Synthesis and antimycobacterial evaluation of newer 1-cyclopropyl-1,4-dihydro-6-fluoro-7-(substituted secondary amino)-8-methoxy-5-(sub)-4-oxoquinoline-3-carboxylic acids

Article abstract of DOI:10.1016/j.bmc.2007.11.050

Thirty-four newer 1-cyclopropyl-1,4-dihydro-6-fluoro-7-(substituted secondary amino)-8-methoxy-5-(sub)-4-oxoquinoline-3-carboxylic acids were synthesized from 1,2,3,4-tetrafluoro benzene and evaluated for in vitro and in vivo antimycobacterial activities against Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant M. tuberculosis (MDR-TB) and Mycobacterium smegmatis (MC²) and also tested for the ability to inhibit the supercoiling activity of DNA gyrase. Among the synthesized compounds, 7-(1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinolin-2(1H)-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-5-nitro-4-oxoquinoline-3-carboxylic acid (13n) was found to be the most active compound in vitro with MIC of 0.16 and 0.33 μM against MTB and MDR-TB, respectively. In the in vivo animal model 13n decreased the bacterial load in lung and spleen tissues with 2.54 and 2.92 - log10 protections, respectively, at the dose of 50 mg/kg body weight. Compound 13n also inhibited the supercoiling activity of mycobacterial DNA gyrase with IC₅₀ of 30.0 μg/ml.

Full text of DOI:10.1016/j.bmc.2007.11.050

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2558–2569
Synthesis and antimycobacterial evaluation of newer
1-cyclopropyl-1,4-dihydro-6-fluoro-7-(substituted secondary
amino)-8-methoxy-5-(sub)-4-oxoquinoline-3-carboxylic acids
Palaniappan Senthilkumar,a Murugesan Dinakaran,a Debjani Banerjee,a
Ruth Vandana Devakaram,a Perumal Yogeeswari,a Arnab China,b
Valakunja Nagarajab 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 8 October 2007; revised 16 November 2007; accepted 17 November 2007
Available online 22 November 2007
Abstract—Thirty-four newer 1-cyclopropyl-1,4-dihydro-6-fluoro-7-(substituted secondary amino)-8-methoxy-5-(sub)-4-oxoquino-
line-3-carboxylic acids were synthesized from 1,2,3,4-tetrafluoro benzene and evaluated for in vitro and in vivo antimycobacterial
activities against Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant M. tuberculosis (MDR-TB) and Mycobacterium
smegmatis (MC2) and also tested for the ability to inhibit the supercoiling activity of DNA gyrase. Among the synthesized com-
pounds, 7-(1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinolin-2(1H)-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-5-nitro-
4-oxoquinoline-3-carboxylic acid (13n) was found to be the most active compound in vitro with MIC of 0.16 and 0.33 lM against
MTB and MDR-TB, respectively. In the in vivo animal model 13n decreased the bacterial load in lung and spleen tissues with 2.54
and 2.92 ꢀ log10 protections, respectively, at the dose of 50 mg/kg body weight. Compound 13n also inhibited the supercoiling
activity of mycobacterial DNA gyrase with IC50 of 30.0 lg/ml.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
drugs have been examined as potential chemotherapeu-
tics for Mycobacterium tuberculosis infection because of
their favorable pharmacokinetic profiles such as easily
absorbed after oral administration and readily pene-
trated into mammalian cells.2 Fluoroquinolones exhibit
potent in vitro and in vivo antimycobacterial activity.3
Quinolones inhibit both bacterial type II topoisomerase,
DNA gyrase and topoisomerase IV,4 which are essential
enzymes catalyzing DNA supercoiling and decatenation
reactions, respectively. There is also a considerable effort
to discover and develop newer fluoroquinolones, and
some of them might have value in the treatment of
TB.5 Several of the quinolone antibacterial drugs have
been examined as inhibitors of MTB as well as other
mycobacterial infections.6 As a result, gatifloxacin and
moxifloxacin (8-methoxy quinolones) are drugs in pipe-
line and to be approved by FDA for the treatment of TB
by the year 2010. Herewith we report the synthesis of
newer 8-methoxy quinolone analogues containing unre-
ported bulky secondary amino function at 7th position
and nitro group at 5th position of 8-methoxy quinolone
and the study of the influence of lipophilic character and
nitro group on activity against MTB.
Tuberculosis (TB) continues to be a major health con-
cern. The World Health Organisation (WHO) declared
TB a global emergency in 1993.1 The emergence of
AIDS and decline of socioeconomic standards have con-
tributed to the disease’s resurgence in industrialized
countries. In most developing countries, although the
disease has always been endemic, its severity has in-
creased because of the global HIV pandemic and exten-
sive social restructuring due to rapid industrialization
and conflicts. 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. During the past
decade, several of the fluoroquinolone antibacterial
Keywords: Antimycobacterial activity; Antitubercular activity; 8-Met-
hoxyquinolone carboxylic acids.
*
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.2007.11.050

P. Senthilkumar et al. / Bioorg. Med. Chem. 16 (2008) 2558–2569
2559
2. Results and discussion
formed with short reaction times (3–6 min), with ease
and was environment friendly. Compounds 13c and 13n
were further reduced by catalytic reduction of the nitro
group to yield 14c and 14n. The purity of the synthesized
compounds was monitored by thin-layer chromatogra-
phy (TLC) and elemental analyses and the structures
were identified by spectral data.
2.1. Synthesis
Thirty-four new 8-methoxyquinolone carboxylic acids
with variation at C5 and C7 positions were synthesized
from 1,2,3,4-tetrafluoro benzene (1) according to the lit-
erature method7–9 with modification in some steps.
Briefly, compound 1 was converted to 1-bromo-2,3,4,5-
tetrafluorobenzene (2) using bromine and aluminium
bromide in the presence of oleum, which was further
methoxylated using sodium methoxide in methanol to
yield 1-bromo-3-methoxy-2,4,5-trifluoro benzene (3).
Compound 3 on treatment with cuprous cyanide and
N-methyl-2-pyrrollidone gave the corresponding cyano
derivative 3-methoxy-2,4,5-trifluorobenzonitrile (4).
Compound 4 was reduced to give the corresponding
amide (5) which was further treated with sulfuric acid
to form 3-methoxy-2,4,5-trifluoro benzoic acid (6). Com-
pound 6 on reaction with 1,10-carbonyldiimidazole in tet-
rahydrofuran 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-(2,4,5-trifluoro-3-methoxy-
phenyl)-3-oxopropanoate (7). Ethyl 3-(cyclopropyla-
mino)-2-(2,4,5-trifluoro-3-methoxybenzoyl)acrylate (9)
was prepared by a two-step one-pot reaction. First treat-
ment of the keto ester 7 with triethyl orthoformate in
acetic anhydride gave the one-carbon homologue enol
ether intermediate ethyl 3-ethoxy-2-(2,4,5-trifluoro-3-
methoxybenzoyl)acrylate (8) as an oil, which on reaction
with cyclopropylamine at 0 °C affords 67% of 9 as an oily
residue. Compound 9 on cyclization with the base potas-
sium carbonate in dimethylsulfoxide yielded ethyl
1-cyclopropyl-6,7-difluoro-1,4-dihydro-8-methoxy-4-oxo-
quinoline-3-carboxylate (10), when compared to earlier
report,7 this step proceeds smoothly without using vio-
lently reactive sodium hydride. Ethyl ester was finally
hydrolysed in acidic condition to yield 1-cyclopropyl-
6,7-difluoro-1,4-dihydro-8-methoxy-4-oxoquinoline-3-
carboxylic acid (11a), which on nitration at C5 position
with sulfuric acid and potassium nitrate yielded 1-cyclo-
propyl-6,7-difluoro-1,4-dihydro-8-methoxy-5-nitro-4-oxo-
quinoline-3-carboxylic acid (11b). The titled compounds
12 and 13a–p were prepared by treating 11a and 11b with
appropriate secondary amines in the presence of potas-
sium carbonate under microwave irradiation in DMSO
(Scheme 1). When compared to conventional method7
of 12 h process, microwave-assisted synthesis was per-
2.2. Antimycobacterial activity
The compounds were screened for their in vitro antimy-
cobacterial activity against MTB, MDR-TB and Myco-
bacterium smegmatis ATCC 14468 (MC2) by agar
dilution method for the determination of MIC in dupli-
cate.10 The MDR-TB clinical isolate was resistant to iso-
niazid, rifampicin, ethambutol and ofloxacin. The
minimum inhibitory concentration (MIC) is defined as
the minimum 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 presented in Table 1.
In the first phase of screening against MTB, all the com-
pounds showed excellent in vitro activity against MTB
with MIC less than 15 lM. Five compounds (13c, 13g,
12l, 13l and 13n) inhibited MTB with MIC of less than
1 lM and were more potent than standard gatifloxacin
(MIC: 1.04 lM). When compared to isoniazid (MIC:
0.36 lM), one compound (13n) was found to be more
active against MTB and one compound (13c) was found
to be equally active to isoniazid. Compound 7-(1-(4-
methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinolin-2(1H)-
yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-5-ni-
tro-4-oxoquinoline-3-carboxylic acid (13n) was found to
be the most active compound in vitro with MIC of
0.16 lM against MTB and was 2.25 and 6.5 times more
potent than isoniazid and gatifloxacin, respectively. Sub-
sequently some of the compounds were evaluated against
MDR-TB, and among the 22 compounds screened, all
the compounds inhibited MDR-TB with MIC ranging
from 0.33 to 3.99 lM and were found to be more active
than isoniazid (MIC: 45.57 lM) and gatifloxacin (MIC:
8.34 lM). Five compounds (13c, 12e, 13l, 13n and 13p)
inhibited MDR-TB with MIC of less than 1 lM. Com-
pound 13n was found to be the most active in vitro with
MIC of 0.33 lM against MDR-TB and was 25 and 138
times more potent than gatifloxacin and isoniazid, respec-
tively. The compounds were also evaluated against MC2
in which all the compounds inhibited MC2 with MIC
R
O
N
O
R
O
N
O
F
F
F
OH
OH
Var. Sec. amines, DMSO,
MWI
R1
N
H2, Ran.Ni, DMF
11 a,b
a: R=H
b: R=NO2
R1
OCH3
OCH3
12a-q: R=H
13a-q: R=NO2
14c,n: R=NH2
Scheme 1. Synthetic protocol of the compounds.

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P. Senthilkumar et al. / Bioorg. Med. Chem. 16 (2008) 2558–2569
Table 1. Physical constants, in vitro antimycobacterial activities and cytotoxicity
R
O
O
F
OH
R
1
N
O
Compound
R
R1
IC50 (lM)
MIC (lM)
MTB
MDRTB
MC2
Cl
12a
13a
H
NO2
NT
102.96
11.12
1.28
NT
1.28
44.48
10.29
N
N
O
C
12b
13b
H
NO2
NT
NT
13.72
6.25
NT
NT
13.72
12.49
N
N
O
12c
13c
14c
H
126.14
115.63
NT
1.57
0.35
3.06
1.57
0.35
NT
3.15
1.44
6.13
H2C
N
N
O
NO2
NH2
O
N
H3C
N
12d
13d
H
NO2
NT
125.88
13.84
3.14
NT
3.14
27.69
6.30
C6H5
12e
13e
H
NO2
119.39
109.94
1.49
1.37
0.74
2.74
1.49
2.74
O
O
C
N
N
O
12f
13f
H
NO2
165.16
147.61
2.06
1.84
2.06
1.84
4.12
3.68
S
N
12g
13g
H
NO2
160.09
143.55
3.99
0.89
3.99
1.79
16.01
1.79
O
N
12h
13h
H
NO2
NT
NT
7.06
6.41
NT
NT
14.09
12.79
N
N
HO
12i
13i
H
NO2
NT
NT
6.43
5.88
NT
NT
12.84
11.75
Cl
N

P. Senthilkumar et al. / Bioorg. Med. Chem. 16 (2008) 2558–2569
2561
Table 1 (continued)
Compound
R
R1
IC50 (lM)
MIC (lM)
MTB
MDRTB
MC2
N
12j
13j
H
NO2
NT
NT
5.94
5.47
NT
NT
5.94
10.93
N
O
N
H
Cl
O
C
(C H ) N
2
5 2
12k
13k
H
NO2
136.01
123.88
1.69
1.55
3.39
1.55
0.85
12.39
N
O
O
12l
13l
H
NO2
149.37
134.87
0.93
0.84
1.86
0.41
1.86
6.75
N
12m
13m
H
NO2
NT
113.11
12.31
1.41
NT
2.82
49.26
2.82
N
O
C
NHC(CH )
3 3
N
12n
13n
14n
H
117.35
108.21
114.13
1.46
0.16
2.85
1.46
0.33
2.85
2.93
0.68
5.72
NO2
NH2
H2C
OCH3
N
12o
13o
H
NO2
141.27
128.23
3.53
3.120
1.76
1.60
7.08
6.42
HOOC
N
N
H C
3
N
O
12p
13p
H
NO2
166.06
148.32
4.14
1.85
2.07
0.93
16.61
3.70
H C
3
Gati
INH
—
—
—
—
>155.3
>455.8
1.04
0.36
8.34
45.57
2.08
45.57
NT indicates not tested.
ranging from 0.68 to 49.26 lM; 33 compounds were
found to be more active than isoniazid (MIC: 45.57 lM)
and six compounds were more active than gatifloxacin
(MIC: 2.08 lM).
tution pattern at C7 demonstrated that the order of
activity was fused piperazines and piperidines P (thio)
morpholines > substituted
piperazines > substituted
piperidines. By introducing bulky lipophilic secondary
amines at C7, enhanced the antimycobacterial activity
was enchanced which might be due to more penetration
of these compounds into mycobacterial cells.
With respect to structure–MTB activity relationship, the
results demonstrated that the antimycobacterial activity
was enhanced to varying degrees (upto 9-fold) by the
introduction of nitro group at C5 position. Reduction
of nitro group at C5 position to amino group (14c and
14n) reduces the activity indicating that favourable sub-
stitution at C5 was NO2 > H > NH2. At C7 position we
have studied with various substituted piperazines (12
and 13a–e), (thio) morpholines (12 and 13f–g), substi-
tuted piperidines (12 and 13h–k), fused piperazines and
piperidines (12 and 13l–p). A comparison of the substi-
2.3. In vitro cytotoxicity
Some compounds were further examined for toxicity
(IC50) in a mammalian Vero cell line upto 62.5 lg/ml
concentrations.11 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. Twenty-one compounds when tested showed IC50

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P. Senthilkumar et al. / Bioorg. Med. Chem. 16 (2008) 2558–2569
values ranging from 102.96 to 166.06 lM. A comparison
of the substitution pattern at C5 demonstrated that nitro
group was more cytotoxic than the unsubstituted deriv-
atives. These results are important as the C5 nitro
substituted compounds with their increased cytoliability
are much less attractive in the development of a quino-
lone 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 mar-
gin of safety becomes a primary concern. The IC50 value
of compound 13n was found to be 108.21 lM and it
showed selectivity index (IC50/MIC) of 676.31.
and studied in this report were tested for their ability
to inhibit supercoiling activity of DNA gyrase. Earlier
studies have revealed that DNA gyrase from MTB and
that from MC2 share many of the properties.13 The
supercoiling assay results with various compounds using
MC2 DNA gyrase are presented in Fig. 1a–d. The IC50
values are presented in Table 3. Of all the newly synthe-
sized compounds, compound 13n inhibited supercoiling
reaction with an IC50value of 30 lg/ml. The compound
13n also showed highest in vivo efficacy against MTB,
MDR-TB and MC2 amongst all the compounds tested
in this report (Table 1). The other compounds that
showed significant supercoiling inhibition include com-
pounds 12l and 13f, with IC50 values of 40 and 50 lg/
ml, respectively. Rest of the compounds tested did not
show comparable inhibition of the enzyme (IC50 val-
ues > 50lg/ml).
2.4. In vivo antimycobacterial activity
Subsequently, compounds 12l and 13n were tested
for in vivo efficacy against MTB at a dose of 50 mg/kg
(Table 2) in CD-1 mice.12 The mice were infected
intravenously with M. tuberculosis ATCC 35801. Drug
treatment by intra-peritoneal 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 0.16 and in spleen: 9.02 0.21). Compound
12l decreased the bacterial load in lung and spleen
tissues with 1.87 and 2.61 ꢀ log10 protections, respec-
tively, and was considered to be promising in reducing
bacterial count in lung and spleen tissues. Compound
13n decreased the bacterial load in lung and spleen
tissues with 2.54 and 2.92 ꢀ log10 protections, respec-
tively, and was considered to be promising in reducing
bacterial count in lung and spleen tissues. When
compared to gatifloxacin at the same dose level 12l
decreased the bacterial load with 0.51 ꢀ log10 protec-
tions and 13n with 0.61 and 0.82 ꢀ log10 protections
in lung and spleen tissues, respectively.
2.6. Phototoxic evaluation
Quinolones in general have favorable safety profiles;
phototoxicity has become a significant factor in the clin-
ical use of some. Indeed, the first quinolone, nalidixic
acid, caused light-induced dermal effects. This type of re-
sponse has now been demonstrated for almost all fluoro-
quinolones, although the relative phototoxic potential
varies greatly among compounds. Phototoxicity is con-
sidered to be an acute, light-induced irritation response
characterized by dermal inflammation, with erythema
and oedema as primary clinical endpoints. Phototoxicity
with the quinolones is generally thought to result from
the absorption of light by the parent compound or a
metabolite in tissue. This photosensitized chromophore
may then transfer its absorbed photoenergy 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.
Five (13c, 12e, 13g, 12k and 13n) compounds were eval-
uated for potential phototoxicity in a standardized
in vivo test system that has been used previously to
assess quinolone antibiotics.14 The test compounds
(140 mg/kg) and the positive control lomefloxacin
hydrochloride (140 mg/kg) were evaluated for phototox-
icity and both ears of each mouse were evaluated for
changes indicative of a positive response: erythema,
oedema or a measurable increase in ear thickness.
Change from baseline was calculated separately for each
animal and time point and analyzed for statistical signif-
icance and are presented in Table 4. The drug and time
factors were analyzed by separate univariate 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
baseline ear thickness was significantly different from
zero. The results indicated that lomefloxacin showed
significant increase in ear thickness from 4–96 to
24–96 h when compared within time points and with
the control, respectively. The test compounds were
found to show a significant difference in ear thickness
at various time-points when compared with the pre-drug
reading (0 h) but were less toxic when compared with the
2.5. DNA gyrase inhibition
DNA gyrase is a target for different classes of natural
and synthetic compounds. The compounds are broadly
classified into (1) quinolones and fluoroquinolones,
which target GyrA subunit of gyrase, (2) coumarins
and (3) cyclothialidines, both of which inhibit the
ATPase activity associated with GyrB subunit of
DNA gyrase. The 1-cyclopropyl-1,4-dihydro-6-fluoro-
7-(substituted secondary amino)-8-methoxy-5-(sub)-4-
oxoquinoline-3-carboxylic acid derivatives synthesized
Table 2. In vivo activity data of 12l, 13n, gatifloxacin and isoniazid
against M. tuberculosis ATCC 35801 in mice
Compound
Lungs Spleen
(log CFU SEM) (log CFU SEM)
Control 7.99 0.16
Gatifloxacin (50 mg/kg) 6.02 0.23
9.02 0.21
6.92 0.07
4.71 0.10
6.41 0.12
6.10 0.18
Isoniazid (25 mg/kg)
12l
13n
5.86 0.23
6.12 0.11
5.41 0.21

P. Senthilkumar et al. / Bioorg. Med. Chem. 16 (2008) 2558–2569
2563
Gyrase
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
a
b
Controls
Compounds
DMSO Cipro -
-
-
-
-
-
-
-
-
-
-
-
-
12c 12e 12k 12l 12n 13e 13f 13g 13m 13n 13q
R
S
1
2
3
4
5
6
7
8
9
10 11 12 13 14
Gyrase
Controls
-
-
-
+
+
+
-
+
-
+
-
DMSO Cipro
Compounds
-
-
12l 13f 13n
R
S
1
2
3
4
5
6
c
d
Gyrase
Controls
Compounds
-
-
-
+
+
+
-
+
-
+
-
Gyrase
-
-
-
+
+
+
-
13n
DMSO Cipro
Controls
Compounds
DMSO Cipro
-
-
12l 13f 13n
-
-
R
S
R
S
1
2
3
4
5
6
1
2
3
4
Figure 1. DNA gyrase supercoiling assay. The assays were performed as described in the text. DNA gyrase was pre-incubated with the indicated
concentrations of the compounds and then rest of the components of the reaction including relaxed circular DNA were added. (a) Lane 1, relaxed
circular DNA; lane 2, supercoiling reaction in presence of solvent control, 5% DMSO; lane 3, ciprofloxacin at 10 lg/ml concentration, used as a
positive control of gyrase inhibition; lanes 4–14, reactions in the presence of 50 lg/ml of compounds 12c, 12e, 12k, 12l, 12n, 13e, 13f, 13g, 13m, 13n
and 13q, respectively. (b) Lane 1, relaxed circular DNA; lane 2, supercoiling reaction in the presence of 5% DMSO; lane 3, ciprofloxacin at 10 lg/ml
concentration; lanes 4–6, reactions in the presence of 40 lg/ml of compounds 12l, 13f and 13n, respectively. (c) Lane 1, relaxed circular DNA; lane 2,
supercoiling reaction in presence of 5% DMSO; lane 3, ciprofloxacin at 10 lg/ml concentration; lanes 4–6, reactions in the presence of 30 lg/ml of
compounds 12l, 13f and 13n, respectively. (d) Lane 1, relaxed circular DNA; lane 2, supercoiling reaction in the presence of 5% DMSO; lane 3,
ciprofloxacin at 10 lg/ml concentration; lanes 4, reaction in the presence of 20 lg/ml of compound 13n. R and S indicate relaxed and supercoiled
DNA, respectively.
Table 3. IC50 values for DNA gyrase inhibition
3. Materials and methods
Compounds
IC50 (lg/ml)
Melting points were taken on an electrothermal melting
point apparatus (Buchi BM530) in open capillary tubes
and are uncorrected. Infrared spectra (KBr disc) were
12c
12e
12k
12l
>50
30
>50
>50
40
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. 13C NMR spectra were recorded on Bruker AC
200/DPX 400 MHz. Elemental analyses (C, H and N)
were performed on Perkin-Elmer model 240 C analyzer
and the data were within 0.4% of the theoretical
values.
12n
13e
13f
>50
>50
50
13g
13m
13n
13q
>50
30
>50
negative (vehicle-treated) and positive controls (lome-
floxacin). Compounds 13c and 13g showed a slight in-
crease in ear thickness from 48–96 to 24–96 h,
respectively, but were less toxic than the positive control
(lomefloxacin). No erythema occurred in mice dosed
with 140 mg/kg of 12e and 13n throughout the 96 h
study, while compounds 13g and 12k showed a signifi-
cant erythema after irradiation until 4 h only and com-
pound 13c developed erythema from 48 h.
3.1. Synthesis of 1-cyclopropyl-6,7-difluoro-1,4-dihydro-
8-methoxy-5-nitro-4-oxoquinoline-3-carboxylic acid (11b)
A solution of 10 (3 g, 1.00 equiv) in concentrated sulfu-
ric acid (30 ml) was treated portionwise with potassium
nitrite (1.5 equiv) over 30 min. The reaction mixture was
stirred at room temperature for 6 h. Then poured into
ice-cold water and the solid obtained was filtered and
washed with water and dissolved in dichloromethane.

2564
P. Senthilkumar et al. / Bioorg. Med. Chem. 16 (2008) 2558–2569
Table 4. Phototoxic evaluation of newer 8-methoxy quinolones
Group
Ear thickness (mm)a
Time (approximately) after start of irradiation (h)c
Erythemab
0
4
24
48
72
96
0
4
24
48
72
96
Controld
13c
0.37 0.03
0.26 0.01
0.33 0.01
0.28 0.02
0.39 0.04
0.32 0.01
0.31 0.01
0.36 0.02
0.27 0.02
0.31 0.01
0.29 0.01
0.38 0.04
0.33 0.01
0.40 0.02
0.38 0.03
0.28 0.01
0.32 0.01
0.35 0.02
0.36 0.05
0.32 0.02
0.48 0.02
0.37 0.03
0.30 0.01
0.33 0.01
0.36 0.01
0.37 0.06
0.33 0.01
0.53 0.02
0.37 0.03
0.33 0.01
0.33 0.01
0.34 0.01
0.39 0.07
0.33 0.01
0.64 0.04
0.38 0.03
0.31 0.02
0.32 0.02
0.33 0.01
0.39 0.07
0.32 0.02
0.60 0.06
0
0
0
0
0
0
6
0
0
0
2
2
0
6
0
0
0
0
0
0
6
0
2
0
0
0
0
6
0
3
0
0
0
0
6
0
4
0
0
0
0
6
12e
13g
12k
13n
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.
The DCM layer was washed successively with water and
5% sodium bicarbonate, dried over sodium sulfate, dis-
tilled recrystallised from methanol (Yield: 88%, mp
>250 °C).
(m, 4H, cyclopropyl), 1.35 (m, 1H, cyclopropyl), 2.59
(t, 4H, 3,5-CH2 of piperazine), 3.32 (t, 4H, 2,6-CH2 of
piperazine), 3.77 (s, 3H, OCH3), 5.2 (s, 1H, CH of
diphenylmethyl), 7.0–7.18 (m, 9H, Ar-H), 8.79 (s, 1H,
C2-H), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) d
ppm: 5.6, 36.0, 49.6, 50.6, 56.1, 73.6, 107.6, 109.5,
118.8, 126.3, 128.3, 129.4, 130.8, 131.9, 132.8, 142.8,
146.5, 148.7, 166.3, 177.5. Anal. (C31H28ClFN4O6) C,
H, N. Calculated C, 61.34; H, 4.65; N, 9.23. Found:
C, 61.34; H, 4.66; N, 9.22.
3.2. Synthesis of 1-cyclopropyl-6,7-difluoro-1,4-dihydro-8-
methoxy-4-oxoquinoline-3-carboxylic acid (11a)
The product 10 (3 g) was suspended in 6 N HCl (50 ml)
and refluxed for 3 h and then cooled to 0 °C. The precip-
itate obtained was filtered, washed with water and dried
(Yield: 90%, mp >250 °C).
3.3.3. 1-Cyclopropyl-1,4-dihydro-6-fluoro-7-(4-(2-furoyl)-
piperazin-1-yl)-8-methoxy-4-oxoquinoline-3-carboxylic
acid (12b). Yield: 65%; mp 215–217°C; 1H NMR
(DMSO-d6) d ppm: 0.28–0.48 (m, 4H, cyclopropyl),
1.35 (m, 1H, cyclopropyl), 3.16 (t, 4H, 3,5-CH2 of piper-
azine), 3.26 (t, 4H, 2,6-CH2 of piperazine), 3.52 (s, 3H,
OCH3), 6.5–7.28 (m, 3H, Ar-H), 7.91 (s, 1H, C5-H),
8.81 (s, 1H, C2-H), 14.6 (s, 1H, COOH); 13C NMR
(DMSO-d6) d ppm: 5.6, 36.2, 47.5, 49.5, 56.2, 107.7,
109.3, 111.4, 113.5, 130.6, 131.8, 133.0, 147.1, 148.1,
155.2, 166.3, 177.5. Anal. (C23H22FN3O6) C, H, N. Cal-
culated C, 60.66; H, 4.87; N, 9.23. Found: C, 60.64; H,
4.88; N, 9.21.
3.3. Synthesis of 1-cyclopropyl-1,4-dihydro-6-fluoro-7-
(substituted secondary amino)-8-methoxy-5-(sub)-4-oxo-
quinoline-3-carboxylic acid (12 and 13a–p)
To a solution of 11a,b (0.295 g, 0.34 g, 1.0 equiv) in
dimethylsulfoxide (10 ml) and substituted secondary
amines (1.1 equiv), potassium carbonate (0.165 g,
1.2 equiv) was added and placed in microwave and al-
lowed to react at 320 V for a period of 2 min. After com-
pletion of the reaction, the mixture was poured into ice-
cold water and kept overnight, filtered and washed with
water to yield 12 and 13a–p.
3.3.4. 1-Cyclopropyl-1,4-dihydro-6-fluoro-7-(4-(2-furoyl)-
piperazin-1-yl)-8-methoxy-5-nitro-4-oxoquinoline-3-car-
boxylic acid (13b). Yield: 66%; mp 118–120 °C; 1H
NMR (DMSO-d6) d ppm: 0.28–0.48 (m, 4H, cyclopro-
pyl), 1.35 (m, 1H, cyclopropyl), 3.16 (t, 4H, 3,5-CH2
of piperazine), 3.26 (t, 4H, 2,6-CH2 of piperazine),
3.52 (s, 3H, OCH3), 6.5–7.28 (m, 3H, Ar-H), 8.81 (s,
1H, C2-H), 14.6 (s, 1H, COOH); 13C NMR (DMSO-
d6) d ppm: 5.6, 36.2, 47.5, 49.5, 56.2, 107.7, 109.3,
111.4, 113.5, 130.6, 131.8, 133.0, 145.0, 147.1, 148.1,
155.2, 166.3, 177.5. Anal. (C23H21FN4O8) C, H, N. Cal-
culated C, 55.20; H, 4.23; N, 11.20. Found: C, 55.1; H,
4.23; N, 11.21.
3.3.1. 7-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-
yl)-1-cyclopropyl-1,4-dihydro-6-fluoro-8-methoxy-4-oxo-
quinoline-3-carboxylic acid (12a). Yield: 69%; mp 115–
117°C; 1H NMR (DMSO-d6) d ppm: 0.28–0.48 (m,
4H, cyclopropyl), 1.35 (m, 1H, cyclopropyl), 2.59 (t,
4H, 3,5-CH2 of piperazine), 3.32 (t, 4H, 2,6-CH2 of
piperazine), 3.77 (s, 3H, OCH3), 5.2 (s, 1H, CH of
diphenylmethyl), 7.0–7.18 (m, 9H, Ar-H), 7.9 (s, 1H,
C5-H), 8.79 (s, 1H, C2-H), 14.6 (s, 1H, COOH); 13C
NMR (DMSO-d6) d ppm: 5.6, 36.0, 49.6, 50.6, 56.1,
73.6, 108.6, 109.5, 118.8, 126.3, 128.3, 129.4, 131.9,
132.8, 142.8, 146.5, 145.3, 148.7, 166.3, 177.5. Anal.
(C31H29ClFN3O4) C, H, N. Calculated C, 66.25; H,
5.20; N, 7.48. Found: C, 66.22; H, 5.19; N, 7.48.
3.3.5. 7-(4-((Benzo[d][1,3]dioxol-5-yl)methyl)piperazin-1-
yl)-1-cyclopropyl-1,4-dihydro-6-fluoro-8-methoxy-4-oxo-
quinoline-3-carboxylic acid (12c). Yield: 68%; mp 95–
97 °C; 1H NMR (DMSO-d6) d ppm: 0.28–0.48 (m,
4H, cyclopropyl), 1.35 (m, 1H, cyclopropyl), 2.82 (t,
4H, 3,5-CH2 of piperazine), 3.1 (t, 4H, 2,6-CH2 of
3.3.2. 7-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-
yl)-1-cyclopropyl-1,4-dihydro-6-fluoro-8-methoxy-5-nitro-
4-oxoquinoline-3-carboxylic acid (13a). Yield: 62%; mp
168–170°C; 1H NMR (DMSO-d6) d ppm: 0.28–0.48

P. Senthilkumar et al. / Bioorg. Med. Chem. 16 (2008) 2558–2569
2565
piperazine), 3.55 (s, 3H, OCH3), 3.6 (s, 2H, CH2 of pip-
eranoyl), 5.86 (s, 2H, –OCH2O–), 6.42–6.62 (m, 3H, Ar-
H), 7.8 (s, 1H, C5-H), 8.8 (s, 1H, C2-H), 14.6 (s, 1H,
COOH); 13C NMR (DMSO-d6) d ppm: 5.6, 36.1, 50.3,
52.2, 56.1, 60.4, 108.2, 109.5, 101.3, 113.9, 115.1,
119.4, 122.2, 128.8, 129.9, 132.5, 145.8, 147.3, 148.5,
148.1, 166.3, 177.5. Anal. (C26H26FN3O6) C, H, N. Cal-
culated C, 63.02; H, 5.29; N, 8.48. Found: C, 63.00; H,
5.29; N, 8.46.
piperazine), 3.51 (s, 3H, OCH3), 4.6 (d, 2H, 3-CH2 of
dihydrobenzodioxinyl), 5.14 (t, 1H, 2-CH of dihydro-
benzodioxinyl), 6.5–6.71 (m, 4H, Ar-H), 7.75 (s, 1H,
C5-H), 8.79 (s, 1H, C2-H), 14.6 (s, 1H, COOH); 13C
NMR (DMSO-d6) d ppm: 5.6, 36.1, 47.4, 48.5, 66.2,
85.9, 108.9, 109.3, 115.0, 119.2, 121.0, 129.8, 132.4,
145.6, 146.7, 148.1, 166.3, 168.7, 177.5. Anal.
(C27H26FN3O7) C, H, N. Calculated C, 61.95; H, 5.01;
N, 8.03. Found: C, 61.95; H, 5.00; N, 8.02.
3.3.6. 7-(4-((Benzo[d][1,3]dioxol-5-yl)methyl)piperazin-1-
yl)-1-cyclopropyl-1,4-dihydro-6-fluoro-8-methoxy-5-nitro-
4-oxoquinoline-3-carboxylic acid (13c). Yield: 80%; mp
188–190 °C; 1H NMR (DMSO-d6) d ppm: 0.28–0.48
(m, 4H, cyclopropyl), 1.35 (m, 1H, cyclopropyl), 2.82
(t, 4H, 3,5-CH2 of piperazine), 3.1 (t, 4H, 2,6-CH2 of
piperazine), 3.55 (s, 3H, OCH3), 3.6 (s, 2H, CH2 of pip-
eranoyl), 5.86 (s, 2H, –OCH2O–), 6.42–6.62 (m, 3H, Ar-
H), 8.8 (s, 1H, C2-H), 14.6 (s, 1H, COOH); 13C NMR
(DMSO-d6) d ppm: 5.6, 36.1, 50.3, 52.2, 56.1, 60.4,
108.2, 109.5, 101.3, 113.9, 115.1, 119.4, 122.2, 128.8,
129.9, 130.7, 131.8, 132.5, 147.3, 148.5, 148.1, 166.3,
177.5. Anal. (C26H25FN4O8) C, H, N. C, H, N. Calcu-
lated C, 57.78; H, 4.66; N, 10.37. Found: C, 57.81; H,
4.64; N, 10.37.
3.3.10. 1-Cyclopropyl-1,4-dihydro-7-(4-(2,3-dihydrobenzo[b]
[1,4]dioxin-2-oyl))(piperazin-1-yl)-6-fluoro-8-methoxy-5-
nitro-4-oxoquinoline-3-carboxylic acid (13e). Yield: 83%;
mp 172–174 °C; 1H NMR (DMSO-d6) d ppm: 0.28–0.48
(m, 4H, cyclopropyl), 1.35 (m, 1H, cyclopropyl), 3.26 (t,
4H, 2,6-CH2 of piperazine), 3.4 (t, 4H, 3,5-CH2 of piper-
azine), 3.51 (s, 3H, OCH3), 4.6 (d, 2H, 3-CH2 of
dihydrobenzodioxinyl), 5.14 (t, 1H, 2-CH of dihydro-
benzodioxinyl), 6.5–6.71 (m, 4H, Ar-H), 8.79 (s, 1H,
C2-H), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) d
ppm: 5.6, 36.1, 47.4, 48.5, 66.2, 85.9, 107.9, 109.3,
115.0, 119.2, 121.0, 129.8, 130.5, 131.7, 132.4, 146.7,
148.1, 152.5, 166.3, 168.7, 177.5. Anal. (C27H25FN4O9)
C, H, N. Calculated C, 57.04; H, 4.43; N, 9.86. Found:
C, 57.07; H, 4.46; N, 9.86.
3.3.7. 1-Cyclopropyl-1,4-dihydro-6-fluoro-8-methoxy-7-
(4-methyl-3-phenylpiperazin-1-yl)-4-oxoquinoline-3-car-
boxylic acid (12d). Yield: 66%; mp 115–117 °C; 1H
NMR (DMSO-d6) d ppm: 0.28–0.52 (m, 4H, cyclopro-
pyl), 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), 3.53 (s,
3H, OCH3), 4.12 (t, 1H, 3-CH of piperazine), 7.0–7.2
(m, 5H, Ar-H), 7.81 (s, 1H, C5-H), 8.5 (s, 1H, C2-H),
14.4 (s, 1H, COOH); 13C NMR (DMSO-d6) d ppm:
5.6, 36.0, 40.5, 52.2, 56.2, 59.0, 63.4, 108.4, 109.4,
119.8, 127.3, 128.5, 131.5, 135.4, 137.4, 145.3, 148.2,
152.7, 166.3, 177.5. Anal. (C25H26FN3O4) C, H, N. Cal-
culated C, 66.51; H, 5.08; N, 9.31. Found: C, 66.52; H,
5.08; N, 9.31.
3.3.11. 1-Cyclopropyl-1,4-dihydro-6-fluoro-8-methoxy-
7-thiomorpholinoquinoline-4-oxoquinoline-3-carboxylic
acid (12f) (CAS No. 114213-99-9). Yield: 70%; mp 168–
170 °C.
3.3.12. 1-Cyclopropyl-1,4-dihydro-6-fluoro-8-methoxy-5-
nitro-7-thiomorpholinoquinoline-4-oxoquinoline-3-carbox-
1
ylic acid (13f). Yield: 81%; mp 163–165 °C; H NMR
(DMSO-d6) d ppm: 0.28–0.54 (m, 4H, cyclopropyl),
1.36 (m, 1H, cyclopropyl), 2.64 (t, 4H, 3,5-CH2 of thi-
omorpholine), 3.38 (t, 4H, 2,6-CH2 of thiomorpholine),
3.51 (s, 3H, OCH3), 8.9 (s, 1H, C2-H), 14.4 (s, 1H,
COOH); 13C NMR (DMSO-d6) d ppm: 5.6, 28.2, 36.3,
50.2, 56.2, 107.8, 109.5, 130.7, 131.7, 133.6, 148.2,
152.7, 166.3, 177.5. Anal. (C18H18FN3O6S) C, H, N.
Calculated C, 51.06; H, 4.28; N, 9.92. Found: C,
51.02; H, 4.25; N, 9.91.
3.3.8. 1-Cyclopropyl-1,4-dihydro-6-fluoro-8-methoxy-7-
(4-methyl-3-phenylpiperazin-1-yl)-5-nitro-4-oxoquinoline-
1
3-carboxylic acid (13d). Yield: 80%; mp 154–156 °C; H
3.3.13. 1-Cyclopropyl-1,4-dihydro-7-(2,6-dimethylmor-
pholino)-6-fluoro-8-methoxy-4-oxoquinoline-3-carboxylic
acid (12g). Yield: 65%; mp 229–231 °C; 1H NMR
(DMSO-d6) d ppm: 0.28–0.48 (m, 4H, cyclopropyl),
1.35 (m, 1H, cyclopropyl), 1.2 (d, 6H, 2,6-CH3 of mor-
pholino), 3.0 (d, 4H, 2,6-CH2 of morpholine), 3.56 (s,
3H, OCH3), 3.9 (m, 2H, 3,5-CH of morpholine), 7.1
(s, 1H, C5-H), 7.9 (s, 1H, C2-H), 14.6 (s, 1H, COOH);
13C NMR (DMSO-d6) d ppm: 5.6, 36.3, 56.3, 69.5,
65.4, 108.9, 109.3, 119.5, 129.9, 132.6, 145.9, 148.2,
166.3, 177.5. Anal. (C20H23FN2O5) C, H, N. Calculated
C, 61.53; H, 5.94; N, 7.18. Found: C, 61.53; H, 5.96; N,
7.19.
NMR (DMSO-d6) d ppm: 0.28–0.52 (m, 4H, cyclopro-
pyl), 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), 3.53 (s,
3H, OCH3), 4.12 (t, 1H, 3-CH of piperazine), 7.0–7.2
(m, 5H, Ar-H), 8.5 (s, 1H, C2-H), 14.4 (s, 1H, COOH);
13C NMR (DMSO-d6) d ppm: 5.6, 36.0, 40.5, 52.2, 56.2,
59.0, 63.4, 107.8, 109.4, 127.3, 128.5, 130.8, 131.5, 135.4,
137.4, 148.2, 152.7, 166.3, 177.5. Anal. (C25H25FN4O6)
C, H, N. Calculated C, 60.48; H, 5.08; N, 11.28. Found:
C, 60.46; H, 5.05; N, 11.30.
3.3.9. 1-Cyclopropyl-1,4-dihydro-7-(4-(2,3-dihydrobenzo[b]
[1,4]dioxin-2-oyl))(piperazin-1-yl)-6-fluoro-8-methoxy-4-
oxoquinoline-3-carboxylic acid (12e). Yield: 64%; mp
171–172 °C; 1H NMR (DMSO-d6) d ppm: 0.28–0.48
(m, 4H, cyclopropyl), 1.35 (m, 1H, cyclopropyl), 3.26
(t, 4H, 2,6-CH2 of piperazine), 3.4 (t, 4H, 3,5-CH2 of
3.3.14. 1-Cyclopropyl-1,4-dihydro-7-(2,6-dimethylmorpho-
lino)-6-fluoro-8-methoxy-5-nitro-4-oxoquinoline-3-carbox-
ylic acid (13g). Yield: 84%; mp 124–126 °C; H NMR
(DMSO-d6) d ppm: 0.28–0.48 (m, 4H, cyclopropyl),
1.35 (m, 1H, cyclopropyl), 1.2 (d, 6H, 2,6-CH3 of mor-
1

2566
P. Senthilkumar et al. / Bioorg. Med. Chem. 16 (2008) 2558–2569
pholino), 3.0 (d, 4H, 2,6-CH2 of morpholine), 3.56 (s,
3H, OCH3), 3.9 (m, 2H, 3,5-CH of morpholine), 7.9
(s, 1H, C2-H), 14.6 (s, 1H, COOH); 13C NMR
(DMSO-d6) d ppm: 5.6, 36.3, 56.3, 69.5, 65.4, 107.9,
109.3, 119.5, 129.9, 130.9, 131.5, 133.4, 148.2, 152.3,
166.3, 177.5. Anal. (C20H22FN3O7) C, H, N. Calculated
C, 55.17; H, 5.09; N, 9.65. Found: C, 55.17; H, 5.09; N,
9.65.
3.3.19. 7-(4-(6-Chloro-1,2-dihydro-2-oxobenzo[d]imida-
zol-3-yl)piperidin-1-yl)-1-cyclopropyl-1,4-dihydro-6-fluoro-
8-methoxy-4-oxoquinoline-3-carboxylic acid (12j). Yield:
70%; mp 259–261 °C; 1H NMR (DMSO-d6) d ppm:
0.28–0.48 (m, 4H, cyclopropyl), 1.35 (m, 1H, cyclo-
propyl), 1.6–2.4 (m, 8H, 4-CH2 of piperidine), 3.52
(s, 3H, OCH3), 4.1 (br m, 1H, CH of piperidine),
6.8–7.7 (m, 3H, Ar-H), 7.88 (s, 1H, C5-H), 8.3 (s,
1H, C2-H), 10.8 (s, 1H, NH), 14.6 (s, 1H, COOH);
3.3.15. 1-Cyclopropyl-1,4-dihydro-6-fluoro-8-methoxy-7-
(4-(piperidin-1-yl)piperidin-1-yl)-4-oxoquinoline-3-carbox-
13C NMR (DMSO-d6)
d ppm: 5.7, 36.2, 26.9,
46.3, 50.3, 108.8, 109.3, 122.2, 123.4, 124.7, 129.8,
130.1, 131.3, 132.3, 145.6, 148.1, 151.8, 166.3, 177.5.
Anal. (C26H24ClFN4O5) C, H, N. Calculated C,
59.26; H, 4.59; N, 10.63. Found: C, 59.26; H, 4.61;
N, 10.61.
1
ylic acid (12h). Yield: 63%; mp 163–165 °C; H NMR
(DMSO-d6) d ppm: 0.28–0.48 (m, 4H, cyclopropyl),
1.35 (m, 1H, cyclopropyl), 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), 3.57 (s, 3H, OCH3), 7.32 (s, 1H, C5-H), 8.2 (s,
1H, C2-H), 14.6 (s, 1H, COOH); 13C NMR (DMSO-
d6) d ppm: 5.6, 36.2, 25.7, 26.7, 28.5, 47.1, 52.8, 56.2,
58.5, 109.3, 107.8, 108.9, 129.8, 132.5, 145.6, 146.6,
148.1, 166.3, 177.5. Anal. (C24H30FN3O4) C, H, N. Cal-
culated C, 64.99; H, 6.82; N, 9.47. Found: C, 65.01; H,
6.80; N, 9.47.
3.3.20. 7-(4-(6-Chloro-1,2-dihydro-2-oxobenzo[d]imida-
zol-3-yl)piperidin-1-yl)-1-cyclopropyl-1,4-dihydro-6-fluoro-
8-methoxy-5-nitro-4-oxoquinoline-3-carboxylic acid (13j).
Yield: 78%; mp 178–180 °C; 1H NMR (DMSO-d6) d
ppm: 0.28–0.48 (m, 4H, cyclopropyl), 1.35 (m, 1H,
cyclopropyl), 1.6–2.4 (m, 8H, 4-CH2 of piperidine),
3.52 (s, 3H, OCH3), 4.1 (br m, 1H, CH of piperidine),
6.8–7.7 (m, 3H, Ar-H), 8.3 (s, 1H, C2-H), 10.8 (s, 1H,
NH), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) d
ppm: 5.7, 36.2, 26.9, 46.3, 50.3, 107.8, 109.3, 122.2,
123.4, 124.7, 130.1, 131.3, 133.3, 148.1, 151.8, 166.3,
177.5. Anal. (C26H23ClFN5O7) C, H, N. Calculated C,
54.60; H, 4.05; N, 12.24. Found: C, 54.61; H, 4.06; N,
12.23.
3.3.16. 1-Cyclopropyl-1,4-dihydro-6-fluoro-8-methoxy-5-
nitro-7-(4-(piperidin-1-yl)piperidin-1-yl)-4-oxoquinoline-3-
1
carboxylic acid (13h). Yield: 69%; mp 167–169 °C; H
NMR (DMSO-d6) d ppm: 0.28–0.48 (m, 4H, cyclopro-
pyl), 1.35 (m, 1H, cyclopropyl), 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), 3.57 (s, 3H, OCH3), 8.2 (s, 1H, C2-H), 14.6 (s,
1H, COOH); 13C NMR (DMSO-d6) d ppm: 5.6, 36.2,
25.7, 26.7, 28.5, 47.1, 52.8, 56.2, 58.5, 109.3, 107.8,
130.7, 131.8, 133.5, 148.1, 152.7, 166.3, 177.5. Anal.
(C24H29FN4O6) C, H, N. Calculated C, 59.01; H, 5.98;
N, 11.47. Found: C, 59.01; H, 5.97; N, 11.49.
3.3.21. 1-Cyclopropyl-7-(3-(diethylcarbamoyl)piperidin-1-
yl)-1,4-dihydro-6-fluoro-8-methoxy-4-oxoquinoline-3-car-
1
boxylic acid (12k). Yield: 70%; mp 85–87 °C; H NMR
(DMSO-d6) d ppm: 0.28–0.48 (m, 4H, cyclopropyl),
1.35 (m, 1H, cyclopropyl), 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), 3.53 (s, 3H, OCH3), 7.8 (s, 1H, C5-H), 8.94
(s, 1H, C2-H), 14.6 (s, 1H, COOH); 13C NMR
(DMSO-d6) d ppm: 5.6, 36.3, 12.9, 22.3, 28.2, 41.3,
42.6, 51.0, 54.4, 108.9, 109.3, 118.8, 129.8, 132.6,
145.4, 146.2, 148.1, 166.3, 177.5. Anal. (C24H30FN3O5)
C, H, N. Calculated C, 62.73; H, 6.58; N, 9.14. Found:
C, 62.72; H, 6.60; N, 9.14.
3.3.17. 7-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)-1-
cyclopropyl-1,4-dihydro-6-fluoro-8-methoxy-4-oxoquino-
line-3-carboxylic acid (12i). Yield: 68%; mp 112–114 °C;
1H NMR (DMSO-d6) d ppm: 0.28–0.46 (m, 4H, cyclo-
propyl), 1.33 (m, 1H, cyclopropyl), 2.0 (t, 4H, 3,5-CH2
of piperidine), 2.7 (t, 4H, 2,6-CH2 of piperidine), 3.51
(s, 3H, OCH3), 7.1–7.18 (m, 4H, Ar-H), 7.82 (s, 1H,
C5-H), 8.88 (s, 1H, C2-H), 10.0 (br s, 1H, OH), 14.6
(s, 1H, COOH); 13C NMR (DMSO-d6) d ppm: 5.6,
36.0, 38.2, 42.6, 56.1, 74.5, 108.9, 109.3, 118.8, 129.6,
131.5, 138.1, 145.7, 148.2, 166.3, 177.5. Anal.
(C25H24ClFN2O5) C, H, N. Calculated C, 61.67; H,
4.97; N, 5.75. Found: C, 61.69; H, 4.99; N, 5.74.
3.3.22. 1-Cyclopropyl-7-(3-(diethylcarbamoyl)piperidin-1-
yl)-1,4-dihydro-6-fluoro-8-methoxy-5-nitro-4-oxoquinoline-
1
3-carboxylic acid (13k). Yield: 75%; mp 128–130 °C; H
NMR (DMSO-d6) d ppm: 0.28–0.48 (m, 4H, cyclopro-
pyl), 1.35 (m, 1H, cyclopropyl), 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), 3.53 (s, 3H, OCH3), 8.94 (s, 1H,
C2-H), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) d
ppm: 5.6, 36.3, 12.9, 22.3, 28.2, 41.3, 42.6, 51.0, 54.4,
107.9, 109.3, 118.8, 129.8, 130.6, 131.7, 133.6, 148.1,
152.5, 166.3, 177.5. Anal. (C24H29FN4O7) C, H, N. Cal-
culated C, 57.14; H, 5.79; N, 11.11. Found: C, 57.15; H,
5.81; N, 11.11.
3.3.18. 7-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)-1-
cyclopropyl-1,4-dihydro-6-fluoro-8-methoxy-5-nitro-4-oxo-
quinoline-3-carboxylic acid (13i). Yield: 76%; mp 136–
138 °C; 1H NMR (DMSO-d6) d ppm: 0.28–0.46 (m,
4H, cyclopropyl), 1.33 (m, 1H, cyclopropyl), 2.0 (t,
4H, 3,5-CH2 of piperidine), 2.7 (t, 4H, 2,6-CH2 of piper-
idine), 3.51 (s, 3H, OCH3), 7.1–7.18 (m, 4H, Ar-H), 8.88
(s, 1H, C2-H), 10.0 (br s, 1H, OH), 14.6 (s, 1H, COOH);
13C NMR (DMSO-d6) d ppm: 5.6, 36.0, 38.2, 42.6, 56.1,
74.5, 107.9, 109.3, 118.8, 129.6, 130.6, 131.5, 138.1,
148.2, 152.4, 166.3, 177.5. Anal. (C25H23ClFN3O7) C,
H, N. Calculated C, 56.45; H, 4.36; N, 7.90. Found:
C, 56.47; H, 4.33; N, 7.91.
3.3.23. 1-Cyclopropyl-1,4-dihydro-7-(1,4-dioxa-8-azaspi-
ro[4.5]dec-8-yl)-6-fluoro-8-methoxy-4-oxoquinoline-3-car-
boxylic acid (12l) (CAS No 848070-83-7). Yield: 73%;
mp 254–256 °C.

P. Senthilkumar et al. / Bioorg. Med. Chem. 16 (2008) 2558–2569
2567
3.3.24. 1-Cyclopropyl-1,4-dihydro-7-(1,4-dioxa-8-azaspi-
ro[4.5]dec-8-yl)-6-fluoro-8-methoxy-5-nitro-4-oxoquinoline-
3-carboxylic acid (13l). Yield: 69%; mp 152–154 °C; H
ppm: 0.28–0.54 (m, 4H, cyclopropyl), 1.36 (m, 1H,
cyclopropyl), 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), 3.52 (s, 3H, OCH3), 3.73 (s, 3H, –OCH3), 6.8–
7.1 (m, 4H, Ar-H), 8.56 (s, 1H, C2-H), 14.4 (s, 1H,
COOH); 13C NMR (DMSO-d6) d ppm: 5.6, 24.3, 25.5,
28.6, 30.4, 36.0, 37.9, 42.9, 56.4, 107.7, 109.3, 114.1,
123.8, 129.1, 130.5, 131.9, 135.4, 148.2, 152.7, 158.4,
166.3, 177.5. Anal. (C31H32FN3O7) C, H, N. Calculated
C, 64.46; H, 5.58; N, 7.27. Found: C, 64.44; H, 5.55; N,
7.27.
1
NMR (DMSO-d6) d ppm: 0.28–0.52 (m, 4H, cyclopro-
pyl), 1.38 (m, 1H, cyclopropyl), 1.78–2.4 (m, 8H, 4-CH2
of azaspirodecane), 3.51 (s, 3H, OCH3), 3.96 (m, 4H, 2-
CH2 of azaspirodecane), 8.9 (s, 1H, C2-H), 14.4 (s, 1H,
COOH); 13C NMR (DMSO-d6) d ppm: 5.6, 34.0, 36.0,
39.8, 56.2, 64.3, 107.8, 109.6, 130.6, 131.7, 133.5, 148.1,
152.7, 166.3, 177.5. Anal. (C21H22FN3O8) C, H, N. Calcu-
lated C, 54.43; H, 4.79; N, 9.07. Found: C, 54.43; H, 4.78;
N, 9.05.
3.3.29. 7-(2-Carboxy-5,6-dihydroimidazo[1,2-a]pyrazin-
7(8H)-yl)-1-cyclopropyl-1,4-dihydro-6-fluoro-8-methoxy-4-
oxoquinoline-3-carboxylic acid (12o). Yield: 79%; mp 169–
171 °C; H NMR (DMSO-d6) d ppm: 0.28–0.48 (m, 4H,
cyclopropyl), 1.35 (m, 1H, cyclopropyl), 3.1–3.8 (m, 6H,
3-CH2), 3.55 (s, 3H, OCH3), 7.6 (s, 1H, CH), 7.63 (s,
1H, C5-H), 8.78 (s, 1H, C2-H), 12.12 (s, 1H, 2-COOH),
14.6 (s, 1H, 3-COOH); 13C NMR (DMSO-d6) d ppm:
5.7, 36.3, 37.4, 50.5, 55.9, 57.2, 109.3, 118.8, 130.6,
132.2, 139.4, 145.6, 148.2, 158.4, 166.3, 167.9, 177.5. Anal.
(C21H19FN4O6) C, H, N. Calculated C, 57.01; H, 4.33; N,
12.66. Found: C, 57.01; H, 4.33; N, 12.66.
3.3.25. 7-(1-(tert-Butylcarbamoyl)-3,4-dihydroisoquino-
lin-2(1H)-yl)-1-cyclopropyl-1,4-dihydro-6-fluoro-8-methoxy-
4-oxoquinoline-3-carboxylic acid (12m). Yield: 63%; mp
70–72 °C; H NMR (DMSO-d6) d ppm: 0.28–0.48 (m,
4H, cyclopropyl), 1.35 (m, 1H, cyclopropyl), 1.3 (s, 9H,
3-CH3), 2.66–2.9 (m, 4H, 2-CH2 of isoquinoline), 3.53 (s,
3H, OCH3), 4.85 (s, 1H, CH of isoquinoline), 6.7–7.1 (m,
4H, Ar-H), 7.8 (s, 1H, C5-H), 8.8 (s, 1H, C2-H), 10.2 (s,
1H, NH), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) d
ppm: 5.6, 36.3, 25.9, 47.4, 48.3, 56.1, 65.5, 108.8, 109.3,
118.8, 127.0, 127.9, 128.6, 129.5, 132.1, 135.4, 138.1,
145.7, 146.2, 148.1, 166.3, 168.7, 177.5. Anal.
(C28H30FN3O5) C, H, N. Calculated C, 66.26; H, 5.96; N,
8.28. Found: C, 66.26; H, 5.95; N, 8.28.
1
1
3.3.30. 7-(2-Carboxy-5,6-dihydroimidazo[1,2-a]pyrazin-
7(8H)-yl)-1-cyclopropyl-1,4-dihydro-6-fluoro-8-methoxy-
5-nitro-4-oxoquinoline-3-carboxylic acid (13o). Yield:
84%; mp 208–210 °C; 1H NMR (DMSO-d6) d ppm:
0.28–0.48 (m, 4H, cyclopropyl), 1.35 (m, 1H, cyclopro-
pyl), 3.1–3.8 (m, 6H, 3-CH2), 3.55 (s, 3H, OCH3), 7.6
(s, 1H, CH), 8.78 (s, 1H, C2-H), 12.12 (s, 1H, 2-COOH),
14.6 (s, 1H, 3-COOH); 13C NMR (DMSO-d6) d ppm:
5.7, 36.3, 37.4, 50.5, 55.9, 57.2, 107.7, 109.3, 130.6,
131.7, 132.2, 133.6, 139.4, 148.2, 152.5, 158.4, 166.3,
167.9, 177.5. Anal. (C21H18FN5O8) C, H, N. Calculated
C, 51.75; H, 3.72; N, 14.37. Found: C, 51.75; H, 3.73; N,
14.35.
3.3.26. 7-(1-(tert-Butylcarbamoyl)-3,4-dihydroisoquinolin-
2(1H)-yl)-1-cyclopropyl-1,4-dihydro-6-fluoro-8-methoxy-
5-nitro-4-oxoquinoline-3-carboxylic acid (13m). Yield:
82%; mp 172–174 °C; 1H NMR (DMSO-d6) d ppm:
0.28–0.48 (m, 4H, cyclopropyl), 1.35 (m, 1H, cyclopro-
pyl), 1.3 (s, 9H, 3-CH3), 2.66–2.9 (m, 4H, 2-CH2 of iso-
quinoline), 3.53 (s, 3H, OCH3), 4.85 (s, 1H, CH of
isoquinoline), 6.7–7.1 (m, 4H, Ar-H), 8.8 (s, 1H, C2-H),
10.2 (s, 1H, NH), 14.6 (s, 1H, COOH); 13C NMR
(DMSO-d6) d ppm: 5.6, 36.3, 25.9, 47.4, 48.3, 56.1, 65.5,
108.8, 109.3, 118.8, 127.0, 127.9, 128.6, 130.6, 131.7,
133.5, 135.4, 138.1, 148.1, 152.3, 166.3, 168.7, 177.5.
Anal. (C28H29FN4O7) C, H, N. Calculated C, 60.86; H,
5.29; N, 10.14. Found C, 60.86; H, 5.30; N, 10.17.
3.3.31. 1-Cyclopropyl-1,4-dihydro-7-(4,4-dimethyloxaz-
olidin-3-yl)-6-fluoro-8-methoxy-4-oxoquinoline-3-carbox-
1
ylic acid (12p). Yield: 62%; mp 180–182 °C; H NMR
(DMSO-d6) d ppm: 0.28–0.48 (m, 4H, cyclopropyl),
1.35 (m, 1H, cyclopropyl), 1.16 (s, 6H, 2-CH3), 3.41 (s,
2H, 5-CH2 of oxazolidinyl), 3.57 (s, 3H, OCH3), 4.6
(s, 2H, 2CH of oxazolidinyl), 7.69 (s, 1H, C5-H), 8.85
(s, 1H, C2-H), 14.6 (s, 1H, COOH); 13C NMR
(DMSO-d6) d ppm: 5.6, 36.5, 55.9, 65.5, 83.6, 84.4,
108.8, 109.1, 119.3, 129.7, 131.4, 132.4, 135.4, 145.6,
146.5, 149.4, 166.3, 177.5. Anal. (C19H21FN2O5) C, H,
N. Calculated C, 60.63; H, 5.62; N, 7.44. Found: C,
60.60; H, 5.65; N, 7.47.
3.3.27. 1-Cyclopropyl-7-(8-(4-methoxybenzyl)-3,4,5,6,7,8-
hexahydroisoquinolin-2(1H)-yl)-1,4-dihydro-6-fluoro-8-
methoxy-4-oxoquinoline-3-carboxylic acid (12n). Yield:
62%; mp 176–178 °C; 1H NMR (DMSO-d6) d ppm:
0.28–0.54 (m, 4H, cyclopropyl), 1.36 (m, 1H, cyclopro-
pyl), 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),
3.52 (s, 3H, OCH3), 3.73 (s, 3H, –OCH3), 6.8–7.1 (m,
4H, Ar-H), 7.88 (s, 1H, C5-H), 8.56 (s, 1H, C2-H),
14.4 (s, 1H, COOH); 13C NMR (DMSO-d6) d ppm:
5.6, 24.3, 25.5, 28.6, 30.4, 36.0, 37.9, 42.9, 56.4, 108.7,
109.3, 114.1, 119.2, 123.8, 129.1, 135.4, 145.6, 146.5,
148.2, 158.4, 166.3, 177.5. Anal. (C31H33FN2O5) C, H,
N. Calculated C, 69.91; H, 6.25; N, 5.26. Found C,
69.90; H, 6.26; N, 5.25.
3.3.32. 1-Cyclopropyl-1,4-dihydro-7-(4,4-dimethyloxaz-
olidin-3-yl)-6-fluoro-8-methoxy-5-nitro-4-oxoquinoline-3-
1
carboxylic acid (13p). Yield: 77%; mp 160–162 °C; H
NMR (DMSO-d6) d ppm: 0.28–0.48 (m, 4H, cyclopro-
pyl), 1.35 (m, 1H, cyclopropyl), 1.16 (s, 6H, 2-CH3),
3.41 (s, 2H, 5-CH2 of oxazolidinyl), 3.57 (s, 3H,
OCH3), 4.6 (s, 2H, 2-CH of oxazolidinyl), 8.85 (s, 1H,
C2-H), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) d
ppm: 5.6, 36.5, 55.9, 65.5, 83.6, 84.4, 107.8, 109.1,
131.4, 135.4, 149.4, 152.9, 166.3, 177.5. Anal.
3.3.28. 1-Cyclopropyl-7-(8-(4-methoxybenzyl)-3,4,5,6,7,8-
hexahydroisoquinolin-2(1H)-yl)-1,4-dihydro-6-fluoro-8-
methoxy-5-nitro-4-oxoquinoline-3-carboxylic acid (13n).
Yield: 60%; mp 108–110 °C; 1H NMR (DMSO-d6) d

2568
P. Senthilkumar et al. / Bioorg. Med. Chem. 16 (2008) 2558–2569
(C19H20FN3O7) C, H, N. Calculated C, 54.16; H, 4.78;
N, 9.97. Found: C, 54.14; H, 4.77; N, 9.99.
sessed on the basis of cellular conversion of MTT into
a formazan product using the Promega Cell Titer 96
non-radioactive cell proliferation assay.
3.4. Synthesis of 5-amino-1-cyclopropyl-1,4-dihydro-6-flu-
oro-7-(substituted secondary amines)-8-methoxy-4-oxo-
quinoline-3-carboxylic acid (14c,n)
3.7. In vivo antimycobacterial activity
One compound was tested for efficacy against MTB at a
dose of 25 mg/kg in 6-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 continued for
10 days. After 35 days post-infection the spleens and
right lungs were aseptically removed and ground in a tis-
sue homogenizer, the number of viable organisms was
determined by serial 10-fold dilutions and subsequent
inoculation onto 7H10 agar plates. Cultures were incu-
bated at 37 °C in ambient air for 4 weeks prior to count-
ing. Bacterial counts were measured and compared with
the counts from negative controls (vehicle treated) in
lung and in spleen.
Compounds 13c and 13n were further reduced by cata-
lytic reduction of the nitro group to yield 14c and 14n.
3.4.1. 5-Amino-1-cyclopropyl-1,4-dihydro-6-fluoro-7-(4-
((benzo[d][1,3]dioxol-5-yl)methyl)piperazin-1-yl)-8-meth-
oxy-4-oxoquinoline-3-carboxylic acid (14c). Yield: 76%;
mp 125–127 °C; 1H NMR (DMSO-d6) d ppm: 0.28–
0.48 (m, 4H, cyclopropyl), 1.35 (m, 1H, cyclopropyl),
2.82 (t, 4H, 3,5-CH2 of piperazine), 3.1 (t, 4H, 2,6-
CH2 of piperazine), 3.55 (s, 3H, OCH3), 3.6 (s, 2H,
CH2 of piperanoyl), 5.86 (s, 2H, –OCH2O–), 6.42–6.62
(m, 3H, Ar-H), 11.8 (br s, 2H, NH2), 8.8 (s, 1H, C2-
H), 14.6 (s, 1H, COOH); 13C NMR (DMSO-d6) d
ppm: 177.5, 166.3, 148.1, 148.5, 147.3, 145.8, 132.5,
129.9, 128.8, 122.2, 119.4, 115.1, 113.9, 101.3, 109.5,
108.2, 60.4, 56.1, 52.2, 50.3, 36.1, 5.6. Anal.
(C26H27FN4O6) C, H, N. Calculated C, 61.17; H, 5.33;
N, 10.97. Found: C, 61.16; H, 5.35; N, 10.95.
3.8. DNA gyrase supercoiling assay
The enzyme was isolated from MC2 cells and purified as
described.10 The compounds tested were dissolved in
DMSO and pre-incubated with the enzyme. The super-
coiling assays were carried out as described previously.10
Briefly, supercoiling assays were carried out by incubat-
ing 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, 2 mM
ATP, 50 lg/ml bovine serum albumin and 90 lg/ml
yeast t-RNA in 5% (v/v) glycerol) for 30 min. Ciproflox-
acin at final concentration of 10 lg/ml was used as a po-
sitive control and another control reaction having 5%
DMSO in absence of the compounds was also per-
formed. 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 ethidium bromide to visualize
the DNA topoisomers.
3.4.2.
5-Amino-1-cyclopropyl-7-(8-(4-methoxybenzyl)-
3,4,5,6,7,8-hexahydroisoquinolin-2(1H)-yl)-1,4-dihydro-6-
fluoro-8-methoxy-4-oxoquinoline-3-carboxylic acid (14n).
Yield: 67%; mp 152–153 °C; 1H NMR (DMSO-d6) d
ppm: 0.28–0.54 (m, 4H, cyclopropyl), 1.36 (m, 1H,
cyclopropyl), 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), 3.52 (s, 3H, OCH3), 3.73 (s, 3H, –OCH3), 6.8–
7.1 (m, 4H, Ar-H), 8.56 (s, 1H, C2-H), 12.1 (br s, 2H,
NH2), 14.4 (s, 1H, COOH); 13C NMR (DMSO-d6) d
ppm: 5.6, 24.3, 25.5, 28.6, 30.4, 36.0, 37.9, 42.9, 56.4,
99.3, 109.3, 114.1, 123.8, 128.9, 130.5, 131.9, 135.4,
136.8, 148.2, 158.4, 166.3, 177.5. Anal. (C31H34FN3O5)
C, H, N. Calculated C, 67.99; H, 6.26; N, 7.67. Found:
C, 68.00; H, 6.26; N, 7.67.
3.5. 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.
3.9. 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 ga-
vage. A second group received a single dose of 140 mg
of lomefloxacin HCl/kg. This lomefloxacin dose is one
that, in preliminary experiments in this test system, pro-
duced a consistent erythema and ear thickening re-
sponse. The final 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 described previously. Animals were
irradiated for 4 h, equal to a total UV light irradiation
of approximately 18 J/cm2. Before dosing, at the end
3.6. 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-

P. Senthilkumar et al. / Bioorg. Med. Chem. 16 (2008) 2558–2569
2569
Bharath, S.; Balasubramanian, V. Antimicrob. Agents
Chemother. 2007, 51, 576.
of the irradiation period and at approximately 24, 48, 72
and 96 h after dosing, both ears of each mouse were
evaluated for changes indicative of a positive response:
erythema, oedema or a measurable increase in ear
thickness.
4. Maxwell, A. Trends Microbiol. 1997, 5, 102.
5. Anquetin, G.; Greiner, J.; Mahmoudi, N.; Santillana-
Hayat, M.; Gozalbes, R.; Farhati, K.; Derouin, F.; Aubry,
A.; Cambau, E.; Vierling, P. Eur. J. Med. Chem. 2006, 41,
1478.
6. Sato, K.; Tomioka, H.; Sano, C.; Shimizu, T.; Sano, K.;
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Acknowledgments
The authors are thankful to Dr. Vanaja Kumar, Deputy
Director, Tuberculosis Research Center, Chennai, India,
for the assistance in biological screening. The work in
BITS-Pilani is supported by grants from Council of Sci-
entific and Industrial Research [01 (1979)/05/EMR/-II]
and University Grant Commission F.30-266/2004(SR)
New Delhi, India. Work in IISc is supported by a grant
from Department of Biotechnology, Government of In-
dia entitled Center for Excellence in mycobacteria
research.
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