Synthesis and?in?vitro antibacterial activity of?novel heterocyclic derivatives of?18-nor-equilenin

Article abstract of DOI:10.1016/j.ejmech.2006.01.018

Syntheses of novel heterocyclic derivatives of 18-nor-equilenin, namely, (12H-11-oxa-17-thia-15-aza-cyclopenta[a]phenanthrene-16-yl)-hydrazine (4a/b) and its fused [1,2,4]triazolo derivatives6H-5-oxa-7-thia-8,9,10a-triaza-pentaleno[4,5-a]phenanthrene (5a/b), 10-methyl-6H-5-oxa-7-thia-8,9,10a-triaza-pentaleno[4,5-a]phenanthrene (6a/b) and tetrazolo derivatives 1-substituted-6H-5-oxa-7-thia-8,9,10,10a-tetraaza-pentaleno[4,5-a]phenan threne (7a/b) along with their antibacterial activities are reported.

Full text of DOI:10.1016/j.ejmech.2006.01.018

European Journal of Medicinal Chemistry 41 (2006) 891–895
http://france.elsevier.com/direct/ejmech
Short communication
Synthesis and in vitro antibacterial activity of novel
heterocyclic derivatives of 18-nor-equilenin
A.V. Karnik a,*, N.J. Malviya a, A.M. Kulkarni a, B.L. Jadhav b
a Department of Chemistry, University of Mumbai, Vidyanagari, Kalina, Mumbai 400098, India
b Department of Life Sciences, University of Mumbai, Vidyanagari, Kalina, Mumbai 400098, India
Received 20 August 2005; received in revised form 3 January 2006; accepted 12 January 2006
Available online 26 May 2006
Abstract
Syntheses of novel heterocyclic derivatives of 18-nor-equilenin, namely, (12H-11-oxa-17-thia-15-aza-cyclopenta[a]phenanthrene-16-yl)-hy-
drazine (4a/b) and its fused [1,2,4]triazolo derivatives6H-5-oxa-7-thia-8,9,10a-triaza-pentaleno[4,5-a]phenanthrene (5a/b), 10-methyl-6H-5-oxa-
7-thia-8,9,10a-triaza-pentaleno[4,5-a]phenanthrene (6a/b) and tetrazolo derivatives 1-substituted-6H-5-oxa-7-thia-8,9,10,10a-tetraaza-pentaleno
[4,5-a]phenanthrene (7a/b) along with their antibacterial activities are reported.
© 2006 Elsevier SAS. All rights reserved.
Keywords: 1,2,3,4-Tetrazol; 1,3,4-Triazole; Heterocyclic analog of 18-nor-equilenin; Heterosteroid; Azasteroid; Thiasteroid; Oxasteroid and antibacterial activity
1. Introduction
present antibiotic resistant bacteria’s, the discovery and develop-
ment of newer antibacterial compounds have got importance and
many groups are working towards the development of novel
antibacterial agents [16]. The present paper discusses the synth-
esis of 2-hydrazino heterocyclic derivatives of 18-nor-equilinin
namely, 1-substituted-16-(12H-11-oxa-17-thia-15-aza-cyclopen-
ta[a]phenanthrene-16-yl)-hydrazine (4a, b) and its [1,2,4]triazolo
derivatives: 1-substituted-6H-5-oxa-7-thia-8,9,10a-triaza-penta-
leno[4,5-a]phenanthrene (5a, b), 1-substituted-10-methyl-6H-5-
oxa-7-thia-8,9,10a-triaza-pentaleno[4,5-a]phenanthrene (6a, b)
and tetrazolo derivatives: 1-substituted-6H-5-oxa-7-thia-
8,9,10,10a-tetraaza-pentaleno[4,5-a]phenanthrene (7a, b).
Steroids are widely distributed in nature and its members dis-
play much diversity in molecular structure [1]. Heterosteroids
comprise of molecules with structural features characteristic of
steroids such as tetra-cyclic cyclopenta[a]phenanthrene frame-
work constituting ABCD ring system, also compounds where
additional rings are fused to the main tetra-cyclic framework
[2]. Heteroatom/s can be a part of ABCD ring system or other
fused ring/s. Heterosteroids are widely distributed in nature [3].
The heterosteroidal compounds containing various combinations
of heteroatoms are reported to exhibit diverse bioactivities [4–
11] and have led to the synthesis of many modified steroidal
hormones, some of which are now in use as drugs [12,13]. Azas-
teroids are by far the most common of the heterocyclic steroids
[13]. Incorporation of oxygen in some steroidal system has re-
sulted in enhanced bioactivity [14] in many cases, while the in-
troduction of sulfur instead of methylene group in the steroidal
system brings about electronic and steric changes in the structure
and was considered to play an important role in the properties of
the molecules [14,15]. Due to development of large number of
2. Chemistry
3-Bromonaphthopyran (2a) and its 3,7-dibromo derivative
(2b) were found to be the right precursors for the synthesis of
4a, b as they provided the ABC ring of the steroidal system.
4a/b could be easily obtained in a high yield process by treat-
ment of 2a/b with thiosemicarbazides. The hydrazine moiety in
4a/b was effectively used for the construction of triazolo and
tatrazolo fused systems in 5a/b, 6a/b and 7a/b, respectively.
The reaction of formic acid with 4a/b gave 5a/b in 3 h with
excellent yields (90–92%). The reaction of acetic anhydride
* Corresponding author.
E-mail address: avkarnik@hotmail.com (A.V. Karnik).
0223-5234/$ - see front matter © 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.01.018

892
A.V. Karnik et al. / European Journal of Medicinal Chemistry 41 (2006) 891–895
with 4a/b gave 6a/b in 5 h with similar excellent yields (87–
89%). Whereas reaction of 4a/b with nitrous acid gave 7a/b at
r.t. in less than 1 h with equally excellent yields (89–91%).
4. Results and discussion
The anti bacterial activity of compounds 4a/b, 5a/b, 6a/b
and 7a/b were assessed in side-by-side comparison with amox-
icillin and vancomycin against some gram-positive [S. aureus,
B. subtilis] and gram-negative [E. coli, P. aeruginosa] bacteria
using agar cup method [17] and results are summarized in Ta-
bles 1 and 2 while the result of MIC of standards have been
reported in Table 3. The antibacterial activity data indicate that
compounds 4a/b, 5a/b, 6a/b and 7a/b showed good activity
against all the tested bacteria’s. Compound 5b was found to
be most potent against all the organisms tested at MIC
50 μg ml−1 except P. aeruginosa where the MIC was found
out to be 100 μg ml−1. Compounds 5a and 7b were also found
to be very potent as well. Synthesis of newer bioactive hetero-
cycles have received greater imputes due to development of
various present time drug resistant pathogens and discovery
of various newer diseases [16]. Some of the desired character-
istics of potential drug candidates are the feasibility of chemi-
cal synthesis at the gram level (and preferably at the kilogram
level), and demonstrated potency as measured in in vitro cell
culture assay [20]. The newer antibacterial agents which have
structural similarity to eqilenin/nor-equilenin [13], with demon-
3. Pharmacology
The in vitro antibacterial activity of these compounds, 4a/b,
5a/b, 6a/b and 7a/b (1000, 500, 250, 100, 50 μg ml–1) were
tested against gram-positive [Staphylococcus aureus, Bacillus
subtilis] and gram-negative bacteria [Escherichia coli, Pseudo-
monas aeruginosa] which were clinical isolates from Hafkkins
Laboratory, Mumbai (India). They were maintained on nutrient
agar (Himedia Lab. Ltd., India) slants at 4 °C prior to use for
antimicrobial susceptibility test. The bioassay of these com-
pounds was carried out by agar cup method [17] against test
microorganism. All these tests were carried out in triplicate and
the average values for zone of inhibition in millimeters (mm)
were taken after 24 h of incubation. The compounds were dis-
solved in methanol. Amoxicillin [18] and vancomycin [19]
were used as positive control for both gram-positive gram-
negative bacteria’s. The zone of inhibition and minimum inhi-
bitory concentration (MIC) were noted. The results of such
studies are presented in Tables 1–3.
Table 1
Antibacterial screening data
Serial number
Compound
Zone of inhibition (mm) activity at concentration of 1000 μg ml−1
S. aureus
15
14
18
19
16
15
14
14
B. subtilis
E. coli
18
17
18
16
15
14
12
11
P. aeruginosa
1
2
3
4
5
6
7
8
4a
4b
5a
5b
6a
6b
7a
7b
14
13
15
16
12
11
14
12
16
18
09
08
11
10
09
07
06
07
11
19
Standard
Amoxicillin
Vancomycin
17
19
17
17
Table 2
Antibacterial screening data
Serial number
Compound
MIC in μg ml−1
S. aureus
500
250
100
50
250
100
250
100
B. subtilis
250
250
50
E. coli
500
100
50
P. aeruginosa
1
2
3
4
5
6
7
8
4a
4b
5a
5b
6a
6b
7a
7b
500
500
100
100
500
500
500
500
50
50
100
250
250
50
250
250
100
50
Table 3
Antibacterial screening data of standards used
Serial number
Compound
Zone of inhibition (mm) [MIC in μg ml−1
]
S. aureus
10 (100)
11 (50)
B. subtilis
11 (100)
14 (50)
E. coli
14 (100)
15 (50)
P. aeruginosa
13 (100)
12 (50)
Std 1
Std 2
Amoxicillin
Vancomycin

A.V. Karnik et al. / European Journal of Medicinal Chemistry 41 (2006) 891–895
893
Scheme 1.
strated potency were successfully synthesized by practically
simple and high yielding reactions (Scheme 1).
succinimide in the solid form was separated by filtration. The
organic layer was then concentrated to 1/4th the original vo-
lume and after over night refrigeration compound 2a/2b crys-
tallized out from the reaction mixture.
5. Experimental section
5.2. 3-Bromo-2,3-dihydro-4H-naphtho[1,2-b]pyran-4-one (2a)
[21]
Melting points reported are uncorrected. IR spectra were re-
corded on a Shimadzu FTIR-4200 spectrometer and H NMR
1
spectra were recoded on Varian EM-360L (60 MHz) using
TMS as an internal standard. Mass spectra were recorded on
GC-MSQP-1000. Elemental analyses were carried on Carlo
Enra EA-1108 elemental analyzer. TLC checked the homoge-
neity of compounds on silica gel.
Yield 2.63 (92%), m.p. 154 °C, IR (KBr): C=C 1560, 1600,
1
C=O 1680 cm−1. H NMR (CDCl3): δ 3.5(m, 3H, CH and
CH2) and 7.1–8.0 (m, 6H, Ar). Anal. Calcd. C 56.35, H 3.27,
Br 28.83. Found C 56.00, H 2.98, Br 28.60.
2,3-Dihydro-4H-naphtho[1,2-b]pyran-4-one [21] (1a) and
its 7-bromo-2,3-dihydro-4H-naphtho[1,2-b]pyran-4-one [22]
(1b) derivative were synthesized as reported in the literature.
5.3. 3,7-Dibromo-2,3-dihydro-4H-naphtho[1,2-b]pyran-4-
one (2b)
Yield 3.41 g (96%), m.p. 220 °C, IR (KBr): C=C 1580,
5.1. Synthesis of 3-bromo-2,3-dihydro-4H-naphtho[1,2-b]
pyran-4-one (2a) and 3,7-dibromo-2,3-dihydro-4H-naphtho
[1,2-b]pyran-4-one (2b)
1
1610, C=O 1685 cm−1. H NMR(CDCl3): δ 3.8(m, 3H, CH
and CH2) and 7.2–7.9 (m, 5H, Ar). Anal. Calcd. C 43.86 H
2.27, Br 44.89. Found C 43.50, H 1.98, Br 44.62.
A mixture containing 2,3-dihydro-4H-naphtho[1,2-b]pyran-
4-one 1a (1.96 g, 0.01 mol)/7-bromo-2,3-dihydro-4H-naphtho
[1,2-b]pyran-4-one 1b (2.77 g, 0.01 mol) and N-bromo succi-
nimide (1.78 g, 0.01 mol) along with catalytic amount of ben-
zoyl peroxide (20 mg) was refluxed in carbon tetrachloride
(30 ml) for 4 h. The reaction mixture was then cooled and
5.4. (2H-11-oxa-17-thia-15-aza-cyclopenta[a]phenanthrene-
16-yl)-hydrazine (4a)
3-Bromo-2,3-dihydro-4H-naphtho[1,2-b]pyran-4-one (2a)
(2.77 g, 10 mmol) was heated with thiaosemicarbazide (3)

894
A.V. Karnik et al. / European Journal of Medicinal Chemistry 41 (2006) 891–895
(0.91 g, 10 mmol) in 1,4-dioxane (10 ml) at 75–80 °C 4 h to
form the hydrobromide salt of 4a which precipitated out. The
reaction mixture was then cooled and vacuum filtered. The so-
lid was then stirred for 30 min with saturated solution of so-
dium bicarbonate. The solid was then filtered and heated with
an activated animal charcoal (0.5 g) in ethanol (25 ml) for
20 min. The filtrate on cooling to room temperature gave pale
yellow solid of (2H-11-oxa-17-thia-15-aza-cyclopenta[a]phe-
nanthrene-16-yl)-hydrazine (4a). Yield 2.47 g (92%), m.p.
220 °C (dec.), IR (KBr) (C=C) 1580, NH2 3200, 3250 and NH
5.8. 10-Methyl-6H-5-oxa-7-thia-8,9,10a-triaza-pentaleno[4,5-
a]phenanthrene (6a)
(12H-11-oxa-17-thia-15-aza-cyclopenta[a]phenanthrene-16-
yl)-hydrazine (4a) (2.69 g, 10 mmol) was refluxed with acetic
anhydride (25 ml) for 5 h. The reaction mixture was then
cooled and neutralized with saturated solution of sodium bicar-
bonate. The solid obtained was then boiled with 0.5 g of acti-
vated animal charcoal and ethanol (50 ml) for 30 min. The
filtrate on cooling gave light brown colored 10-methyl-6H-5-
oxa-7-thia-8,9,10a-triaza-pentaleno[4,5-a]phenanthrene (6a).
Yield 2.54 g (87%), m.p. 265 °C (dec.), IR (KBr) 1592,
1617, 2977, 3050 cm−1. 1H NMR (CDCl3) δ 2.5 (s, 3H,
CH3), 5.6 (s, 2H, CH2) and 7.1–8.0 (m, 6H, 6H-Ar), ms spec-
trum showed m/z 293 (M+), 294 (M + 1)+ and 295 (M + 2)+.
Anal. Calcd. C 65.53, H 3.75, N 14.33, S 10.92. Found C
65.38, H 3.50, N 14.03, S 10.72.
1
3400 cm−1. H NMR (CDCl3) δ 1.6 (s, 1H, NH), 1.9 (s, 2H,
NH2), 5.4 (s, 2H, CH2) and 7.3–8.3 (m, 6H, Ar), MS spectrum
showed m/z 269 (M+), 270 (M + 1)+ and 271 (M + 2)+. Anal.
Calcd. C 62.45, H 4.09, N 15.61, S 11.90. Found C 62.25, H
3.89, N 15.41, S 11.62.
5.5. 4-Bromo-(12H-11-oxa-17-thia-15-aza-cyclopenta[a]
phenanthrene-16-yl)-hydrazine (4b)
5.9. 1-Bromo-10-methyl-6H-5-oxa-7-thia-8,9,10a-triaza-
pentaleno[4,5-a]phenanthrene (6b)
Yield 3.33 g (96%) m.p. 241 °C (dec.), IR (KBr) 1580,
1
1620 NH2 3150, 3200 and NH 3300 cm−1. H NMR (CDCl3)
Yield 3.29 (89%), m.p. 292 °C (dec.), IR (KBr) 1595, 1627,
2930, 3070 cm−1. 1H NMR (CDCl3) δ 2.6 (s, 3H, CH3), 5.5 (s,
2H, CH2) and 7.1–7.9 (m, 5H, 5H-Ar), MS spectrum showed
m/z 370 (M+), 371 (M + 1)+, 372 (M + 2)+, 373 (M + 3)+, 374
(M + 4)+. Anal. Calcd. C 51.61, H 2.69, Br 21.51, N 11.29, S
8.60. Found C 51.36, H 2.42, Br 21.28, N 11.01, S 8.32.
δ 1.7 (s, 1H, NH), 1.9 (s, 2H, NH2), 5.3 (s, 2H, CH2) and 7.2–
8.1 (m, 5H, Ar). Anal. Calcd. C 48.28, H 2.87, Br 22.99, N
12.07, S 9.20. Found C 48.00, H 2.61, Br 22.72, N 11.80, S
9.00.
5.6. 6H-5-oxa-7-thia-8,9,10a-triaza-pentaleno[4,5-a]
phenanthrene (5a)
5.10. 6H-5-oxa-7-thia-8,9,10,10a-tetraaza-pentaleno[4,5-a]
phenanthrene (7a)
(12H-11-oxa-17-thia-15-aza-cyclopenta[a]phenanthrene-16-
yl)-hydrazine (4a) (2.69 g, 10 mmol) was refluxed with formic
acid (25 ml) for 3 h. The reaction mixture was then cooled and
neutralized with saturated solution of sodium bicarbonate. The
solid obtained was then boiled with 0.5 g of activated animal
charcoal and ethanol (50 ml) for 30 min. The filtrate on cooling
gave light brown colored compound 6H-5-oxa-7-thia-8,9,10a-
triaza-pentaleno[4,5-a]phenanthrene (5a). Yield 2.51 g (90%),
m.p. 250 °C (dec.), IR (KBr) 1554, 1596, 1621, 2919,
(12H-11-oxa-17-thia-15-aza-cyclopenta[a]phenanthrene-16-
yl)-hydrazine (4a) (2.69 g, 10 mmol) was added to 10% acetic
acid solution (30 ml) under stirring at r.t. To this reaction mix-
ture was added sodium nitrate (1.38 g, 20 mmol) in small lots.
The reaction was then stirred for further 1 h. The reaction mix-
ture was then neutralized with saturated solution of sodium bi-
carbonate. The solid obtained was then filtered, dried and then
boiled with 0.5 g of activated animal charcoal and ethanol
(50 ml) for 30 min. The filtrate on cooling gave light brown
colored 6H-5-oxa-7-thia-8,9,10,10a-tetraaza-pentaleno[4,5-a]
phenanthrene (7a). Yield 2.49 (89%), m.p. 270 °C (dec.), IR
1
3053 cm−1. H NMR (CDCl3) δ 5.6 (s, 2H, CH2) and 7.2–8.2
(m, 7H, 6H-Ar and C-10 H), ms spectrum showed m/z 279
(M+), 280 (M + 1)+ and 281 (M + 2)+. Anal. Calcd. C 64.52,
H 3.23, N 15.05, S 11.47. Found C 64.32, H 3.00, N 14.80, S
11.27.
(KBr) 1593, 1610, 2977, 3050 cm−1. H NMR (CDCl3) δ 5.4
1
(s, 2H, CH2) and 7.1–7.9 (m, 6H, 6H-Ar), ms spectrum
showed m/z 280 (M+), 281 (M + 1)+ and 282 (M + 2)+. Anal.
Calcd. C 60.00, H 2.86, N 20.00, S 11.43. Found C 59.80, H
2.62, N 19.72, S 11.19.
5.7. 11-Bromo-6H-5-oxa-7-thia-8,9,10a-triaza-pentaleno[4,5-
a]phenanthrene (5b)
5.11. 1-Bromo-6H-5-oxa-7-thia-8,9,10,10a-tetraaza-pentaleno
[4,5-a]phenanthrene (7b)
Yield 3.28 g (92%), m.p. 265 °C (dec.), IR (KBr) 1594,
1619, 2763, 2881 cm−1. 1H NMR (CDCl3) δ 5.4 (s, 2H,
CH2) and 7.2–8.0 (m, 6H, 5H-Ar and C-10 H), ms spectrum
showed m/z 357 (M+), 358 (M + 1)+, 359 (M + 2)+, 360 (M +
3)+, 361 (M + 4)+. Anal. Calcd. C 50.28, H 2.23, Br 22.35, N
11.73, S: 8.94. Found C 50.00, H 2.03, Br 22.07, N 11.48, S
8.69.
Yield 3.25 (91%), m.p. 283 °C (dec.), IR (KBr) 1597, 2924,
1
3067 cm−1. H NMR (CDCl3) δ 5.6 (s, 2H, CH2) and 7.2–8.1
(m, 5H, 5H-Ar), ms spectrum showed m/z 358 (M+), 359 (M +
1)+, 360 (M + 2)+, 361 (M + 3)+ and 362 (M + 4)+. Anal.

A.V. Karnik et al. / European Journal of Medicinal Chemistry 41 (2006) 891–895
895
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46.54, H 1.68, Br 22.00, N 15.34, S 8.64.
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(1991) 223–300.
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