Synthesis and antimycobacterial activities of novel 6-nitroquinolone-3-carboxylic acids

Article abstract of DOI:10.1016/j.ejmech.2008.02.031

Various 1-(substituted)-1,4-dihydro-6-nitro-4-oxo-7-(sub-secondary amino)-quinoline-3-carboxylic acids were synthesized from 2,4-dichlorobenzoic acid by six step synthesis. The compounds were evaluated for antimycobacterial in vitro and in vivo against My

Full text of DOI:10.1016/j.ejmech.2008.02.031

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 345e358
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antimycobacterial activities of novel
6-nitroquinolone-3-carboxylic acids
Palaniappan Senthilkumar a, Murugesan Dinakaran a, Perumal Yogeeswari a,
b
b
Dharmarajan Sriram a, , Arnab China , Valakunja Nagaraja
*
a Medicinal Chemistry Research Laboratory, Pharmacy group, Birla Institute of Technology and Science, Pilani 333031, India
b Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, Karnataka, India
Received 23 August 2007; received in revised form 15 February 2008; accepted 29 February 2008
Available online 7 March 2008
Abstract
Various 1-(substituted)-1,4-dihydro-6-nitro-4-oxo-7-(sub-secondary amino)-quinoline-3-carboxylic acids were synthesized from 2,4-
dichlorobenzoic acid by six step synthesis. The compounds were evaluated for antimycobacterial in vitro and in vivo 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 48 synthesized compounds, 7-(4-((ben-
zo[d][1,3]dioxol-5-yl)methyl)piperazin-1-yl)-1-cyclopropyl-1,4-dihydro-6-nitro-4-oxoquinoline-3-carboxylic acid (8c) was found to be the
most active compound in vitro with MIC of 0.08 and 0.16 mM against MTB and MDR-TB, respectively. In the in vivo animal model 8c decreased
the bacterial load in lung and spleen tissues with 2.78 and 4.15-log 10 protections, respectively, at the dose of 50 mg/kg body weight.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Nitroquinolones; Quinolones; Antitubercular; Antimycobacterial; Tuberculosis
1. Introduction
market was insufficient to guarantee return on investment.
There is now recognition that new drugs to treat TB are urgently
required, specifically for use in shorter treatment regimens than
are possible with the current agents and which can be employed
to treat multi-drug-resistant and latent disease. Recently World
Health Organization (WHO) recommended third-line regimen
for the treatment of TB which includes quinolone antibacterial
ofloxacin. Quinolones act by interfering with the action of the
bacterial DNA gyrase, resulting in double strand breaks in
DNA which in turn leads to the degradation of chromosomal
DNA. Breaks in the genome caused by quinolones also leads
to termination of chromosomal replication and interference
with cell division and gene expression [2]. Various nitrohetero-
cycles like nitrofuranylamides [3,4], 6-nitro-2,3-dihydroimi-
dazo[2,1-b]oxazoles [5], nitroimidazopyran [6], 5-nitro-2,
3-dihydroimidazo-oxazole [7] and nitroimidazole [8] inhibited
Mycobacterium tuberculosis H37Rv (MTB) in vitro and in vivo.
Till date few 6-nitroquinolones were studied [9,10] and were
mostly bearing identical substituents at positions 1 and 7 of
Tuberculosis (TB) is one of the most prevalent infectious
diseases known to man. About 32% of the world’s population
is infected with TB. Every year, approximately 8 million of
these infected people develop active TB. It is the leading cause
of mortality and morbidity among the infectious diseases with
about 2.2 million deaths every year worldwide [1]. In the recent
past, TB is receiving the increased attention that this global
public health problem deserves. Governments, nongovernmen-
tal organizations, and philanthropic organizations have begun
investing funds required to control and eventually eliminate
this scourge. Until now, progress in TB drug development has
been impeded by two major factors: (a) the belief that there
was little need for new agents, and (b) the high cost of develop-
ment coupled with the perception that the potential global
* Corresponding author. Tel.: þ91 1596 244684; fax: þ91 1596 244183.
E-mail address: dsriram@bits-pilani.ac.in (D. Sriram).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.02.031

346
P. Senthilkumar et al. / European Journal of Medicinal Chemistry 44 (2009) 345e358
the quinolone moiety. Taking into account that quinolones
exhibit fairly good antimycobacterial activity and having in
mind the not fully explored antitubercular potential of nitroqui-
nolone pharmacophore, in the present work effort has been
taken to explore the effect of nitro substitution at 6th position
of quinolone in combination with N1 cyclopropyl/t-butyl/4-
fluorophenyl, with various bulky secondary amino function at
7th position. We report herein the synthesis of newer
1-(substituted)-1,4-dihydro-6-nitro-4-oxo-7-(sub-secondary
amino)-quinoline-3-carboxylic acids and evaluation of their
antimycobacterial activities together with toxicological results.
quinolones 6aec. Ethyl esters were finally hydrolyzed in acidic
condition to yield 1-(cyclopropyl/t-butyl/4-fluorophenyl)-1,
4-dihydro-7-chloro-6-nitro-4-oxo-quinoline-3-carboxylic acid
(7aec). The titled compounds 8e10aep were prepared by
treating 7aec with appropriate secondary amines under micro-
wave irradiation in DMSO in closed system. When compared to
conventional method [11] of 24 h process, microwave assisted
synthesis was performed with short reaction times (2e3 min),
with ease and was environment friendly.
The purity of the synthesized compounds was monitored by
thin layer chromatography (TLC) and elemental analyses and
the structures were identified by spectral data. In general, in-
frared spectra (IR) showed C]O stretching peak of carboxylic
acid at 1720e1730 cmꢁ1; C]O stretching peak of pyridine
carbonyl at 1612e1620 cmꢁ1 and absorption of aryl nitro
group at 1470e1370 cmꢁ1. In the nuclear magnetic resonance
spectra (1H NMR) the signals of the respective protons of the
prepared derivatives were verified on the basis of their chem-
ical shifts, multiplicities and coupling constants. The spectra
showed a broad singlet at d 14.6 ppm corresponding to
COOH proton; singlet at d 9.52 ppm corresponding to C2
proton; singlet at d 8.3 ppm corresponding to C8 proton; sin-
glet at d 9.34 ppm corresponding to C5 proton; N1 cyclopropyl
protons showed multiplets at d 1.42 (1H) and 0.28e0.48 (4H)
ppm; N1 t-butyl protons showed singlet at d 1.62 (9H) ppm;
and N1 4-fluorophenyl protons showed multiplet at
d 6.50e6.72 (4H) ppm. The elemental analysis results were
within ꢂ0.4% of the theoretical values.
2. Synthesis
The synthesis of various 6-nitroquinolone (8e10aep) is out-
lined in Scheme 1. 2,4-Dichlorobenzoic acid (1) was converted
to 2,4-dichloro-5-nitrobenzoic acid (2) by treatment with nitric
acid in the presence of sulphuric acid at 0e5 ꢀC. Compound 2
on reaction with 1,10-carbonyldiimidazole (CDI) in tetrahydro-
furan afforded the corresponding imidazolide, which, in situ
was treated with neutral magnesium salt of ethyl potassium
malonate in the presence of tri-ethyl amine to yield ethyl
3-(2,4-dichloro-5-nitrophenyl)-3-oxopropanoate (3). Ethyl 1-
(cyclopropyl/t-butyl/4-fluorophenyl)-7-chloro-6-nitro-4-oxo-1,
4-dihydro-quinoloine-3-carboxylates (6aec) were prepared by
a three-step one-pot reaction. First treatment of the keto ester 3
with tri-ethylorthoformate in acetic anhydride gave the one-
carbon homologue enol ether intermediate ethyl 2-(2,4-
dichloro-5-nitrobenzoyl)-3-ethoxyacrylate (4), which upon
evaporation to dryness was allowed to react with slight excess
of appropriate amines under nitrogen atmosphere in a mixture
of ether and ethanol at 0 ꢀC gave the enamino ester ethyl
2-(2,4-dichloro-5-nitrobenzoyl)-3-(cyclopropyl/4-fluorophenyl/
t-butyl amino)acrylate (5aec), and base catalyzed cyclization
of 5aec with potassium carbonate in DMSO yielded
3. Results and discussion
3.1. Antimycobacterial activity
The compounds were screened for their in vitro antimyco-
bacterial activity against MTB, MDR-TB and Mycobacterium
O
O
O
O
O N
O N
2
2
OH
OH
O
a
b
c
Cl
Cl
Cl
O
Cl
Cl
Cl
3
2
1
O
O
O
O N
2
O N
2
O
O
e
d
NH
Cl
Cl
5a-c
Cl
Cl
OC H
2
5
4
R
1
O
O
O
N
O
O
O
O N
2
O N
2
O N
O
2
OH
OH
g
f
R
N
R
Cl
2
Cl
N
R
N
8a-q
9a-q
10a-q
6a-c
7a-c
R
1
1
R
1
3
R1: Cyclopropyl, 4-F-phenyl and t-butyl; -NR2R3: various secondary amines
Scheme 1. (a) HNO3, H2SO4; (b) CDI, C2H5OOCCH2COOK, MgCl2, (C2H5)3N; (c) (C2H5O)3CH, (CH3CO)2O; (d) R1NH2; (e) K2CO3.

P. Senthilkumar et al. / European Journal of Medicinal Chemistry 44 (2009) 345e358
347
smegmatis ATCC 14468 (MC2) by agar dilution method for
the determination of MIC in duplicate. The MDR-TB clinical
isolate was resistant to isoniazid, rifampicin, ethambutol and
ofloxacin. The minimum inhibitory concentration (MIC) is
defined as the concentration of the compound required to
give complete inhibition of bacterial growth and MICs of
the synthesized compounds along with the standard drugs
for comparison are reported in Table 1.
piperidines ꢃ (thio) morpholines. The results demonstrated
that the contribution of the C7 position to antimycobacterial ac-
tivity was dependent on the substituent at N1 and was in the order
of substituted piperazines > substituted piperidines > fused pi-
perazines and piperidines > (thio) morpholines when N1 was
cyclopropyl; substituted piperazines ꢃ substituted piperidines
and (thio) morpholines > fused piperazines and piperidines
when N1 was 4-fluorophenyl; substituted piperazines ꢃ substi-
tuted piperidines, fused piperazines and piperidines and (thio)
morpholines when N1 was tert-butyl.
Some compounds were further examined for toxicity
(CC50) in a mammalian Vero cell line till 62.50 mg/ml concen-
trations. After 72 h of exposure, viability was assessed on the
basis of cellular conversion of MTT into a formazan product
and the results are reported in Table 1. Nine compounds
when tested showed CC50 values ranging from 54.39 to
142.30 mM. A comparison of the substitution pattern at N1
demonstrated that 4-fluorophenyl group was more cytotoxic
than the cyclopropyl group. These results are important as
the N1 phenyl substituted compounds with their increased cy-
toliability, are much less attractive in the development of a qui-
nolone for the treatment of TB. This is primarily due to the
fact that the eradication of TB requires a lengthy course of
treatment, and the need for an agent with a high margin of
safety becomes a primary concern. Compound 8c was found
to be non-toxic up to 62.50 mg/ml (126.90 mM) and showed se-
lectivity index (CC50/MIC) of more than 1586.
Subsequently, compound 8c was tested for in vivo efficacy
against MTB at the dose of 50 mg/kg (Table 2) in CD-1 mice.
The mice were infected intravenously with M. tuberculosis
ATCC 35801. Drug treatment by intraperitoneal route began
after 10 days of inoculation of the animal with microorganism
and continued for 10 days. After 35 days post-infection the
spleens and right lungs were aseptically removed, and the
number of viable organisms was determined and compared
with the counts from negative (vehicle-treated) controls
(mean culture forming units (CFU) in lung: 7.99 ꢂ 0.16 and
in spleen: 9.02 ꢂ 0.21). Compound 8c decreased the bacterial
load in lung and spleen tissues with 2.78 and 4.15-log 10
protections, 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 8c decreased
the bacterial load with 0.81 and 2.05-log 10 protections in lung
and spleen tissues, respectively. Compound 8c was found to be
less active than isoniazid in the in vivo study on dose basis.
In the first phase of screening against MTB, all the com-
pounds showed good in vitro activity against MTB with MIC
less than 15 mM. Five compounds (8bed, 8i and 8j) inhibited
MTB with MIC of less than 1 mM and were relatively more po-
tent than standard fluoroquinolone gatifloxacin (MIC: 1.04 mM).
When compared to isoniazid (MIC: 0.36 mM), two compounds
(8c and 8d) were found to be more active against MTB.
Compound 7-(4-((benzo[d][1,3]dioxol-5-yl)methyl)pipera-
zin-1-yl)-1-cyclopropyl-1,4-dihydro-6-nitro-4-oxoquinoline-
3-carboxylic acid (8c) was found to be the most active
compound in vitro with MIC of 0.08 mM against MTB and
was 4.50 and 13 times more potent than isoniazid and gatiflox-
acin, respectively. Subsequently some of the compounds were
evaluated against MDR-TB, and among the 18 compounds
screened, all the compounds inhibited MDR-TB with MIC
ranging from 0.16 to 6.01 mM and were found to be more active
than isoniazid (MIC: 45.57 mM), and gatifloxacin (MIC:
8.34 mM). Eight compounds (8bed, 9e, 10f, 8i, 8j and 8o)
inhibited MDR-TB with MIC of less than 1 mM. Compound
8c was found to be the most active compound in vitro with
MIC of 0.16 mM against MDR-TB and was 52 and 284 times
more potent than gatifloxacin and isoniazid, respectively. The
compounds were also evaluated against MC2 strain in which
all the compounds inhibited the bacteria with MIC ranging
from 0.79 to 64.36 mM and 45 compounds were found to be
more active than isoniazid (MIC: 45.57 mM).
With respect to structureeMTB activity relationship, the
results demonstrated that the antimycobacterial activity
imparted by the in N1 substituent was in the order of cyclo-
propyl > 4-flurophenyl > tert-butyl. This result correlated
with the other antibacterial fluoroquinolones, wherein cyclo-
propyl group was the favorable substituent. This result also
correlated well with the previous report on the importance of cy-
clopropyl group as antimycobacterial pharmacophore [12]. The
enhanced activity of the cyclopropyl group could be supported
by a hypothesis based on the belief that the antibacterial activity
of quinolones is related to the amount of un-ionized drug that is
able to penetrate the cell membranes. This in turn is associated
with a highly acidic carboxyl group and a less basic C7 amino
substituent [13]. The carboxyl group becomes more acidic if
the N1 substituent is electron donating in nature. The greater ac-
tivity of cyclopropyl-substituted quinolones was therefore asso-
ciated with the electron donating effect of this moiety. At C7
position we have studied with various substituted piperazines
(8e10aef), (thio) morpholines (8e10g and 10h), substituted
piperidines (8e10iel), fused piperazines and piperidines (8e
10mep). A comparison of the substitution pattern at C7 demon-
strated that the order of activity was substituted piperazi-
3.2. Inhibition of DNA gyrase activity
The 6-nitroquinolone-3-carboxylic acid derivatives de-
scribed above were evaluated for their ability to inhibit DNA
supercoiling activity of DNA gyrase isolated from M. smegma-
tis. Quinolones and fluoroquinolones are synthetic compounds
which inhibit bacterial type II DNA topoisomerases. These
enzymes facilitate DNA replication, transcription and other
DNA transaction processes. They do so by transiently creating
a double strand break in the DNA followed by religation of the
broken strands after the passage of intact duplex DNA through
nes > substituted piperidines > fused
piperazines
and

348
P. Senthilkumar et al. / European Journal of Medicinal Chemistry 44 (2009) 345e358
Table 1
Physical constants, in vitro antimycobacterial activities and cytotoxicity
O
O
O N
2
OH
R
1
N
R
No.
R
R1
Yield (%)
M.p. (ꢀC)
CC50 (mM)
MIC (mM)
MTB
MDRTB
MC2
Cl
8a
9a
Cyclopropyl
4-F-phenyl
t-Butyl
68
62
71
192
134
128
NT
NT
NT
5.59
10.19
5.44
NT
NT
NT
5.59
40.78
10.87
N
N
10a
O
C
N
N
8b
9b
Cyclopropyl
4-F-Phenyl
t-Butyl
73
66
68
237
127
222
111.2
NT
NT
0.69
6.18
6.68
0.34
NT
NT
1.39
24.68
13.34
O
O
10b
H C
N
N
2
O
8c
9c
Cyclopropyl
4-F-phenyl
t-Butyl
73
80
71
148
108
182
126.9
NT
NT
0.08
2.85
3.07
0.16
0.71
1.53
0.79
22.87
24.58
10c
N
N
H C
3
8d
9d
Cyclopropyl
4-F-phenyl
t-Butyl
69
80
67
198
126
147
136.36
NT
NT
0.20
6.23
0.20
NT
NT
0.87
24.88
64.36
C H
6
5
10d
16.09
O
O
8e
9e
Cyclopropyl
4-F-phenyl
t-Butyl
70
83
71
134
70
NT
54.39
NT
2.99
2.72
5.83
6.01
0.68
NT
12.01
10.88
23.29
N
N
C
O
10e
231
F
O
8f
Cyclopropyl
4-F-phenyl
t-Butyl
70
84
81
241
223
NT
NT
2.69
4.94
2.62
2.69
NT
0.65
10.79
19.73
10.49
N
N
C
9f
10f
N
>250
104.9
CH
3
O
F
8g
9g
Cyclopropyl
4-F-phenyl
t-Butyl
77
81
71
208
114
NT
NT
NT
4.17
14.55
7.99
NT
NT
NT
33.29
29.11
31.93
S
N
10g
>250
8h
9h
Cyclopropyl
4-F-phenyl
t-Butyl
O
73
84
74
172
103
108
NT
70.79
NT
2.01
3.53
7.76
4.03
1.77
NT
8.08
14.16
30.98
N
10h
8i
Cyclopropyl
4-F-Phenyl
t-Butyl
N
74
81
78
173
108
156
141.9
NT
NT
0.43
6.33
6.86
0.43
NT
NT
1.77
25.28
27.41
N
9i
10i

P. Senthilkumar et al. / European Journal of Medicinal Chemistry 44 (2009) 345e358
349
Table 1 (continued)
No.
R
R1
Yield (%)
M.p. (ꢀC)
CC50 (mM)
MIC (mM)
MTB
MDRTB
MC2
HO
8j
Cyclopropyl
4-F-Phenyl
t-Butyl
70
76
75
197
145
127
129.2
NT
NT
0.81
5.82
6.26
0.39
NT
3.22
23.24
25.00
Cl
N
9j
10j
NT
N
N
8k
9k
Cyclopropyl
4-F-Phenyl
t-Butyl
67
78
69
209
181
NT
NT
NT
1.49
5.42
5.79
2.97
NT
2.97
43.26
23.11
O
N
H
Cl
10k
>250
NT
O
C
(C H ) N
2
5 2
8l
Cyclopropyl
4-F-phenyl
t-Butyl
72
73
70
174
75
NT
NT
NT
3.42
6.13
6.62
3.42
NT
6.86
24.49
26.45
N
9l
10l
152
NT
O
O
8m
9m
Cyclopropyl
4-F-phenyl
t-Butyl
64
69
71
182
110
136
NT
NT
NT
3.76
6.67
7.45
1.88
NT
15.05
26.63
28.97
N
10m
NT
N
8n
9n
Cyclopropyl
4-F-phenyl
t-Butyl
75
82
72
206
112
135
NT
NT
NT
1.55
5.60
1.55
NT
3.09
22.38
24.01
O
10n
C
NHC(CH )
3
12.01
NT
3
N
HOOC
8o
9o
Cyclopropyl
4-F-phenyl
t-Butyl
78
84
72
210
179
142.2
NT
NT
1.78
12.67
6.87
0.43
NT
7.12
50.67
54.89
N
N
10o
>250
NT
N
8p
9p
Cyclopropyl
4-F-phenyl
t-Butyl
e
71
76
73
e
208
114
69
NT
NT
3.04
5.36
5.74
1.04
0.36
NT
NT
24.34
21.42
22.91
2.08
OCH
H C
3
2
10p
Gati
INH
NT
>155.3
>455.8
NT
8.34
45.57
e
e
e
e
e
e
45.57
NT indicates not tested.
the cleaved enzymeeDNA complex. This leads to the change
in linking number of DNA in an ATP dependent reaction cycle
[14]. DNA gyrase, an essential enzyme in bacteria, is unique
in catalyzing the negative supercoiling of DNA and as a result,
the enzyme is vital for almost all cellular processes that in-
volve duplex DNA. In all the species of mycobacteria
including M. tuberculosis, DNA gyrase is the sole type II
topoisomerase and topoisomerase IV is not found in these
genomes. Our earlier studies have revealed that DNA gyrase
from M. tuberculosis and M. smegmatis are highly similar in
antigenic and biochemical properties [15]. The supercoiling
assay results with various compounds using M. smegmatis
DNA gyrase are presented in Fig. 1a and b. The IC50 values
presented in Table 3 show that compounds 8b, 8j, 8i, 8n,
8h, 8k, 8c, 8d and 10f inhibit DNA gyrase at 30 mg/ml concen-
tration. Compounds 9e, 9h and 10b have IC50 of 20 mg/ml and
compound 10a inhibits the enzyme activity with an IC50 value
as low as 5 mg/ml.
3.3. Phototoxic evaluation
Quinolones in general have favorable safety profiles; photo-
toxicity has become a significant factor in the clinical use of
some. Phototoxicity with the quinolones is generally thought
to result from the absorption of light by the parent compound
or a metabolite in tissue. Three (8c, 8d and 8i) compounds
were evaluated for potential phototoxicity in a standardized in
vivo test system that has been used previously to assess quino-
lone antibiotics. The test compounds (140 mg/kg) and the pos-
itive control lomefloxacin hydrochloride (140 mg/kg) were
evaluated for phototoxicity and both ears of each mouse were
evaluated for changes indicative of a positive response: ery-
thema, edema or a measurable increase in ear thickness. Change
from baseline was calculated separately for each animal and
time point and analyzed for statistical significance and pre-
sented in Table 4. The drug and time factors were analyzed
by separate univariate methods. Orthogonal contrasts were

350
P. Senthilkumar et al. / European Journal of Medicinal Chemistry 44 (2009) 345e358
Table 2
In vivo activity data of 8c gatifloxacin and isoniazid against M. tuberculosis
ATCC 35801 in mice
8i showed a significant erythema after irradiation at the 72 h
time point only while lomefloxacin hydrochloride showed
erythema after irradiation throughout the period of testing.
Compound
Lungs
(log CFU ꢂ SEM)
Spleen
(log CFU ꢂ SEM)
4. Conclusion
Control
7.99 ꢂ 0.16
6.02 ꢂ 0.23
5.86 ꢂ 0.23
5.21 ꢂ 0.17
9.02 ꢂ 0.21
6.92 ꢂ 0.07
4.71 ꢂ 0.10
4.87 ꢂ 0.13
Gatifloxacin (50 mg/kg)
Isoniazid (25 mg/kg)
8c (50 mg/kg)
Screening of the antimycobacterial activity of these novel
series identified newer potent antitubercular 6-nitroquino-
lone-3-carboxylic acids endowed with high activity toward
MDR-TB, with MIC99 ranging from 0.16 to 6.01 mM. 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 structureeactivity and mechanistic studies
should prove fruitful.
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 baseline ear thickness was significantly different from
zero. The results indicated that lomefloxacin showed significant
increase in ear thickness from 4 to 96 h and from 24 to 96 h
when compared within time points and with the control, respec-
tively. The test compounds were found to show a significant dif-
ference in ear thickness at various time points when compared
with the pre-drug reading (0 h) but were less or not toxic except
8c (72 h) and 8i (72 and 96 h) when compared with the negative
(vehicle-treated) and positive controls (lomefloxacin hydro-
chloride). No erythema occurred in mice dosed with 140 mg/
kg of 8c and 8d throughout the 96 h study, while compound
5. Experimental protocols
5.1. Chemistry
Melting points were determined in open capillary tubes on
a Buchi 530 melting point apparatus and are uncorrected.
Fig. 1. DNA gyrase supercoiling assay. The assays were performed as described in Section 5. DNA gyrase was incubated with indicated concentrations of the
compounds before the addition of rest of the components. (a) Lane 1: relaxed DNA, lane 2: supercoiling reaction, lane 3: supercoiling reaction in the presence
of 5% DMSO. Ciprofloxacin at concentration of 10 mg/ml (lane 4) was used as the positive control for inhibition of enzyme. Lanes 5e10 have 30 mg/ml concen-
trations of compounds 8bed, 8iek, respectively. (b) Lane 1: relaxed DNA, lane 2: supercoiling reaction, lane 3: supercoiling reaction in the presence of 5%
DMSO. Ciprofloxacin at concentration of 10 mg/ml (lane 4) was used as the positive control for inhibition of enzyme. Lane 5: 30 mg/ml 8n, lane 6: 20 mg/ml
9e, lane 7: 30 mg/ml 9h, lane 8: 5 mg/ml 10a, lane 9: 20 mg/ml 10b and lane 10: 30 mg/ml 10f. R and S indicate relaxed and supercoiled pUC18 DNA, respectively.

P. Senthilkumar et al. / European Journal of Medicinal Chemistry 44 (2009) 345e358
351
Table 3
IC50 values for DNA gyrase inhibition
5.1.2. Synthesis of ethyl 3-(2,4-dichloro-5-nitrophenyl)-3-
oxopropanoate (3)
Compounds
IC50 (mg/ml)
To a solution of 2 (1.00 equiv, 2.36 g) in tetrahydrofuran,
CDI (1.20 equiv, 1.94 g) was added for 30 min at the temper-
ature of 70 ꢀC. The resulting crude imidazolide was used
without further purification in the next step. To a solution
of potassium salt of ethyl malonate (1.30 equiv, 2.21 g) in
acetonitrile was added dropwise magnesium chloride (2.00
equiv, 1.90 g) and tri-ethyl amine (4.0 equiv, 4.04 g) at 0
ꢀC and stirred at room temperature for 2.5 h. To this
solution, the imidazolide prepared above was added and
the reaction mixture was refluxed at 70 ꢀC for 2.5 h. After
completion of reaction, the solvent was distilled off and
poured into ice water and acidified to pH 5e6 with 20%
HCl, then extracted with ethyl acetate (3 ꢄ 25 ml) and dried
over magnesium sulphate and distilled the solvent to give 3
(yield: 82%; m.p.: 141 ꢀC).
Yield: 82%; m.p.: 141 ꢀC; IR (KBr) cmꢁ1: 2900, 1726, 1705,
1470e1370; 1H NMR (DMSO-d6) d ppm: 1.45 (t, 3H, e
CH2CH3), 3.40 (s, 2H, CH2), 4.2 (m, 2H, eCH2CH3), 7.66 (s,
1H, AreH), 8.64 (s, 1H, AreH); 13C NMR (DMSO-d6) d ppm:
194.3, 168.4, 146.9, 141.8, 137.2, 136.6, 134.2, 132.0, 130.9,
128.6, 126.3, 61.3, 50.6, 14.5; Anal (C11H9Cl2NO5) C, H, N.
8b
8c
30
30
30
30
30
30
30
20
20
5
8d
8i
8j
8k
8n
9e
9h
10a
10b
10f
20
30
Infrared (IR) and proton nuclear magnetic resonance (1H NMR)
spectra were recorded for the compounds on JASCO IR Report
100 (KBr) and Brucker Avance (300 MHz) instruments, respec-
tively. Chemical shifts are reported in parts per million (ppm) us-
ing tetramethyl silane (TMS) as an internal standard. All
exchangeable protons were confirmed by the addition of D2O.
13C NMR spectra were recorded on Bruker AC 200/DPX
400 MHz. Elemental analyses (C, H, and N) were undertaken
with PerkineElmer model 240C analyzer and all analyses
were consistent with theoretical values (within ꢂ0.4%) unless
indicated. The homogeneity of the compounds was monitored
by ascending thin layer chromatography (TLC) on silica gel-G
(Merck)-coated aluminium plates, visualized by iodine vapor
and UV light. Microwave oven used was Catalyst system, India.
5.1.3. Synthesis of ethyl 1-(substituted)-7-chloro-6-nitro-4-
oxo-1,4-dihydro-quinoloine-3-carboxylates (6aec)
A mixture of 3 (1.00 equiv, 3.06 g), tri-ethylorthofor-
mate (1.50 equiv, 2.18 g) and acetic anhydride (2.50 equiv,
2.55 g) were refluxed at 140 ꢀC for 1 h. The ethyl acetate
produced as a byproduct was distilled simultaneously under
atmospheric pressure. After completion of reaction the
reaction mixture was concentrated under reduced pressure
to yield ethyl 2-(2,4-dichloro-5-nitrobenzoyl)-3-ethoxya-
crylate (4). The above residue was dissolved in a mixture
of ether (20 ml) and ethanol (20 ml). Added corresponding
primary amine (1.10 equiv) at 0 ꢀC and stirred for 30 min
under nitrogen atmosphere, followed by distillation yielded
ethyl 2-(2,4-dichloro-5-nitrobenzoyl)-3-(cyclopropyl/4-fluoro-
phenyl/t-butyl amino)acrylate (5aec). The above crude solid
(1.00 equiv) was dissolved in DMSO (30 ml) and added an-
hydrous potassium carbonate (1.60 equiv) and refluxed at
60 ꢀC for 3 h. After completion of reaction, the mixture
5.1.1. Synthesis of 2,4-dichloro-5-nitrobenzoic acid (2)
To a solution of 2,4-dichlorobenzoic acid (1) (1.00 equiv,
1.91 g) in 96% sulphuric acid (40 ml), 65% nitric acid
(1.50 equiv, 0.95 g) was added slowly at0e5 ꢀC and the resulting
mixturewas then stirred at room temperature for 1 h. The mixture
is then poured into ice-cold water and extracted with ethyl acetate
(3 ꢄ 50 ml). The combined extracts were dried over magnesium
sulphate and evaporated under reduced pressure to yield 2.
Yield: 98%; m.p.: 138 ꢀC; IR (KBr) cmꢁ1: 2900, 1726,
1470e1370; 1H NMR (DMSO-d6) d ppm: 7.80 (s, 1H,
ArH), 8.84 (s, 1H, AreH), 14.60 (s, 1H, COOH); 13C NMR
(DMSO-d6) d ppm: 169.4, 146.9, 141.8, 136.6, 132.0, 130.9,
128.6; Anal (C7H3Cl2NO4) C, H, N.
Table 4
Phototoxic evaluation of the titled compounds
Group
Ear thickness (mm)a
Erythemab
Time (approximately) after start of irradiaton (h)c
0
4
24
48
72
96
0
4
24
48
72
96
Controld
8c
8d
0.37 ꢂ 0.03
0.25 ꢂ 0.01
0.30 ꢂ 0.02
0.24 ꢂ 0.01
0.31 ꢂ 0.01
0.36 ꢂ 0.02
0.27 ꢂ 0.01
0.31 ꢂ 0.02
0.26 ꢂ 0.02
0.40 ꢂ 0.02
0.38 ꢂ 0.03
0.28 ꢂ 0.003
0.30 ꢂ 0.01
0.26 ꢂ 0.01
0.48 ꢂ 0.02
0.37 ꢂ 0.03
0.28 ꢂ 0.01
0.32 ꢂ 0.02
0.26 ꢂ 0.01
0.53 ꢂ 0.02
0.37 ꢂ 0.03
0.31 ꢂ 0.01
0.32 ꢂ 0.01
0.29 ꢂ 0.01
0.64 ꢂ 0.04
0.38 ꢂ 0.03
0.29 ꢂ 0.02
0.34 ꢂ 0.02
0.28 ꢂ 0.01
0.60 ꢂ 0.06
0
0
0
0
0
0
0
0
0
6
0
0
0
0
0
0
6
0
0
0
3
6
0
0
0
0
6
2e
0
8i
Lomefloxacin
6
a
Mean ear thickness ꢂ SEM; left and right ears were averaged.
b
c
d
e
Number of mice with erythema.
Time zero ¼ pre-dose (mice exposed to UVA light immediately after dosing); 4 h ¼ end of irradiation period.
Control ¼ 0.5% aqueous solution of sodium carboxymethylcellulose (4 N s/m2) dosed at 10 ml/kg.
Very slight erythema, not considered to be drug-related.

352
P. Senthilkumar et al. / European Journal of Medicinal Chemistry 44 (2009) 345e358
was diluted with ice-cold water and neutralized with 20%
HCl to a pH of 5e6. The precipitate thus formed was filtered
and washed with water followed by 30% ethylacetate:hexane
mixture to yield ethyl 1-(substituted)-7-methoxy-6-nitro-4-
oxo-1,4-dihydro-quinoloine-3-carboxylates (6aec).
14.68 (s, 1H, COOH); 13C NMR (DMSO-d6) d ppm: 177.5,
166.3, 152.4, 151.6, 148.1, 145.3, 137.7, 135.5, 127.0, 126.1,
120.8, 116.4, 115.3, 109.3; Anal (C16H8 ClFN2O5) C, H, N.
5.1.4.3. 1-tert-Butyl-7-chloro-1,4-dihydro-6-nitro-4-oxoquino-
line-3-carboxylic acid (7c). Yield: 85%; m.p.: 245 ꢀC; IR
1
5.1.3.1. Ethyl 1-(cyclopropyl)-7-chloro-6-nitro-4-oxo-1,4-
dihydro-quinoloine-3-carboxylates (6a). Yield: 90%; m.p.:
>280 ꢀC; IR (KBr) cmꢁ1: 2900, 1726, 1705, 1616, 1470e
1370; 1H NMR (DMSO-d6) d ppm: 0.28e0.44 (m, 4H,
cyclopropyl), 1.36 (m, 1H, cyclopropyl), 1.45 (t, 3H, e
CH2CH3), 4.2 (m, 2H, eCH2CH3), 8.3 (s, 1H, C8eH), 9.34 (s,
1H, C5eH), 9.52 (s, 1H, C2eH); 13C NMR (DMSO-d6)
d ppm: 177.5, 165.5, 151.6, 145.3, 135.5, 137.7, 127.1, 126.1,
115.3, 110.2, 61.4, 36.2, 14.2, 5.6; Anal (C13H9ClN2O5) C, H, N.
(KBr) cmꢁ1: 2900, 1726, 1705, 1620, 1470e1370; H NMR
(DMSO-d6) d ppm: 1.6 (s, 9H, t-butyl), 8.3 (s, 1H, C8eH),
9.34 (s, 1H, C5eH), 9.56 (s, 1H, C2eH), 14.6 (s, 1H,
COOH); 13C NMR (DMSO-d6) d ppm: 177.5, 166.3, 151.6,
148.1, 145.3, 137.7, 136.5, 127.0, 126.1, 115.3, 109.3, 65.1,
28.5; Anal (C14H13ClN2O5) C, H, N.
5.1.5. Synthesis of 1-(substituted)-1,4-dihydro-6-nitro-7-
(substituted secondary amines)-4-oxoquinoline-3-
carboxylic acid (8e10aep)
5.1.3.2. Ethyl 1-(4-fluorophenyl)-7-methoxy-6-nitro-4-oxo-1,4-
dihydro-quinoloine-3-carboxylates (6b). Yield: 92%; m.p.:
>280 ꢀC; IR (KBr) cmꢁ1: 2900, 1726, 1705, 1616, 1470e
Compound 7aec (1.0 equiv) in dimethyl sulphoxide (2.5 ml)
and appropriate secondary amines (1.1 equiv) were irradiated in
a microwave oven at an intensity of 80% (560 W) with 30 s/cy-
cle. The number of cycles 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.
1
1370; H NMR (DMSO-d6) d ppm: 1.45 (t, 3H, eCH2CH3),
4.2 (m, 2H, eCH2CH3), 6.48e6.68 (m, 4H, Ar-H), 8.3 (s,
1H, C8eH), 9.34 (s, 1H, C5eH), 9.52 (s, 1H, C2eH); 13C
NMR (DMSO-d6) d ppm: 177.5, 165.5, 152.4, 151.6, 145.3,
135.5, 136.3, 137.7, 127.1, 126.1, 120.8, 116.4, 115.3, 110.2,
61.4, 14.2; Anal (C13H9ClN2O5) C, H, N.
5.1.5.1. 7-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-
1-cyclopropyl-1,4-dihydro-6-nitro-4-oxoquinoline-3-carboxylic
acid (8a). Yield: 68%; m.p.: 192 ꢀC; IR (KBr) cmꢁ1: 2900,
5.1.3.3. Ethyl 1-(t-butyl)-7-methoxy-6-nitro-4-oxo-1,4-dihydro-
quinoloine-3-carboxylates (6c). Yield: 86%; m.p.: 276 ꢀC; IR
1
1
(KBr) cmꢁ1: 2900, 1726, 1705, 1616, 1470e1370; H NMR
1724, 1708, 1620, 1460e1370; H NMR (DMSO-d6) d ppm:
(DMSO-d6) d ppm: 1.45 (t, 3H, eCH2CH3), 1.6 (s, 9H, t-bu-
tyl), 4.2 (m, 2H, eCH2CH3), 8.3 (s, 1H, C8eH), 9.34 (s, 1H,
C5eH), 9.52 (s, 1H, C2eH); 13C NMR (DMSO-d6) d ppm:
177.5, 165.5, 151.6, 145.3, 136.3, 137.7, 127.1, 126.1,
115.3, 110.2, 65.1, 61.4, 28.5, 14.2; Anal (C13H9ClN2O5) C,
H, N.
0.28e0.48 (m, 4H, cyclopropyl), 1.35 (m, 1H, cyclopropyl),
2.59e3.12 (m, 8H, CH2 of piperazine), 4.2 (s, 1H, CH of
diphenylmethyl), 7.0e7.18 (m, 9H, Ar-H), 8.32 (s, 1H, C8e
H), 9.34 (s, 1H, C5eH), 9.56 (s, 1H, C2eH), 14.6 (s, 1H,
COOH); 13C NMR (DMSO-d6) d ppm: 177.5, 166.3, 151.1,
148.1, 146.2, 142.8, 140.9, 131.8, 129.7, 129.3, 128.3,
126.4, 121.9, 117.5, 109.3, 99.3, 73.6, 49.6, 49.3, 36.0, 5.6;
Anal (C30H27ClN4O5) C, H, N.
5.1.4. Synthesis of 1-(substituted)-1,4-dihydro-7-chloro-6-
nitro-4-oxo-quinoline-3-carboxylic acid (7aec)
Compound 6aec (1.00 equiv) was suspended in 6 N HCl
(5 ml) and refluxed for 6 h and then cooled to 0 ꢀC. The
precipitate obtained was filtered, washed with water followed
by 20% ethyl acetate yielded 7aec.
5.1.5.2. 1-t-Butyl-7-(4-((4-chlorophenyl)(phenyl)methyl)pipera-
zin-1-yl)-1,4-dihydro-6-nitro-4-oxoquinoline-3-carboxylic acid
(9a). Yield: 62%; m.p.: 134 ꢀC; IR (KBr) cmꢁ1: 2900, 1724,
1708, 1620, 1460e1370; 1H NMR (DMSO-d6) d ppm:
2.59e3.12 (m, 8H, CH2 of piperazine), 4.2 (s, 1H, CH of di-
phenylmethyl), 6.3e7.18 (m, 13H, Ar-H), 8.32 (s, 1H, C8e
H), 9.34 (s, 1H, C5eH), 9.56 (s, 1H, C2eH), 14.6 (s, 1H,
COOH); 13C NMR (DMSO-d6) d ppm: 177.5, 166.3, 152.4,
149.4, 147.7, 146.3, 142.8, 140.9, 131.8, 136.3, 129.7,
129.3, 128.3, 126.6, 126.3, 123.1, 120.7, 116.4, 109.3,
104.9, 73.6, 49.6, 49.3; Anal (C33H26ClFN4O5) C, H, N.
5.1.4.1. 7-Chloro-1-cyclopropyl-1,4-dihydro-6-nitro-4-oxoqui-
noline-3-carboxylic acid (7a). Yield: 82%; m.p.: 249 ꢀC; IR
1
(KBr) cmꢁ1: 2900, 1726, 1705, 1616, 1470e1370; H NMR
(DMSO-d6) d ppm: 0.28e0.44 (m, 4H, cyclopropyl), 1.36 (m,
1H, cyclopropyl), 8.3 (s, 1H, C8eH), 9.34 (s, 1H, C5eH), 9.52
(s, 1H, C2eH), 14.60 (s, 1H, COOH); 13C NMR (DMSO-d6)
d ppm: 177.5, 166.3, 151.6, 148.1, 137.7, 135.5, 127.0, 126.1,
115.3, 109.3, 36.3, 5.6; Anal (C13H9ClN2O5) C, H, N.
5.1.5.3. 7-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-
1,4-dihydro-1-(4-fluorophenyl)-6-nitro-4-oxoquinoline-3-car-
5.1.4.2. 7-Chloro-1-(4-fluorophenyl)-1,4-dihydro-6-nitro-4-
oxoquinoline-3-carboxylic acid (7b). Yield: 86%; m.p.:
>280 ꢀC; IR (KBr) cmꢁ1: 2900, 1728, 1710, 1620, 1460e
1370; 1H NMR (DMSO-d6) d ppm: 6.48e6.68 (m, 4H, Ar-H),
8.3 (s, 1H, C8eH), 9.34 (s, 1H, C5eH), 9.54 (s, 1H, C2eH),
boxylic acid (10a). Yield: 71%; m.p.: 128 ꢀC; IR (KBr) cmꢁ1
:
1
2900, 1724, 1708, 1620, 1460e1370; H NMR (DMSO-d6)
d ppm: 1.76 (s, 9H, t-butyl), 2.59e3.12 (m, 8H, CH2 of piper-
azine), 4.2 (s, 1H, CH of diphenylmethyl), 7.0e7.18 (m, 9H,
Ar-H), 8.32 (s, 1H, C8eH), 9.34 (s, 1H, C5eH), 9.56 (s, 1H,

P. Senthilkumar et al. / European Journal of Medicinal Chemistry 44 (2009) 345e358
353
C2eH), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) d ppm:
177.5, 166.3, 151.1, 148.1, 146.2, 142.8, 140.9, 131.8,
129.7, 129.3, 128.3, 126.4, 121.9, 117.5, 109.3, 99.3, 73.6,
65.0, 49.6, 49.3, 28.5; Anal (C31H31ClN4O5) C, H, N.
3.6 (s, 2H, CH2 of piperanoyl), 5.86 (s, 2H, eOCH2Oe),
6.42e7.62 (m, 7H, Ar-H), 8.34 (s, 1H, C8eH), 9.34 (s, 1H,
C5eH), 9.56 (s, 1H, C2eH), 14.6 (s, 1H, COOH); 13C NMR
(DMSO-d6) d ppm: 177.5, 166.3, 152.4, 149.4, 148.6, 147.7,
146.3, 136.3, 128.9, 126.6, 123.1, 122.2, 120.7, 116.4,
115.0, 113.9, 109.3, 104.9, 101.3, 60.4, 52.2, 49.0; Anal
(C28H23FN4O7) C, H, N.
5.1.5.4.
1-(Cyclopropyl)-7-(4-(2-furoyl)piperazin-1-yl)-1,4-
dihydro-6-nitro-4-oxoquinoline-3-carboxylic acid (8b). Yield:
73%; m.p.: 237 ꢀC; IR (KBr) cmꢁ1: 2890, 1724, 1710, 1624,
1460e1360; 1H NMR (DMSO-d6) d ppm: 0.28e0.48 (m,
4H, cyclopropyl), 1.35 (m, 1H, cyclopropyl), 3.16e3.3 (m,
8H, CH2 of piperazine), 7.1e7.48 (m, 3H, Ar-H), 8.34 (s,
1H, C8eH), 9.32 (s, 1H, C5eH), 9.56 (s, 1H, C2eH), 14.6
(s, 1H, COOH); 13C NMR (DMSO-d6) d ppm: 177.5, 166.3,
155.2, 151.1, 148.1, 147.1, 146.2, 126.5, 121.9, 117.5,
113.5, 111.7, 109.3, 99.3, 48.5, 47.5, 36.0, 5.6; Anal
(C22H20N4O7) C, H, N.
5.1.5.9. 7-(4-((Benzo[d][1,3]dioxol-5-yl)methyl)piperazin-1-yl)-1-
tert-butyl-1,4-dihydro-6-nitro-4-oxoquinoline-3-carboxylic acid
(10c). Yield: 71%; m.p.: 182 ꢀC; IR (KBr) cmꢁ1: 3250, 2890,
1
1724, 1710, 1624, 1464e1360; H NMR (DMSO-d6) d ppm:
1.76 (s, 9H, t-butyl), 2.82e3.12 (m, 8H, CH2 of piperazine), 3.6
(s, 2H, CH2 of piperanoyl), 5.86 (s, 2H, eOCH2Oe), 6.42e6.62
(m, 3H, Ar-H), 8.34 (s, 1H, C8eH), 9.34 (s, 1H, C5eH), 9.56 (s,
1H, C2eH), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) d ppm:
177.5, 166.3, 151.1, 148.6, 148.1, 147.8, 146.2, 128.9, 126.5,
121.9, 122.2, 117.5, 115.0, 113.9, 109.3, 101.3, 99.3, 65.0, 60.4,
52.2, 49.0, 28.5; Anal (C26H28N4O7) C, H, N.
5.1.5.5. 1-(4-Fluorophenyl)-7-(4-(2-furoyl)piperazin-1-yl)-1,4-
dihydro-6-nitro-4-oxoquinoline-3-carboxylic acid (9b). Yield:
66%; m.p.: 127 ꢀC; IR (KBr) cmꢁ1: 2890, 1724, 1710, 1624,
1
1460e1360; H NMR (DMSO-d6) d ppm: 3.16e3.3 (m, 8H,
5.1.5.10. 1-Cyclopropyl-1,4-dihydro-7-(4-methyl-3-phenylpiper-
azin-1-yl)-6-nitro-4-oxoquinoline-3-carboxylic acid (8d). Yield:
69%; m.p.: 198 ꢀC; IR (KBr) cmꢁ1: 2890, 1724, 1710, 1620,
1460e1368; 1H NMR (DMSO-d6) d ppm: 0.28e0.52 (m,
4H, cyclopropyl), 1.38 (m, 1H, cyclopropyl), 2.2 (s, 3H,
CH3), 2.6 (t, 2H, 5-CH2 of piperazine), 3.15 (t, 2H, 6-CH2
of piperazine), 3.4 (d, 2H, 2-CH2 of piperazine), 4.12 (t, 1H,
3-CH of piperazine), 7.0e7.2 (m, 5H, Ar-H), 8.34 (s, 1H,
C8eH), 9.32 (s, 1H, C5eH), 9.56 (s, 1H, C2eH), 14.4 (s,
1H, COOH); 13C NMR (DMSO-d6) d ppm: 177.5, 166.3,
151.1, 148.1, 146.2, 137.4, 128.9, 128.4, 127.5, 126.5,
121.9, 117.5, 109.3, 99.3, 63.4, 59.0, 52.2, 49.0, 40.5, 36.0,
5.6; Anal (C24H24N4O5) C, H, N.
CH2 of piperazine), 6.5e7.68 (m, 7H, Ar-H), 8.34 (s, 1H,
C8eH), 9.32 (s, 1H, C5eH), 9.56 (s, 1H, C2eH), 14.6 (s,
1H, COOH); 13C NMR (DMSO-d6) d ppm: 177.5, 166.3,
155.2, 152.4, 149.4, 147.1, 147.7, 146.3, 146.0, 136.3,
126.6, 123.1, 120.7, 116.4, 113.5, 111.7, 109.3, 104.9, 48.5,
47.5; Anal (C25H19FN4O7) C, H, N.
5.1.5.6. 1-tert-Butyl-7-(4-(2-furoyl)piperazin-1-yl)-1,4-dihydro-
6-nitro-4-oxoquinoline-3-carboxylic acid (10b). Yield: 68%;
m.p.: 222 ꢀC; IR (KBr) cmꢁ1: 2890, 1724, 1710, 1624,
1460e1360; 1H NMR (DMSO-d6) d ppm: 1.76 (s, 9H, t-
butyl), 3.16e3.3 (m, 8H, CH2 of piperazine), 7.1e7.48 (m,
3H, Ar-H), 8.34 (s, 1H, C8eH), 9.32 (s, 1H, C5eH), 9.56 (s,
1H, C2eH), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6)
d ppm: 177.5, 166.3, 155.2, 151.1, 148.1, 147.1, 146.1,
126.5, 121.9, 117.5, 113.5, 111.7, 109.3, 99.3, 65.0, 48.5,
47.5, 28.5; Anal (C23H24N4O7) C, H, N.
5.1.5.11. 1,4-Dihydro-1-(4-fluorophenyl)-7-(4-methyl-3-phenyl-
piperazin-1-yl)-6-nitro-4-oxoquinoline-3-carboxylic acid (9d).
Yield: 80%; m.p.: 126 ꢀC; IR (KBr) cmꢁ1: 2890, 1724,
1
1710, 1620, 1460e1368; H NMR (DMSO-d6) d ppm: 2.2
(s, 3H, CH3), 2.6 (t, 2H, 5-CH2 of piperazine), 3.15 (t, 2H,
6-CH2 of piperazine), 3.4 (d, 2H, 2-CH2 of piperazine), 4.12
(t, 1H, 3-CH of piperazine), 6.45e7.4 (m, 9H, Ar-H), 8.34
(s, 1H, C8eH), 9.32 (s, 1H, C5eH), 9.56 (s, 1H, C2eH),
14.4 (s, 1H, COOH); 13C NMR (DMSO-d6) d ppm: 177.5,
166.3, 152.4, 149.4, 147.7, 146.3, 137.4, 136.3, 128.9,
128.4, 127.5, 126.6, 123.1, 120.7, 116.4, 109.3, 104.9, 63.4,
59.0, 52.2, 49.0, 40.5; Anal (C27H23FN4O5) C, H, N.
5.1.5.7. 7-(4-((Benzo[d][1,3]dioxol-5-yl)methyl)piperazin-1-yl)-
1-cyclopropyl-1,4-dihydro-6-nitro-4-oxoquinoline-3-carboxylic
acid (8c). Yield: 73%; m.p.: 148 ꢀC; IR (KBr) cmꢁ1: 3250,
1
2890, 1724, 1710, 1624, 1464e1360; H NMR (DMSO-d6)
d ppm: 0.28e0.48 (m, 4H, cyclopropyl), 1.35 (m, 1H, cyclo-
propyl), 2.82e3.12 (m, 8H, CH2 of piperazine), 3.6 (s, 2H,
CH2 of piperanoyl), 5.86 (s, 2H, eOCH2Oe), 6.42e6.62 (m,
3H, Ar-H), 8.34 (s, 1H, C8eH), 9.34 (s, 1H, C5eH), 9.56 (s,
1H, C2eH), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6)
d ppm: 177.5, 166.3, 151.1, 148.6, 148.1, 147.8, 146.2, 128.9,
126.5, 122.2, 121.9, 117.5, 115.0, 113.9, 109.3, 101.3, 99.3,
60.4, 52.2, 49.0, 36.0, 5.6; Anal (C25H24N4O7) C, H, N.
5.1.5.12. 1-tert-Butyl-1,4-dihydro-7-(4-methyl-3-phenylpipera-
zin-1-yl)-6-nitro-4-oxoquinoline-3-carboxylic acid (10d). Yield:
67%; m.p.: 147 ꢀC; IR (KBr) cmꢁ1: 2890, 1724, 1710, 1620,
1460e1368; 1H NMR (DMSO-d6) d ppm: 1.76 (s, 9H, t-
butyl), 2.2 (s, 3H, CH3), 2.6 (t, 2H, 5-CH2 of piperazine),
3.15 (t, 2H, 6-CH2 of piperazine), 3.4 (d, 2H, 2-CH2 of piper-
azine), 4.12 (t, 1H, 3-CH of piperazine), 7.0e7.2 (m, 5H,
Ar-H), 8.34 (s, 1H, C8eH), 9.32 (s, 1H, C5eH), 9.56 (s, 1H,
C2eH), 14.4 (s, 1H, COOH); 13C NMR (DMSO-d6) d ppm:
177.5, 166.3, 151.1, 148.1, 146.2, 137.4, 128.9, 128.4,
5.1.5.8. 7-(4-((Benzo[d][1,3]dioxol-5-yl)methyl)piperazin-1-yl)-
1,4-dihydro-1-(4-fluorophenyl)-6-nitro-4-oxoquinoline-3-carbox-
ylic acid (9c). Yield: 80%; m.p.: 108 ꢀC; IR (KBr) cmꢁ1
:
3250, 2890, 1724, 1710, 1624, 1464e1360; 1H NMR
(DMSO-d6) d ppm: 2.82e3.12 (m, 8H, CH2 of piperazine),

354
P. Senthilkumar et al. / European Journal of Medicinal Chemistry 44 (2009) 345e358
1
127.5, 126.5, 121.9, 117.5, 109.3, 99.3, 65.0, 63.4, 59.0, 52.2,
49.0, 40.5, 28.5; Anal (C25H28N4O5) C, H, N.
(KBr) cmꢁ1: 2890, 1724, 1710, 1624, 1464e1360, 1208; H
NMR (DMSO-d6) d ppm: 2.3 (s, 3H, 5-CH3 of isoxazolyl),
3.12e3.28 (m, 8H, CH2 of piperazine), 6.58e7.08 (m, 7H,
Ar-H), 8.34 (s, 1H, C8eH), 9.32 (s, 1H, C5eH), 9.54 (s, 1H,
C2eH), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) d ppm:
177.5, 172.5, 168.9, 166.3, 160.3, 152.4, 149.4, 147.7, 146.3,
136.3, 132.0, 126.6, 123.1, 120.7, 116.4, 111.6, 110.5, 109.3,
104.9, 48.5, 47.1, 6.0; Anal (C31H22F3N5O7) C, H, N.
5.1.5.13. 1-Cyclopropyl-7-(4-(2,3-dihydrobenzo[b][1,4]dioxin-
2-oyl)(piperazin-1-yl))-1,4-dihydro-6-nitro-4-oxoquinolineꢁ-31-
carboxylic acid (8e). Yield: 70%; m.p.: 134 ꢀC; IR (KBr) cm
:
1
2890, 1724, 1710, 1624, 1460e1360; H NMR (DMSO-d6)
d ppm: 0.28e0.48 (m, 4H, cyclopropyl), 1.35 (m, 1H, cyclo-
propyl), 3.26e3.4 (m, 8H, CH2 of piperazine), 4.6 (d, 2H,
3-CH2 of dihydrobenzodioxinyl), 5.14 (t, 1H, 2-CH of dihydro-
benzodioxinyl), 6.62e6.98 (m, 4H, Ar-H), 8.34 (s, 1H, C8eH),
5.1.5.18. 1-tert-Butyl-7-(3-(2,6-difluorophenyl)-5-methylisoxa-
zol-4-oyl)(piperazin-1-yl)-1,4-dihydro-6-nitro-4-oxoquinoline-
3-carboxylic acid (10f). Yield: 81%; m.p.: >250 ꢀC; IR (KBr)
cmꢁ1: 2890, 1724, 1710, 1624, 1464e1360, 1208; H NMR
(DMSO-d6) d ppm: 1.74 (s, 9H, t-butyl), 2.3 (s, 3H, 5-CH3
of isoxazolyl), 3.12e3.28 (m, 8H, CH2 of piperazine), 6.82e
7.08 (m, 3H, Ar-H), 8.34 (s, 1H, C8eH), 9.32 (s, 1H,
C5eH), 9.54 (s, 1H, C2eH), 14.6 (s, 1H, COOH); 13C
NMR (DMSO-d6) d ppm: 177.5, 172.5, 168.9, 166.3, 160.3,
151.1, 148.1, 146.2, 132.0, 126.5, 121.9, 117.5, 111.6, 110.5,
109.3, 99.3, 65.0, 48.5, 47.1, 28.5, 6.0; Anal (C29H27F2N5O7)
C, H, N.
9.3 (s, 1H, C5eH), 9.56 (s, 1H, C2eH), 14.6 (s, 1H, COOH); 13
C
1
NMR (DMSO-d6) d ppm: 177.5, 168.7, 166.3, 151.1, 148.1,
146.7, 146.2, 126.5, 121.9, 121.0, 117.5, 115.0, 109.3, 99.3,
85.9, 66.2, 48.5, 47.4, 36.0, 5.6; Anal (C26H24N4O8) C, H, N.
5.1.5.14. 7-(4-(2,3-Dihydrobenzo[b][1,4]dioxin-2-oyl)(pipera-
zin-1-yl))-1-(4-fluorophenyl)-1,4-dihydro-6-nitro-4-oxoquino-
line-3-carboxylic acid (9e). Yield: 83%; m.p.: 70 ꢀC; IR (KBr)
cmꢁ1: 2890, 1724, 1710, 1624, 1460e1360; 1H NMR (DMSO-
d6) d ppm: 3.26e3.4 (m, 8H, CH2 of piperazine), 4.6 (d, 2H, 3-
CH2 of dihydrobenzodioxinyl), 5.14 (t, 1H, 2-CH of dihydroben-
zodioxinyl), 6.5e6.98 (m, 8H, Ar-H), 8.34 (s, 1H, C8eH), 9.3 (s,
1H, C5eH), 9.56 (s, 1H, C2eH), 14.6 (s, 1H, COOH); 13C NMR
(DMSO-d6) d ppm: 177.5, 168.7, 166.3, 152.4, 149.4, 147.7,
146.7, 146.3, 136.3, 126.6, 123.1, 121.0, 120.7, 116.4, 115.0,
109.3, 104.9, 85.9, 66.2, 48.5, 47.4; Anal (C29H23FN4O8) C, H, N.
5.1.5.19. 1-Cyclopropyl-1,4-dihydro-6-nitro-4-oxo-7-thiomor-
pholinoquinoline-3-carboxylic acid (8g). Yield: 77%; m.p.:
208 ꢀC; IR (KBr) cmꢁ1: 2890, 1724, 1710, 1620, 1460e1368;
1H NMR (DMSO-d6) d ppm: 0.28e0.54 (m, 4H, cyclopropyl),
1.36 (m, 1H, cyclopropyl), 2.64e3.4 (m, 8H, CH2 of thiomor-
pholine), 8.34 (s, 1H, C8eH), 9.32 (s, 1H, C5eH), 9.56 (s, 1H,
C2eH), 14.4 (s, 1H, COOH); 13C NMR (DMSO-d6) d ppm:
177.5, 166.3, 151.1, 148.1, 146.2, 126.5, 121.9, 117.5, 109.3,
99.3, 50.2, 36.0, 28.1, 5.6; Anal (C17H17N3O5S) C, H, N.
5.1.5.15. 1-tert-Butyl-7-(4-(2,3-dihydrobenzo[b][1,4]dioxin-2-
oyl)(piperazin-1-yl))-1,4-dihydro-6-nitro-4-oxoquinoline-3-car-
boxylic acid (10e). Yield: 71%; m.p.: 231 ꢀC; IR (KBr) cmꢁ1
:
1
2890, 1724, 1710, 1624, 1460e1360; H NMR (DMSO-d6)
d ppm: 1.76 (s, 9H, t-butyl), 3.26e3.4 (m, 8H, CH2 of piper-
azine), 4.6 (d, 2H, 3-CH2 of dihydrobenzodioxinyl), 5.14 (t,
1H, 2-CH of dihydrobenzodioxinyl), 6.62e6.98 (m, 4H,
Ar-H), 8.34 (s, 1H, C8eH), 9.3 (s, 1H, C5eH), 9.56 (s, 1H,
C2eH), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) d ppm:
177.5, 168.7, 166.3, 151.1, 148.1, 146.7, 146.2, 126.5,
121.9, 121.0, 117.5, 115.0, 109.3, 99.3, 85.9, 66.2, 65.0,
48.5, 47.4, 28.5; Anal (C27H28N4O8) C, H, N.
5.1.5.20. 1,4-Dihydro-1-(4-fluorophenyl)-6-nitro-4-oxo-7-thio-
morpholinoquinoline-3-carboxylic acid (9g). Yield: 81%; m.p.:
114 ꢀC; IR (KBr) cmꢁ1: 2890, 1724, 1710, 1620, 1460e1368;
1H NMR (DMSO-d6) d ppm: 2.64e3.4 (m, 8H, CH2 of thiomor-
pholine), 8.34 (s, 1H, C8eH), 6.5e6.98 (m, 4H, Ar-H), 9.32 (s,
1H, C5eH), 9.56 (s, 1H, C2eH), 14.4 (s, 1H, COOH); 13C NMR
(DMSO-d6) d ppm: 177.5, 166.3, 152.4, 149.4, 147.7, 146.3,
136.3, 126.6, 123.1, 120.7, 116.4, 109.3, 104.9, 50.2, 28.1;
Anal (C20H16FN3O5S) C, H, N.
5.1.5.16. 1-Cyclopropyl-7-(3-(2,6-difluorophenyl)-5-methyli-
soxazol-4-oyl)(piperazin-1-yl)-1,4-dihydro-6-nitro-4-oxoquino-
line-3-carboxylic acid (8f). Yield: 70%; m.p.: 241 ꢀC; IR
5.1.5.21. 1-tert-Butyl-1,4-dihydro-6-nitro-4-oxo-7-thiomorpho-
linoquinoline-3-carboxylic acid (10g). Yield: 71%; m.p.:
>250 ꢀC; IR (KBr) cmꢁ1: 2890, 1724, 1710, 1620, 1460e1368;
1H NMR (DMSO-d6) d ppm: 1.76 (s, 9H, t-butyl), 2.64e3.4 (m,
8H, CH2 of thiomorpholine), 8.34 (s, 1H, C8eH), 9.32 (s, 1H,
C5eH), 9.56 (s, 1H, C2eH), 14.4 (s, 1H, COOH); 13C NMR
(DMSO-d6) d ppm: 177.5, 166.3, 151.1, 148.1, 146.2, 126.5,
121.9, 117.5, 109.3, 99.3, 65.0, 50.2, 28.5, 28.1; Anal
(C18H21N3O5S) C, H, N.
1
(KBr) cmꢁ1: 2890, 1724, 1710, 1624, 1464e1360, 1208; H
NMR (DMSO-d6) d ppm: 0.28e0.48 (m, 4H, cyclopropyl),
1.35 (m, 1H, cyclopropyl), 2.3 (s, 3H, 5-CH3 of isoxazolyl),
3.12e3.28 (m, 8H, CH2 of piperazine), 6.82e7.08 (m, 3H,
Ar-H), 8.34 (s, 1H, C8eH), 9.32 (s, 1H, C5eH), 9.54 (s, 1H,
C2eH), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) d ppm:
177.5, 172.5, 168.9, 166.3, 160.3, 151.1, 148.1, 146.2,
132.0, 126.5, 121.9, 117.5, 111.6, 110.5, 109.3, 99.3, 48.5,
47.1, 36.0, 5.6, 6.0; Anal (C28H23F2N5O7) C, H, N.
5.1.5.22. 1-Cyclopropyl-1,4-dihydro-7-(2,6-dimethylmorpho-
lino)-6-nitro-4-oxoquinoline-3-carboxylic acid (8h). Yield:
73%; m.p.: 172 ꢀC; IR (KBr) cmꢁ1: 2892, 1724, 1710, 1628,
1460e1360; 1H NMR (DMSO-d6) d ppm: 0.28e0.48 (m,
4H, cyclopropyl), 1.2 (d, 6H, 2,6-CH3 of morpholino), 1.35
5.1.5.17. 7-(3-(2,6-Difluorophenyl)-1-(4-fluorophenyl)-5-meth-
ylisoxazol-4-oyl)(piperazin-1-yl)-1,4-dihydro-6-nitro-4-oxoqui-
noline-3-carboxylic acid (9f). Yield: 84%; m.p.: 223 ꢀC; IR

P. Senthilkumar et al. / European Journal of Medicinal Chemistry 44 (2009) 345e358
355
(m, 1H, cyclopropyl), 3.0 (d, 4H, 2,6-CH2 of morpholine), 3.9
(m, 2H, 3,5-CH of morpholine), 8.34 (s, 1H, C8eH), 9.3 (s,
1H, C5eH), 9.56 (s, 1H, C2eH), 14.6 (s, 1H, COOH); 13C
NMR (DMSO-d6) d ppm: 177.5, 166.3, 151.1, 148.1, 146.2,
126.5, 121.9, 117.5, 109.3, 99.3, 69.2, 65.1, 36.0, 20.5, 5.6;
Anal (C19H21N3O6) C, H, N.
5CH2), 1.78 (s, 9H, t-butyl), 2.2 (t, 4H, 2CH2), 2.7 (m, 1H,
CH), 2.8 (t, 4H, 2CH2), 8.34 (s, 1H, C8eH), 9.32 (s, 1H,
C5eH), 9.54 (s, 1H, C2eH), 14.6 (s, 1H, COOH); 13C NMR
(DMSO-d6) d ppm: 177.5, 166.3, 151.1, 148.1, 146.2, 126.5,
121.9, 117.5, 109.3, 99.3, 65.0, 58.2, 52.4, 46.9, 28.5, 28.2,
26.2, 25.9; Anal (C24H32N4O5) C, H, N.
5.1.5.23. 1-(4-Fluorophenyl)-1,4-dihydro-7-(2,6-dimethylmor-
pholino)-6-nitro-4-oxoquinoline-3-carboxylic acid (9h). Yield:
84%; m.p.: 103 ꢀC; IR (KBr) cmꢁ1: 2892, 1724, 1710, 1628,
1460e1360; 1H 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 morpholine), 6.4e6.7 (m, 4H, Ar-H), 8.34 (s,
1H, C8eH), 9.3 (s, 1H, C5eH), 9.56 (s, 1H, C2eH), 14.6 (s,
1H, COOH); 13C NMR (DMSO-d6) d ppm: 177.5, 166.3,
152.4, 149.4, 147.7, 146.3, 136.3, 126.6, 123.1, 120.7, 116.4,
109.3, 104.9, 69.2, 65.1, 20.5; Anal (C22H20FN3O6) C, H, N.
5.1.5.28. 7-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)-1-cy-
clopropyl-1,4-dihydro-6-nitro-4-oxoquinoline-3-carboxylic acid
(8j). Yield: 70%; m.p.: 197 ꢀC; IR (KBr) cmꢁ1: 2900, 1724,
1708, 1620, 1460e1370; 1H NMR (DMSO-d6) d ppm:
0.28e0.46 (m, 4H, cyclopropyl), 1.32 (m, 1H, cyclopropyl),
2.0e2.7 (m, 8H, 3,5-CH2 of piperidine), 7.1e7.18 (m, 4H,
Ar-H), 8.34 (s, 1H, C8eH), 9.30 (s, 1H, C5eH), 9.56 (s, 1H,
C2eH), 10.0 (br s, 1H, OH), 14.6 (s, 1H, COOH); 13C NMR
(DMSO-d6) d ppm: 177.5, 166.3, 151.1, 148.1, 146.2, 138.2,
131.5, 129.6, 129.1, 126.5, 121.9, 117.5, 109.3, 99.3, 74.3,
42.6, 38.2, 36.0, 5.6; Anal (C24H22ClN3O6) C, H, N.
5.1.5.24. 1-tert-Butyl-1,4-dihydro-7-(2,6-dimethylmorpholino)-
6-nitro-4-oxoquinoline-3-carboxylic acid (10h). Yield: 74%;
m.p.: 108 ꢀC; IR (KBr) cmꢁ1: 2892, 1724, 1710, 1628, 1460e
5.1.5.29. 7-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)-1,4-di-
hydro-1-(4-fluorophenyl)-6-nitro-4-oxoquinoline-3-carboxylic
acid (9j). Yield: 76%; m.p.: 145 ꢀC; IR (KBr) cmꢁ1: 2900,
1
1360; H NMR (DMSO-d6) d ppm: 1.2 (d, 6H, 2,6-CH3 of
1
morpholino), 1.76 (s, 9H, t-butyl), 3.0 (d, 4H, 2,6-CH2 of mor-
pholine), 3.9 (m, 2H, 3,5-CH of morpholine), 8.34 (s, 1H,
C8eH), 9.3 (s, 1H, C5eH), 9.56 (s, 1H, C2eH), 14.6 (s, 1H,
COOH); 13C NMR (DMSO-d6) d ppm: 177.5, 166.3, 151.1,
148.1, 146.2, 126.5, 121.9, 117.5, 109.3, 99.3, 69.2, 65.1,
20.5, 28.5; Anal (C20H25N3O6) C, H, N.
1724, 1708, 1620, 1460e1370; H NMR (DMSO-d6) d ppm:
2.0e2.7 (m, 8H, 3,5-CH2 of piperidine), 6.52e7.18 (m, 8H,
Ar-H), 8.34 (s, 1H, C8eH), 9.30 (s, 1H, C5eH), 9.56 (s, 1H,
C2eH), 10.0 (br s, 1H, OH), 14.6 (s, 1H, COOH); 13C NMR
(DMSO-d6) d ppm: 177.5, 166.3, 152.4, 149.4, 147.7, 146.3,
138.2, 136.3, 131.5, 129.6, 129.1, 126.6, 123.1, 120.7,
116.4, 109.3, 104.9, 74.3, 42.6, 38.2; Anal (C27H21ClFN3O6)
C, H, N.
5.1.5.25. 1-Cyclopropyl-1,4-dihydro-6-nitro-4-oxo-7-(4-(piperi-
din-1-yl)piperidin-1-yl)quinoline-3-carboxylic acid (8i). Yield:
74%; m.p.: 173 ꢀC; IR (KBr) cmꢁ1: 2890, 1724, 1710, 1624,
1464e1360; 1H NMR (DMSO-d6) d ppm: 0.28e0.48 (m,
4H, cyclopropyl), 1.35 (m, 1H, cyclopropyl), 1.5e1.6 (m,
10H, 5CH2), 2.2 (t, 4H, 2CH2), 2.7 (m, 1H, CH), 2.8 (t, 4H,
2CH2), 8.34 (s, 1H, C8eH), 9.32 (s, 1H, C5eH), 9.54 (s,
1H, C2eH), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6)
d ppm: 177.5, 166.3, 151.1, 148.1, 146.2, 126.5, 121.9,
117.5, 109.3, 99.3, 58.2, 52.4, 46.9, 36.0, 28.2, 26.2, 25.9,
5.6; Anal (C23H28N4O5) C, H, N.
5.1.5.30. 1-tert-Butyl-7-(4-(4-chlorophenyl)-4-hydroxypiperi-
din-1-yl)-1,4-dihydro-6-nitro-4-oxoquinoline-3-carboxylic acid
(10j). Yield: 75%; m.p.: 127 ꢀC; IR (KBr) cmꢁ1: 2900, 1724,
1708, 1620, 1460e1370; 1H NMR (DMSO-d6) d ppm: 1.76 (s,
9H, t-butyl), 2.0e2.7 (m, 8H, 3,5-CH2 of piperidine), 7.1e7.18
(m, 4H, Ar-H), 8.34 (s, 1H, C8eH), 9.30 (s, 1H, C5eH), 9.56 (s,
1H, C2eH), 10.0 (br s, 1H, OH), 14.6 (s, 1H, COOH); 13C
NMR (DMSO-d6) d ppm: 177.5, 166.3, 151.1, 148.1, 146.2,
138.2, 131.5, 129.6, 129.1, 126.5, 121.9, 117.5, 109.3, 99.3,
74.3, 65.0, 42.6, 38.2, 28.5; Anal (C25H26ClN3O6) C, H, N.
5.1.5.26. 1,4-Dihydro-1-(4-fluorophenyl)-6-nitro-4-oxo-7-(4-
(piperidin-1-yl)piperidin-1-yl)quinoline-3-carboxylic acid (9i).
Yield: 81%; m.p.: 108 ꢀC; IR (KBr) cmꢁ1: 2890, 1724,
1710, 1624, 1464e1360; 1H NMR (DMSO-d6) d ppm:
1.5e1.6 (m, 10H, 5CH2), 1.78 (s, 9H, t-butyl), 2.2 (t, 4H,
2CH2), 2.7 (m, 1H, CH), 2.8 (t, 4H, 2CH2), 6.5e6.88 (m,
4H, Ar-H), 8.34 (s, 1H, C8eH), 9.32 (s, 1H, C5eH), 9.54 (s,
1H, C2eH), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6)
d ppm: 177.5, 166.3, 152.4, 149.4, 147.7, 146.3, 136.3,
126.6, 123.1, 120.7, 116.4, 109.3, 104.9, 58.2, 52.4, 46.9,
28.2, 26.2, 25.9; Anal (C26H27FN4O5) C, H, N.
5.1.5.31. 7-(4-(6-Chloro-1,2-dihydro-2-oxobenzo[d]imidazol-
3-yl)piperidin-1-yl)-1-cyclopropyl-1,4-dihydro-6-nitro-4-oxo-
quinoline-3-carboxylic acid (8k). Yield: 67%; m.p.: 209 ꢀC; IR
(KBr) cmꢁ1: 3110, 2896, 1728, 1712, 1710, 1624, 1460e1360;
1H NMR (DMSO-d6) d ppm: 0.28e0.48 (m, 4H, cyclopropyl),
1.35 (m, 1H, cyclopropyl), 1.6e2.4 (m, 8H, 4CH2 of piperidine),
4.1 (br m, 1H, CH of piperidine), 7.2e7.56 (m, 3H, Ar-H), 8.34
(s, 1H, C8eH), 9.26 (s, 1H, C5eH), 9.56 (s, 1H, C2eH), 10.8 (s,
1H, NH), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) d ppm:
177.5, 166.3, 151.7, 151.1, 148.1, 146.2, 131.4, 130.1, 126.5,
124.7, 123.2, 122.2, 121.9, 117.5, 109.3, 99.3, 50.5, 46.2, 36.0,
26.8, 5.6; Anal (C25H22ClN5O6) C, H, N.
5.1.5.27. 1-tert-Butyl-1,4-dihydro-6-nitro-4-oxo-7-(4-(piperidin-
1-yl)piperidin-1-yl)quinoline-3-carboxylic acid (10i). Yield:
78%; m.p.: 156 ꢀC; IR (KBr) cmꢁ1: 2890, 1724, 1710, 1624,
5.1.5.32. 7-(4-(6-Chloro-1,2-dihydro-2-oxobenzo[d]imidazol-
3-yl)piperidin-1-yl)-1-(4-fluorophenyl)-1,4-dihydro-6-nitro-4-
1
1464e1360; H NMR (DMSO-d6) d ppm: 1.5e1.6 (m, 10H,

356
P. Senthilkumar et al. / European Journal of Medicinal Chemistry 44 (2009) 345e358
oxoquinoline-3-carboxylic acid (9k). Yield: 78%; m.p.:
181 ꢀC; IR (KBr) cmꢁ1: 3110, 2896, 1728, 1712, 1710,
1624, 1460e1360; 1H NMR (DMSO-d6) d ppm: 1.6e2.4
(m, 8H, 4CH2 of piperidine), 4.1 (br m, 1H, CH of piperidine),
6.4e6.9 (m, 7H, Ar-H), 8.34 (s, 1H, C8eH), 9.26 (s, 1H, C5e
H), 9.56 (s, 1H, C2eH), 10.8 (s, 1H, NH), 14.6 (s, 1H,
COOH); 13C NMR (DMSO-d6) d ppm: 177.5, 166.3, 152.4,
151.7, 149.4, 147.7, 146.3, 136.3, 131.4, 130.1, 126.6,
124.7, 123.1, 122.2, 120.7, 116.4, 109.3, 104.9, 50.5, 46.2,
26.8; Anal (C28H21ClFN5O6) C, H, N.
109.3, 99.3, 65.0, 54.4, 51.0, 42.6, 41.3, 28.5, 28.2, 22.3,
12.9; Anal (C24H32N4O6) C, H, N.
5.1.5.37.
1-Cyclopropyl-1,4-dihydro-7-(1,4-dioxa-8-azaspiro
[4.5]dec-8-yl)-6-nitro-4-oxoquinoline-3-carboxylic acid (8m).
Yield: 78%; m.p.: 126 ꢀC; IR (KBr) cmꢁ1: 2890, 1724, 1712,
1
1618, 1466e1368; H NMR (DMSO-d6) d ppm: 0.28e0.52
(m, 4H, cyclopropyl), 1.38 (m, 1H, cyclopropyl), 1.78e2.4 (m,
8H, 4-CH2 of azaspirodecane), 3.96 (m, 4H, 2-CH2 of azaspiro-
decane), 8.32 (s, 1H, C8eH), 9.30 (s, 1H, C5eH), 9.56 (s, 1H,
C2eH), 14.4 (s, 1H, COOH); 13C NMR (DMSO-d6) d ppm:
177.5, 166.3, 151.1, 148.1, 146.2, 126.5, 121.9, 117.5, 109.3,
99.3, 64.4, 39.6, 36.0, 34.4, 5.6; Anal (C20H21N3O7) C, H, N.
5.1.5.33. 1-tert-Butyl-7-(4-(6-chloro-1,2-dihydro-2-oxobenzo
[d]imidazol-3-yl)piperidin-1-yl)-1,4-dihydro-6-nitro-4-oxoqui-
noline-3-carboxylic acid (10k). Yield: 69%; m.p.: >250 ꢀC;
IR (KBr) cmꢁ1: 3110, 2896, 1728, 1712, 1710, 1624, 1460e
5.1.5.38. 1,4-Dihydro-7-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-
1-(4-fluorophenyl)-6-nitro-4-oxoquinoline-3-carboxylic acid
(9m). Yield: 69%; m.p.: 110 ꢀC; IR (KBr) cmꢁ1: 2890, 1724,
1
1360; H NMR (DMSO-d6) d ppm: 1.6e2.4 (m, 8H, 4CH2
of piperidine), 1.76 (s, 9H, t-butyl), 4.1 (br m, 1H, CH of pi-
peridine), 7.2e7.56 (m, 3H, Ar-H), 8.34 (s, 1H, C8eH), 9.26
(s, 1H, C5eH), 9.56 (s, 1H, C2eH), 10.8 (s, 1H, NH), 14.6 (s,
1H, COOH); 13C NMR (DMSO-d6) d ppm: 177.5, 166.3,
151.7, 151.1, 148.1, 146.2, 131.4, 130.1, 126.5, 124.7,
123.2, 122.2, 121.9, 117.5, 109.3, 99.3, 65.0, 50.5, 46.2,
26.8, 28.5; Anal (C26H26ClN5O6) C, H, N.
1
1712, 1618, 1466e1368; H NMR (DMSO-d6) d ppm: 1.78e
2.4 (m, 8H, 4-CH2 ofazaspirodecane),3.96(m,4H,2-CH2 ofazas-
pirodecane), 6.5e6.98 (m, 4H, Ar-H), 8.32 (s, 1H, C8eH), 9.30 (s,
1H, C5eH), 9.56 (s, 1H, C2eH), 14.4 (s, 1H, COOH); 13C NMR
(DMSO-d6) d ppm: 177.5, 166.3, 152.4, 149.4, 147.7, 146.3,
136.3, 126.6, 123.1, 120.7, 116.4, 109.3, 104.9, 64.4, 39.6, 34.4;
Anal (C23H20FN3O7) C, H, N.
5.1.5.34. 1-Cyclopropyl-7-(3-(diethylcarbamoyl)piperidin-1-
yl)-1,4-dihydro-6-nitro-4-oxoquinoline-3-carboxylic acid (8l).
Yield: 72%; m.p.: 174 ꢀC; IR (KBr) cmꢁ1: 2890, 1724,
5.1.5.39. 1-tert-Butyl-1,4-dihydro-7-(1,4-dioxa-8-azaspiro[4.5]-
dec-8-yl)-6-nitro-4-oxoquinoline-3-carboxylic
acid
(10m).
1
1710, 1624, 1464e1360, 1208; H NMR (DMSO-d6) d ppm:
Yield: 71%; m.p.: 136 ꢀC; IR (KBr) cmꢁ1: 2890, 1724,
1
1712, 1618, 1466e1368; H NMR (DMSO-d6) d ppm: 1.76
0.28e0.48 (m, 4H, cyclopropyl), 1.2 (t, 6H, 2-CH3 of ethyl),
1.35 (m, 1H, cyclopropyl), 1.78e2.7 (m, 9H, H of piperidine),
3.24 (q, 4H, 2-CH2 of ethyl), 8.32 (s, 1H, C8eH), 9.28 (s, 1H,
C5eH), 9.54 (s, 1H, C2eH), 14.6 (s, 1H, COOH); 13C NMR
(DMSO-d6) d ppm: 177.5, 175.3, 166.3, 151.1, 148.1, 146.2,
126.5, 121.9, 117.5, 109.3, 99.3, 54.4, 51.0, 42.6, 41.3, 36.0,
28.2, 22.3, 12.9, 5.6; Anal (C23H28N4O6) C, H, N.
(s, 9H, t-butyl), 1.78e2.4 (m, 8H, 4-CH2 of azaspirodecane),
3.96 (m, 4H, 2-CH2 of azaspirodecane), 8.32 (s, 1H, C8eH),
9.30 (s, 1H, C5eH), 9.56 (s, 1H, C2eH), 14.4 (s, 1H,
COOH); 13C NMR (DMSO-d6) d ppm: 177.5, 166.3, 151.1,
148.1, 146.2, 126.5, 121.9, 117.5, 109.3, 99.3, 65.0, 64.4,
39.6, 34.4, 28.5; Anal (C21H25N3O7) C, H, N.
5.1.5.35. 7-(3-(Diethylcarbamoyl)piperidin-1-yl)-1,4-dihydro-
1-(4-fluorophenyl)-6-nitro-4-oxoquinoline-3-carboxylic acid
(9l). Yield: 73%; m.p.: 75 ꢀC; IR (KBr) cmꢁ1: 2890, 1724,
5.1.5.40.
7-(1-(tert-Butylcarbamoyl)-3,4-dihydroisoquinolin-
2(1H )-yl)-1-cyclopropyl-1,4-dihydro-6-nitro-4-oxoquinolineꢁ-31-
carboxylicacid(8n). Yield: 75%; m.p.: 206 ꢀC; IR (KBr) cm
:
1
1
1710, 1624, 1464e1360, 1208; H NMR (DMSO-d6) d ppm:
2890, 1724, 1712, 1626, 1468e1360; H NMR (DMSO-d6)
d ppm: 0.28e0.48 (m, 4H, cyclopropyl), 1.3 (s, 9H, 3CH3),
1.35 (m, 1H, cyclopropyl), 2.66e2.9 (m, 4H, 2CH2 of isoquino-
line), 4.85 (s, 1H, CH of isoquinoline), 6.7e7.1 (m, 4H, Ar-H),
8.32 (s, 1H, C8eH), 9.28 (s, 1H, C5eH), 9.56 (s, 1H, C2eH),
10.2 (s, 1H, NH), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6)
d ppm: 177.5, 168.7, 166.3, 151.1, 148.1, 146.2, 137.5, 135.6,
129.6, 128.3, 127.6, 126.5, 121.9, 117.5, 109.3, 99.3, 65.4,
48.1, 47.3, 36.0, 30.4, 26.3, 5.6; Anal (C27H28N4O6) C, H, N.
1.2 (t, 6H, 2-CH3 of ethyl), 1.78e2.7 (m, 9H, H of piperidine),
3.24 (q, 4H, 2-CH2 of ethyl), 6.5e6.98 (m, 4H, Ar-H), 8.32 (s,
1H, C8eH), 9.28 (s, 1H, C5eH), 9.54 (s, 1H, C2eH), 14.6 (s,
1H, COOH); 13C NMR (DMSO-d6) d ppm: 177.5, 175.3,
166.3, 152.4, 149.4, 147.7, 146.3, 136.3, 126.6, 123.1,
120.7, 116.4, 109.3, 104.9, 54.4, 51.0, 42.6, 41.3, 28.2, 22.3,
12.9; Anal (C26H27FN4O6) C, H, N.
5.1.5.36. 7-(3-(Diethylcarbamoyl)piperidin-1-yl)-1-tert-butyl-
1,4-dihydro-6-nitro-4-oxoquinoline-3-carboxylic acid (10l).
Yield: 70%; m.p.: 152 ꢀC; IR (KBr) cmꢁ1: 2890, 1724,
5.1.5.41. 7-(1-(tert-Butylcarbamoyl)-3,4-dihydroisoquinolin-
2(1H )-yl)-1-(4-fluorophenyl)-1,4-dihydro-6-nitro-4-oxoquino-
line-3-carboxylic acid (9n). Yield: 82%; m.p.: 112 ꢀC; IR
1
1710, 1624, 1464e1360, 1208; H NMR (DMSO-d6) d ppm:
1
1.2 (t, 6H, 2-CH3 of ethyl), 1.7 (s, 9H, t-butyl), 1.78e2.7
(m, 9H, H of piperidine), 3.24 (q, 4H, 2-CH2 of ethyl), 8.32
(s, 1H, C8eH), 9.28 (s, 1H, C5eH), 9.54 (s, 1H, C2eH),
14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) d ppm: 177.5,
175.3, 166.3, 151.1, 148.1, 146.2, 126.5, 121.9, 117.5,
(KBr) cmꢁ1: 2890, 1724, 1712, 1626, 1468e1360; H NMR
(DMSO-d6) d ppm: 1.3 (s, 9H, 3CH3), 2.66e2.9 (m, 4H,
2CH2 of isoquinoline), 4.85 (s, 1H, CH of isoquinoline),
6.7e7.1 (m, 8H, Ar-H), 8.32 (s, 1H, C8eH), 9.28 (s, 1H,
C5eH), 9.56 (s, 1H, C2eH), 10.2 (s, 1H, NH), 14.6 (s, 1H,

P. Senthilkumar et al. / European Journal of Medicinal Chemistry 44 (2009) 345e358
357
1
COOH); 13C NMR (DMSO-d6) d ppm: 177.5, 168.7, 166.3,
152.4, 149.4, 147.7, 146.3, 137.5, 136.3, 135.6, 129.6,
128.3, 127.6, 126.6, 123.1, 120.7, 116.4, 109.3, 104.9, 65.4,
48.1, 47.3, 30.4, 26.3; Anal (C30H27FN4O6) C, H, N.
(KBr) cmꢁ1: 3210, 2890, 1724, 1710, 1620, 1460e1368; H
NMR (DMSO-d6) d ppm: 0.28e0.54 (m, 4H, cyclopropyl),
1.36 (m, 1H, cyclopropyl), 1.6e1.95 (m, 8H, 4-CH2 of isoqui-
nolinyl), 2.2e3.4 (m, 7H, 2-CH2 and 1-CH of isoquinolinyl,
and CH2), 3.73 (s, 3H, eOCH3), 6.8e7.1 (m, 4H, Ar-H),
8.34 (s, 1H, C8eH), 9.32 (s, 1H, C5eH), 9.56 (s, 1H, C2eH),
14.4 (s, 1H, COOH); 13C NMR (DMSO-d6) d ppm: 177.5,
166.3, 157.9, 151.1, 148.1, 146.2, 131.8, 128.8, 126.5, 123.7,
121.9, 117.5, 114.2, 109.3, 99.3, 61.1, 55.9, 42.8, 37.6, 36.0,
30.2, 28.5, 25.8, 24.2, 5.6; Anal (C30H31N3O6) C, H, N.
5.1.5.42. 1-tert-Butyl-7-(1-(tert-butylcarbamoyl)-3,4-dihydroi-
soquinolin-2(1H )-yl)-1,4-dihydro-6-nitro-4-oxoquinoline-3-
carboxylic acid (10n). Yield: 72%; m.p.: 135 ꢀC; IR (KBr)
cmꢁ1: 2890, 1724, 1712, 1626, 1468e1360; 1H NMR
(DMSO-d6) d ppm: 1.3 (s, 9H, 3CH3), 1.76 (s, 9H, t-butyl),
2.66e2.9 (m, 4H, 2CH2 of isoquinoline), 4.85 (s, 1H, CH of
isoquinoline), 6.7e7.1 (m, 4H, Ar-H), 8.32 (s, 1H, C8eH),
9.28 (s, 1H, C5eH), 9.56 (s, 1H, C2eH), 10.2 (s, 1H, NH),
14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) d ppm: 177.5,
168.7, 166.3, 151.1, 148.1, 146.2, 137.5, 135.6, 129.6,
128.3, 127.6, 126.5, 121.9, 117.5, 109.3, 99.3, 65.4, 65.0,
48.1, 47.3, 30.4, 28.5, 26.3; Anal (C28H32N4O6) C, H, N.
5.1.5.47. 1,4-Dihydro-1-(4-fluorophenyl)-7-(8-(4-methoxyben-
zyl)-3,4,5,6,7,8-hexahydroisoquinolin-2(1H )-yl)-6-nitro-4-oxo-
quinoline-3-carboxylic acid (9p). Yield: 76%; m.p.: 114 ꢀC; IR
(KBr) cmꢁ1: 3210, 2890, 1724, 1710, 1620, 1460e1368; H
NMR (DMSO-d6) d ppm: 1.6e1.95 (m, 8H, 4-CH2 of isoquino-
linyl), 2.2e3.4 (m, 7H, 2-CH2 and 1-CH of isoquinolinyl, and
CH2), 3.73 (s, 3H, eOCH3), 6.5e7.1 (m, 8H, Ar-H), 8.34 (s,
1H, C8eH), 9.32 (s, 1H, C5eH), 9.56 (s, 1H, C2eH), 14.4 (s,
1H, COOH); 13C NMR (DMSO-d6) d ppm: 177.5, 166.3,
157.9, 152.4, 149.4, 147.7, 146.3, 136.3, 131.8, 128.8, 126.6,
123.7, 123.1, 120.7, 116.4, 114.2, 109.3, 104.9, 61.1, 55.9,
42.8, 37.6, 30.2, 28.5, 25.8, 24.2; Anal (C33H30FN3O6) C, H, N.
1
5.1.5.43.
7-(2-Carboxy-5,6-dihydroimidazo[1,2-a]pyrazin-
7(8H )-yl)-1-cyclopropyl-1,4-dihydro-6-nitro-4-oxoquinoline-3-
carboxylic acid (8o). Yield: 78%; m.p.: 210 ꢀC; IR (KBr)
1
cmꢁ1: 3200, 2890, 1724, 1710, 1624, 1464e1360, 1208; H
NMR (DMSO-d6) d ppm: 0.28e0.48 (m, 4H, cyclopropyl),
1.35 (m, 1H, cyclopropyl), 3.1e3.8 (m, 6H, 3-CH2), 7.6 (s,
1H, CH), 8.32 (s, 1H, C8eH), 9.32 (s, 1H, C5eH), 9.54 (s,
1H, C2eH), 12.12 (s, 1H, 2-COOH), 14.6 (s, 1H, 3-COOH);
13C NMR (DMSO-d6) d ppm: 177.5, 167.5, 166.3, 157.6,
151.1, 148.1, 146.2, 130.3, 129.5, 126.5, 121.9, 117.5, 109.3,
99.3, 57.1, 50.7, 37.1, 36.0, 5.6; Anal (C20H17N5O7) C, H, N.
5.1.5.48. 1-tert-Butyl-1,4-dihydro-7-(8-(4-methoxybenzyl)-3,
4,5,6,7,8-hexahydroisoquinolin-2(1H )-yl)-6-nitro-4-oxoquino-
line-3-carboxylic acid (10p). Yield: 73%; m.p.: 69 ꢀC; IR
(KBr) cmꢁ1: 3210, 2890, 1724, 1710, 1620, 1460e1368; H
NMR (DMSO-d6) d ppm: 1.6e1.95 (m, 8H, 4-CH2 of isoqui-
nolinyl), 1.76 (s, 9H, t-butyl), 2.2e3.4 (m, 7H, 2-CH2 and
1-CH of isoquinolinyl, and CH2), 3.73 (s, 3H, eOCH3),
6.8e7.1 (m, 4H, Ar-H), 8.34 (s, 1H, C8eH), 9.32 (s, 1H,
C5eH), 9.56 (s, 1H, C2eH), 14.4 (s, 1H, COOH); 13C NMR
(DMSO-d6) d ppm: 177.5, 166.3, 157.9, 151.1, 148.1, 146.2,
131.8, 128.8, 126.5, 123.7, 121.9, 117.5, 114.2, 109.3, 99.3,
65.0, 61.1, 55.9, 42.8, 37.6, 30.2, 28.5, 25.8, 24.2; Anal
(C31H35N3O6) C, H, N.
1
5.1.5.44.
7-(2-Carboxy-5,6-dihydroimidazo[1,2-a]pyrazin-
7(8H )-yl)-1,4-dihydro-1-(4-fluorophenyl)-6-nitro-4-oxoquino-
line-3-carboxylic acid (9o). Yield: 84%; m.p.: 179 ꢀC; IR
(KBr) cmꢁ1: 3200, 2890, 1724, 1710, 1624, 1464e1360,
1208; 1H NMR (DMSO-d6) d ppm: 3.1e3.8 (m, 6H, 3-
CH2), 6.5e6.9 (m, 4H, Ar-H), 7.6 (s, 1H, CH), 8.32 (s, 1H,
C8eH), 9.32 (s, 1H, C5eH), 9.54 (s, 1H, C2eH), 12.12 (s,
1H, 2-COOH), 14.6 (s, 1H, 3-COOH); 13C NMR (DMSO-
d6) d ppm: 177.5, 167.5, 166.3, 157.6, 152.4, 149.4, 147.7,
146.3, 136.3, 130.3, 129.5, 126.6, 123.1, 120.7, 116.4,
109.3, 104.9, 57.1, 50.7, 37.1; Anal (C23H16FN5O7) C, H, N.
5.2. MIC determination
All compounds were screened for their in vitro antimyco-
bacterial activity against MTB, MDR-TB and MC2 in Middle-
brook 7H11 agar medium supplemented with OADC by agar
dilution method similar to that recommended by the National
Committee for Clinical Laboratory Standards for the determi-
nation of MIC in duplicate [16]. 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 compound
required to give complete inhibition of bacterial growth.
5.1.5.45. 1-tert-Butyl-7-(2-carboxy-5,6-dihydroimidazo[1,2-
a]pyrazin-7(8H )-yl)-1,4-dihydro-6-nitro-4-oxoquinoline-3-
carboxylic acid (10o). Yield: 72%; m.p.: >250 ꢀC; IR (KBr)
1
cmꢁ1: 3200, 2890, 1724, 1710, 1624, 1464e1360, 1208; H
NMR (DMSO-d6) d ppm: 1.74 (s, 9H, t-butyl), 3.1e3.8 (m,
6H, 3-CH2), 7.6 (s, 1H, CH), 8.32 (s, 1H, C8eH), 9.32 (s,
1H, C5eH), 9.54 (s, 1H, C2eH), 12.12 (s, 1H, 2-COOH),
14.6 (s, 1H, 3-COOH); 13C NMR (DMSO-d6) d ppm: 177.5,
167.5, 166.3, 157.6, 151.1, 148.1, 146.2, 130.3, 129.5,
126.5, 121.9, 117.5, 109.3, 99.3, 65.0, 57.1, 50.7, 37.1, 28.5;
Anal (C21H21N6O7) C, H, N.
5.3. Cytotoxicity
5.1.5.46.
1-Cyclopropyl-1,4-dihydro-7-(8-(4-methoxybenzyl)-
Some compounds were further examined for toxicity (CC50)
in a mammalian Vero cell line at the concentration of 62.5 mg/ml
[17]. After 72 h of exposure, viability was assessed on the basis
3,4,5,6,7,8-hexahydroisoquinolin-2(1H )-yl)-6-nitro-4-oxoqui-
noline-3-carboxylic acid (8p). Yield: 71%; m.p.: 208 ꢀC; IR

358
P. Senthilkumar et al. / European Journal of Medicinal Chemistry 44 (2009) 345e358
of cellular conversion of MTT into a formazan product using the
Promega Cell Titer 96 non-radioactive cell proliferation assay.
evaluated for changes indicative of a positive response:
erythema, edema or a measurable increase in ear thickness.
5.4. In vivo studies
Acknowledgements
One compound was tested for efficacy against MTB at the
dose of 50 mg/kg in six-week-old female CD-1 mice six per
group. In this model [18,19], the mice were infected intrave-
nously through caudal vein with approximately 107 viable M. tu-
berculosis ATCC 35801. Drug treatment by intraperitoneal
route began after 10 days of inoculation of the animal with
microorganism and continued for 10 days. After 35 days post-
infection the spleen and right lung were aseptically removed
and ground in a tissue homogenizer, the number of viable organ-
isms were 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 controls (vehicle-treated) in lung and in spleen.
The authors are thankful to Council of Scientific and Indus-
trial Research [01 (1979)/05/EMR/-II] and University Grant
Commissions F.30-266/2004(SR) New Delhi, India for their
financial assistances. The authors are also thankful to Dr.
Vanaja Kumar, Deputy Director, Tuberculosis Research Cen-
ter, Chennai, India for their assistance in biological screening
and A. Nambi for technical assistance in gyrase assays. The
work in VN’s laboratory is supported by COE grant from
Department of Biotechnology, Government of India.
References
[1] C. Dye, S. Scheele, P. Dolin, V. Pathania, M.C. Raviglione, J. Am. Med.
Assoc. 282 (1999) 677e686.
[2] N.R. Cozzarelli, Science 207 (1980) 953e960.
5.5. DNA gyrase supercoiling assay
[3] R.P. Tangallapally, R. Yendapally, R.E. Lee, K. Hevener, V.C. Jones,
A.J.M. Lenaerts, M.R. McNeil, Y. Wang, S. Franzblau, R.E. Lee,
J. Med. Chem. 47 (2004) 5276e5283.
In order to carry out gyrase inhibition assays DNA gyrase
purified from M. smegmatis was used. The enzyme was prepared
and stored by standardized procedures [15]. The compounds
tested were dissolved in DMSO and pre-incubated with gyrase.
Supercoiling assays were carried out as described previously
[18], by incubating 400 ng of relaxed circular pUC18 in a buffer
[35 mM TriseHCl pH 7.5, 5 mM MgCl2, 25 mM potassium
glutamate, 2 mM spermidine, 2 mM ATP, 50 mg/ml bovine
serum albumin, and 90 mg/ml yeast t-RNA in 5% (v/v) glycerol]
for 30 min at 37 ꢀC. Ciprofloxacin at 10 mg/ml final concentra-
tion was used as a control and a reaction having 5% DMSO in
the absence of compounds was also performed. The reaction
samples were analyzed in agarose gel electrophoresis as
described earlier [20].
[4] R.P. Tangallapally, R. Yendapally, R.E. Lee, A.J.M. Lenaerts, J. Med.
Chem. 48 (2005) 8261e8269.
[5] H. Sasaki, Y. Haraguchi, M. Itotani, H. Kuroda, H. Hashizume,
T. Tomishige, M. Kawasaki, M. Matsumoto, M. Komatsu, H. Tsubouchi,
J. Med. Chem. 49 (2006) 7854e7860.
[6] A.J. Lenaerts, V. Gruppo, K.S. Marietta, C.M. Johnson, D.K. Driscoll,
N.M. Tompkins, J.D. Rose, R.C. Reynolds, I.M. Orme, Antimicrob.
Agents Chemother. 49 (2005) 2294e2301.
[7] D.R. Ashtekar, R. Costa-Perira, K. Nagarajan, N. Vishvanathan, A.D. Bhatt,
W. Ritflel, Antimicrob. Agents Chemother. 37 (1993) 183e186.
[8] C.E. Barry, H.I.M. Boshoff, C.S. Dowd, Curr. Pharm. Des. 10 (2004)
3239e3262.
[9] M. Artico, A. Mai, G. Sbardella, S. Massa, C. Musiu, S. Lostiaj,
F. Demontis, P.L. Colla, Bioorg. Med. Chem. Lett. 9 (1999) 1651e1656.
[10] G. Sbardella, A. Mai, M. Artico, M.G. Setzu, G. Poni, P.L. Colla, Il
Farmaco 59 (2004) 463e471.
[11] T.W. Daniel, W. Chu, P.B. Fernandes, A.K. Claiborne, E. Pihuleac,
C.W. Nordeen, R.E. Maleczka, A.G. Pernet, J. Med. Chem. 28 (1985)
1558e1564.
5.6. Phototoxicity evaluation
[12] T.E. Renau, J.P. Sanchez, J.W. Gage, J.J.A. Dever, M.A. Shapiro,
S.J. Gracheck, J.M. Domagala, J. Med. Chem. 39 (1996) 729e735.
[13] D.C. Hooper, Drug Resist. Update 2 (1999) 38e55.
[14] C.H. Levine, A. Hiasa, K.J. Marians, Biochim. Biophys. Acta 1400
(1998) 29e43.
Female swiss albino mice, approximately 2 months old and
weighing 20e25 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-irradiation period. Eighteen mice
were randomly distributed into three dosing groups. The first
group received a single dose of screened compound at 140 mg/
kg by oral gavage. The second group received a single dose of
140 mg/kg of lomefloxacin HCl. This lomefloxacin dose is one
that, in preliminary experiments in this test system, produced
a consistent erythema and ear thickening response. The final
group served as a vehicle control, and received 10 ml/kg of the
sodium carboxymethylcellulose vehicle only. Test animals
were exposed to UVA light in a manner adapted from that
described previously [21]. 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 period and at approximately
24, 48, 72 and 96 h after dosing, both ears of each mouse were
[15] U.H. Manjunatha, K. Madhusudan, S.Visweswariah Sandhya,
V. Nagaraja, Curr. Sci. 79 (2000) 968e974.
[16] National Committee for Clinical Laboratory Standards, Antimycobacte-
rial Susceptibility Testing for Mycobacterium tuberculosis. Proposed
Standard M24-T, National Committee for Clinical Laboratory Standards,
Villanova, Pa, 1995.
[17] L.L. Gundersen, J. Nissen-Meyer, B. Spilsberg, J. Med. Chem. 45 (2002)
1383e1386.
[18] D. Sriram, P. Yogeeswari, J.S. Basha, D.R. Radha, V. Nagaraja, Bioorg.
Med. Chem. 13 (2005) 5774e5778.
[19] E. Miyazaki, M. Miyazaki, J.M. Chen, R.E. Chaisson, W.R. Bishai,
Antimicrob. Agents Chemother. 43 (1999) 85e89.
[20] U.H. Manjunatha, M. Dalal, M. Chatterji, D.R. Radha, S.S. Visweswariah,
V. Nagaraja, Nucleic Acids Res. 30 (2002) 2144e2152.
[21] T.N. Mayne, N.J. Johnson, W.M. Kluwe, D.L. Lencoski, R.J. Polzer,
J. Antimicrob. Chemother. 39 (1997) 67e73.