Synthesis of steroidal and nonsteroidal vicinal heterocyclic alcohols, N-(1-cycloalkenyl)heterocycles and their antibacterial studies

Article abstract of DOI:10.1016/j.steroids.2014.03.011

A solvent free steroidal and nonsteroidal epoxide ring opening reaction by nitrogen containing heterocycles under microwave irradiation is described. Some of the epoxide ring opening compounds were converted to their corresponding N-(1-cycloalkenyl)heterocycles via an acid catalyzed dehydration reaction. The antimicrobial activities of the epoxide ring opening compounds and N-(1-cycloalkenyl)heterocyclic compounds were tested by agar diffusion assay. Compounds 6, 9-12, 24 and 27 showed moderate inhibition against the growth of pathogenic bacteria Escherichia coli, Pseudomonas syringae, Bacillus subtilis, Proteus vulgaris and Staphylococcus aureus.

Full text of DOI:10.1016/j.steroids.2014.03.011

Steroids 84 (2014) 36–45
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of steroidal and nonsteroidal vicinal heterocyclic alcohols,
N-(1-cycloalkenyl)heterocycles and their antibacterial studies
a
a
a
b
Pallabi Saikia , Partha Pratim Kaishap , Jonalee Goswami , Anil Kumar Singh ,
b
a,
⇑
a,
⇑
Hari Prasanna Deka Boruah , Sanjib Gogoi , Romesh C. Boruah
a
Medicinal Chemistry Division, CSIR-North East Institute of Science and Technology, Jorhat 785006, India
Biotechnology Division, CSIR-North East Institute of Science and Technology, Jorhat 785006, India
b
a r t i c l e i n f o
a b s t r a c t
Article history:
A solvent free steroidal and nonsteroidal epoxide ring opening reaction by nitrogen containing heterocy-
cles under microwave irradiation is described. Some of the epoxide ring opening compounds were con-
verted to their corresponding N-(1-cycloalkenyl)heterocycles via an acid catalyzed dehydration reaction.
The antimicrobial activities of the epoxide ring opening compounds and N-(1-cycloalkenyl)heterocyclic
compounds were tested by agar diffusion assay. Compounds 6, 9–12, 24 and 27 showed moderate inhi-
bition against the growth of pathogenic bacteria Escherichia coli, Pseudomonas syringae, Bacillus subtilis,
Proteus vulgaris and Staphylococcus aureus.
Received 23 August 2013
Received in revised form 11 March 2014
Accepted 15 March 2014
Available online 28 March 2014
Keywords:
Heterosteroid
Epoxide
Ó 2014 Elsevier Inc. All rights reserved.
Microwave
Antimicrobial
N-(1-Cycloalkenyl)heterocycle
1
. Introduction
The incorporation of a heterocycle in the steroid backbone often
abiraterone acetate (VI) and VN/124-1 (VII) are potent CYP17
inhibitors [7,8] (Fig. 1).
As a part of our ongoing research on development of new meth-
odology for the synthesis of novel steroidal and nonsteroidal het-
erocyclic compounds [9], herein we report a new solvent free
green strategy [10] for the steroidal and nonsteroidal epoxide ring
opening reaction by imidazoles, benzimidazole for the synthesis of
hydroxy-(1H-imidazol-yl)/hydroxy-(1H-benzimidazol-yl) steroids
and 2-(1H-imidazol-1-yl)/2-(1H-benzimidazol-1-yl)cyclohexanols
under microwave irradiation. We extended our methodology for
the epoxide ring opening reactions with other nitrogen containing
heterocycles also. Further, these epoxide ring opening steroidal
and nonsteroidal b-hydroxy heterocyclic compounds were con-
verted to the corresponding N-(1-cycloalkenyl)heterocycles via
an acid catalyzed dehydration reaction in good yields. We also re-
port in vitro antimicrobial activities of these compounds against
five pathogenic strains, they are, Escherichia coli (ATCC 8739),
Proteus vulgaris (MTCC 426), Pseudomonas syringae (MTCC 673),
Bacillus subtilis (ATCC 6633) and Staphylococcus aureus (ATCC
29213) species.
alters the biological activity of the steroidal molecule which serves
as a platform for developing a new pharmaceutical compound for
various applications [1,2]. Vicinal imidazolyl alcohols and their
derivatives are very important class of compounds due to their
wide range of biological activities [3]. For example, non steroidal
vicinal imidazolyl alcohols such as ornidazole, metronidazole
(I, Fig. 1) and secnidazole (II, Fig. 1) are effective antiprotozoal
drugs. At the same time different heterocycle substituted steroidal
compounds having vicinal hydroxyl group are found to have
different biological activities. In Fig. 1, steroidal compounds such as
2b-piperazinyl-5a-androstane-3a,17b-diols (III) and 2,3-dihyd-
rowithaferin A-3b-triazoles (IV) show antiproliferative potency
and cytotoxic activity respectively [4,5].
On the other hand, both nonsteroidal and steroidal N-(1-cyclo-
alkenyl)heterocycles are important classes of compounds which
have potential biological activity, for instance nonsteroidal
compounds
1-[2-(methylsulfanyl)-1-cyclopentenyl]imidazole
(VIII, Fig. 1) and 1-[2-(2-thienyl)-1-cyclopentenyl]imidazole (IX,
Fig. 1) are reported as neuronal injury inhibitors [6] whereas ste-
2. Experimental
roidal N-(1-cycloalkenyl)heterocycles such as abiraterone (V),
2.1. General methods
⇑
Corresponding authors. Tel.: +91 3762372948; fax: +91 3762370011.
E-mail addresses: skgogoi1@gmail.com (S. Gogoi), rc_boruah@yahoo.co.in
Melting points were measured with a Buchi B-540 melting
(
R.C. Boruah).
point apparatus and are uncorrected. IR spectra were recorded on
http://dx.doi.org/10.1016/j.steroids.2014.03.011
039-128X/Ó 2014 Elsevier Inc. All rights reserved.
0

P. Saikia et al. / Steroids 84 (2014) 36–45
37
OH
O
O
OH
O
MeO
N
O
O
N
N
OH
N
N
R
H
H
O2N
N
N
HO
O
O
H
Metronidazole (I, R = H)
N
OH
Secnidazole (II, R = CH )
3
RM (III, antiproliferative activity)4
KK-12 (IV, cytotoxic)5
(
antiprotozoal drugs)3
N
N
N
N
N
N
S
H
Abiraterone (V), R = H
S
N
RO
H
RO
VN/124-1 (TOK-001, VII)
CYP17 inhibitory and
AR ablative activities)8
1-[2-(2-thienyl)-
1
1
-[2-(methylsulfanyl)-
-cyclopentenyl]imidazole
Abiraterone acetate (VI),
1-cyclopentenyl]imidazole
(
(IX, neuronal injury inhibitors)6
R = COCH3
(VIII, neuronal injury
(
CYP17 inhibitor)7
inhibitors)6
Fig. 1. Example of biologically important steroidal/nonsteroidal heterocyclic alcohol and N-(1-cycloalkenyl)heterocycles.
Elmer FT-IR-2000 spectrometer using KBr pellets or on a thin film
using chloroform. NMR spectra were recorded on Avance DPX
2 4
with DCM and dried over Na SO . The crude product obtained after
removal of the DCM layer was purified by silica gel column chro-
matography using ethyl acetate/hexane (0.5:9.5) as the eluent to
3
00 MHz FT-NMR spectrometer or Bruker Avance III 500 MHz FT-
NMR spectrometer using tetramethylsilane (TMS) as an internal
standard. Mass spectra were recorded on Trace DSQ GCMS instru-
ment. All the commercially available reagents were used as
received. All experiments were monitored by thin layer chroma-
tography (TLC). TLC was performed on pre-coated silica gel plates
afford steroidal 7-keto 3,5-diene 3. Yield 6.36 g (92%); R
f
= 0.7
ꢁ1
(EtOAc/hexane = 1:9); m.p. 117–119 °C. IR (KBr, cm ): 2945,
1
1712, 1580, 1250; H NMR (CDCl
3
, 300 MHz): d 0.71 (s, 3H),
0.85–2.36 (m, 24H), 0.88 (d, J = 6.6 Hz, 6H), 0.92 (d, J = 6.4 Hz,
1
3
3
3H), 1.11 (s, 3H), 5.60 (s, 1H), 6.06–6.22 (m, 2H); C NMR (CDCl ,
(
(
Merck). Column chromatography was performed on silica gel
60–120 mesh, Merck Chemicals). All microwave reactions were
75 MHz) d 12.0, 16.6, 18.9, 21.2, 22.6, 22.9, 23.4, 23.9, 26.4, 26.5,
28.0, 28.6, 32.8, 35.8, 36.3, 39.0, 39.5, 43.4, 46.0, 49.6, 50.7, 54.9,
124.2, 127.7, 136.6, 160.7, 202.3. MS (EI, m/z) = 382; Anal. Calcd.
carried out in a Synthos 3000 (Anton Paar) microwave reactor.
for C27
H42O: C 84.75; H 11.06; found: C 84.77; H 11.28.
2
2
.2. Chemical synthesis
1
3
2
.2.1.3. Preparation of 3
a a
,4 -epoxy-5-en-cholest-7-one (4) . To a
.2.1. Conversion of cholesteroyl acetate (1) to 3
a
,4b-epoxy-5-en-
stirring solution of compound 3 (6.2 g, 16.22 mmol) in chloroform
(15 mL), m-CPBA (4.2 g, 24.33 mmol) was added at 0 °C. The reac-
tion mixture was then stirred for 4 h at room temperature and
cholest-7-one (4)
.2.1.1. Preparation of 3b-acetoxy-cholest-5-en-7-one (2) . To a
solution of cholesteroyl acetate (1, 10.0 g, 23.34 mmol) and RuCl
1
1
2
3
2 3
after completion of the reaction, as indicated by TLC, 4% Na SO
ꢀ
H
2
O (60 mg) in cyclohexane (40 mL), 70% tert-butyl hydroperoxide
was added into it and the whole reaction mixture was kept stirring
for another 4 h. The reaction mixture was then extracted with
(
20 mL) was added at room temperature for 1 h and the reaction
mixture was stirred for another 5 h. The reaction mixture was then
treated with saturated NaHCO solution, washed with water, ex-
tracted with EtOAc and dried over Na SO . The crude product ob-
tained after removal of the EtOAc layer was purified by silica gel
column chromatography using ethyl acetate/hexane (1:9) as the
eluent to afford pure keto compound 2. Yield 8.26 g (80%);
3 2 4
CHCl washed with water, brine and dried over Na SO . The crude
3
product obtained after removal of the solvent was purified by silica
gel column chromatography using ethyl acetate/hexane (1:9) as
the eluent to afford pure steroidal epoxide 4. Yield 4.84 g (75%);
2
4
ꢁ1
f
R = 0.5 (EtOAc/hexane = 1:9); m.p. 128–129 °C. IR (KBr, cm ):
1
2952, 1670, 1462, 1381; H NMR (CDCl
3
, 300 MHz): d 0.69
ꢁ
1
R
2
3
6
f
= 0.5 (EtOAc/hexane = 1:9); m.p. 158–160 °C. IR (KBr, cm )
(s, 3H), 0.85–2.36 (m, 24H), 0.88 (d, J = 6.5 Hz, 6H), 0.92 (d,
1
13
949, 1732, 1670, 1466, 1376, 1248, 758; H NMR (CDCl
3
,
J = 6.5 Hz, 3H), 1.08 (s, 3H), 3.39–3.49 (m, 2H), 6.04 (s, 1H);
C
00 MHz): d 0.68 (s, 3H), 0.65–2.55 (m, 29H), 0.86 (d, J = 6.3 Hz,
NMR (CDCl , 75 MHz) d 12.0, 17.7, 18.8, 20.7, 21.2, 22.6, 22.8,
3
1
3
H), 1.21 (s, 3H), 1.61 (s, 3H), 4.77–4.66 (m, 1H), 5.70 (s, 1H);
, 75 MHz) d 12.0, 17.3, 18.9, 21.2, 21.3, 22.6, 22.8,
3.8, 27.3, 28.0, 26.3, 28.6, 35.7, 36.0, 36.2, 37.7, 38.3, 38.6, 39.5,
3.1, 45.4, 49.8, 49.9, 54.7, 72.2, 126.7, 163.9, 170.3, 202.0; MS
C
23.8, 26.2, 26.4, 28.0, 28.6, 35.6, 35.8, 36.2, 38.8, 39.5, 43.4, 46.5,
49.7, 50.5, 52.0, 52.4, 54.8, 131.4, 160.2, 201.3. MS (EI, m/
z) = 398; Anal. calcd. for C27H O : C 81.35; H 10.62; found: C
42 2
81.33; H 10.59.
NMR (CDCl
2
4
3
+
(
7
EI, m/z) = 382.3 [M ꢁCH
3
COOH]; Anal. Calcd for C29
46 3
H O : C
8.68; H 10.47; Found: C 78.70; H 10.69.
2
4
.2.1.4. Preparation of 3b-acetoxy-cholest-5,6-epoxide (13:14 =
:1) . To a stirring solution of Cholesterol acetate 1 (3.0 g,
1
4
1
2
2
.2.1.2. Preparation of cholesta-3,5-dien-7-one (3) . Compound 2
7.0 mmol) in chloroform (15 mL), m-CPBA (1.81 g, 10.5 mmol)
was added at 0 °C. The reaction mixture was then stirred for 6 h
at room temperature and after completion of the reaction, as indi-
(
8.0 g, 18.09 mmol) was refluxed with hydrochloric acid (20 mL)
in methanol for 1 h. The residue obtained after removal of the sol-
vent was washed with water, neutralised with NaHCO , extracted
3
2 3
cated by TLC, 4% Na SO was added into it and the whole reaction

3
8
P. Saikia et al. / Steroids 84 (2014) 36–45
mixture was kept stirring for another 4 h. The reaction mixture
was then extracted with CHCl washed with water, brine and dried
over Na SO . The crude product obtained after removal of the sol-
vent was purified by silica gel column chromatography using ethyl
acetate/hexane (1:9) as the eluent to afford a mixture of and b
steroidal epoxides 13 and 14 ( /b = 4:1). Yield 2.27 g (73%); m.p.
4–97 °C. IR (KBr, cm ): 2952, 1725; 1H NMR (CDCl
, 300 MHz):
d 0.61 (s, 2.4H), 0.64 (s, 0.6H), 0.82–2.32 (m, 40H), 2.02 (s, 2.4H),
3H), 1.01 (s, 3H), 2.63 (br s, 1H), 4.34 (s, 1H), 5.79 (s, 1H); 13C
NMR (CDCl , 75 MHz) d 12.0, 17.7, 18.8, 20.7, 21.2, 22.6, 22.8,
3
3
2
4
23.8, 26.2, 26.4, 28.0, 28.6, 29.7, 35.6, 35.7, 36.1, 38.8, 39.5, 43.4,
46.5, 49.7, 50.5, 52.0, 52.4, 54.8, 131.4, 160.3, 201.4; MS (EI,
+
+
a
m/z) = 483 (M) , 465 (Mꢁ18) . Anal. calcd. for C32
2
H53NO : C,
a
79.45; H, 11.04; N, 2.90; found: C, 79.38; H, 11.26; N, 2.71.
ꢁ1
9
3
2.2.2.6. 3b-Morpholino-4
a-hydroxy-5-en-cholest-7-one (10). Yellow
ꢁ1
2
4
1
2
3
5
.03 (s, 0.6H), 2.84–2.91 (m, 0.8H), 3.04–3.12 (m, 0.2H), 4.72–
solid; m.p. 121–123 °C; yield 74%; IR (CHCl
3
, cm ): 3422, 2951,
.79 (m, 0.2H), 4.88–5.02 (m, 0.8H); 13C NMR (CDCl
1669, 1453, 756; H NMR (CDCl , 300 MHz) d 0.67 (s, 3H), 0.84–
1
3
, 75 MHz) d
3
1.8, 11.9, 15.9, 17.0, 18.6, 20.6, 21.3, 21.9, 22.6, 22.8, 23.79,
3.84, 24.0, 24.2, 27.2, 28.0, 28.07, 28.14, 28.8, 29.7, 29.9, 32.1,
2.5, 36.7, 38.0, 39.4, 39.5, 39.8, 42.3, 42.4, 51.0, 55.8, 56.2, 56.8,
2.66 (m, 25H), 0.86 (d, J = 6.5 Hz, 6H), 0.92 (d, J = 6.5 Hz, 3H),
1.42 (s, 3H), 2.38–2.42 (m, 4H), 2.66 (d, J = 2.0 Hz, 1H), 3.68 (t,
1
3
J = 6.0 Hz, 4H), 4.33 (s, 1H), 5.81 (s, 1H);
3
C NMR (CDCl ,
9.2, 62.5, 63.6, 65.2, 71.3, 71.4, 170.2, 170.6; MS (EI, m/z) = 398
75 MHz) d 11.9, 17.4, 18.9, 20.6, 22.6, 22.8, 23.8, 26.3, 28.0, 28.5,
+
[
M ].
29.7, 32.9, 35.7, 36.1, 38.6, 39.2, 39.5, 43.1, 45.8, 50.6, 52.3, 54.8,
+
6
6.7, 66.9, 74.7, 132.7, 162.6, 202.2; MS (EI, m/z) = 485 (M) . Anal.
2
.2.2. General procedure for the preparation of steroidal and
nonsteroidal vicinal heterocyclic alcohols (5–12, 15, 16, 18–22)
The epoxide (0.5 mmol) and N-containing heterocycle
0.5 mmol) were mixed intimately and the mixture was irradiated
in a closed vessel in a Synthos 3000 microwave reactor at 600 Watt
140 °C and 12 bar) for 6–16 min. The residue obtained was puri-
calcd. for C31
10.66; N, 2.92.
3
H51NO : C, 76.65; H, 10.58; N, 2.88 found: C, 76.48; H,
4
(
2.2.2.7. 3b-Thiomorpholino-4
a-hydroxy-5-en-cholest-7-one (11).
ꢁ1
Yellow thick oil; yield 69%; IR (CHCl
3
, cm ): 3423, 2954, 1665,
1
(
1457, 754; H NMR (CDCl , 300 MHz) d 0.68 (s, 3H), 0.85–2.64
3
fied by silica gel column chromatography using EtOAc/hexane as
the eluent to afford the vicinal heterocyclic alcohols.
(m, 33H), 0.85 (d, J = 6.6 Hz, 6H), 0.92 (d, J = 6.5 Hz, 3H), 1.40 (s,
3H), 2.65 (d, J = 2.0 Hz, 1H), 4.36 (s, 1H), 5.84 (s, 1H); 13C NMR
(
3
CDCl , 75 MHz) d 11.7, 17.4, 18.9, 20.4, 22.6, 22.8, 23.8, 26.3,
2
.2.2.1. 3b-(1H-Imidazolo)-4
a
-hydroxy-5-en-cholest-7-one (5). Thick
28.5, 28.6, 29.5, 32.9, 35.7, 36.1, 38.7, 38.9, 39.7, 43.6, 45.8, 50.6,
ꢁ1
yellow gum, yield 69%; IR (CHCl
3
, cm ): 3421, 2952, 1672, 1451,
52.3, 55.2, 58.3, 66.7, 66.9, 74.7, 132.9, 162.8, 202.4; MS (EI,
1
+
7
2
2
56; H NMR (CDCl
3
, 300 MHz): d 0.67 (s, 3H), 0.83–2.66 (m,
m/z) = 501 (M) . Anal. calcd. for C31
2
H51NO S: C, 74.20; H, 10.24;
5H), 0.85 (d, J = 6.5 Hz, 6H), 0.92 (d, J = 6.5 Hz, 3H), 1.41 (s, 3H),
N, 2.79. found: C, 74.13; H, 10.35; N, 2.98.
.65–2.67 (m, 1H), 4.34 (br s, 1H), 5.83 (s, 1H), 6.90 (s, 1H), 7.11
1
3
(
s, 1H), 7.58 (s, 1H); C NMR (CDCl
3
, 75 MHz): d 11.7, 17.3, 18.8,
2.2.2.8.
3b-Tetrahydroisoquinolino-4a-hydroxy-5-en-cholest-7-one
ꢁ1
2
3
1
0.5, 22.6, 22.8, 23.8, 26.4, 28.2, 28.5, 29.6, 32.9, 35.7, 35.9, 38.6,
9.2, 39.5, 43.2, 45.8, 50.6, 52.3, 54.7, 66.7, 66.9, 74.8, 132.5,
33.8, 134.5, 162.7, 202.1. MS (EI, m/z) = 466 (M ). Anal. calcd.
(12). White solid; m.p. 194–196 °C; yield 77%; IR (CHCl
3395, 2924, 1670, 1461, 1374, 1156, 771; H NMR (CDCl ,
3
, cm ):
1
3
+
300 MHz) d 0.67 (s, 3H), 0.83–2.60 (m, 29H), 0.86 (d, J = 6.5 Hz,
6H), 0.91 (d, J = 6.5 Hz, 3H), 1.08 (s, 3H), 2.84 (d, J = 2.1 Hz, 1H),
3.41–3.48 (m, 2H), 4.51 (s, 1H), 5.87 (s, 1H), 7.01–7.83 (m, 4H);
46 2 3
for C30H N O : C, 77.21; H, 9.93; N, 6.00; found: C, 77.45; H,
9
.71; N, 6.37.
1
3
3
C NMR (CDCl , 75 MHz) d 12.0, 14.1, 17.7, 18.8, 21.2, 22.6, 22.7,
2
.2.2.2. 3b-(4-Nitro-1H-imidazolo)-4
a
-hydroxy-5-en-cholest-7-one
22.8, 26.2, 26.4, 28.0, 28.6, 29.7, 35.6, 35.7, 36.1, 38.8, 39.4, 43.4,
ꢁ1
(
6). Thick yellow gum, yield 62%; IR (CHCl
3
, cm ): 3424, 2950,
, 300 MHz): d 0.69 (s, 3H), 0.86–2.64
m, 25H), 0.84 (d, J = 6.4 Hz, 6H), 0.90 (d, J = 6.6 Hz, 3H), 1.42 (s,
46.5, 49.7, 50.5, 54.8, 126.2, 127.8, 129.7, 129.9, 131.4, 160.2,
1
+
1
(
672, 754; H NMR (CDCl
3
201.4; MS (EI, m/z) = 531 (M) . Anal. calcd. for C36
H
53NO
2
: C,
81.30; H, 10.05; N, 2.63; Found: C, 81.38; H, 9.97; N, 2.89.
3
8
H), 2.65–2.68 (m, 1H), 4.36 (s, 1H), 5.84 (s, 1H), 8.20 (s, 1H),
+
.23 (s, 1H); MS (EI, m/z) = 511 (M ). Anal. calcd. for C30
H
45
N
3
O
4
:
2.2.2.9. 3b-Acetoxy-5a-hydroxy–cholest-6b-(1H-imidazole) (15). This
C, 70.42; H, 8.86; N, 8.21; found: C, 70.52; H, 8.86; N, 8.35.
compound was further recrystallized from ethanol. White solid,
ꢁ
1
m.p. 225–228 °C, yield 75%; IR (CHCl
3
, cm ): 3205, 2945, 1729,
1
2
.2.2.3. 3b-(4-Formyl-1H-imidazolo)-4
a
-hydroxy-5-en-cholest-7-one
1381, 1245, 771; H NMR (CDCl , 300 MHz): d 0.74 (s, 3H), 0.75 (s,
3
ꢁ1
(
7). Thick brown gum, yield 66%; IR (CHCl
3
, cm ): 3421, 2954,
, 300 MHz): d 0.70 (s, 3H), 0.85–2.64
m, 25H), 0.85 (d, J = 6.4 Hz, 6H), 0.92 (d, J = 6.6 Hz, 3H), 1.40 (s,
3H), 0.87–2.20 (m, 28H), 0.87 (d, J = 6.5 Hz, 6H), 0.92 (d, J = 6.4 Hz,
3H), 2.05 (s, 3H), 2.83 (br s, 1H), 3.97 (d, J = 5.7 Hz, 1H), 5.13–5.24
1
1
676, 752; H NMR (CDCl
3
1
3
(
3
(m, 1H), 7.01 (s, 1H), 7.08 (s, 1H), 7.70 (s, 1H). C NMR (CDCl ,
3
7
C
H), 2.66–2.68 (m, 1H), 4.37 (s, 1H), 5.86 (s, 1H), 7.54 (s, 1H),
.61 (s, 1H), 9.93 (s, 1H); MS (EI, m/z) = 494 (M ). Anal. calcd. for
125 MHz): d 12.2, 15.6, 18.5, 21.0, 21.4, 22.5, 22.7, 23.8, 24.0, 27.9,
28.1, 30.6, 30.9, 32.4, 32.9, 35.7, 36.0, 38.3, 38.5, 39.4, 39.6, 42.7,
+
31
H
46
N
2
3
O : C, 75.26; H, 9.37; N, 5.66; found: C, 75.60; H, 9.71;
45.0, 55.9, 56.0, 62.1, 70.2, 76.6, 119.9, 128.0, 137.7, 171.0. MS (EI,
+
N, 5.43.
m/z) = 512.4 (M ). Anal. calcd. for C32
H
52
N
2
O
3
: C, 74.95; H, 10.22.
N, 5.46; found: C, 74.81; H, 10.28; N, 5.84.
2
.2.2.4. 3b-(1H-Benzo[d]imidazolo)-4
a-hydroxy-5-en-cholest-7-one
ꢁ1
(8). Thick yellow gum, yield 65%; IR (CHCl
3
, cm ): 3429, 2955,
2.2.2.10. 3b-Acetoxy-5a-hydroxy-cholest-6b-(1H-benzo[d]imidazole)
1
1
2
2
678; H NMR (CDCl , 300 MHz): d 0.70 (s, 3H), 0.84–2.64 (m,
3
(16). This compound was further recrystallized from ethanol.
ꢁ
1
5H), 0.84 (d, J = 6.5 Hz, 6H), 0.92 (d, J = 6.6 Hz, 3H), 1.40 (s, 3H),
White solid, m.p. 157–159 °C, yield 71%; IR (CHCl
3
, cm ): 3402,
1
.66–2.68 (m, 1H), 4.35 (s, 1H), 5.87 (s, 1H), 7.13–7.91 (m, 5H);
2937, 1727, 1381, 1248, 771; H NMR (CDCl , 300 MHz): d 0.80
3
+
MS (EI, m/z) = 516 (M ). Anal. calcd. for C34
H
48
2
N O
2
: C, 79.02; H,
(s, 3H), 0.87–2.52 (m, 28H), 0.87 (d, J = 6.5 Hz, 6H), 0.94 (d,
J = 6.2 Hz, 3H), 1.01 (s, 3H), 1.95 (s, 3H), 3.41 (br s, 1H), 4.36 (d,
J = 6.7 Hz, 1H), 5.11–5.18 (m, 1H), 7.25–7.30 (m, 2H), 7.45 (m,
9
.36; N, 5.42; found: C, 78.82; H, 9.30; N, 5.67.
1
3
2.2.2.5. 3b-Piperidino-4 -hydroxy-5-en-cholest-7-one (9). Brown so-
a
3
1H), 7.80 (m, 1H), 8.39 (s, 1H). C NMR (CDCl , 75 MHz): d 12.5,
ꢁ
1
lid; m.p. 117–119 °C; yield 68%; IR (CHCl
3
, cm ): 3418, 2929,
, 300 MHz) d 0.66 (s, 3H),
.83–2.60 (m, 35H), 0.86 (d, J = 6.5 Hz, 6H), 0.92 (d, J = 6.5 Hz,
17.1, 18.4, 18.6, 21.2, 21.4, 22.6, 22.8, 24.0, 26.4, 28.0, 28.2, 32.2,
32.9, 33.8, 35.8, 36.2, 37.7, 38.7, 39.5, 39.8, 42.8, 44.8, 56.3, 58.4,
60.4, 71.0, 77.1, 111.2, 120.1, 122.2, 122.9, 136.0, 142.6, 142.9,
1
0
670, 1466, 1382, 757; 1H NMR (CDCl
3

P. Saikia et al. / Steroids 84 (2014) 36–45
39
+
1
7
71.3. MS (EI, m/z) = 562.4 (M ). Anal. calcd. for C36
H
54
N
2
O
3
: C,
50.2, 54.8, 114.1, 120.3, 122.4, 128.6, 133.4, 154.2, 156.5, 201.7.
ꢁ1
+
6.82; H, 9.67; N, 4.98. Found: C, 76.89; H, 9.74; N, 4.73.
IR (CHCl
Anal. calcd. for C30
80.44; H, 9.87; N, 6.39.
3
, cm ): 2926, 1662, 1466, 772; MS (EI, m/z) = 448 [M] .
44 2
H N O: C, 80.31; H, 9.88; N, 6.24; Found: C,
2
.2.2.11. Trans-2-(1H-imidazol-1-yl)cyclohexanol (18). White solid,
m.p. 134–136 °C, yield 79%; IR (CHCl
, 300 MHz): d 1.22–2.14 (m, 8H),
.52–3.74 (m, 2H), 4.94 (br s, 1H), 6.83 (s, 1H), 6.92 (s, 1H), 7.36
, 75 MHz): d 24.3, 25.0, 32.3, 34.4, 63.8,
2.7, 117.3, 127.9, 136.2; MS (EI, m/z) = 166 (M ). Anal. calcd. for
O: C, 65.03; H, 8.49; N, 16.85; Found: C, 65.06; H, 8.52;
ꢁ1
3
, cm ): 3447, 2923, 1437,
1
1
3
260, 1119; H NMR (CDCl
3
2.2.3.2. 3-(4-Nitro-1H-imidazolo)-3,5-dien-cholest-7-one (24). Yel-
1
3
low solid; m.p. 90–92 °C; yield 83%; H NMR (CDCl , 300 MHz) d
1
3
(
s, 1H); C NMR (CDCl
3
0.72 (s, 3H), 0.80–2.59 (m, 24H), 0.86 (d, J = 6.7 Hz, 6H), 0.93 (d,
+
7
C
J = 6.4 Hz, 3H), 1.23 (s, 3H), 5.31 (s, 1H), 6.54 (t, J = 4.1 Hz, 1H),
1
3
H
9 14
N
2
8.22 (s, 2H); C NMR (CDCl
3
, 75 MHz) d 11.9, 13.0, 18.7, 22.6,
N, 16.88.
22.8, 23.8, 24.2, 28.0, 28.2, 28.5, 29.0, 29.7, 35.7, 35.8, 36.2, 38.3,
3
9.5, 42.4, 51.1, 53.7, 56.2, 56.3, 114.3, 120.8, 122.6, 129.2, 134.4,
ꢁ1
2
.2.2.12. Trans-2-(4-nitro-1H-imidazol-1-yl)cyclohexanol (19). Yel-
156.1, 156.0, 201.2; IR (CHCl
m/z) = 493 [M ]. Anal. calcd. for C30
3
, cm ): 2930, 1672, 1466; MS (EI,
: C, 72.99; H, 8.78; N,
ꢁ1
1
+
low oil, yield 75%; IR (CHCl
3
, cm ): 3443, 2925, 1262, 1120;
H
H
43
N
3
O
3
NMR (CDCl
3
, 300 MHz): d 0.84–2.20 (m, 8H), 3.29–3.82 (m, 2H),
8.51; found: C, 72.79; H, 8.61; N, 8.64.
+
7
.47 (s, 1H), 7.79 (s, 1H); MS (EI, m/z) = 211 (M ). Anal. calcd. for
C
9
H
13
N
3
O
3
: C, 51.18; H, 6.20; N, 19.89; Found: C, 51.40; H, 6.28;
2.2.3.3. 3-(4-Formyl-1H-imidazolo)-3,5-dien-cholest-7-one (25). Yel-
1
N, 19.63.
low solid; m.p. 108–110 °C; yield 75%; H NMR (CDCl
.72 (s, 3H), 0.81–2.55 (m, 24H), 0.86 (d, J = 6.6 Hz, 6H), 0.93 (d,
J = 6.4 Hz, 3H), 1.27 (s, 3H), 5.33 (s, 1H), 6.33 (t, J = 4.3 Hz, 1H),
3
, 300 MHz) d
0
2.2.2.13.
Trans-1-(2-hydroxycyclohexyl)-1H-imidazole-4-carbalde-
ꢁ
1
13
hyde (20). Colorless liquid, yield 76%; IR (CHCl
923, 1723, 1434, 1262; 1H NMR (CDCl
, 300 MHz): d 0.88–2.19
m, 8H), 3.67–3.82 (m, 2H), 7.72 (s, 1H), 7.87 (s, 1H), 9.66 (s, 1H);
3
, cm ): 3451,
7.53 (d, J = 0.8 Hz, 1H), 7.58 (d, J = 1.0 Hz, 1H), 9.90 (s, 1H);
C
2
3
NMR (CDCl , 75 MHz) d 12.0, 16.7, 18.9, 21.4, 22.6, 22.8, 23.8,
3
(
28.0, 28.5, 29.7, 31.0, 32.2, 35.7, 36.1, 37.3, 38.6, 39.5, 43.2, 45.8,
1
3
C NMR (CDCl
25.0, 139.2, 141.2, 184.9; MS (EI, m/z) = 194 (M ). Anal. calcd.
for C10 : C, 61.84; H, 7.27; N, 14.42; Found: C, 61.96; H,
3
, 75 MHz): d 24.2, 24.9, 31.8, 34.1, 64.5, 72.9,
49.0, 50.1, 54.8, 122.4, 125.3, 132.8, 134.6, 139.0, 142.2, 155.7,
+
ꢁ1
1
186.0, 201.3; IR (CHCl
3
, cm ): 2952, 1671, 1536, 1466; MS (EI,
+
H
14
N
2
O
2
m/z) = 476 [M ]. Anal. calcd. for C31
44 2 2
H N O : C, 78.11; H, 9.30; N,
7
.18; N, 14.40.
5.88; found: C, 78.14; H, 9.33; N, 5.91.
2
.2.2.14. Trans-2-(1H-benzimidazol-1-yl)cyclohexanol (21). Brown
2.2.3.4. 3-(1H-Benzo[d]imidazolo)-3,5-dien-cholest-7-one (26). Brown
solid; m.p. 126–128 °C; yield 88%; H NMR (CDCl , 300 MHz) d 0.73
3
(s, 3H), 0.80–2.60 (m, 24H), 0.86 (d, J = 6.6 Hz, 6H), 0.93 (d, J = 6.5 Hz,
ꢁ1
1
solid, m.p. 163–165 °C. yield 78%; IR (CHCl
1
3
, cm ): 3090, 2926,
, 300 MHz): d
615, 1494, 1458, 1253, 1075, 742; 1H NMR (CDCl
3
1
2
.42–1.60 (m, 3H); 1.74–1.93 (m, 3H), 2.03–2.09 (m, 1H), 2.23–
3H), 1.25 (s, 3H), 5.21 (s, 1H), 6.38 (s, 1H), 7.10–7.86 (m, 4H), 7.87 (s,
1
3
.29 (m, 1H), 3.92–4.08 (m, 2H), 7.14 (t, J = 7.3 Hz, 1H), 7.24 (t,
3
1H); C NMR (CDCl , 75 MHz) d 12.0, 16.9, 18.9, 22.6, 22.8, 26.2,
13
J = 7.8 Hz, 1H), 7.50 (m, 2H), 7.60 (s, 1H).
3
C NMR (CDCl ,
28.0, 29.7, 35.8, 36.2, 38.7, 39.5, 42.8, 51.8, 56.0, 56.6, 114.1, 120.5,
7
5 MHz): d 24.5, 25.3, 31.9, 34.4, 62.5, 72.4, 110.6, 119.5, 122.2,
121.9, 122.6, 122.8, 129.2, 134.4, 156.2, 156.7, 201.7; IR (CHCl
cm ): 3418, 2929, 1670, 1466, 1382, 757; MS (EI, m/z) = 498 [M] .
3
+
,
+
ꢁ1
1
22.7, 133.8, 140.8, 142.8; MS (EI, m/z) = 216 (M ). Anal. calcd.
O: C, 72.19; H, 7.46; N, 12.95; Found: C, 72.21; H,
.48; N, 12.99.
for C13
H
16
N
2
Anal. calcd. for C34
46 2
H N O: C, 81.88; H, 9.30; N, 5.62; found: C,
7
81.98; H, 9.21; N, 5.75.
2
.2.2.15. Trans-2-(1H-pyrazol-1-yl)cyclohexanol (22). White solid,
2.2.3.5. 3-(Morpholino)-3,5-dien-cholest-7-one (27). Thick yellow li-
quid; yield 68%; H NMR (CDCl , 300 MHz) d 0.67 (s, 3H), 0.80–2.45
3
(m, 24H), 0.86 (d, J = 6.6 Hz, 6H), 0.92 (d, J = 6.4 Hz, 3H), 1.41(s, 3H),
ꢁ1
1
m.p. 65–66 °C; yield 80%; IR (CHCl3, cm ): 3447, 2931, 1245,
1
1
123; H NMR (CDCl
3
, 300 MHz): d 1.29–1.35 (m, 3H), 1.70–1.77
(
6
m, 3H), 2.00–2.07 (m, 2H), 3.69–3.85 (m, 2H), 4.07 (br s, 1H),
2.46 (t, J = 4.0 Hz, 4H), 3.67 (t, J = 4.2 Hz, 4H), 5.27 (s, 1H), 5.79 (s,
1H); C NMR (CDCl , 75 MHz) d 12.0, 17.4, 18.9, 22.6, 22.7, 22.8,
3
1
3
13
.17 (d, J = 2 Hz, 1H), 7.39 (d, J = 2.1 Hz, 1H), 7.43 (s, 1H);
C
NMR (CDCl
28.5, 139.1; MS (EI, m/z) = 166 (M ). Anal. calcd. for C
C, 65.03; H, 8.49; N, 16.85; Found: C, 65.25; H, 8.58; N, 16.99.
3
, 75 MHz): d 24.1, 24.8, 31.2, 33.6, 66.9, 72.9, 105.0,
22.9, 26.3, 28.0, 29.7, 35.7, 36.1, 38.5, 38.9, 39.0, 39.5, 43.0, 45.9,
3
50.0, 52.3, 54.8, 66.7, 71.9, 123.9, 132.8, 159.7, 201.8; IR (CHCl ,
+
1
H
9 14
N
2
O:
ꢁ1
cm ): 2952, 1672, 1457, 1355, 1177, 923; MS (EI, m/z) = 467
+
[
M] . Anal. calcd. for C31
2
H49NO : C, 79.60; H, 10.56; N, 2.99; found:
2.2.3. Preparation of N-(1-cycloalkenyl)heterocycles (6)
C, 79.81; H, 10.42; N, 3.27.
A stirring solution of vicinal hydroxy compound (200 mg) and
catalytic amount of sulfuric acid in acetic acid (0.5 mL) was heated
at 80 °C for 4–6 h. Acetic acid was removed in vacuo and ethylace-
2.2.3.6. 3b-Acetyl-6-(1H-imidazolo)-cholest-5-en (28). Yellow solid;
m.p. 99–102 °C; yield 81%; H NMR (CDCl , 300 MHz) d 0.71
3
1
tate (30 mL) and saturated solution of NaHCO
3
were added into the
(s, 3H), 0.80–2.40 (m, 28H), 0.85 (d, J = 6.6 Hz, 6H), 0.92 (d,
residue. The ethylacetate layer was washed with brine and the res-
idue obtained after removal of the ethylacetate layer was purified
by silica gel column chromatography using EtOAc/hexane as the
eluent to afford the steroidal/nonsteroidal N-(1-cycloalkenyl)het-
erocycle derivative.
J = 6.5 Hz, 3H), 1.24 (s, 3H), 2.00 (s, 3H), 4.47–4.55 (m, 1H), 6.85
(s, 1H), 7.10 (s, 1H), 7.43 (s, 1H); C NMR (CDCl , 75 MHz) d
3
1
3
11.8, 18.7, 19.6, 21.2, 22.5, 22.8, 23.8, 24.1, 27.4, 28.0, 28.1, 31.9,
35.7, 36.1, 36.9, 37.4, 39.5, 42.3, 49.5, 56.0, 56.2, 72.8, 119.2,
ꢁ
1
3
129.0, 129.3, 136.7, 136.9, 170.2. IR (CHCl , cm ): 2928, 1733,
+
1
C
584, 1020; MS (EI, m/z) = 494.4 [M] . Anal. calcd. for
: C, 77.68; H, 10.19; N, 5.66; found: C, 77.51; H,
2
.2.3.1. 3-(1H-Imidazolo)-3,5-dien-cholest-7-one (23). Yellow solid;
32 50 2 2
H N O
1
m.p. 182–186 °C; yield 88%; H NMR (CDCl
H), 0.80–2.50 (m, 24H), 0.85 (d, J = 6.7 Hz, 6H), 0.93 (d,
J = 6.4 Hz, 3H), 1.22 (s, 3H), 5.09 (s, 1H), 6.25 (t, J = 4.2 Hz, 1H),
3
, 300 MHz) d 0.72 (s,
10.04; N, 5.84.
3
2.2.3.7. 3b-Acetyl-6-(1H-benzo[d]imidazolo)-cholest-5-en (29). Yel-
.88 (s, 1H), 7.10 (s, 1H), 7.51 (s, 1H); 13C NMR (CDCl
low solid; m.p. 105–108 °C; yield 83%; H NMR (CDCl
1
6
1
2
3
, 75 MHz) d
3
, 300 MHz)
2.0, 12.6, 14.1, 16.6, 18.9, 21.4, 22.6, 22.7, 22.8, 23.8, 26.2, 28.0,
8.5, 29.4, 29.7, 31.9, 32.4, 35.7, 36.1, 37.3, 39.5, 43.2, 45.7, 49.1,
d 0.70 (s, 3H), 0.80–2.42 (m, 28H), 0.85 (d, J = 6.6 Hz, 6H), 0.92
(d, J = 6.5 Hz, 3H), 1.23 (s, 3H), 2.11 (s, 3H), 4.74–4.85 (m, 1H),

4
0
P. Saikia et al. / Steroids 84 (2014) 36–45
7
.00–7.23 (m, 3H), 7.64–7.68 (m, 1H), 7.70 (s, 1H); IR (CHCl
3
,
3. Results and discussion
ꢁ
1
+
cm ): 2948, 1733, 1462, 1225; MS (EI, m/z) = 544.4 [M] . Anal.
calcd. for C36 : C, 79.36; H, 9.62; N, 5.14; found: C, 79.42;
H, 9.67; N, 5.06.
H
52
N
2
O
2
3.1. Chemistry
In general, epoxide rings can be opened by different nucleo-
philes in presence of acidic or basic catalysts [16]. Epoxide ring
opening by the nucleophiles imidazoles and benzimidazoles has
medicinal importance because the products 1-(2-hydroxyal-
kyl)imidazoles and 1-(2-hydroxyalkyl)benzimidazoles obtained
are biologically important compounds due to their antifungal prop-
erties [17]. Epoxide ring opening with imidazole nucleophiles can
2
7
.2.3.8. 1-(Cyclohex-1-en-1-yl)-1H-imidazole (30). Brown oil, yield
9%; H NMR (CDCl , 300 MHz): d 1.64–1.72 (m, 2H), 1.78–1.86
3
1
(
(
2
m, 2H), 2.17–2.22 (m, 2H), 2.41–2.43 (m, 2H), 5.83 (s, 1H), 7.07
s, 1H), 7.09 (s, 1H), 7.66 (s, 1H). 13C NMR (CDCl
, 75 MHz): d
1.6, 22.3, 24.0, 27.3, 116.4, 116.5, 129.2, 133.7, 134.5; IR (CHCl
3
3
,
ꢁ
1
+
cm ): 2927, 1661, 773; MS (EI, m/z) = 148 (M ). Anal. calcd. for
C
1
be performed using LiBr [3], strong base [18], Lewis acid [Yb(OTf)
3
]
9 12 2
H N : C, 72.94; H, 8.16; N, 18.90; found: C, 72.98; H, 8.01; N,
8.72.
[
19] and same epoxide ring opening with benzimidazole can be
performed under strong basic conditions [18]. In addition, the
synthesis of pharmaceutically important compounds N-(1-cyclo-
hex-1-enyl)imidazole (30) and N-(1-cyclohex-1-enyl)pyrazole
(31) was carried out by Katritzky and co-workers via elimination
of benzotriazole or 5-phenyltetrazole from the corresponding
2
.2.3.9. 1-(Cyclohex-1-en-1-yl)-1H-pyrazole (31). Brown liquid,
1
yield 72%; H NMR (CDCl
.89 (m, 2H), 2.18–2.27 (m, 2H), 2.53–2.60 (m, 2H), 6.11–6.13
m, 1H), 6.31–6.32 (m, 1H), 7.59 (d, J = 2.0 Hz, 1H), 7.61 (d,
3
, 300 MHz): d 1.61–1.71 (m, 2H), 1.80–
1
(
1
-[1-(heterocycyl)cycloalkyl]-benzotriazoles or 1-[1-(heterocycyl)
1
3
cyclohexyl]-5-phenyltetrazole [20]. Moreover, Ogata and co-
workers found N-(1-cyclohex-1-enyl)imidazole (30) as a byproduct
in the preparation of 1,1-bis-(1H-imidazol-1-yl)cyclohexane [21].
We started the synthesis of epoxide 4a from commercially
available cholesterol acetate (1) via allylic oxidation, acid catalyzed
elimination and epoxidation pathway [11–13] (Scheme 1). This
3
J = 2.4 Hz, 1H); C NMR (CDCl , 75 MHz): d 21.8, 22.4, 24.1, 25.9,
1
7
ꢁ
1
05.9, 113.8, 125.8, 136.5, 139.7; IR (CHCl
3
, cm ): 2926, 1657,
+
68; MS (EI, m/z) = 148 (M ). Anal. calcd. for C : C, 72.94;
9
12 2
H N
H, 8.16; N, 18.90; found: C, 72.81; H, 8.11; N, 18.58.
epoxidation afforded only
a-epoxide 4 due to the presence of
2.3. Biology
angular methyl group at C-10 position of the steroid moiety [13].
Finally, the microwave irradiation of an equimolar mixture of
epoxide 4 and imidazole in a closed vessel in a Synthos 3000
microwave reactor at 600 Watt (140 °C and 12 bar) for 6 min affor-
ded compound 3-(1H-imidazol-yl)-4-hydroxy-5-en-cholest-7-one
The antibacterial assay was performed following standard pro-
tocols and using test pathogens E. coli, P. syringae, B. subtilis, P. vul-
garis and S. aureus. The antibacterial activity of all the compounds
was tested using agar diffusion assay method [15] by observing
presence of inhibition zone (in mm) around the well. All tested
bacteria were grown in Mueller Hinton broth (MHB) at 37 °C for
(
5) in 69% yield (Table 1, entry 1). Similarly, microwave reaction
of steroidal epoxide 4 with different imidazole derivatives and
benzimidazole afforded -hydroxy-3b-heterosteroids 6–8
Table 1, entry 2–4). This reaction condition was also effective for
4
a
2
4 h. Whole experiments were carried out in a laminar flow to
(
strictly maintain aseptic conditions. 100 L of fresh inoculum from
l
opening of steroidal epoxide with different N-containing heterocy-
cles. For example, the ring opening reaction of steroidal epoxide 4
with different heterocycles such as pyrrolidine, morpholine, thi-
omorpholine and tetrahydroisoquinoline under the above reaction
each culture was added on the plates and spread using sterilized
spreader. Wells were punctured on freshly spread bacterial culture
on MHA using sterile cork borer. The solution of test compound
was prepared in DMSO solvent (Stock concentration 25 mg/mL)
condition afforded 4
yields (Table 1, entry 5–8). On the other hand, cholesterol acetate
1) on epoxidation with mCPBA provided a mixture of epoxides
3 ( ) and 14 (b) (13/14 = 4:1) (Scheme 2) [14]. The and b ratio
a-hydroxy-3b-heterosteroids 9–12 in good
and the bored wells were filled with test compound (50 lg). Four
wells were bored on each plate, each filled with same compound
and two plates for each test compound were taken. All the plates
were incubated at 37 °C for 24 h. Growth inhibition of test organ-
isms was measured with standard scale, the mean values of inhibi-
tion zones were taken and the data were compared with standard
drug kanamycin acid sulphate.
(
1
a
a
of compounds 13 and 14 was determined by the integration of the
1
C-6 proton signals in the H NMR spectra of the crude mixture of
epoxides 13 and 14 (d = 2.84–2.91 for the
a-epoxide and
i
ii
iii
AcO
AcO
O
O
Chloesterol acetate (1)
2
3
iv
Het
O
O
O
OH
4
5-12
t
Scheme 1. Reagents and conditions: (i) BuOOH, RuCl
3
, cyclohexane, rt, 4 h; (ii) HCl, MeOH, reflux, 1 h; (iii) mCPBA, CHCl
3
, rt, 5 h; (iv) N-heterocycles, microwave, neat,
6
–16 min.

P. Saikia et al. / Steroids 84 (2014) 36–45
41
Table 1
Opening of steroidal/nonsteroidal epoxide rings by N-containing heterocycles.
Entry
1
N-Heterocycle + epoxide
Vicinal heterocyclic alcohol
Time (min)
6
Yielda (%)
69
H
N
+
4
N
N
O
OH
N
5
2
H
N
6
62
+
4
N
O N
2
N
O
O N
2
OH
N
6
3
H
6
66
N
+
4
N
OHC
N
O
OHC
OH
N
7
4
N
6
65
+
4
N
H
N
O
N
OH
8
9
5
10
68
+
4
N
H
N
O
OH
6
O
10
74
+
+
4
N
H
N
O
O
S
OH
1
0
7
S
10
69
4
N
H
N
O
OH
11
8
10
77
NH
+
4
N
O
OH
12
(
continued on next page)

4
2
P. Saikia et al. / Steroids 84 (2014) 36–45
Table 1 (continued)
Entry
9
N-Heterocycle + epoxide
Vicinal heterocyclic alcohol
Time (min)
10
Yielda (%)
75b
H
N
+
(13 + 14)
N
AcO
HO
N
15
N
1
0
N
10
71b
+
(13 + 14)
N
H
AcO
HO N
N
N
1
6
1
1
10
79
N
O
+
N
N
H
1
7
OH
1
8
1
1
2
3
NO2
N
N
10
10
75
76
N
N
NO2
N
+
17
N
H
OH
1
9
CHO
CHO
N
+
17
N
H
OH
2
0
1
1
4
5
N
16
10
78
80
+
17
N
H
N
N
OH
21
N
+
17
N
N
H
N
OH
2
2
a
Referring to the amount of product isolated by chromatography.
Recrystallized from ethanol.
b
i
+
AcO
AcO
AcO
O
13 (α)
O
Chloesterol acetate (1)
14 (β)
13 (α): 14 (β) = 4:1
OH
Het
ii
ii
O
AcO
HO
Het
1
5-16
Cyclohexene oxide (17)
18-22
Scheme 2. Reagents and conditions: (i) m-CPBA, CHCl
3
, rt, 5 h; (ii) N-heterocycles, microwave, neat, 10 min, recrystallization.

P. Saikia et al. / Steroids 84 (2014) 36–45
43
(
δ = 0.75)
benzimidazolyl alcohols, which on recrystalliztion in ethanol
provided pure imidazolyl alcohol 15 and benzimidazolyl alcohol
1
8
H
1
7
1
1
6 (Table 1, entries 9 and 10). In the NOESY spectrum of compound
1
1
1
9
13
0
5, the presence of correlation of CH
3
-19 protons with H-2 and
1
16
14
H
2'
9
0
H-5 also indicated that imidazole group of 15 was b oriented
1
0 H
H
(Fig. 2). Similarly, microwave reaction of nonsteroidal epoxide 17
3
5
7
with different imidazole derivatives, benzimidazole and pyrazole
provided compounds 18–22 in good yield (Table 1, entries 11–15).
In the next step, the microwave induced dehydration reaction of
compound 5 was studied. We observed that increasing the time of
the epoxide opening reaction from 6 to 25 min for the synthesis of
compounds 5–8 afforded the water eliminated compounds 23–26
in moderate yields (23 = 66%, 24 = 55%, 25 = 56% and 26 = 61%,
Scheme 3). Surprisingly, all of our attempts to carry out the dehy-
dration reaction of all other compounds by increasing the time of
the reaction met with failure. Then we found that stirring a solu-
tion of compound 5 and catalytic amount of sulfuric acid in acetic
AcO
δ = 7.01)
OH
1
'
N
(
H
H
(δ = 7.70)
5
'
N
Fig. 2. Key NOESY correlations and relative stereochemistry of compound 15.
d = 3.04–3.12 for the b-epoxide) [22]. When equimolar amount of
mixture of epoxides 13 and 14 was treated with imidazole and
benzimidazole by following above condition under microwave
we obtained
a
diastereomeric mixture of imidazolyl and
i
ii
Het
O
Het
O
Het
O
OH
23-27
5-8, 10
23-26
Scheme 3. Reagents and conditions: (i) H
2
SO
4
3
(cat.), CH COOH, 80 °C, 4; (ii) N-hyterocycles, microwave(140 °C and 12 bar), neat, 25 min.
Table 2
Synthesis of steroidal and nonsteroidal N-(1-cycloalkenyl)heterocycles.
Entry
1
Vicinal heterocyclic alcohol
Steroidal/nonsteroidal N-(1-cycloalkenyl)heterocycles
Time (h)
4
Yielda (%)
88
5
N
O
N
23
2
3
4
6
7
8
4
4
4
83
75
88
N
O
O N
2
N
2
4
N
O
OHC
N
2
5
N
O
N
2
6
(
continued on next page)

4
4
P. Saikia et al. / Steroids 84 (2014) 36–45
Table 2 (continued)
Entry
5
Vicinal heterocyclic alcohol
Steroidal/nonsteroidal N-(1-cycloalkenyl)heterocycles
Time (h)
6
Yielda (%)
68
10
N
O
O
27
6
15
4
81
AcO
N
28
N
7
16
4
83
AcO
N
N
2
9
8
9
18
22
N
4
6
79
72
N
N
30
N
31
a
Referring to the amount of product isolated by chromatography.
OH
Het
i
i
AcO
AcO
Het
HO
Het
Het
18, 22
30-31
28-29
15-16
Scheme 4. Reagents and conditions: (i) H
2 4 3
SO (cat.), CH COOH, 80 °C, 4–6 h.
sulphate (Table 3). As shown in Table 3 only few of the synthesized
steroidal N-heterocyclic derivatives (6, 9–12, 24 and 27) showed
moderate in vitro antimicrobial activity against the tested microor-
ganisms. The steroidal derivatives 9, 10 and 11 were effective in
inhibiting all the tested bacterial strains. The tetrahydroisoquino-
line substituted steroidal derivative 12 showed inhibition against
three Gram-negative bacterial strains E. coli, P. syringe and P. vulga-
ris. Among the steroidal imidazoles only nitro group substituted
steroidal imidazole 6 showed inhibition against S. aureus and B.
subtilis. Removal of hydroxyl group of this steroidal derivative led
to decrease in the antimicrobial activity of the compounds as seen
for compounds 24 and 27. Compound 24 showed inhibition only
against bacterial strain S. aureus whereas compound 27 showed
inhibition only against three Gram-negative bacterial strains
acid at 80 °C for 4 h also provided compound 23 in good yield
(Table 2, 88%, entry 1).
This reaction condition was efficiently used for the dehydration
reactions of other hydroxyl compounds 6–8, 10, 15–16, 18 and 22
to obtain steroidal and nonsteroidal N-(1-cycloalkenyl)heterocy-
cles 24–31 in good yields (Table 2, entries 2–9), although the dehy-
dration reaction of compounds 10 and 22 took longer time (6 h) to
provide dehydrated compounds 27 and 31 respectively. (see
Scheme 4).
3.2. Biology
The antimicrobial activity of compounds 5–12, 15–16, 18–31
was evaluated and compared with standard drug kanamycin acid

P. Saikia et al. / Steroids 84 (2014) 36–45
Bioorg Med Chem 2006;14:4341–52;
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9
1
1
1
2
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–
–
–
18.0 ± 0.5
12.06 ± 0.6
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