line assay of various compounds. In general, it was observed that
16-azolyl group in steroids represents a reasonable pharmaco-
phore for cytotoxic activity. Introduction of a pyrrolidine group
at 3 position of 16-azolyl steroid skeleton did not bring any signif-
icant change in cytotoxicity profile of the compounds.
4
a
a
a
-
bromo-17-oxosteroids to the 16b- isomer in alkaline medium has
previously been reported [33]. To allow irreversible binding of ste-
roid to the aromatase enzyme, double bond was inserted between
C
1
and C
quinone (DDQ) in refluxing dry dioxane. This afforded a mixture
of 16 -bromoandrosta-1,4-diene-3,17-dione (4a, 30%) and 16b-
2
by oxidation of 3 using 2,3-dichloro-5,6-dicyanobenzo-
The aromatase inhibitory activity data of the newly synthesized
steroids is presented in Table 1. Of these compounds, 16b-(imida-
zol-1-yl)-4-androstene-3,17-dione (6) (IC50 = 0.180 lM) and 16b-
a
bromoandrosta-1,4-diene-3,17-dione (4b, 70%), which were sepa-
(imidazol-1-yl)-androsta-1,4-diene-3,17-dione (7) (IC50 = 0.168
lM)
rated using fractional crystallization. A little variability in the ratio
exhibited strong inhibition of the enzyme.
of 16a- and 16b-bromo isomers in compound 4 in comparison to
16-Bromo compounds 3 (IC50 = 2.65 lM) and 4 (IC50 = 13.2
lM)
compound 3 may be attributed to the crystallization process. The
synthesis of 4 could also be achieved by direct DDQ oxidation of
exhibited moderate inhibition of the enzyme. 17-keto-3-hydroxy
imidazolyl substituted derivative 5 with IC50 = 3.3 M displayed
l
3
-hydroxy derivative 2, but it took 4 days to complete the reaction
substantial binding with aromatase enzyme as compared to dihy-
for the formation of 4. The target 16-imidazolyl substituted ste-
roids 5–7 were obtained by thermal fusion of corresponding 16-
bromosteroids 2–4 with powdered imidazole. Only 16b-imidazolyl
derivative could be isolated by crystallization. The configuration of
imidazolyl moiety at C16 has been assigned b as is evident from H
NMR spectral data.
droxy 8 and diacetoxy steroid 9, which produced only 53% inhibi-
tion at 36 lM indicating the importance of oxidation in D ring for
enzyme binding. 16-Imidazolyl steroids 6 and 7 were found to be
approximately 160 and 170 times more potent in comparison to
aminoglutethimide and 1.2 and 1.4 times more potent in assess-
ment to standard drug exemestane (IC50 = 230 nM), respectively.
The exchange of the bromine by an imidazole ring resulted into
potent inhibition of the aromatase enzyme. Substitution of pyrrol-
idine at 3 position in compound 11 resulted in loss of binding affin-
ity for the enzyme, however introduction of 17-acetoxy as in 12
improved the activity profile. It is assumed that the observed var-
iation in aromatase inhibitory profile of these derivatives might
have resulted due to changes in three dimensional attachments
of the compounds with the enzyme. As is evident from irreversible
binding studies (Table 2), steroidal derivatives except bromo
substituted 1,4-diene 4 are acting as competitive inhibitors that
compete with substrate androstenedione for noncovalent binding
to the active site of the enzyme. It is also anticipated that imidazole
group containing steroids interfere with steroid hydroxylations by
the binding of the sterically available N with the heme Fe (III) iron
of cytochrome P450. Of all only compound 4 is behaving as an
irreversible inhibitor although to a lesser extent in comparison to
exemestane (Table 2). Despite the structural similarity of com-
pounds 4, 7 and exemestane with respect to ring A, it seems that
the nitrogen of the imidazolyl-substituted compound 7 is able to
complex the heme–iron of the enzyme leading to a different bind-
ing mode than the bromo-substituted compound 4, which is prob-
ably binding like exemestane. The present findings are potentially
useful for understanding the spatial and electronic nature of the
binding site of aromatase as well as for developing effective steroi-
dal aromatase inhibitors.
1
Repeated efforts to carry out Oppenauer oxidation of 3-hydro-
xy-5-ene derivative 5 for the synthesis of imidazolyl substituted
3
-keto-4-ene steroid 6, as well as DDQ reduction of 3-keto-4-ene
steroid 6 to prepare target compound 7 remained unsuccessful.
Therefore synthetic route, where fusion with imidazole is the last
step, was adopted for the synthesis of target compounds as shown
in Scheme 1. To improve the yield, microwave assisted synthesis
[
34] was also performed for synthesizing highly potent aromatase
inhibitor 7 by fusion of bromo product 4 with imidazole. Although
this method resulted in reduced reaction time from 30 (conven-
tional) to 15 min (microwave technique) but no significant change
in yield was observed.
To study structure activity relationship in this particular series
of compounds, further modifications of steroidal nucleus were car-
ried out. Treatment of 16b-imidazolyl steroid 5 with sodium boro-
hydride in methanol at room temperature afforded 3b,17b-diol 8,
which upon subsequent acetylation with acetic anhydride and
dry pyridine in a steam bath yielded 3b,17b-diacetoxy derivative 9.
To create two bistertiary nitrogens with a central steroid nu-
cleus, 16b-(imidazol-1-yl)-4-androstene-3,17-dione (6) was
heated under reflux with freshly distilled pyrrolidine in methanol
to furnish the unstable dienamine 10, which upon immediate
reduction with sodium borohydride in methanol at room temper-
ature afforded 3b-pyrrolidinyl derivative 11 as shown in Scheme 2.
Further acetylation of 11 using acetic anhydride and dry pyridine
in a steam bath yielded 16b-(imidazol-1-yl)-3b-pyrrolidino-5-
androsten-17b-yl acetate (12).
Both the bisquaternary steroidal salts 13 and 14, when given
intravenously produced flaccid paralysis in chicks. The mean onset
of action for 13 at 0.125 mg/kg was 5.55 min and mean duration of
action was 29.52 min. 17-Acetoxy bisquaternary steroid 14 exhib-
ited quicker onset of action of 2.20 min and a longer duration of
Bisquaternary ammonium steroids 13 and 14 were prepared by
treating the bistertiary amines 11 and 12 with methyl iodide in
dichloromethane at room temperature. 2-CH of imidazole

R. Bansal et al. / Steroids 77 (2012) 621–629
627
(6)
a, b
OH
N
N
d
N
(11)
c
R
1
OCOCH
N
3
R
N
N
N
d
N
N
-
X
2
R
(12)
(13) R = CH , R = OH, X= I
3 1
(
14) R = CH , R = OCOCH , X= I
3 1 3
4 3
Scheme 2. Synthesis of compounds 10–14 Reagents and conditions: (a) Pyrrolidine/methanol; (b) NaBH /methanol; (c) Acetic anhydride in dry pyridine; and (d) CH I,
Dichloromethane.
Table 2
Irreversible aromatase inhibition of various compounds.
Table 1
Aromatase inhibitory data of various compounds.
Inhibition on CYP 19a IC50
(lM)
RPb
RPc
S. No. Compound
Inhibition of aromatase after incubation for
irreversible binding
S. No.
Compound
a
1
2
3
4
5
6
7
8
9
3
4
5
2.65
13.20
3.30
0.18
0.16
11
2
9
160
170
0.09
0.02
0.07
1.27
1.43
Inhibitor concentration [
lM]
% inhibition
1
2
3
4
5
3
4
5
2.5
13
3
0.2
0.2
0.2
n. i.b
6 (DPJ-RG-1241)
7 (RB-401)
8
9
11
12
27.6 ± 3.6
n. i.b
No inhibition at 36 lM
6 (DPJ-RG-1241)
7 (RB-401)
Exemestane
n. i.b
n. i.b
53% inhibition at 36
40% inhibition at 36
4.90
l
M
lM
45.9 ± 6.8
Aminoglutethimide 30
n. i.b
6
0.05
a
b
c
3
a
3
[
1b- H ] androstenedione.
[1b- H] androstenedione.
n. i.: inhibition 610%.
b
Relative potency = Relative to aminoglutethimide (RP = 1; IC50 = 28.5
Relative potency = Relative to exemestane (RP = 1; IC50 = 0.23 M).
lM).
l
Table 3
NMJ blocking activity of compounds 13 and 14 on frog rectus abdominis muscle
preparation.
action of 42 min at the same dose. The minimum lethal dose
observed for 13 and 14 was 0.5 mg/kg. d-Tubocurarine at a dose
of 1 mg/kg exhibited flaccid paralysis. The mean onset of action
for d-tubocurarine (2 mg/kg) was 2.33 min and mean duration of
action was 40 min. The minimum lethal dose observed for d-
tubocurarine was 2 mg/kg.
Rabbit head drop method has been used to demonstrate the po-
tency of neuromuscular blocking agents. This method has been ac-
cepted by the USP-1975 and the method for calculation of potency
and confidence interval should not exceed 0.08. Administration of
Acetylcholine Response mm ± SD
Dose (
l
M)
Normal tissue
response
In presence of compound 13
(0.68 M)
l
1.7
13 ± 4.92
15 ± 5.17
24 ± 7.80
32 ± 9.31
00 ± 0.00
01 ± 1.2
04 ± 3.71
13 ± 4.79
3
6
1
2
.4
.8
3.6
7.2
24 ± 3.91
Normal tissue
response
In presence of compound 14
(0.0047
lM)
1
3 and 14 produced complete head drop. The potency of 13 was
found to be 41.25 times than that of d-tubocurarine and confidence
interval was found to be 0.600 which lies well within the limits of
USP-1975 specification. The potency of 14 was found to be 149.26
times than that of d-tubocurarine and confidence interval was
1.7
09 ± 0.94
16 ± 3.29
29 ± 3.74
41 ± 6.16
05 ± 2.16
3
6
1
.4
.8
3.6
12 ± 33.29
22 ± 4.784
36 ± 6.54

6
28
R. Bansal et al. / Steroids 77 (2012) 621–629
5
4
3
0
0
0
5
4
3
2
1
0
0
0
0
0
0
20
10
0
ACh pre compd
Ach Post compd
14
14
ACh Pre compd 13
Ach Post compd
13
-
6.5
-6.0
-5.5
-5.0
-4.5
-4.0
-6.5
-6.0
-5.5
-5.0
-4.5
-4.0
Log Concentration (M)
Log Concentration (M)
Fig. 3. Effects of bisquaternary steroids 13 and 14 on isolated frog rectus abdominis muscle preparation.
found to be 0.054, which lies well within the limits of USP-1975
specification.
In case of frog rectus abdominis muscle preparation, acetycho-
line at 1.7, 3.4, 6.8 and 13.6 lM produced concentration dependent
molecules. Suitable structural exploration of the steroid skeleton
might lead to formation of molecules with potent cytotoxic,
anti-aromatase, NMJ-blocking and acetylcholinesterase inhibitory
properties. The present series represents a new class of steroidal
derivatives of potential medicinal significance.
contractions. Administration of both 13 and 14 resulted in relaxa-
tion of muscle. Responses of the muscle to acetylcholine were re-
duced in presence of 13 (0.68 lM) and 14 (0.0047 lM) as shown
Acknowledgements
in Table 3. Parallel shift to the right indicated competitive antago-
nism as depicted in Fig. 3. From the shift of acetylcholine dose re-
sponse curves, the affinity constant of the compounds are
The authors are thankful to Department of Science and Technol-
ogy and Council for Scientific and Industrial Research, India for
providing financial assistance. The authors express appreciation
to National Cancer Institute, Bethesda, Maryland, USA for evalua-
tion of compounds for antineoplastic activity. The generous supply
of steroids by Cipla Ltd., India is gratefully acknowledged. We
dedicate this work to the memory of Prof. D. P. Jindal on his 70th
birth anniversary.
calculated as 0.14 lM for 13 and 0.007 lM for 17-acetoxy bisqua-
ternary salt 14. Compound 13 is not only acting as an antagonist
at acetylcholine receptors but is also inhibiting acetylcholinestrase
activity at low concentrations. When subjected to the anticholines-
terase assay, a high inhibitory activity (K
approximately 115-fold more in comparison to galanthamine
= 2.9 M) was revealed. Thus, there are two functionally oppo-
i
= 25 nM), which was
(K
i
l
site effects: reduction of neuromuscular transmission because of
the block of acetylcholine receptors and augmentation of transmis-
sion because of the reduced metabolism of the released acetylcho-
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