Antimycobacterial activities of novel 2-(sub)-3-fluoro/nitro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6 -carboxylic acid

Article abstract of DOI:10.1016/j.bmc.2007.11.016

Various 2-(sub)-3-fluoro/nitro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6 -carboxylic acid derivatives were synthesized from 2-aminothiophenol by a five-step reaction, evaluated for in-vitro and in-vivo antimycobacterial activities against Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant Mycobacterium tuberculosis (MDR-TB), and Mycobacterium smegmatis (MC2), and also tested for the ability to inhibit the supercoiling activity of DNA gyrase from M. smegmatis. Among the thirty-four synthesized compounds, 2-(3-(diethylcarbamoyl)piperidin-1-yl)-)-3-fluoro-5,12-dihydro-5-oxobenz othiazolo[3,2-a]quinoline-6-carboxylic acid (7l) was found to be the most active compound in vitro with MIC of 0.18 and 0.08 μM against MTB and MTR-TB, respectively. Compound 7l was found to be 2 and 570 times more potent than isoniazid against MTB and MDR-TB, respectively. In the in-vivo animal model 7l decreased the bacterial load in lung and spleen tissues with 2.78 and 3.12 - log 10 protections, respectively, at the dose of 50 mg/kg body weight.

Full text of DOI:10.1016/j.bmc.2007.11.016

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3408–3418
Antimycobacterial activities of novel 2-(sub)-3-fluoro/nitro-5,
12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid
Murugesan Dinakaran,a Palaniappan Senthilkumar,a Perumal Yogeeswari,a
Arnab China,b Valakunja Nagarajaa and Dharmarajan Srirama,*
aMedicinal Chemistry Research Laboratory, Pharmacy group, Birla Institute of Technology and Science, Pilani 333031, India
bDepartment of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India
Received 15 September 2007; revised 2 November 2007; accepted 3 November 2007
Available online 9 November 2007
Abstract—Various 2-(sub)-3-fluoro/nitro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid derivatives were syn-
thesized from 2-aminothiophenol by a five-step reaction, evaluated for in-vitro and in-vivo antimycobacterial activities against
Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant Mycobacterium tuberculosis (MDR-TB), and Mycobacterium
smegmatis (MC2), and also tested for the ability to inhibit the supercoiling activity of DNA gyrase from M. smegmatis. Among
the thirty-four synthesized compounds, 2-(3-(diethylcarbamoyl)piperidin-1-yl)-)-3-fluoro-5,12-dihydro-5-oxobenzothiazolo[3,2-
a]quinoline-6-carboxylic acid (7l) was found to be the most active compound in vitro with MIC of 0.18 and 0.08 lM against
MTB and MTR-TB, respectively. Compound 7l was found to be 2 and 570 times more potent than isoniazid against MTB and
MDR-TB, respectively. In the in-vivo animal model 7l decreased the bacterial load in lung and spleen tissues with 2.78 and
3.12 À log10 protections, respectively, at the dose of 50 mg/kg body weight.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
new compounds employed in the chemotherapy of
tuberculosis,3,4 we also identified benzothiazolo[3,2-
a]quinoline-6-carboxylic acid derivatives which inhibited
in-vitro Mycobacterium tuberculosis H37 Rv (MTB) and
multi-drug resistant Mycobacterium tuberculosis (MDR-
TB). We report herein the results concerning the synthe-
sis of newer benzothiazolo[3,2-a]quinoline-6-carboxylic
acid derivatives by modifying 2nd position with various
unreported bulky secondary amino functions to study
the influence of lipophilic character at 2nd position on
activity against MTB and also to study the antimyco-
bacterial effect of fluoro/nitro groups at 3rd position,
in-vitro and in-vivo antituberculous activity of first rep-
resentative compounds of this family together with tox-
icological results.
Tuberculosis (TB) is a worldwide pandemic; about 2 bil-
lion people, equal to one-third of the world’s total pop-
ulation, are infected with Mycobacterium tuberculosis
(MTB), the microbes that cause TB. TB is a leading kill-
er among HIV-infected people with weakened immune
systems; about 200,000 people living with HIV/AIDS
die from TB every year. Multidrug-resistant TB
(MDR-TB) is a form of TB that does not respond to
the standard treatments using first-line drugs; MDR-
TB is present in virtually all countries recently surveyed
by World Health Organization (WHO) and partners.
450,000 new MDR-TB cases are estimated to occur
every year; the highest rates of MDR-TB are in coun-
tries of the former Soviet Union and in China.1 The
WHO declared TB a global health emergency in 1993,
and the Stop TB Partnership proposed a Global Plan
to stop TB which aims to save 14 million lives between
2006 and 2015.2 In the course of screening to discover
2. Results and discussion
2.1. Synthesis
Several of the quinolone antibacterials, such as moxi-
floxacin and gatifloxacin, have been examined as
inhibitors of MTB.5 However, no large systematic
study has been undertaken to either optimize the fused
quinolone antibacterials against MTB or examine
Keywords: Antimycobacterial activity; Antitubercular activity; Tuber-
culosis; Benzothiazoloquinolone.
*
Corresponding author. Tel.: +91 1596 244684; fax: +91 1596
244183; e-mail: dsriram@bits-pilani.ac.in
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.016

M. Dinakaran et al. / Bioorg. Med. Chem. 16 (2008) 3408–3418
3409
specific structure–activity relationship of the fused quin-
olone against MTB. Lipophilic phenyl substituents at N-
1 position of simple fluoroquinolone were reported ear-
lier6 and also substitution of a sulfur atom at the 2 posi-
tion of 1,4-dihydro-4-oxoquinoline-3-carboxylic acid
derivatives can lead to active compounds.7 Hence, a
conformationally restricted rigid analog formed by
bridging the phenyl and quinolone rings by a sulfur
atom would provide a useful indication of the relevance
of the planarity of the ring system to antimycobacterial
activity. The titled compounds were synthesized from 2-
aminothiophenol by a five-step synthesis. 2-Aminothi-
ophenol (1), on reaction with ethyl malonyl chloride
(2) in presence of triethylamine, yielded ethyl 2-(ben-
zothiazol-2-yl)acetate (3) via two intermediates ethyl 2-(2-
mercaptophenylcarbamoyl)acetate and its enol tautomer.
Compound 3 on reaction with 2,4-dichloro-5-fluoro ben-
zoyl chloride/2,4-dichloro-5-nitro benzoyl chloride in pres-
ence of sodium hydride yielded ethyl 2-(benzothiazol-2-yl)-
3-(2,4-dichloro-5-fluoro/nitrophenyl)-3-oxopropanoates
(4a–b). Compounds 4a–b undergo cyclization in pres-
ence of sodium hydride in ethylene glycol dimethyl ether
yielding ethyl 2-Chloro-3-fluoro/nitro-5,12-dihydro-5-
oxobenzothiazolo[3,2-a]quinoline-6-carboxylates (5a–
b), which on hydrolysis with 2 N sodium hydroxide
yielded 2-chloro-3-fluoro/nitro-5,12-dihydro-5-oxobenzo-
thiazolo [3,2-a]quinoline-6-carboxylic acids (6a–b). The
titled compounds 7–8a–s were prepared by treating
6a–b with appropriate secondary amines in presence of
potassium carbonate under microwave irradiation in
DMSO. When compared to conventional method8 of
2–3 h process, microwave-assisted synthesis was per-
formed with short reaction times (2–3 min) with ease
and was environment friendly. The purity of the synthe-
sized compounds was monitored by thin layer chroma-
tography (TLC) and elemental analyses, and the
structures were identified by spectral data.
(7l) was found to be the most active compound
in vitro with MIC of 0.18 lM against MTB and was 2
and 5.8 times more potent than isoniazid and gatifloxa-
cin, respectively. Subsequently some of the compounds
were evaluated against MDR-TB, and among the
twenty-two compounds screened all the compounds
inhibited MDR-TB with MIC ranging from 0.08 to
7.55 lM and were found to be more active than isonia-
zid (MIC: 45.57 lM) and gatifloxacin (MIC: 8.34 lM).
Elevan compounds (7a, 7i, 7j, 7l, 8l, 7m, 8m, 7o, 8o,
7s, and 8s) inhibited MDR-TB with MIC of less than
1 lM. Compound 7l was found to be the most active
compound in vitro with MIC of 0.08 lM against
MDR-TB and was 104 and 570 times more potent than
gatifloxacin and isoniazid, respectively. The compounds
were also evaluated against MC2 in which all the com-
pounds inhibited MC2 with MIC ranging from 2.79 to
113.26 lM and sixteen compounds were found to be
more active than isoniazid (MIC: 45.57 lM).
With respect to structure-MTB activity relationship, at
C2 position we have studied with various substituted
piperazines (7–8a–f), (thio) morpholines (7–8g–h),
substituted piperidines (7–8i–l), fused piperazines, piper-
idines, and pyrrolidine (7–8m–r). A comparison of the
substitution pattern at C7 demonstrated that the order
of activity was substituted piperidines > fused pipera-
zines, pyrrolidine, and piperidines > substituted pipera-
zines, (thio) morpholines. With respect to C3 position
among the fluoro (7a–r) and nitro (8a–r) substituents,
in most of the cases compounds with fluoro substituents
were found to be more active against MTB.
2.3. In-vitro cytotoxicity
Some compounds were further examined for toxicity
(IC50) in a mammalian Vero cell line upto 62.5 lg/ml con-
centrations. After 72 h of exposure, viability was assessed
on the basis of cellular conversion of MTT into a forma-
zan product using the Promega Cell Titer 96 non-radioac-
tive cell proliferation assay10 and the results are reported
in Tables 1 and 2. Twenty compounds when tested
showed IC50 values ranging from 50.0 to 130.3 lM. A
comparison of the substitution pattern at C3 demon-
strated that nitro substituted analogs were more cytotoxic
than the fluoro substituted analogs. These results are
important as the nitro group substituted compounds with
their increased cytoliability are much less attractive in the
development of a quinolone for the treatment of TB. This
is primarily due to the fact that the eradication of TB re-
quires a lengthy course of treatment, and the need for
an agent with a high margin of safety becomes a primary
concern. The IC50 for the compound 7l was found to be
126.1 lM and showed selectivity index (IC50/MIC) of
1576 against MDR-TB.
2.2. Antimycobacterial activity
The compounds were screened for their in-vitro antimy-
cobacterial activity against MTB, MDR-TB, and M.
smegmatis ATCC 14468 (MC2) by agar dilution method
for the determination of MIC in duplicate.9 The MDR-
TB clinical isolate was resistant to isoniazid, rifampicin,
ethambutol, and ofloxacin. The minimum inhibitory
concentration (MIC) is defined as the minimum concen-
tration of compound required to give complete inhibi-
tion of bacterial growth, and MICs of the synthesized
compounds along with the standard drugs for compari-
son are reported in Tables 1 and 2.
In the first phase of screening against MTB, all the com-
pounds showed excellent in-vitro activity against MTB
with MIC of less than 12.82 lM. Five compounds (7i,
7j, 7l, 7m, and 7s) inhibited MTB with MIC of less than
1 lM and were more potent than standard fluoroquino-
lone gatifloxacin (MIC: 1.04 lM). When compared to
isoniazid (MIC: 0.36 lM), three compounds (7i, 7j,
and 7s) were equally active and one compound (7l)
was found to be more active against MTB. Compound
2-(3-(diethylcarbamoyl)piperidin-1-yl)-)-3-fluoro-5,12-dihy-
dro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid
2.4. In-vivo antimycobacterial activity
Subsequently, compound 7l was tested for efficacy
against MTB at a dose of 50 mg/kg (Table 3) in CD-1
mice. In this model,11 the mice were infected intrave-
nously with M. tuberculosis ATCC 35801. Drug treat-
ment by intraperitoneal route began after 10 days of

3410
M. Dinakaran et al. / Bioorg. Med. Chem. 16 (2008) 3408–3418
Table 1. Physical constants, in-vitro antimycobacterial activities, and cytotoxicity
O
O
R
OH
R1
N
S
Compound
R
R1
Yield (%)
Mp (°C)
IC50 (lM)
MIC (lM)
MTB
MDRTB
MC2
Cl
7a
F
70
253–255
104.5
2.61
0.65
10.45
N
N
8a
7b
NO2
F
75
77
209–210
267–268
50.0
NT
5.01
6.37
NT
NT
39.99
50.87
O
C
N
N
O
8b
7c
8c
7d
8d
7e
8e
7f
NO2
76
73
72
75
73
77
75
75
72
207–210
181–182
209–210
259–261
287–290
237–238
233–235
>300
NT
6.04
1.47
NT
5.89
NT
NT
NT
5.59
NT
5.06
NT
48.22
5.89
H2C
N
N
O
F
>117.6
111.9
NT
O
NO2
5.60
44.76
102.55
97.17
2.79
N
H3C
N
F
12.82
12.15
1.39
C6H5
NO2
NT
O
O
C
F
111.7
NT
N
N
O
F
NO2
10.66
2.52
85.24
40.42
77.45
O
F
NT
C
N
N
N
CH3
O
F
8f
NO2
281–283
NT
4.85

M. Dinakaran et al. / Bioorg. Med. Chem. 16 (2008) 3408–3418
3411
Table 1 (continued)
Compound
R
R1
Yield (%)
Mp (°C)
IC50 (lM)
MIC (lM)
MTB
3.76
MDRTB
MC2
7g
8g
F
S
N
76
74
277–278
268–271
NT
NT
7.55
NT
7.55
NO2
7.09
7.34
113.26
29.31
7h
F
O
N
70
256–260
NT
NT
8h
7i
NO2
F
79
72
237–238
260–262
NT
6.90
0.39
NT
110.26
6.53
130.3
0.19
N
N
8i
NO2
75
294–296
61.7
3.08
1.54
98.71
NT, Not tested.
inoculation of the animal with microorganism and was
continued for 10 days. After 35 days post infection the
spleens and right lungs were aseptically removed, the
number of viable organisms was determined and com-
pared with the counts from negative (vehicle treated)
controls (Mean culture forming units (CFU) in lung:
7.99 0.16 and in spleen: 9.02 0.21). Compound 7l de-
creased the bacterial load in lung and spleen tissues with
2.78 and 3.12 À log10 protections, respectively and was
considered to be promising in reducing bacterial count
in lung and spleen tissues. When compared to gatifloxa-
cin at the same dose level 7l decreased the bacterial load
with 0.81 and 1.02 À log10 protections in lung and
spleen tissues, respectively. Compound 7l was found to
be less active than isoniazid in the in-vivo study. The
reason for this less in-vivo activity might be the low bio-
availability of the compound (Scheme 1).
ase are presented in Figure 1. The IC50 values presented
in Table 4 show that the compounds 7j, 7l, and 7r inhibit
supercoiling activity with a IC50 value of 40 lg/ml. The
other two compounds tested, that is, 7e and 7i, have
IC50 values >50 lg/ml, which is much higher than that
of the positive control moxifloxacin though these com-
pounds show comparable cell killing activity against
MTB, MDR-MTB and MC2.
2.6. Phototoxic evaluation
Quinolones in general have favorable safety profiles;
phototoxicity has become a significant factor in the clini-
cal use of some14 quinolones. Indeed, the first quinolone,
nalidixic acid, caused light-induced dermal effects. This
type of response has now been demonstrated for almost
all Fluoroquinolones,15 although the relative phototoxic
potential varies greatly among compounds. Phototoxicity
is considered to be an acute, light-induced irritation
response characterized by dermal inflammation, with ery-
thema and edema as primary clinical endpoints. Photo-
toxicity with the quinolones is generally thought to result
from the absorption of light by the parent compound or a
metabolite in tissue.16 This photosensitized chromophore
may then transfer its absorbed photo energy to oxygen
molecules, creating an environment for the production
of reactive oxygen species such as singlet oxygen. These
reactive species are then thought to attack cellular lipid
membranes, initiating the inflammatory process.
2.5. DNA gyrase inhibition
The benzthiazoloquinolone derivatives synthesized and
studied in this report were tested for their ability to in-
hibit supercoiling activity of DNA gyrase. The bacterial
targets for quinolones and fluoroquinolones are the type
II DNA topoisomerases, DNA gyrase, and topoisomer-
ase IV. These ATP-dependent enzymes act by a tran-
sient double-stranded DNA break, followed by strand
passage and religation reactions to facilitate DNA trans-
action processes.12 DNA gyrase is unique in catalyzing
the negative supercoiling of DNA and is essential for
DNA replication, transcription, and recombination. In
all the species of mycobacteria including MTB, DNA
gyrase is the sole type II topoisomerase carrying out
the reactions of both the type II topoisomerases. Fur-
ther, our earlier studies have revealed that DNA gyrase
from M. tuberculosis and that from M. smegmatis are
highly similar at protein level, antigenic properties,
and catalytic activities.13 The supercoiling assay results
with various compounds using M. smegmatis DNA gyr-
Three (7j, 7l, 7m) compounds were evaluated for poten-
tial phototoxicity in a standardized in-vivo test system
that has been used previously to assess quinolone antibi-
otics.17 The test compounds (140 mg/kg) and the positive
control lomefloxacin hydrochloride (140 mg/kg) were
evaluated for phototoxicity and both ears of each mouse
were evaluated for changes indicative of a positive re-
sponse: erythema, edema or a measurable increase in
ear thickness. Change from baseline was calculated sep-

3412
M. Dinakaran et al. / Bioorg. Med. Chem. 16 (2008) 3408–3418
Table 2. Physical constants, in-vitro antimycobacterial activities, and cytotoxicity
O
O
R
OH
R1
N
S
Compound
R
R1
Yield (%)
Mp (°C)
IC50 (lM)
MIC (lM)
MTB
0.36
MDRTB
MC2
HO
7j
8j
F
79
70
229–232
214–215
119.5
113.6
0.36
2.84
2.98
Cl
N
NO2
2.84
2.77
90.91
44.41
N
N
7k
F
75
233–235
NT
5.56
O
N
H
Cl
8k
7l
NO2
F
74
70
224–225
256–257
NT
5.31
0.18
NT
84.75
3.15
O
C
(C2H5)2N
126.1
0.08
N
8l
NO2
F
77
78
74
71
76
80
227–230
280–283
198–199
271–272
202–205
263–265
59.8
2.99
0.86
1.62
2.87
2.73
3.26
0.75
0.86
0.39
1.43
5.49
0.39
95.68
6.89
O
O
7m
8m
7n
8n
7o
N
>137.5
129.8
115.0
NT
NO2
F
103.85
45.99
43.81
52.25
N
O
C
NHC(CH3)3
NO2
F
N
HOOC
>130.6
N
N
8o
NO2
79
255–258
123.65
1.54
0.77
98.92
N
7p
8p
F
71
79
264–266
219–220
NT
NT
5.50
NT
NT
43.96
83.94
H2C
OCH3
NO2
10.49

M. Dinakaran et al. / Bioorg. Med. Chem. 16 (2008) 3408–3418
3413
Table 2 (continued)
Compound
R
R1
Yield (%)
Mp (°C)
IC50 (lM)
MIC (lM)
MTB
1.55
MDRTB
MC2
H3CO
H3CO
7q
8q
F
74
76
>300
>123.9
117.6
3.09
2.93
6.20
N
NO2
237–240
1.47
0.37
47.04
t BOC
NH
7r
F
258–259
>122.7
0.18
6.14
N
8r
NO2
—
239–240
—
—
58.24
>155.3
>455.8
2.91
1.04
0.36
0.73
8.34
45.57
93.19
2.08
45.57
Gati
INH
—
—
—
—
—
Table 3. In vivo activity data of 7l, gatifloxacin, and isoniazid against
M. tuberculosis ATCC 35801 in mice
on Perkin-Elmer model 240 C analyzer and the data
were within 0.4% of the theoretical values.
Compound
Lungs (log
CFU SEM)
Spleen (log
CFU SEM)
3.1. Synthesis of ethyl 2-(benzothiazol-2-yl)acetate (3)
Control
7.99 0.16
6.02 0.23
5.86 0.23
5.21 0.12
9.02 0.21
6.92 0.07
4.71 0.10
5.9 0.06
Ethyl malonyl chloride (5.0 g) (2) is added to a solution
of 2-aminothiophenol (3.6 ml) (1) and triethylamine
(4.6 ml) in anhydrous ether, and the reaction mixture
is stirred for 3 h at room temperature. The triethylamine
hydrochloride salt is filtered and washed with diethyl
ether. The product is purified by distillation yielding
5.4 g of compound 3 (mp: 190–192 °C).
Gatifloxacin (50 mg/kg)
Isoniazid (25 mg/kg)
7l (50 mg/kg)
arately for each animal and time point and analyzed for
statistical significance and are presented in Table 5. The
drug and time factors were analyzed by separate univar-
iate methods. Orthogonal contrasts were used to test for
both linear and quadratic trends over time in each group
by Student’s t-tests to test whether the change from base-
line ear thickness was significantly different from zero.
The results indicated that lomefloxacin showed signifi-
cant increase in ear thickness from 4 to 96 h and 24 to
96 h when compared within time points and with the
control, respectively. No significant difference in ear
thickness was observed with the test compounds at vari-
ous time-points when compared with the pre-drug read-
ing (0 h) and were non-toxic when compared with the
negative (vehicle-treated) and positive controls (lome-
floxacin). No erythema occurred in mice dosed with
140 mg/kg of 7j, 7l, and 7m throughout the 96 h study.
3.2. Synthesis of ethyl 3-(benzothiazol-2-yl)-4-(2,4-dichloro-
5-fluoro/nitrophenyl)-2,4-dioxobutanoate (4a–b)
Compound 3 (5.4 g) dissolved in anhydrous tetrahydro-
furan (100 ml) is added slowly to a cold solution of so-
dium hydride (60% in mineral oil) (1.1 g) suspended in
anhydrous tetrahydrofuran. After stirring for 1 h, a
solution of 2,4-dichloro-5-fluoro benzoyl chloride/2,4-
dichloro-5-nitro benzoyl chloride in anhydrous tetrahy-
drofuran is added dropwise over a period of 15 min. The
reaction mixture is stirred at 25 °C for 2 h. The solvent is
removed under reduced pressure, and the residue is
redissolved in ethyl acetate. The organic layer is washed
with water and saturated aqueous sodium chloride, and
then dried over magnesium sulfate. Removal of solvent
in vacuo yields a solid which is recrystallized to give
compounds 4a–b. (4a: Yield: 83%; mp: 210–213 °C, 4b:
Yield: 79%; mp: 219–222 °C).
3. Materials and methods
Melting points were taken on an electrothermal melting
point apparatus (Buchi BM530) in open capillary tubes
and are uncorrected. Infrared spectra (KBr disk) were
3.3. Synthesis of ethyl 2-chloro-3-fluoro/nitro-5,12-dihy-
dro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid
(5a–b)
1
run on Jasco IR Report 100 spectrometer. H NMR
spectra were scanned on a JEOL Fx 300 MHz NMR
spectrometer using DMSO-d6 as solvent. Chemical
shifts are expressed in d (ppm) relative to tetramethylsil-
ane. Elemental analyses (C, H, and N) were performed
Compounds 4a–b (approximately 4.0 g) dissolved in eth-
ylene glycol dimethyl ether (75 ml) are added slowly to a
solution of sodium hydride (60% in mineral oil) (0.42 g)

3414
M. Dinakaran et al. / Bioorg. Med. Chem. 16 (2008) 3408–3418
COOC2H5
COOC2H5
COOC2H5
OC2H5
O
OH
SH
H2N
SH
HN
SH
N
N
S
O
a
+
O
Cl
2
1
3
O
O
O
O
S
R
R
b
OC2H5
d
OC2H5
c
Cl
N
S
Cl
Cl
N
5a-b
4a-b
O
O
O
O
R
R
OH
OH
e
R1
N
Cl
S
N
N
S
6a-b
7-8a-s
R1
Scheme 1. Synthetic protocol of compounds. Reagents: (a) (C2H5)3N; (b) NaH, 2,4-dichloro-5-fluoro benzoyl chloride/2,4-dichloro-5-nitro benzoyl
chloride; (c) NaH; (d) 2N NaOH; (e) MWI.
in ethylene glycol dimethy ether (75 ml). The mixture is
heated at 160 °C for 30 min. The reaction mixture is al-
lowed to cool to room temperature and then treated
with cold water. The precipitate is collected and washed
with water yielding compounds 5a–b (5a: Yield: 62%;
mp: 220–221 °C, 5b: Yield: 67%; mp: 230–234 °C).
were irradiated in a microwave oven at an intensity
of 80% with 30 s/cycle. The number of cycle in turn
depended on the completion of the reaction, which
was checked by TLC. The reaction timing varied from
1.5 to 3 min. After completion of the reaction, the
mixture was poured into ice-cold water and washed
with water and isopropanol to give titled products
7–8a–r.
3.4. Synthesis of 2-chloro-3-fluoro/nitro-5,12-dihydro-5-
oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid (6a–
b)
3.5.1. 2-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-
yl)-3-fluoro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quino-
line-6-carboxylic acid (7a). Yield: 70%; mp: 253–255 °C;
Compounds 5a–b (approximately 0.5 g) in tetrahydrofu-
ran (100 ml) were treated with 2 N sodium hydroxide
(15 ml) and the mixture was heated at 85 °C for 6 h
and was cooled. The precipitate was filtered and washed
with ether followed by cold water. The solid was dis-
solved in trifluoroacetic acid (15 ml) and water
(500 ml) was added, and the precipitate was filtered
yielding 6a–b (6a: Yield: 92%; mp: >300 °C, 6b: Yield:
94%; mp: >300 °C).
1
IR (KBr) cmÀ1: 2910, 1722, 1710, 1620, 1460–1370; H
NMR (DMSO-d6) d ppm: 2.59–3.12 (m, 8H, CH2 of
piperazine), 4.22 (s, 1H, CH of diphenylmethyl), 5.9 (s,
1H, C1-H), 6.3–7.15 (m, 13H, Ar-H), 7.26 (s, 1H, C4-
H), 14.2 (s, 1H, COOH); Anal. (C33H25ClFN3O3S) C,
H, N.
3.5.2. 2-(4-(2-Furoyl)piperazin-1-yl)-3-nitro-5,12-dihy-
dro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid
3.5. Synthesis of 2-(sub)-3-fluoro/nitro-5,12-dihydro-5-
oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid (7–
8a–s)
(8b). Yield: 75%; mp: 207–210 °C; IR (KBr) cmÀ1: 2890,
1
1710, 1724, 1624, 1460–1360; H NMR (DMSO-d6) d
ppm: 3.16–3.26 (m, 8H, CH2 of piperazine), 5.9 (s,
1H, C1-H), 6.32–7.68 (m, 7H, Ar-H), 7.26 (s, 1H, C4-
H), 14.2 (s, 1H, COOH); Anal. (C25H18N4O7S) C, H, N.
Compounds 6a–b (1.0 equiv) in dimethylsulfoxide
(2.5 ml) and appropriate secondary amines (4 equiv)

M. Dinakaran et al. / Bioorg. Med. Chem. 16 (2008) 3408–3418
3415
Gyrase
-
+
+
+
+
+
+
+
a
Controls
Compounds
-
-
DMSO Mox
-
-
7j
-
7i
-
7l
-
7e
-
8r
-
R
S
1
2
3
4
5
6
7
8
Gyrase
-
+
+
+
-
+
b
Controls
-
-
DMSO Mox
-
Compounds
-
-
7l
7e
R
S
1
2
3
4
5
Figure 1. DNA gyrase supercoiling assay. The assays were carried out as described in Section 3. DNA gyrase was pre-incubated with the indicated
concentrations of compounds in ice and then rest of the components of the reaction including relaxed DNA was added. (a) Lane 1: relaxed circular
DNA, lane 2: supercoiling reaction in presence of 5% DMSO, lane 3: positive control for gyrase inhibition, moxifloxacin at 5 lg/ml concentration.
Lanes 4–8: supercoiling reaction in presence of 50 lg/ml of compounds 7j, 7i, 7l, 7e, and 7r, respectively. (b) Lane 1: relaxed circular DNA, lane 2:
supercoiling reaction in presence of 5% DMSO, lane 3: moxifloxacin at 5 lg/ml concentration. Lanes 4 and 5: supercoiling reaction in presence of
40 lg/ml of compounds 7l and 7e, respectively. R and S indicate relaxed and supercoiled DNA, respectively.
Table 4. IC50 values for DNA gyrase inhibition
(m, 7H, Ar-H), 7.28 (s, 1H, C4-H), 14.2 (s, 1H, COOH);
Anal. (C28H22FN3O5S) C, H, N.
Compounds
IC50 (lg/ml)
7e
7i
7j
7l
7r
>50
>50
40
3.5.4. 2-(4-Methyl-3-phenylpiperazin-1-yl)-3-nitro-5,12-
dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic
acid (8d). Yield: 73%; mp: 287–290 °C; IR (KBr) cmÀ1
:
40
50
1
2890, 1710, 1724, 1620, 1460–1368; H NMR (DMSO-
d6) d ppm: 2.2 (s, 3H, CH3), 2.6 (t, 2H, 5-CH2 of piper-
azine), 3.15 (t, 2H, 6-CH2 of piperazine), 3.4 (d, 2H, 2-
CH2 of piperazine), 4.12 (t, 1H, 3-CH of piperazine),
5.9 (s, 1H, C1-H), 6.3–7.2 (m, 9H, Ar-H), 7.3 (s, 1H,
C4-H), 14.4 (s, 1H, COOH); Anal. (C27H22N4O5S) C,
H, N.
3.5.3. 2-(4-((Benzo[d][1,3]dioxol-6-yl)methyl)piperazin-1-
yl)-3-fluoro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quino-
line-6-carboxylic acid (7c). Yield: 75%; mp: 181–182 °C;
IR (KBr) cmÀ1: 3250, 2890, 1710, 1724, 1624, 1464–
1360; H NMR (DMSO-d6) d ppm: 2.82–3.12 (m, 8H,
CH2 of piperazine), 3.6 (s, 2H, CH2 of piperanoyl),
5.86 (s, 2H, –OCH2O–), 5.9 (s, 1H, C1-H), 6.40–7.1
1
3.5.5. 2-(4-(2,3-Dihydrobenzo[b][1,4]dioxin-2-oyl)(pipera-
zin-1-yl)-3-fluoro-5,12-dihydro-5-oxobenzothiazolo[3,2-
a]quinoline-6-carboxylic acid (7e). Yield: 77%; mp: 237–
Table 5. Phototoxic evaluation of newer compounds
Group
Ear thickness (mm)a
Time (approximately) after start of irradiaton (h)c
Erythemab
0
4
24
48
72
96
0
4
24
48
72
96
Controld
0.29 0.02
0.29 0.01
0.27 0.01
0.29 0.01
0.31 0.01
0.29 0.01
0.32 0.03
0.29 0.02
0.30 0.02
0.40 0.02
0.32 0.02
0.31 0.02
0.29 0.02
0.31 0.01
0.48 0.02
0.31 0.01
0.29 0.02
0.29 0.01
0.30 0.01
0.53 0.02
0.34 0.03
0.32 0.02
0.30 0.01
0.28 0.02
0.64 0.04
0.34 0.03
0.30 0.02
0.28 0.01
0.30 0.02
0.60 0.06
0
0
0
0
0
0
0
2
0
6
0
0
0
0
6
0
0
0
0
6
0
0
0
0
6
0
0
0
0
6
7j
7l
7m
Lomefloxacin
a Mean ear thickness SEM; left and right ears were averaged.
b Number of mice with erythema.
c Time zero, pre-dose (mice exposed to UV light immediately after dosing); 4 h, end of irradiation period.
d Control, 0.5% aqueous solution of sodium carboxymethylcellulose (4 Ns/m2) dosed at 10 ml/kg.

3416
M. Dinakaran et al. / Bioorg. Med. Chem. 16 (2008) 3408–3418
238 °C; IR (KBr) cmÀ1: 2890, 1710, 1724, 1624, 1460–
(m, 7H, Ar-H), 7.3 (s, 1H, C4-H), 10.8 (s, 1H, NH),
14.2 (s, 1H, COOH); Anal. (C28H20ClN5O6S) C, H, N.
1
1360; H NMR (DMSO-d6) d ppm: 3.26–3.4 (m, 8H,
CH2 of piperazine), 4.6 (d, 2H, 3-CH2 of dihydrobenzo-
dioxinyl), 5.14 (t, 1H, 2-CH of dihydrobenzodioxinyl),
5.9 (s, 1H, C1-H), 6.3–7.12 (m, 8H, Ar-H), 7.3 (s, 1H,
C4-H), 14.2 (s, 1H, COOH); Anal. (C29H22FN3O6S) C,
H, N.
3.5.12. 2-(3-(Diethylcarbamoyl)piperidin-1-yl)-3-fluoro-
5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-car-
boxylic acid (7l). Yield: 70%; mp: 256–257 °C; IR (KBr)
cmÀ1: 2890, 1710, 1724, 1624, 1464–1360, 1208; 1H
NMR (DMSO-d6) d ppm: 1.2 (t, 6H, 2-CH3 of ethyl),
1.78–2.7 (m, 9H, H of piperidine), 3.24 (q, 4H, 2-CH2
of ethyl), 5.9 (s, 1H, C1-H), 6.2–6.8 (m, 4H, Ar-H), 7.3
(s, 1H, C4-H), 14.2 (s, 1H, COOH); Anal.
(C26H26FN3O4S) C, H, N.
3.5.6. 2-(3-(2,6-Difluorophenyl)-5-methlisoxazol-4-oyl)(pip-
erazin-1-yl)-3-fluoro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]-
quinoline-6-carboxylic acid (7f). Yield: 75%; mp: >300 °C;
IR (KBr) cmÀ1: 2890, 1710, 1724, 1624, 1464–1360,
1
1208; H NMR (DMSO-d6) d ppm: 2.3 (s, 3H, 5-CH3
of isoxazolyl), 3.12–3.28 (m, 8H, CH2 of piperazine),
5.9 (s, 1H, C1-H), 6.32–7.08 (m, 7H, Ar-H), 7.3 (s, 1H,
C4-H), 14.2 (s, 1H, COOH); Anal. (C31H21F3N4O5s)
C, H, N.
3.5.13.
2-(1,4-Dioxa-8-azaspiro[4.5]dec-8-yl)-3-nitro-
5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-car-
boxylic acid (8m). Yield: 74%; mp: 198–199 °C; IR
(KBr) cmÀ1: 2890, 1712, 1724, 1618, 1466–1368; 1H
NMR (DMSO-d6) d ppm: 1.78-2.4 (m, 8H, 4-CH2 of
azaspirodecane), 3.96 (m, 4H, 2-CH2 of azaspirode-
cane), 5.9 (s, 1H, C1-H), 6.2–6.82 (m, 4H, Ar-H), 7.32
(s, 1H, C4-H), 14.4 (s, 1H, COOH); Anal.
(C23H19N3O7S) C, H, N.
3.5.7. 2-Thiomorpholino-3-nitro-5,12-dihydro-5-oxobenzo-
thiazolo[3,2-a]quinoline-6-carboxylic acid (8g). Yield:
74%; mp: 268–271 °C; IR (KBr) cmÀ1: 2890, 1710,
1
1724, 1620, 1460–1368; H NMR (DMSO-d6) d ppm:
2.64 (t, 4H, 3,5-CH2 of thiomorpholine), 3.38 (t, 4H,
2,6-CH2 of thiomorpholine), 5.9 (s, 1H, C1-H), 6.32–
6.88 (m, 4H, Ar-H), 7.3 (s, 1H, C4-H), 14.2 (s, 1H,
COOH); Anal. (C20H15N3O5S2) C, H, N.
3.5.14. 2-(1-(tert-Butylcarbamoyl)-3,4-dihydroisoquino-
lin-2(1H)-yl)-3-fluoro-5,12-dihydro-5-oxobenzothiazol-
o[3,2-a]quinoline-6-carboxylic acid (7n). Yield: 71%;
mp: 271–272 °C; IR (KBr) cmÀ1: 2890, 1712, 1724,
1
3.5.8. 2-(2,6-Dimethylmorpholino)-3-fluoro-5,12-dihy-
dro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid
(7h). Yield: 70%; mp: 258–260 °C; IR (KBr) cmÀ1: 2892,
1626, 1468–1360; H NMR (DMSO-d6) d ppm: 1.3 (s,
9H, 3 CH3), 2.66–2.9 (m, 4H, 2 CH2 of isoquinoline),
4.85 (s, 1H, CH of isoquinoline), 5.88 (s, 1H, C1-H),
6.28–7.1 (m, 8H, Ar-H), 7.32 (s, 1H, C4-H), 10.2 (s,
1H, NH), 14.2 (s, 1H, COOH); Anal. (C30H26FN3O4S)
C, H, N.
1
1710, 1724, 1628, 1460–1360; H NMR (DMSO-d6) d
ppm: 1.2 (d, 6H, 2,6 CH3 of morpholino), 3.0 (d, 4H,
2,6-CH2 of morpholine), 3.9 (m, 2H, 3,5-CH of morpho-
line), 5.9 (s, 1H, C1-H), 6.4–6.9 (m, 4H, Ar-H), 7.3 (s,
1H, C4-H), 14.2 (s, 1H, COOH); Anal. (C22H19FN2O4S)
C, H, N.
3.5.15. 2-(2-Carboxy-5,6-dihydroimidazo[1,2-a]pyrazin-
7(8H)-yl)-3-nitro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quin-
oline-6-carboxylic acid (8o). Yield: 79%; mp: 255–
258 °C; IR (KBr) cmÀ1: 3200, 2890, 1710, 1724, 1624,
3.5.9. 2-(4-(Piperidin-1-yl)piperidin-1-yl)-3-nitro-5,12-
dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic
1
1464–1360, 1208; H NMR (DMSO-d6) d ppm: 3.1–3.8
acid (8i). Yield: 75%; mp: 294–296 °C; IR (KBr) cmÀ1
:
(m, 6H, 3-CH2), 5.88 (s, 1H, C1-H), 6.28–7.0 (m, 4H,
Ar-H), 7.6 (s, 1H, CH), 7.32 (s, 1H, C4-H), 12.12 (s,
1H, 2-COOH), 14.6 (s, 1H, 3-COOH); Anal.
(C23H15N5O7S) C, H, N.
1
2890, 1710, 1724, 1624, 1464–1360; H NMR (DMSO-
d6) d ppm: 1.5–1.6 (m, 10H, 5 CH2), 2.2 (t, 4H, 2
CH2), 2.7 (m, 1H, CH), 2.8 (t, 4H, 2 CH2), 5.9 (s, 1H,
C1-H), 6.4–6.88 (m, 4H, Ar-H), 7.3 (s, 1H, C4-H), 14.2
(s, 1H, COOH); Anal. (C26H26N4O5S) C, H, N.
3.5.16. 2-(8-(4-Methoxybenzyl)-3,4,5,6,7,8-hexahydroiso-
quinolin-2(1H)-yl)-3-fluoro-5,12-dihydro-5-oxobenzothiaz-
olo[3,2-a]quinoline-6-carboxylic acid (7p). Yield: 79%;
mp: 219–220 °C; IR (KBr) cmÀ1: 3210, 2890, 1710,
3.5.10. 2-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)-3-
fluoro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-
6-carboxylic acid (7j). Yield: 79%; mp: 229–232 °C; IR
(KBr) cmÀ1: 2900, 1708, 1724, 1620, 1460–1370; 1H
NMR (DMSO-d6) d ppm: 2.0 (t, 4H, 3,5-CH2 of piper-
idine), 2.7 (t, 4H, 2,6-CH2 of piperidine), 5.9 (s, 1H, C1-
H), 6.28–7.18 (m, 8H, Ar-H), 7.3 (s, 1H, C4-H), 10.0 (bs,
1H, OH), 14.62 (s, 1H, COOH); Anal. (C27H20-
ClFN2O4S) C, H, N.
1
1724, 1620, 1460–1368; H NMR (DMSO-d6) d ppm:
1.6–1.95 (m, 8H, 4-CH2 of isoquinolinyl), 2.2-3.4 (m,
7H, 2-CH2 and 1-CH of isoquinolinyl, and CH2), 3.73
(s, 3H, –OCH3), 5.88 (s, 1H, C1-H), 6.28–7.1 (m, 8H,
Ar-H), 7.32 (s, 1H, C4-H), 14.2 (s, 1H, COOH); Anal.
(C33H29FN2O4S) C, H, N.
3.5.17. 2-(3,4-Dihydro-6,7-dimethoxyisoquinolin-2(1H)-
yl)-3-nitro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quino-
line-6-carboxylic acid (8q). Yield: 76%; mp: 237–240 °C;
IR (KBr) cmÀ1: 3210, 2890, 1710, 1724, 1620, 1460–
3.5.11. 2-(4-(6-Chloro-1,2-dihydro-2-oxobenzo[d]imida-
zol-3-yl)piperidin-1-yl)-3-nitro-5,12-dihydro-5-oxobenzo-
thiazolo[3,2-a]quinoline-6-carboxylic acid (8k). Yield:
74%; mp: 224–225 °C; IR (KBr) cmÀ1: 3110, 2896,
1
1368; H NMR (DMSO-d6) d ppm: 2.72–3.6 (m, 6H,
1
1712, 1728, 1710, 1624, 1460–1360; H NMR (DMSO-
CH2 of isoquinolinyl), 3.7 (s, 6H, –OCH3), 5.88 (s, 1H,
C1–H), 6.3–6.93 (m, 6H, Ar-H), 7.32 (s, 1H, C4-H),
14.2 (s, 1H, COOH); Anal. (C27H21N3O7S) C, H, N.
d6) d ppm: 1.6–2.4 (m, 8H, 4 CH2 of piperidine), 4.1
(bm, 1H, CH of piperidine), 5.9 (s, 1H, C1-H), 6.2–6.9

M. Dinakaran et al. / Bioorg. Med. Chem. 16 (2008) 3408–3418
3417
3.5.18. 2-(6-tert-Butoxycarbonylamino-3-azabicyclo[3.1.0]-
hex-3-yl)-3-nitro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]-
quinoline-6-carboxylic acid (8r). Yield: 76%; mp: 239–
240 °C; IR (KBr) cmÀ1: 3210, 2890, 1710, 1724, 1620,
2 mM ATP, 50 lg/ml bovine serum albumin, and 90 lg/
ml yeast t-RNA in 5% (v/v) glycerol) for 30 min. The
compounds tested were dissolved in DMSO and pre-
incubated with gyrase in reaction buffer prior to the
addition of DNA. Moxifloxacin at final concentration
of 5 lg/ml was used as a positive control and another
control reaction having 5% DMSO in absence of com-
pounds was also performed. The reaction samples were
heat-inactivated at 65 °C for 15 min and applied onto
1% agarose gel for electrophoresis in Tris–acetate–
EDTA buffer for 12 h. The gels were stained with ethi-
dium bromide.
1
1460–1368; H NMR (DMSO-d6) d ppm: 1.43 (s, 9H,
tert-butyl), 1.78–3.8 (m, 7H, azabicyclohexane ring H),
4.76 (bs, 1H, NH-t-Boc), 5.88 (s, 1H, C1-H), 6.3–6.8
(m, 4H, Ar-H), 7.32 (s, 1H, C4-H), 14.2 (s, 1H, COOH);
Anal. (C26H24N4O7S) C, H, N.
3.6. In-vitro antimycobacterial activity
All compounds were screened for their in-vitro antimy-
cobacterial activity against MTB, MDR-TB, and MC2
in Middlebrook 7H11agar medium supplemented with
OADC by agar dilution method similar to that recom-
mended by the National Committee for Clinical Labora-
tory Standards for the determination of MIC in
duplicate. The MDR-TB clinical isolate was obtained
from Tuberculosis Research Center, Chennai, India,
and was resistant to isoniazid, rifampicin, ethambutol,
and ofloxacin. The minimum inhibitory concentration
(MIC) is defined as the minimum concentration of com-
pound required to give complete inhibition of bacterial
growth.
3.10. Phototoxicity evaluation
Female swiss albino mice, approximately 2 months old
and weighing 20–25 g, were used in this study. Before
oral dosing, they were fasted overnight for at least
18 h. Food was returned at the end of the 4 h photo-irra-
diation period. Eighteen mice were randomly distributed
into three dosing groups. First group received a single
dose of screened compound at 140 mg/kg by oral gavage.
A second group received a single dose of 140 mg of lome-
floxacin HCl/kg. This lomefloxacin dose is one that, in
preliminary experiments in this test system, produced a
consistent erythema and ear thickening response. The fi-
nal group served as a vehicle control and received 10 ml/
kg of the methylcellulose vehicle only. Test animals were
exposed to UV light in a manner adapted from that de-
scribed previously. Animals were irradiated for 4 h,
equal to a total UV light irradiation of approximately
18 J/cm2. Before dosing, at the end of the irradiation per-
iod, and at approximately 24, 48, 72, and 96 h after dos-
ing, both ears of each mouse were evaluated for changes
indicative of a positive response: erythema, edema or a
measurable increase in ear thickness.
3.7. Cytotoxicity
Some compounds were further examined for toxicity
(IC50) in a mammalian Vero cell line at concentrations
of 62.5 lg/ml. After 72 h of exposure, viability was as-
sessed on the basis of cellular conversion of MTT into
a formazan product using the Promega Cell Titer 96
non-radioactive cell proliferation assay.
3.8. In-vivo antimycobacterial activity
One compound was tested for efficacy against MTB at a
dose of 25 mg/kg in six-week-old female CD-1 mice six
per group. In this model, the mice were infected intrave-
nously through caudal vein with approximately 107 via-
ble M. tuberculosis ATCC 35801. Drug treatment by
intraperitoneal route began after 10 days of inoculation
of the animal with microorganism and was continued
for 10 days. After 35 days post infection the spleens
and right lungs were aseptically removed and ground
in a tissue homogenizer, the number of viable organisms
was determined by serial 10-fold dilutions and subse-
quent 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 controls (vehicle treated)
in lung and in spleen.
Acknowledgments
The authors are thankful to Department of Biotechnol-
ogy (BT/01/COE/05/06/01), Government of India, for
their financial assistances. The authors are also thankful
to Dr. Vanaja Kumar, Deputy Director, Tuberculosis
Research Center, Chennai, India, for their assistance
in biological screening.
References and notes
1. World Health Organization, Tuberculosis fact sheet 2007.
2. Zignol, M.; Hosseini, M. S.; Wright, A.; Weezenbeek, C.
L.; Nunn, P.; Watt, C. J.; Williams, C. G.; Dye, C. J.
Infect. Dis. 2006, 194, 479.
3. Sriram, D.; Yogeeswari, P.; Thirumurugan, R.; Pavana,
R. K. J. Med. Chem. 2006, 49, 3448.
3.9. DNA gyrase supercoiling assay
4. Sriram, D.; Yogeeswari, P.; Dinakaran, M.; Thirumur-
ugan, R. J. Antimicrob. Chemother. 2007, 59, 1194.
5. Kubendiran, G.; Paramasivan, C. N.; Sulochana, S.;
Mitchison, D. A. J. Chemother. 2006, 18, 617.
6. Renau, T. E.; Sanchez, J. P.; Shapiro, M. A.; Dever, J. A.;
Gracheck, S. J.; Domagala, J. M. J. Med. Chem. 1995, 38,
2974.
DNA gyrase was purified from M. smegmatis cells using
the method described by Manjunatha et al.18 The super-
coiling assays were carried out by the procedure already
described.11 Briefly, supercoiling assays were carried out
by incubating 400 ng of relaxed circular pUC18 in
supercoiling buffer (35 mM Tris–HCl, pH 7.5, 5 mM
MgCl2, 25 mM potassium glutamate, 2 mM spermidine,
7. Kondo, H.; Taguchi, H.; Inoue, Y.; Sakamoto, F.;
Tsukamoto, G. J. Med. Chem. 1990, 33, 2012.

3418
M. Dinakaran et al. / Bioorg. Med. Chem. 16 (2008) 3408–3418
8. Tsuzuki, Y.; Tomita, K.; Shibamori, K.; Sato, Y.;
13. Manjunatha, U. H.; Madhusudan, K.; Sandhya, S.
Visweswariah.; Nagaraja, V. Curr. Sci. 2000, 79, 968.
14. Ferguson, J.; Walker, E. M.; Johnson, B. E. British
J. Dermat. 1989, 120, 291.
15. Domagala, J. M. J. Antimicrob. Chemother. 1994, 33, 685–
706.
Kashimoto, S.; Chiba, K. J. Med. Chem. 2004, 47, 2097.
9. National Committee for Clinical Laboratory Standards.
Antimycobacterial susceptibility testing for Mycobacte-
rium tuberculosis. Proposed standard M24-T. National
Committee for Clinical Laboratory Standards, Villanova,
Pa., 1995.
10. Gundersen, L. L.; Nissen-Meyer, J.; Spilsberg, B. J. Med.
Chem. 2002, 45, 1383.
11. Sriram, D.; Yogeeswari, P.; Basha, J. S.; Radha, D. R.;
Nagaraja, V. Bioorg. Med. Chem. 2005, 13, 5774.
12. Levine, C. H.; Hiasa, A.; Marians, K. J. Biochim. Biophys.
Acta 1998, 1400, 29.
16. Takayama, S.; Hirohashi, M.; Kato, M.; Shimada, H.
J. Toxicol. Environ. Health 1995, 45, 1.
17. Mayne, T. N.; Johnson, N. J.; Kluwe, W. M.; Lencoski,
D. L.; Polzer, R. J. J. Antimicrob. Chemother. 1997, 39, 67.
18. Manjunatha, U. H.; Dalal, M.; Chatterji, M.; Radha, D.
R.; Visweswariah, S. S.; Nagaraja, V. Nucleic Acids Res.
2002, 30, 2144.