Synthesis and biological evaluation of some thiazolidinone derivatives of steroid as antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2008.09.004

Steroidal thiazolidinone derivatives were prepared by the multi-step reactions of steroid. It is prepared from steroidal thiosemicarbazones with ethyl bromoacetate in dioxane. Steroidal thiosemicarbazones were prepared by the reaction of thiosemicarbazide with steroidal ketones. The structures of these compounds were elucidated by IR, ¹H NMR, Fab mass spectrometries and their purities were confirmed by elemental analyses. The antibacterial activity of these compounds was evaluated by the disk diffusion assay against two Gram-positive and two Gram-negative bacteria and then the minimum inhibitory concentration (MIC) of compounds was determined. The results showed that steroidal thiazolidinone derivatives are better in inhibiting the growth as compared to steroidal thiosemicarbazone derivatives of both types of the bacteria (Gram-positive and Gram-negative). Compounds 7 and 8 are better antibacterial agents as compared to standard drug Amoxicillin.

Full text of DOI:10.1016/j.ejmech.2008.09.004

European Journal of Medicinal Chemistry 44 (2009) 2597–2600
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and biological evaluation of some thiazolidinone derivatives
of steroid as antibacterial agents
*
Salman Ahmad Khan , Mohammed Yusuf
Department of Chemistry, Punjabi University, Patiala, Punjab 147002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 March 2008
Received in revised form 20 July 2008
Accepted 1 September 2008
Available online 16 September 2008
Steroidal thiazolidinone derivatives were prepared by the multi-step reactions of steroid. It is prepared
from steroidal thiosemicarbazones with ethyl bromoacetate in dioxane. Steroidal thiosemicarbazones
were prepared by the reaction of thiosemicarbazide with steroidal ketones. The structures of these
compounds were elucidated by IR, 1H NMR, Fab mass spectrometries and their purities were confirmed
by elemental analyses. The antibacterial activity of these compounds was evaluated by the disk diffusion
assay against two Gram-positive and two Gram-negative bacteria and then the minimum inhibitory
concentration (MIC) of compounds was determined. The results showed that steroidal thiazolidinone
derivatives are better in inhibiting the growth as compared to steroidal thiosemicarbazone derivatives of
both types of the bacteria (Gram-positive and Gram-negative). Compounds 7 and 8 are better antibac-
terial agents as compared to standard drug Amoxicillin.
Keywords:
Thiosemicarbazone
Thiazolidinone
Antibacterial activity
Amoxicillin
Ó 2008 Published by Elsevier Masson SAS.
1. Introduction
application in drug development for the treatment of HIV-infection,
hypertension and inflammation [19]. Several thiazolidinone deriva-
The treatment of infectious diseases still remains an important
and challenging problem. Despite search of novel antimicrobial
agents is a field of current and growing interest. Many compounds
have been synthesized with this aim, their clinical use has been
limited by their relatively high risk of toxicity, bacterial resistance
and/or pharmacokinetic deficiencies. A major research emphasis to
counter this growing problem is the development of antimicrobials
structurally unrelated to the existing molecules. One possibility to
achieve this goal is the combination of a steroid molecule with
structural elements possessing appropriate biological activities [1–3].
In addition, considerable attention has been focused on substituted
thiosemicarbazone derivatives due to their interesting biological
activity. Compounds with a thiosemicarbazone structure are known
to possess tranquilizing, muscle relaxant, psychoanaleptic, hypnotic,
ulcerogenic, antidepressant, antibacterial, antifungal and analgesic
anti-inflammatory properties [4–11]. Steroidal thiosemicarbazones
dramatically increase the diversity of certain biological properties
[12–14]. The presence of thiazolidinone moiety in the structure of
several naturally occurring molecules with important antibiotic,
immunosuppressive and antitumor activities has been known for
several years [15–18]. The aminothiazole ring system has found
tives have been shown to exhibitexcellent bactericidal[20] fungicidal
[21,22] and anthelmintic [23] activities. Recently thiazolidinones
have been synthesized and screened for possible antimicrobial
activities [24,25]. Thiazolidinone as evident from the literature, it was
noted that lot of research has been carried out on thiazolidinone
derivatives have been antibacterial but no work has been done on
steroidal (cholesterol) derivatives screeningon bacterial. Inthis paper
the steroidal thiazolidinone derivatives have been synthesized by the
reaction of steroidal thiosemicarbazones and ethyl bromoacetate in
dioxane. The activities of these compounds were screened in vitro
against bacteria such as Staphylococcus aureus, Streptococcus pyo-
genes, and Salmonella typhimurium and Escherichia coli.
2. Results and discussion
Thiosemicarbazones prepared by condensing the steroidal
ketones with cyclopentyl thiosemicarbazide in the presence of
catalytic amount of conc. HCl gave yield 63–69%. 3
b-acetoxycholest-
6-one [26], 3 -chloro-cholest-6-one [27], 5 -cholest-6-one [28]
b
a
were prepared according to the published methods. The thio-
semicarbazone derivatives were used as starting material for the
preparation of thiazolidinone derivatives. The thiazolidinone
derivatives were synthesized by the literature procedure [29] as
indicated in Scheme 1. Thiosemicarbazones were refluxed with
ethyl bromoacetate in dioxan for 12 h and after that solvent was
removed under reduced pressure and crystallization was done in
ethanol. All the compounds were soluble in DMSO and ethanol. The
* Corresponding author. Department of Chemistry, Integral University Dasauli,
P.O. Bas-ha Kursi Road, Lucknow 226026, India. Mobile: þ91 9335620811, þ91
9305352751.
E-mail address: sahmad_phd@yahoo.co.in (S.A. Khan).
0223-5234/$ – see front matter Ó 2008 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2008.09.004

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S.A. Khan, M. Yusuf / European Journal of Medicinal Chemistry 44 (2009) 2597–2600
Cholesterol
pyridine
acetic anhydride
thionyl chloride
(2a)
(3a)
(1a)
Fuming HNO3
Fuming HNO3
Fuming HNO3
Cl
CH3COO
(3b)
NO2
NO2
NO2
(2b)
(1b)
Zn dust
Acetic acid
Acetic acid
Zn dust
Acetic acid
Zn dust
CH3COO
O
Cl
O
S
S
O
(3c)
(1c)
S
(2d)
H2N-NH-C-NH-
H2N-NH-C-NH-
H2N-NH-C-NH-
S
S
Cl
O
CH3COO
S
N-NH-C-NH-
(2f)
N-NH-C-NH-
(3f)
N-NH-C-NH-
(6)
(5)
O
O
(4)
Br
O
dioxane
Br
Br
dioxane
dioxane
O
O
S
S
S
CH3COO
Cl
O
O
O
N-N
N
N
R
N-N
N-N
N
R
R
(7)
Where R =
(8)
(9)
Scheme 1. Schematic diagram showing the synthesis of compounds 7–9. Where compound (1a): 3
(3a): cholest-5-ene.
b-acetoxycholest-5-ene, compound (2b): 3b-chlorocholest-5-ene, compound
structures of all the compounds were established by means of their
IR, 1H NMR, FAB mass spectra and the elemental analyses were
carried out to check the purity of the compounds.
Characteristic peaks were observed in the mass spectra of
compounds 7–9, which followed the similar fragmentation pattern.
ꢁ
The spectrum of compound 7 showed a molecular ion peak (Mþ
)
ꢁ
Assignments of selected characteristic IR band positions provide
significant indication for the formation of the cyclized thiazolidi-
none analogues of thiosemicarbazones. All the compounds showed
v (C]N) stretch at 1562–1572 cmꢀ1 due to the ring closure. In
addition, the absorption band at 1162–1185 cmꢀ1 was attributed to
the v (C–N) stretch vibrations. The compounds showed intense
bands at 624–642 cmꢀ1 due to v (C–S) stretch, which also confirm
the formation of thiazole ring in all the compounds.
Further evidence for the formation of thiazolidinone
compounds was obtained from the 1H 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 thiazole protons of all the compounds are
shown as singlet in the range 3.80–4.21 ppm.
at m/z 626, compound 8 showed a molecular ion peak (Mþ ) at m/z
ꢁ
601/603 and compound 9 showed a molecular ion peak (Mþ ) at
m/z 569. Further fragmentation pattern of these compounds is
given in the in Section 3.
2.1. Antibacterial activity
The compounds (4–9) were tested for their antibacterial activ-
ities by disc-diffusion method [30] using nutrient broth medium
[contained (g/L): beef extract 3 g; peptone 5 g; pH 7.0]. The Gram-
positive bacteria and Gram-negative bacteria utilized in this study
consisted of S. aureus, S. pyogenes, S. typhimurium and E. coli. In the
disc-diffusion method, sterile paper discs (05 mm) impregnated
with compound dissolved in dimethylsulfoxide (DMSO) at

S.A. Khan, M. Yusuf / European Journal of Medicinal Chemistry 44 (2009) 2597–2600
2599
Table 2
concentration 100 mg/mL were used. Then, the paper discs
Minimum inhibition concentration (MIC) of steroidal thiazolidinone derivatives,
positive Amoxicillin control.
impregnated with the solution of the compound tested were placed
on the surface of the media inoculated with the microorganism.
The plates were incubated at 35 ꢂC for 24 h. After incubation, the
growth inhibition zone are shown in Table 1. The thiazolidinone
derivatives were further checked by 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 in their biological
effects which could be helpful in designing more potent antibac-
terial agents for therapeutic use.
Strain
MIC (
m
g mLꢀ1
)
Compounds
Positive control
7
8
9
S. aureus
64
32
64
32
32
32
64
64
128
128
128
128
32
32
32
32
S. pyogenes
S. typhimurium
E. coli
ꢁ
protons); MS (Mþ ) at m/z 586, 527 (M-AcO), 517 (M-C5H9), 502 (M-
C5H10N), 458 (M-C6H10NS), 443 (M-C6H11N2S).
3.1.2. 3
Yields: 69%; Mp. 174–176 ꢂC; Anal. Calcd. for C33H56N3SCl: C,
70.46; H, 9.96; N, 7.47. Found: C, 69.32; H, 8.47; N, 6.35; IR (nmax
cmꢀ1 KBr): 3385 (N–H), 1570 (C]N), 1165 (C]S), 718 (C–Cl); 1H
NMR:
b-Chloro-cholest-6-onecyclopentyl thiosemicarbazone (5)
3. Experimental
;
The entire chemicals were purchased from Aldrich Chemical
Company (U.S.A) and were used without further purification. The
reactions were monitored by precoated aluminium silica gel 60F
254 thin layer plates procured from Merck (Germany). All melting
points were measured with a capillary apparatus and are uncor-
rected. All the compounds were routinely checked by IR, 1H NMR
and mass spectrometries. IR spectra were recorded in KBr on
a Perkin–Elmer model 1620 FTIR spectrophotometer. 1H NMR
spectra were recorded at ambient temperature using a Brucker
spectroscopin DPX-300 MHz spectrophotometer in CDCl3 and
DMSO. The following abbreviations were used to indicate the peak
multiplicity s- singlet, d- doublet, t- triplet, m- multiplet. 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.
d
9.84 (s, 1H, NH), 7.6 (d, 1H), 4.6 (br, m, 1H, w1/2 ¼15 Hz, C3
a
axial), 4.4 (m, 8H, –CH2) 1.22 (C10–CH3), 0.72 (C13–CH3), 1.02, 0.87
ꢁ
(remaining methyl protons); Mass spectra (Mþ ) at m/z 561/563,
528 (M-Cl), 492/494 (M-C5H9), 477/478 (M-C5H10N), 433/435
(M-C6H10NS), 418/420 (M-C6H11N2S).
3.1.3. Cholest-6-onecyclopentyl thiosemicarbazone (6)
Yields: 63%; Mp.156–158 ꢂC; Anal. Calcd. for C33H57N3S: C, 75.14;
H, 10.81; N, 7.96. Found: C, 74.95; H, 9.26; N, 6.95; IR (nmax; cmꢀ1
KBr): 3386 (NH), 1628 (C]C), 1568 (C]N) 1185 (C]S), 2928 (C–H);
1H NMR:
d
9.7 (s, 1H, NH), 5.9 (d, 1H, NH), 0.07 (C10–CH3), 0.66
ꢁ
(C13–CH3), 0.90 and 0.82 (remaining proton); Mass spectra (Mþ ) at
m/z 528, 459 (M-C5H9), 444 (M-C5H10N), 400 (M-C6H10NS), 385
(M-C6H11N2S).
3.1. Synthesis of thiosemicarbazones: a general method
3.2. General procedure for the syntheses of the steroidal
thiazolidinone
Steroidal thiosemicarbazones were synthesized (Scheme 1) by
refluxing the solution of cyclopentyl thiosemicarbazide (0.03 mol)
and the alcoholic solution of steroidal ketones (0.03 mol) in ethanol
in the presence of few drops of HCl at 80 ꢂC for 5 h with continuous
stirring. After cooling the compounds were filtered and recrystal-
lized from methanol.
To a suspension of the steroidal thiosemicarbazone (4–6)
(1.5 mmol) in dioxane (20 mL), an equivalent amount of ethyl
bromoacetate (1.5 mmol) was added. The reaction mixture was
heated under reflux for 12 h, concentrated to approximately half its
volume and allowed to cool to room temperature. The separated
solid product was filtered, washed with ethanol, dried and crys-
tallized from ethanol.
3.1.1. 3
Yields: 64%; Mp. 186–188 ꢂC; Anal. Calcd. for C35H59N3O2S: C,
71.79; H, 10.08; N, 7.17. Found: C, 70.08; H, 9.25; N, 6.58; IR (nmax
cmꢀ1 KBr): 3385 (N–H), 1738 (OCOCH3), 1625 (weak, C]C), 1586
(C]N), 1158 (C]S); 1H NMR
: 9.82 (s, 1H, N–H), 7.32 (d, 1H, –NH),
-H, axial), 4.7 (m, 8H, –CH2) 2.4 (s, 3H,
1.22 (C10–CH3), 0.75 (C13–CH3) 0.95, 0.89 (other methyl
b-Acetoxycholest-6-one cyclopentyl thiosemicarbazone (4)
;
3.2.1. 3
Yields: 73%; Mp.148–152 ꢂC; Anal. Calcd. for C37H59N3SO3: C,
71.04; H, 9.42; N, 6.72. Found: C, 69.32; H, 9.46; N, 5.85; IR (nmax
cmꢀ1 KBr): 2936 (C–H), 722 (OCOCH3), 1682 (C]O), 572 (C]N),
1185 (C–N), 624 (C–S); 1H NMR:
4.6 (m, 10H, –CH2), 4.5 (br, m,
axial), 4.21 (s, 2H, thiazole-H), 2.3 (s, 3H,
b-Acetoxycholest-6-diazo(4-thiazolidinon)cholestane (7)
d
;
4.5 (Br, m, w1/2 ¼ 17 Hz, C3
a
OCOCH3),
d
d
1H, w1/2 ¼ 15 Hz, C3
a
OCOCH3), 1.20 (C10–CH3), 0.76 (C13–CH3), 0.98 and .88 (remain-
Table 1
ꢁ
ing proton); Mass spectra (Mþ ) at m/z 626, 567 (M-AcO), 598
Antibacterial activity of steroidal derivatives positive control (Amoxicillin) and
negative control (DMSO) measured by the halo zone test (unit, mm).
(M-CO), 584 (M-C2H2O), 557 (M-C5H9), 552 (M-C2H2OS), 443
(M-C8H11N2SO).
Compounds
Corresponding effect on microorganisms
S. aureus
S. pyogenes
S. typhimurium
E. coli
3.2.2. 3
Yields: 75%; Mp. 134–136 ꢂC; Anal. Calcd. for C35H56N3SOCl: C,
69.76; H, 9.30; N, 6.72. Found: C, 68.50; H, 8.88, N, 6.62; IR (nmax
cmꢀ1 KBr): 2922 (C–H), 1568 (C]N), 1162 (C–N), 715 (C–Cl), 632
(C–S); 1H NMR (DMSO-d6) (
b-Chloro-cholest-6-diazo(4-thiazolidinon)cholestane (8)
4
5
6
7
8
9
12.6 ꢃ 0.5
11.5 ꢃ 0.6
09.4 ꢃ 0.3
18.5 ꢃ 0.7
20.4 ꢃ 0.5
14.5 ꢃ 0.5
17.0 ꢃ 0.5
–
11.4 ꢃ 0.4
10.2 ꢃ 0.3
10.2 ꢃ 0.2
21.4 ꢃ 0.4
21.4 ꢃ 0.3
15.2 ꢃ 0.6
18.2 ꢃ 0.4
–
10.4 ꢃ 0.4
11.2 ꢃ 0.3
10.4 ꢃ 0.4
19.6 ꢃ 0.4
19.4 ꢃ 0.5
13.6 ꢃ 0.5
17.2 ꢃ 0.8
–
13.3 ꢃ 0.4
11.7 ꢃ 0.4
12.3 ꢃ 0.5
22.8 ꢃ 0.7
22.5 ꢃ 0.6
14.2 ꢃ 0.6
20.0 ꢃ 0.2
–
;
d): 4.72 (br, m, 1H, w1/2 ¼15 Hz
axial C3
a
-H), 4.11 (s, 2H, thiazole-H) 1.20 (C10–CH3), 0.74 (C10–
ꢁ
CH3), 0.88, 1.08 (other methyl protons); Mass spectra (Mþ ) at m/z
Amoxicillin
DMSO
601/603, 567 (M-Cl), 587/589 (M-CH2), 573/575 (M-CO), 559/561

2600
S.A. Khan, M. Yusuf / European Journal of Medicinal Chemistry 44 (2009) 2597–2600
(M-C2H2O), 532/534 (M-C5H9), 527/529 (M-C2H2OS), 418/420
(M-C8H11N2SO).
acetoxy substituents on the 3b-position of the steroidal thiazoli-
dinone ring increased the antibacterial activity. Among all the six
compounds compounds 7 and 8 showed better antibacterial
activity than their respective drug.
3.2.3. 6-Diazo(4-thiazolidinon)cholestane (9)
Yields: 68%; Mp. 116–118 ꢂC; Anal. Calcd. for (C35H57N3SO): C,
74.07; H, 10.05; N, 7.40. Found: C, 73.52; H, 9.35; N, 6.80; IR (nmax
cmꢀ1 KBr): 2935 (C–H), 1562 (C]N), 1178 (C–N), 1672 (C]O), 642
(C–S); 1H NMR (DMSO-d6) (
): 3.8 (s, 2H, thiazole-H) 1.22, (C10–
;
Acknowledgment
d
CH3), 0.69 (C10–CH3), 0.82, 1.10 (other methyl protons); Mass
Authors are thankful to Head, Department of Chemistry, Punjabi
University Patiala, for providing laboratory facilities for research.
ꢁ
spectra (Mþ ) at m/z 569, 555 (M-CH2), 541 (M-CO), 527 (M-C2H2O),
500 (M-C5H9), 495 (M-C2H2OS), 386 (M-C8H11N2SO).
3.3. Organism culture and in vitro screening
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mL of this suspension was mixed with
10 mL of sterile antibiotic agar at 40 ꢂC and poured onto an agar
plate in a laminar flow cabinet. Five paper disks (05 mm in diam-
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and from stock solution different concentrations 10, 20, 25, 50, and
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of different concentrations were poured over disk plate onto it.
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m
m
m
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mL to each tube was added 100 L of a 24 h old inoculum. The MIC,
m
defined as the lowest concentration of the test compound, which
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