Discovery of new antitubercular oxazolyl thiosemicarbazones

Article abstract of DOI:10.1021/jm060339h

Twenty 4-(5-cyclobutyloxazol-2-yl)thiosemicarbazones were synthesized and evaluated for preliminary in vitro and in vivo activity against Mycobacterium tuberculosis H37Rv (MTB) and multidrug-resistant Mycobacterium tuberculosis (MDR-TB). Among them, (4-br

Full text of DOI:10.1021/jm060339h

3448
J. Med. Chem. 2006, 49, 3448-3450
Scheme 1a
Discovery of New Antitubercular Oxazolyl
Thiosemicarbazones
Dharmarajan Sriram,* Perumal Yogeeswari,
Rathinasababathy Thirumurugan, and Roheet Kumar Pavana
Medicinal Chemistry Research Laboratory, Pharmacy Group, Birla
Institute of Technology & Science, Pilani-333031, India
ReceiVed March 23, 2006
a Reagents: (a) DMSO; (b) DIBAL-H, CH2Cl2; (c) Br2, CH2Cl2; (d)
NH2CONH2, DMF; (e) CS2, NaOH, NH2NH2, C2H5OH, HCl; (f) CH3COONa,
OdCRR1, C2H5OH.
Abstract: Twenty 4-(5-cyclobutyloxazol-2-yl)thiosemicarbazones were
synthesized and evaluated for preliminary in vitro and in vivo activity
against Mycobacterium tuberculosis H37Rv (MTB) and multidrug-
resistant Mycobacterium tuberculosis (MDR-TB). Among them, (4-
bromophenyl)(phenyl)methanone N-(5-cyclobutyl-1,3-oxazol-2-yl)-
thiosemicarbazone 6q was found to be the most active compound in
vitro with minimum inhibitory concentration of 0.05 µg/mL against
MTB and MDR-TB. In the in vivo animal model 6q decreased the
bacterial load in lung and spleen tissues with 2.1 log 10 and 3.72 log 10
protections, respectively, at 50 mg/kg body weight dose.
2-amine8 (4) in 48% yield. The synthesis of thiosemicarbazone
derivatives was carried out in three steps,9 as shown in Scheme
1. First, to a solution of 5-cyclobutyloxazol-2-amine (0.01 mol)
in THF (10 mL) was added potassium hydroxide (0.01 mol)
and carbon disulfide (0.75 mL), and the mixture was stirred at
15-20 °C for 1 h to form a potassium salt of dithiocarbamate.
To the stirred mixture was added hydrazine hydrate (0.01 mol),
and the stirring was continued at 60 °C for 1 h to obtain 4-(5-
cyclobutyloxazol-2-yl)thiosemicarbazide (5) in 90% yield. Thio-
semicarbazide derivative on condensation with various carbonyl
compounds in the presence of glacial acetic acid afforded various
thiosemicarbazones (6a-t) (Table 1) in 62-86% yields. The
purity of the compounds was checked by TLC and elemental
analyses, and the compounds of this study were identified by
spectral data. In the 1H NMR spectra the signals of the respective
protons of the prepared derivatives were verified on the basis
of their chemical shifts, multiplicities, and coupling constants.
The spectra of all the compounds showed a singlet at δ 6.68
ppm corresponding to fourth-position proton of the oxazole ring,
a broad multiplet in the region of 1.76-2.28 corresponding to
six cyclobutyl protons, a quintet with J ) 7.2 corresponding to
a single proton of the cyclobutyl ring, and a D2O exchangeable
singlet at δ 7.26 ppm corresponding to NH protons. The
elemental analysis results were within (0.4% of the theoretical
values.
All compounds were screened for their in vitro antimyco-
bacterial activity against MTB and MDR-TB by an agar dilution
method similar to that recommended by the National Committee
for Clinical Laboratory Standards10 for the determination of
minimum inhibitory concentration (MIC) in duplicate. The
MDR-TB clinical isolate was obtained from Tuberculosis
Research Center, Chennai, India, and was resistant to isoniazid
(INH), rifampicin, ethambutol, and ciprofloxacin. The MIC is
defined as the minimum concentration of compound required
to give 99% inhibition of bacterial growth, and MIC values of
the synthesized compounds along with the standard drugs for
comparison were reported (Table 1). Among the synthesized
compounds, 6q (MIC ) 0.05 µg/mL) was equally active as INH
and was more potent than rifampicin against MTB. Compared
to ethambutol (MIC ) 1.56 µg/mL), eight compounds were
more potent against MTB. Against MDR-TB, when compared
to INH (MIC ) 1.56 µg/mL), eight compounds were more
active with MIC values ranging from 0.05 to 0.78 µg/mL.
Compound 6q was found to be the most potent (MIC ) 0.05
µg/mL) and was 31 times more potent against MDR-TB when
compared to the standard drug INH. Compared to rifampicin
(MIC ) 3.12 µg/mL), nine compounds were more potent and
five compounds were equipotent against MTB. All the com-
pounds were more potent than ethambutol against MDR-TB.
Tuberculosis (TB) remains the leading cause of mortality
because of a bacterial pathogen, Mycobacterium tuberculosis.
It is estimated that 8.2 million new TB cases occurred worldwide
in the year 2000, with approximately 1.8 million deaths in the
same year, and more than 95% of those were in developing
countries.1 The incidence of TB infection has steadily risen in
the past decade, and this increase can be attributed to a similar
increase in human immunodeficiency virus (HIV) infection.2
The association of TB and HIV infections is so dramatic that
in some cases nearly two-thirds of the patients diagnosed with
TB are also HIV-1 seropositive.3 Furthermore, numerous studies
have shown that TB is a cofactor in the progression of HIV
infection.4 The reemergence of TB infection is further compli-
cated by an increase in cases that are resistant to conventional
antitubercular drug therapy. Not only does the increasing rate
of multidrug-resistant TB create problems for the treatment but
also the costs are exploding. Thus, new drugs are necessary to
overcome the current problems of therapy. Earlier we reported
antitubercular N1-(4-acetamido phenyl)-N4-(2-nitrobenzylidene)-
semicarbazone, which inhibited in vitro Mycobacterium tuber-
culosis H37Rv (MTB) and was more potent than the commonly
used antitubercular agents.5 In the course of screening to
discover new compounds employed in the chemotherapy of
tuberculosis, we identified oxazolylthiosemicarbazones deriva-
tives that inhibited in vitro MTB and multidrug resistant
Mycobacterium tuberculosis (MDR-TB). We present the pre-
liminary results concerning the synthesis and the in vitro and
in vivo antituberculous activity of the first representative
compound of this family.
Bromomethylcyclobutane (1) was reacted with sodium cya-
nide in dimethylsulfoxide medium at 70 °C for 3 h to afford
2-cyclobutylacetonitrile (2) in 75% yield.6 2-Cyclobutylaceto-
nitrile was dissolved in dry dichloromethane and was reduced
with diisobutylaluminum hydride (20% weight by solution in
toluene) at -75 °C under nitrogen atmosphere to yield 2-cy-
clobutylacetaldehyde (3) in 70% yield.7 Cyclobutylacetaldehyde,
on treatment with bromine in dichloromethane, yielded 2-bromo-
2-cyclobutylacetaldehyde, which on heating with urea in di-
methyl formamide at 90 °C for 3 h yielded 5-cyclobutyloxazol-
* To whom correspondence should be addressed. Telephone: +91-1596-
244684. Telefax: +91-1596-244183. E-mail: dsriram@bits-pilani.ac.in.
10.1021/jm060339h CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/20/2006

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3449
Table 1. Physical Constants and in Vitro Antimycobacterial and Cytotoxicity of the Synthesized Compounds
MIC in (µg/mL)
compd
R
R1
yield (%)
mp (°C)
IC50 a (µg/mL)
MTBb
MDR-TBc
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
Ison
Rifa
Gati
Etha
H
H
H
H
H
H
H
H
2-hydroxyphenyl
2-nitrophenyl
3-nitrophenyl
62
76
73
81
71
67
86
64
67
70
69
64
70
63
68
60
62
66
78
72
152
205
183
237
155
207
256
166
172
126
111
131
57
NT
NT
<62.5
<62.5
<62.5
NT
NT
NT
NT
NT
NT
NT
NT
62.5
62.5
>62.5
>62.5
NT
6.25
0.78
0.78
0.39
3.12
1.56
3.12
6.25
6.25
0.78
0.78
0.39
3.12
1.56
3.12
6.25
12.5
3.12
3.12
3.12
0.78
0.78
0.20
0.10
0.05
6.25
25.00
12.5
1.56
3.12
6.25
25.00
4-nitrophenyl
4-methylphenyl
4-chlorophenyl
4-dimethylaminophenyl
4-methoxyphenyl
4-hydroxy-3-methoxyphenyl
phenyl
2-hydroxyphenyl
4-hydroxyphenyl
4-methylphenyl
4-aminophenyl
4-nitrophenyl
H
12.5
CH3
CH3
CH3
CH3
CH3
CH3
C6H5
C6H5
C6H5CH2-
H
F
3.12
3.12
3.12
0.78
0.78
0.20
0.10
0.05
6.25
25.00
12.5
0.05
0.1
107
188
41
65
91
phenyl
4-bromophenyl
benzyl
194
168
NT
NT
>62.5
>62.5
>62.5
>62.5
0.2
1.56
a NT: not tested. b Mycobacterium tuberculosis H37Rv. c Multidrug-resistant Mycobacterium tuberculosis.
Table 2. In Vivo Activity Data of 6q, Isoniazid, and Gatifloxacin
against M. tuberculosis in Mice
route. Thirty-five days after infection, the spleens and right lungs
were aseptically removed and ground in a tissue homogenizer
and the number of viable organisms was determined by serial
10-fold dilutions and subsequent inoculation onto 7H10 agar
plates. Cultures were incubated at 37 °C in ambient air for 4
weeks prior to counting. Bacterial counts were measured and
compared with the counts from negative (untreated) controls
(mean culture forming units (CFU) in lung, 7.88; mean CFU
in spleen, 8.84). Compound 6q decreased the bacterial load in
lung and spleen tissues with 2.1 log 10 and 3.72 log 10 protec-
tions, respectively, and was considered to be promising in
reducing bacterial count in lung and spleen tissues. When
compared to gatifloxacin at the same dose level, 6q decreases
the bacterial load with 0.24 log 10 and 1.12 log 10 protections
in lung and spleen tissues, respectively. Compound 6q was
found to be less active than INH in the in vivo study. The reason
for this less in vivo activity might be due to the instability of
the compounds, as it gets hydrolyzed to the inactive intermediate
4-(5-cyclobutyloxazol-2-yl)thiosemicarbazide (5).
Screening of the antimycobacterial activity of these novel
series identified oxazolylthiosemicarbazones as a new lead
endowed with antitubercular activity, exhibiting MIC values
between 0.05 and 12.5 µg/mL. In conclusion, it has been shown
that the potency, selectivity, and low cytotoxicity of these
compounds make them valid leads for synthesizing new
compounds that possess better activity. Further structure-
activity and mechanistic studies should prove to be fruitful.
log CFU ( SEM
compd
lung
spleen
control
6q (50 mg/kg)
gatifloxacin (50 mg/kg)
isoniazid (25 mg/kg)
7.88 ( 0.22
5.78 ( 0.12
6.02 ( 0.18
5.12 ( 0.10
8.84 ( 0.21
5.12 ( 0.10
6.24 ( 0.12
4.11 ( 0.17
Compound (4-bromophenyl)(phenyl)methanone N-(5-cyclobu-
tyl-1,3-oxazol-2-yl)thiosemicarbazone (6q) was found to be the
most active compound in vitro with an MIC of 0.05 µg/mL
against MTB and MDR-TB. Among the benzaldehyde and
actophenone derived thiosemicarbazones (6a-r), substituents
with electron-withdrawing groups such as nitro (6b-d,o) and
chloro (6f) enhanced the activity. With respect to the carbimino
terminal, the order of activity was found to be (sub)dipheyl-
benzophenone > (sub)acetophenone > (sub)benzaldehyde >
dibenzyl ketone > isatin. The intermediate 4-(5-cyclobutylox-
azol-2-yl)thiosemicarbazide (5) did not show any inhibition to
25 µg/mL.
Some compounds were further examined for toxicity (IC50)
in a mammalian Vero cell line at 62.5 µg/mL. After 72 h of
exposure, viability was assessed on the basis of cellular
conversion of MTT into a formazan product using the Promega
Cell Titer 96 nonradioactive cell proliferation assay.11 Com-
pounds 6p and 6q were found to be nontoxic to 62.5 µg/mL.
Compound 6p showed selectivity index (IC50/ MIC) of more
than 1250.
Acknowledgment. The authors are thankful to Dr. Vanaja
Kumar, Deputy Director, Tuberculosis Research Center, Chen-
nai, India, for assistance in biological screening.
Subsequently, 6q was tested for efficacy against MTB at a
dose of 50 mg/kg (Table 2) in 6-week-old female CD-1 mice.
In this model,12 the mice were infected intravenously through
the caudal vein with approximately 107 viable M. tuberculosis
ATCC 35801. Drug treatment began after inoculation of the
animal with microorganism for 10 days by the intraperitoneal
Supporting Information Available: Experimental procedures
for synthesis and analytical data of representative compounds are
available. This material is available free of charge via the Internet
at http://pubs.acs.org.

3450 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
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JM060339H