Synthesis and in vitro antibacterial activity of novel steroidal (6R)-spiro-1,3,4-thiadiazoline derivatives

Article abstract of DOI:10.1002/jhet.1014

Novel steroidal (6R)-spiro-1,3,4-thiadiazoline derivatives were synthesized by the cyclization of steroidal thiosemicarbazones with acetic anhydride, screened in vitro against antibacterial activity using disc-diffusion method and the minimum inhibitory concentration. The results showed that steroidal thiadiazoline derivatives exhibited better antibacterial activity than the steroidal thiosemicarbazone derivatives. Chloro and acetoxy substituents on the 3β-position of the steroidal thiadiazoline ring increased the antibacterial activity. Among all the compounds, compound 7 and 8 were found better inhibitors of both types of bacteria (Gram-positive and Gram-negative) as compared to the respective drug amoxicillin. All the synthesized compounds were well characterized by spectroscopic methods such as IR, ¹H-NMR, ¹³C-NMR mass, and elemental analysis and their stereochemistry was also discussed.

Full text of DOI:10.1002/jhet.1014

1
452
Synthesis and In Vitro Antibacterial Activity of Novel Steroidal
6R)-Spiro-1,3,4-thiadiazoline Derivatives
Vol 49
(
a
a,b
Salman A. Khan * and Abdullah M. Asiri
a
Chemistry Department, Faculty of Science, King Abdul Aziz University, Jeddah 21589,
Saudi Arabia
b
The Center of Excellence for Advanced Materials Research, King Abdul Aziz University,
Jeddah 21589, Saudi Arabia
*
E-mail: sahmad_phd@yahoo.co.in
Received March 2, 2011
DOI 10.1002/jhet.1014
View this article online at wileyonlinelibrary.com.
Novel steroidal (6R)-spiro-1,3,4-thiadiazoline derivatives were synthesized by the cyclization of steroidal
thiosemicarbazones with acetic anhydride, screened in vitro against antibacterial activity using disc-diffusion
method and the minimum inhibitory concentration. The results showed that steroidal thiadiazoline deriva-
tives exhibited better antibacterial activity than the steroidal thiosemicarbazone derivatives. Chloro and acet-
oxy substituents on the 3b-position of the steroidal thiadiazoline ring increased the antibacterial activity.
Among all the compounds, compound 7 and 8 were found better inhibitors of both types of bacteria
(
Gram-positive and Gram-negative) as compared to the respective drug amoxicillin. All the synthesized
1 13
compounds were well characterized by spectroscopic methods such as IR, H-NMR, C-NMR mass, and
elemental analysis and their stereochemistry was also discussed.
J. Heterocyclic Chem., 49, 1452 (2012).
INTRODUCTION
alternate pathways for survival [3,4]. Hence, the present
work is aimed towards developing novel molecules with
improved potential for treating bacterial infections and
with decreased probability for developing drug resistance.
Five member heterocyclic compounds with sepal refer-
ences of sulfur canting such as thiazole, thiazolidinone,
and thiadiazoline are abundant in nature and are of great
significance to life because their structural subunits exist
in many natural products such as vitamins, hormones, anti-
biotics etc. Compounds with a thiadiazoline structure are
known to possess tranquilizing, muscle relaxing, psycho-
analeptic, hypnotic, ulcerogenic, antidepressant, antibacte-
rial, antifungal, analgesic, and antiinflammatory properties
[5–10]. Recently a number of thiazolidinone derivatives
were synthesized and their potential antibacterial activity
has been studied in our laboratory [11]. It is evident from
the literature that no work has been done on steroidal
Infectious diseases are the main cause of mortality in the
world and the resistance of pathogenic bacteria towards
available antibiotics is rapidly becoming a major world-
wide problem. Hence, the design of new compounds to
deal with resistant bacteria has become one of the most im-
portant areas of antibacterial research today. Food poison-
ing, rheumatic, salmonellosis, and diarrhea are the second
leading cause of death from bacterial disease world-wide
[
1]. More than 50 million people worldwide are infected
and up to 150,000 die every year because of these bacterial
infections [2]. Moreover, drug resistance in food poison-
ing, rheumatic, salmonellosis, and diarrhea can be attrib-
uted to the use of drugs (amoxicillin, norfloxacin,
ciprofloxacin, and chloramphenicol) for treatment and to
the adaptation of the bacterial parasite by developing
©
2012 HeteroCorporation

November 2012
Synthesis and In Vitro Antibacterial Activity of Novel Steroidal
6R)-Spiro-1,3,4-thiadiazoline Derivatives
1453
(
(
cholesterol) thiadiazoline derivative screening on bacteria.
pattern of these compounds has given in the in experimen-
tal section.
Considering the facts, that nearly all the classes of the cy-
clic thiosemicarbazones are biologically active, and as a
part of our continuous efforts towards the development of
more potent antibacterial agents, we herein report the syn-
thesis, characterization, and in vitro antibacterial activity of
novel spiro-1,3,4-thiadiazoline derivatives.
Stereochemistry of spiro1,3,4-thiadiazolines. All the
1
,3,4-thiadiazolines derivatives (7–9) have C6─S bond
axially and C6─NAc bond as equatorially oriented.
Although, sulfur atom is more bulky than the nitrogen
but NAc group becomes more bulky than the sulfur
atom, so during cyclization (Scheme 1) the thiadiazoline
ring preferably closes at C-6 from the front (b, axial) side
by the attack of sulfur (C═S) of the thiosemicarbazone
moiety and as the bulky group, already attached at C-6, is
moved toward back (a, equatorial) side to avoid the 1,3-
diaxial interactions mainly because of C10b-Me, and
leaving front (b, axial) side for the attack of incoming
group. So the compounds (7-9) of this reaction have R
geometry at C-6 in which C6─S and C6─NAc bonds are
oriented axially (b) and equatorially (a), respectively.
This arrangement (geometry) provides greater stability to
the molecule as they have less 1,3-diaxial interactions.
The mechanism (Scheme 2) for the formation of 1,3,4-
thiadiazolines can also explained on the basis of the hard
soft acid and base principle [17]. The harder acetylating
reagent reacts with the harder nitrogen atom rather than
the softer sulfur atom and this acetylation favors cycliza-
tion of thiosemicarbazones to 1,3,4-thiazolines.
RESULTS AND DISCUSSION
Chemistry.
The starting material steroidal thiosemi-
carbazones were prepared by condensing the steroidal
ketones with p-toludinethiosemicarbazide in the presence
of catalytic amount of conc. HCl [12]. 3b-acetoxycholest-
6
6
methods. The 1,3,4-thiadiazoline derivatives were
synthesized according to the literature procedure by the
acetylation of steroidal thiosemicarbazone derivatives as
shown in Scheme 1 [16]. All the compounds were
soluble in DMSO and ethanol. The structures of all the
compounds were established on the basis of spectral
studies such as IR, HNMR, FAB mass spectra, and the
elemental analyses were carried out to check the purity of
the compounds.
-one [13], 3b-chloro-cholest-6-one [14], and 5a-cholest-
-one [15] were prepared according to the published
1
Assignments of selected characteristic IR band positions
provide significant indication for the formation of the
cyclized thiadiazoline analogs of thiosemicarbazones. All
the compounds showed v (C═N) stretch at 1652–1658
Antibacterial activity. Disc–diffusion and micro dilution
assay. The compounds (4–9) were tested for their
antibacterial activities by disc-diffusion method using
nutrient broth medium [contained (g/L): beef extract 3 g;
peptone 5 g; pH 7.0] [18]. The Gram-positive bacteria
and Gram-negative bacteria utilized in this study
consisted of Staphylococcus aureus, Streptococcus
pyogenes, Salmonella typhimurium, and Escherichia
coli. In the disc-diffusion method, sterile paper discs
(0.5 mm) impregnated with compound dissolved in
dimethylsulfoxide (DMSO) at concentration 100 mg/mL
were used. Then, the paper discs impregnated with the
solution of the compound tested were placed on the
surface of the media inoculated with the microorganism.
ꢀ
1
cm due to the ring closure. In addition, the absorption
ꢀ
1
band at 1162–1168 cm was attributed to the v (C─N)
stretch vibrations. The compounds showed intense bands
ꢀ
1
at 642–648 cm due to v (C─S) stretch, which also con-
firm the formation of thiazole ring in all the compounds.
Further evidence for the formation of thiadiazoline com-
1
pounds was obtained from the H-NMR spectra, which
provide diagnostic tools for the positional elucidation of
the protons. Assignments of the signals are based on the
chemical shifts and intensity patterns. The acetylation pro-
ton thiadiazoline ring of all the compounds is shown as sin-
ꢁ
The plates were incubated at 35 C for 24 h. After
1
3
glet in the range 2.12–2.26 ppm. C NMR spectra of the
incubation, the growth inhibition zones are shown in
Table 1. The thiadiazoline derivatives were further
checked by minimum inhibitory concentration (MIC)
method. The results are presented in Table 2. The
molecular structure of these active compounds showed
enhanced activity. The distinct differences in the
antibacterial property of these compounds further justify
the purpose of this study. The importance of such work
lies in the possibility that the new compound might be
more efficacious drugs against bacteria for which a
thorough investigation regarding the structure-activity
relationship, toxicity, and their biological effects, which
could be helpful in designing more potent antibacterial
agents for therapeutic use.
compounds (7-9) were taken in CDCl and the signal
3
obtained further confirmed the proposed structures. All
the compounds showed a signal at (146-7-155.8) ppm
due to (N═C─S) and (47.5–55.8) ppm due to N─C6─S
indicates the cyclization of thiocarbamoyl carbon. The
1
3
characteristic peaks observed within the C NMR spectra
of thiadizazoline derivatives are given in Section 6.Charac-
teristic peaks were observed in the mass spectra of com-
pounds 7–9, which followed the similar fragmentation
pattern. The spectrum of compound 7 showed a molecular
+
ion peak (M ) at m/z 691, compound 8 showed a molecular
+
ion peak (M ) at m/z 668 and compound 9 showed a mo-
+
lecular ion peak (M ) at m/z 634. Further fragmentation
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

1
454
S. A. Khan and A. M. Asiri
Vol 49
Scheme 1. Schematic diagram showing the synthesis of compounds 7, 8, and 9. compound 1a: 3b-acetoxycholest-5-ene; compound 2b: 3b-chloro-
cholest-5-ene; compound 3a: cholest-5-ene.
Cholesterol
pyridine
acetic anhydride
thionyl chloride
(
2a)
(3a)
(1a)
Fuming HNO3
Fuming HNO3
Fuming HNO3
C H
C H
8 17
C H
8
17
8
17
Cl
AcO
(
3b)
(
2b)
NO2
Zn dust
NO2
Zn dust
NO2
(
1b)
Acetic acid
Acetic acid
Acetic acid Zn dust
C H
C H
C
8
H
17
8
17
8
17
Cl
O
(3c)
AcO
O
S
(
2c)
O
(
1c)
S
C
S
C
H N NH
C
NH
2
H N NH
NH
CH3
H N NH
NH
2
2
CH3
CH3
C H
8
17
C H
C H
8
17
8
17
S
C
S
C
S
Cl
AcO
NH
NH
N
N
NH
NH
N
NH
C
NH
CH3
(
6)
(
5)
CH3
(
4)
CH3
Ac O/ Pyridine
2
Ac O/ Pyridine
Ac O/ Pyridine
2
2
C H
8
17
C H
8
17
C H
8
17
AcO
S
Ac
Ac
N
Cl
S
N
Ac
Ac
N
N
S
N
Ac
N
Ac
N
R
N
N
R
R
(
7)
(8)
(9)
Where R =
CH3
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

November 2012
Synthesis and In Vitro Antibacterial Activity of Novel Steroidal
6R)-Spiro-1,3,4-thiadiazoline Derivatives
1455
(
Scheme 2. Mechanism of steroidal thiadiazoline derivatives.
Ac O/Pyridine
2
-AcOH
S
S
R
R
CH3
N-N-C-N-
N-N-C-N-
CH3
R
S
N-Ac
H
H
H
N
H
Ac-N
OAc
H C
3
CONCLUSION
for thin-layer chromatography (TLC) and spots were visualized
under UV-light. All melting points were measured with a capil-
lary apparatus and are uncorrected. All the compounds were rou-
This research involves the synthesis of novel steroidal
6R)-spiro-1,3,4-thiadiazoline derivatives by the cycliza-
1
(
tinely checked by IR, H NMR, and mass spectrometry. IR
tion of steroidal thiosemicarbazones. The antibacterial
activity of these compounds was carried out by disc-
diffusion method and the MIC, against culture of bacteria.
The biological behavior of these compounds revealed that
chloro- and acetoxy-substituents on the 3b-position of the
steroidal thiadiazoline ring increased the antibacterial ac-
tivity. Among all the six compounds compound 7 and 8
showed better antibacterial activity than the respective drug
amoxicillin. These results identified that steroidal (6R)-
spiro-1,3,4-thiadiazoline derivatives are new leads in anti-
bacterial chemotherapy. The study suggests the beneficial
potential of these leads that need to be further explored in
order to discover and develop better and yet safer therapeu-
tic agents for bacterial infections.
spectra were recorded on a Perkin-Elmer model 1620 FTIR spec-
trophotometer as KBr discs. H NMR spectra were recorded on
Brucker spectroscopin DPX-400 MHz spectrophotometer in
1
CDCl and DMSO. The following abbreviations were used to
3
indicate the peak multiplicity s-singlet, d-doublet, t-triplet, and
m-multiple. FAB mass spectra were recorded on a JEOL SX102
mass spectrometer using Argon/Xenon (6 kV, 10 mB gas.
Column chromatography was performed on silica gel (merck).
Anhydrous sodium sulfate was used as a drying agent for the
organic phase.
Synthesis of steroidal thiosemicarbazones (4–6): A general
method. Steroidal thiosemicarbazones was synthesized by refluxing
the solution of thiosemicarbazide (0.03 mol) in methanol and the
ꢁ
alcoholic solution of steroidal ketones (0.03 mol) at 60 C for 5 h
with continuous stirring after cooling the compounds were filtered
and recrystallized from methanol.
3
b-acetoxy-5a-cholestan-6-one-p-toludinethiosemicarbazone
ꢁ
ꢀ1
(
1
4). Yield: 68%; m.p. 156 C; IR (KBr) lmax cm : 3245 (N─H),
EXPERIMENTAL
732 (AcO), 1562 (C═N), 1621 (C═C), 1124 (C─N), 1034
1
All chemicals were purchased from Aldrich Chemical Com-
pany (USA) and were used without further purification. Precoated
aluminum sheets (silica gel 60 F254, Merck Germany) were used
(C═S); H NMR (DMSO) d : 10.46 (2H, s, ─NH), 7.32–8.22
H
(4H, m, aryl protons), 4.76 (br, m, 1H J = 18 Hz, C3a axial),
1.92 (s, 3H, CH ), 1.08 (s, 3H, 10─CH ), 0.96 (s, 3H, 18─CH ),
3
3
3
Table 1
Antibacterial activity of steroidal thiosemicarbazones (4–6) and steroidal thiadiazoline derivatives (7–9) (positive control amoxicillin and negative control
DMSO) measured by the halo zone test (mm).
Corresponding effect on microganisms
Compounds
S. aureus
S. pyogenes
S. typhimurium
E. coli
4
5
6
7
8
9
10.8 ꢂ 0.4
10.6 ꢂ 0.5
9.8 ꢂ 0.5
17.5 ꢂ 0.5
21.7 ꢂ 0.4
13.6 ꢂ 0.5
17.0 ꢂ 0.5
–
11.5 ꢂ 0.3
11.2 ꢂ 0.3
9.4 ꢂ 0.2
21.4 ꢂ 0.5
20.5 ꢂ 0.4
14.4 ꢂ 0.5
18.2 ꢂ 0.4
–
11.2 ꢂ 0.2
10.3 ꢂ 0.2
10.4 ꢂ 0.2
19.4 ꢂ 0.4
22.8 ꢂ 0.3
14.2 ꢂ 0.4
17.2 ꢂ 0.8
–
12.6 ꢂ 0.3
10.2 ꢂ 0.5
9.6 ꢂ 0.3
20.5 ꢂ 0.5
21.6 ꢂ 0.6
13.8. ꢂ 0.4
20.0 ꢂ 0.2
–
Amoxycillin
DMSO
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

1
456
S. A. Khan and A. M. Asiri
Vol 49
+
.
Table 2
128.4, 52.3, 45.2, 29.3, 24.5, 21.2, 18.7; Mass spectra (M ) at
m/z 691, 677 (M─CH ), 601 (M─C H ), 650 (M─CH CO), 545
Minimum inhibition concentration (MIC) of steroidal thiadiazoline
derivatives (positive control amoxicillin).
3
7
7
3
9 13 3 2 3
(M─C H10NO), 428 (M─C12H N SO ), 632 (M─CH COO);
Anal. Calc. for C41
C, 70.95; H, 8.78; N, 6.07.
3b-chloro-5a-cholestan-(6R)-spiro-6,4 -acetyl-2 -(acetylami-
61 3 4
H N O S: C, 71.20; H, 8.82; N, 6.07. Found:
Compounds
Positive
0
0
ꢀ
1
2
0
0
0
MIC (mg mL ), Strain
7
8
9
control
nomethylbenzene)-Δ -1 ,3,4 -thiadiazoline (8). Yield: 62%;
ꢀ
1
greenish semi-solid IR vmax (KBr) cm : 2962 (C─H), 1732,
696 (amide), 1658 (C═N), 1168 (C─N), 722 (C─Cl), 646
S. aureus
32
64
64
32
32
64
32
128
64
128
512
32
32
32
32
1
S. Pyogenes
S. typhimurium
E. coli
1
(
C─S). H NMR (CDCl ): d/ppm: 4.52 (m, W
18 Hz, C3
3
1/2h
a─H), 2.24, 2.14 (each, s, 3H Ac), 7.36–8.34 (m, aryl protons),
128
1
1
.96 (s, 3H, CH
.14 (s, 3H, 10-CH
): d/ppm: 174.4, 165.4, 154.2, 137.2, 136.4, 127.5,
3
), 0.74 (s, 3H, 13-CH
3
), 0.97 (s, H, 18-CH
3
),
1
3
3
), and 0.84 (s, 3H, 19-CH
3
); C NMR
(
CDCl
3
+
.
5
4.3, 46.5, 27.3, 25.7, 22.5, 19.8; Mass spectra (M ) at m/z 668,
0
(
(
.88 (s, 3H, 19─CH
3
) and 0.78 (s, 3H, 13─CH
M ) at m/z 608, 593 (M─CH
M─C H N), 458 (M─C H NS), 443 (M─C H N S), 549
3
); Mass spectra
653 (M─CH3), 577 (M─C H ), 625 (M─CH CO), 520
(M─C H NO), 405 (M─C H N SO ), 533 (M─Cl); Anal.
9 10 12 13 3 2
Calc. for C H O N SCl: C, 70.16; H, 8.69; N, 6.29. Found: C,
39 58 2 3
7 7 3
+.
3
7 7
), 517 (M─C H ), 502
7
8
8
8
8
9
2
(
6
M─AcO) Anal. Calc. for C H N O S: C, 73.14; H, 9.39; N,
.91. Found: C, 73.09; H, 9.25; N, 6.55.
69.98; H, 8.45; N, 6.08.
5a-cholestan-(6R)-spiro-6,4 -acetyl-2 -(acetylaminomethyl-
3
7 57 3 2
0
0
2
0
0
0
3
b-chloro-5a-cholestan-6-one-p-toludinethiosemicarbazone
benzene)-Δ -1 ,3,4 -thiadiazoline (9). Yield: 73%; semi-solid;
ꢁ
ꢀ1
ꢀ1
(
1
5). Yield: 72%; m.p. 136 C; IR (KBr) lmax cm : 3242 (N─H),
566 (C═N), 1628 (C═C), 1122 (C─N), 1032 (C═S), 716
IR vmax (KBr) cm : 2962 (C─H), 1726, 1698 (amide), 1652
1
(C═N), 1166 (C─N), 642 (C─S); H NMR (400 MHz,
1
(
(
C─Cl); H NMR (DMSO) d : 10.42 (2H, s, ─NH), 7.31–8.36
CDCl ): d/ppm: 4.56 (m, W
1/2h
18 Hz, C3 a─H), 2.26, 2.16
H
3
4H, m, aryl protons), 4.54 (br, m, 1H w1/2 = 17 Hz, C3a─H,
(each, s, 3H Ac), 7.38–8.38 (m, aryl protons), 1.92 (s, 3H,
axial), 1.91 (s, 3H, ─CH
8─CH ), 0.86 (s, 3H, 19─CH
Mass spectra (M ) at m/z 584, 569 (M─CH ), 493 (M─C H ),
3
), 1.06 (s, 3H, 10─CH
3
), 0.95 (s, 3H,
CH ), 0.76 (s, 3H, 13-CH ), 0.98 (s, H, 18-CH ), 1.12 (s, 3H,
10-CH ), and 0.86 (s, 3H, 19-CH ); C NMR (CDCl ): d/ppm:
3 3 3
3
3
3
13
1
3
3
), and 0.74 (s, 3H, 13─CH
3
);
+.
3
7 7
175.6, 164.2, 153.5, 135.2, 134.5, 126.5, 53.5, 44.2, 28.6, 23.8,
+
.
4
78 (M─C H N), 458 (M─C H NS), 419 (M─C H N S), 549
19.2, 20.8, Mass spectra (M ) at m/z 634, 619 (M─CH ), 543
7
8
8
8
8
9
2
3
(
M─Cl); Anal. Calc. for C35
54
H N
3
SCl: C, 72.04; H, 9.26; N,
(M─C H ), 591 (M─CH CO), 486 (M─C H NO), 371
7
7
3
9 10
7
.20. Found: C, 71.96; H, 9.16; N, 7.18.
12 13 3 2 39 59 2 3
(M─C H N SO ). Anal. Calc. for C H O N S: C, 73.93; H,
9.30; N, 6.63. Found: C, 73.85; H, 9.18; N, 6.45.
5
a-cholestan-6-one-p-toludinethiosemicarbazone (6). Yield:
ꢁ
ꢀ1
7
(
5%; m.p. 210 C; IR (KBr) l
C═N), 1632 (C═C), 1126 (C─N), 1028 (C═S); H NMR
cm : 3252 (N─H), 1572
Organism culture and in vitro screening. Antibacterial
activity was carried out by the disc-diffusion method with minor
modifications. S. aureus, S. pyogenes, S. typhimurium, and
E. coli were sub cultured in BHI medium and incubated for 18 h
max
1
H
(DMSO) d : 10.46 (2H, s, ─NH), 7.30–8.42 (4H, m, aryl
3 3
protons), 1.91 (s, 3H, ─CH ), 1.05 (s, 3H, 10─CH ), 0.97 (s,
ꢁ
3
H, 18─CH ), 0.86 (s, 3H, 19-CH ) and 0.76 (s, 3H, 13-CH );
at 37 C, and then the bacterial cells were suspended,
3
3
3
+.
Mass spectra (M ) at m/z 550, 535, (M─CH ), 459 (M─C H ),
according to the McFarland protocol in saline solution to
3
7 7
ꢀ
5
ꢀ1
4
41 (M─C
Calc. for C35
5.52; H, 9.96; N, 7.62.
Oxidative cyclization of steriodal 6-ketone thiosemicarbazones
7
H
H
8
N), 400 (M─C
8
H
8
NS), 385 (M─C
8
H
9
N
2
S); Anal.
produce a suspension of about 10 CFU mL : 10 mL of this
55 3
N S: C, 76.50; H, 10.01; N, 7.65. Found: C,
suspension was mixed with 10 mL of sterile antibiotic agar at
ꢁ
7
40 C and poured onto an agar plate in a laminar flow cabinet.
Five paper discs (0.5 mm diameter) were fixed onto nutrient
agar plate. One milligram of each test compound was
dissolved in 100 mL DMSO to prepare stock solution and
from stock solution different concentration 10, 20, 25, 50, and
100 mg/mL of each test compound were prepared. These
compounds of different concentration were poured over disc
plate on to it. amoxicillin (30 mg/disc) was used as standard
drug (positive control). DMSO poured disc was used as
negative control. The susceptibility of the bacteria to the test
compounds was determined by the formation of an inhibitory
0 0 0
(
4-6). Steroidal 6R-Spiro-1 ,3 ,4 -thiadiazolines. General
procedure. Steroidal thiosemicarbazones 4–6 (1.0 mmol)
were dissolved in chloroform (25 mL) and treated with
freshly distilled acetic anhydride (11.0 mmol) and pyridine
(
2.5 mmol) and the mixture was stirred for 3–4 h over an oil
ꢁ
bath at 80 C. Reaction progress was monitored by TLC.
After completion, solvent was removed under reduced
pressure and the residue was purified by column
chromatography over silica gel (light petroleum:diethyl ether,
0
0
0
ꢁ
8
:2) to give the respective steroidal (6R)-spiro-1 ,3 ,4 -
thiadiazolines 7–9.
b-acetoxy-5a-cholestan-(6R)-spiro-6, 4 -acetyl-2 -(acetyla-
zone after 18 h of incubation at 36 C. (Table 1) reports the
inhibition zones (mm) of each compound and the controls. The
minimum inhibitory concentration (MIC) was evaluated by the
0
0
3
2
0
0
0
ꢀ5
ꢀ1
minomethylbenzene)-Δ -1 ,3,4 -thiadiazoline (7). Yield: 74%;
m.p. 132 C; IR v
macro dilution test using standard inoculums of 10 CFL mL .
ꢁ
ꢀ1
(KBr) cm : 2965 (C─H), 1722, 1692
Serial dilutions of the test compounds, previously dissolved
in dimethyl sulfoxide (DMSO) were prepared to final
concentrations of 512, 256, 128, 64, 32, 16, 8, 4, 2, and
1 mg/mL to each tube was added 100 mL of a 24 h old
inoculum. The MIC, defined as the lowest concentration of the
test compound, which inhibits the visible growth after 18 h, was
max
(
(
amide), 1656 (C═N), 1735 (OCOCH ), 1162 (C─N), 648
3
1
3
C─S). H NMR (CDCl ): d/ppm: 4.86 (m, W1/2h 18 Hz, C3
a─H), 2.26, 2.12 (each, s, 3H Ac), 7.34–8.32 (4H, m, aryl
3 3
protons), 1.94 (s, 3H, CH ), 0.75 (s, 3H, 13-CH ), 0.96 (s, H,
1
8-CH ), 1.12 (s, 3H, 10-CH ), and 0.82 (s, 3H, 19-CH );
3
3
3
1
3
ꢁ
C NMR (CDCl ): d/ppm: 173.6, 168.0, 155.2, 136.6, 132.2,
determined visually after incubation for 18 h, at 37 C, and the
3
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

November 2012
Synthesis and In Vitro Antibacterial Activity of Novel Steroidal
6R)-Spiro-1,3,4-thiadiazoline Derivatives
1457
(
result are presented in Table 2. Tests using DMSO and amoxicillin
as negative and positive controls.
[5] Gupta, A.; Mishra, P.; Pandeya, S. N.; Kashaw, S. K.; Kashaw,
V.; Stables, J. P. Eur J Med Chem 2009, 44, 1100.
[
6] Zhan, P.; Liu, X.; Cao, Y.; Wang, Y.; Pannecouque, C.;
Clercq, E. D. Bioorg Med Chem Lett 2006, 18, 5368.
7] Hagiwara, K.; Hashimoto, S.; Shimoda, S. J Pesticide Sci
992, 17, 251.
[
Acknowledgments. The authors are thankful Dr. Kishwar Saleem
Department of Chemistry, Jamia Millia Islamia, New Delhi, India
for useful discussion.
1
[8] Satyanarayana, K.; Rao, M. N. A. J Pharm Sci 1995, 84, 263.
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[
[
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet